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  • 1. JOURNAL OF THE AMERICAN SOCIETY FOR CLINICAL LABORATORY SCIENCE A S C L S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CLINICAL LABORATORY SCIENCE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Focus: Psychostimulants
  • 2. ADDRESS CHANGES ASCLS MEMBER EDITORS Editor-in-Chief Susan J Leclair PhD CLS(NCA) Department of Medical Laboratory Science University of Massachusetts Dartmouth North Dartmouth MA 02747-2715 sleclair@umassd.edu Continuing Education Editor George Fritsma MS MT(ASCP) Department of Pathology 619 South 19th Street West Pavillion, P230 U of Alabama at Birmingham Birmingham AL 35233 gfritsma@path.uab.edu Clinical Practice Editor Bernadette Rodak MS CLS(NCA) Clinical Laboratory Science Indiana University, 409 Fesler 1120 South Avenue Indianapolis IN 46202-5133 brodak@iupui.edu Research and Reports Editor David G Fowler PhD CLS(NCA) University of Mississippi Medical Center Dept of Clinical Laboratory Sciences 2500 North State St Jackson MS 39216 dfowler@shrp.umsmed.edu Clinical Laboratory Science (ISSN 0894-959X) is published quarterly by the American Society for Clinical Laboratory Science, 6701 Democracy Blvd., Suite 300, Bethesda MD 20814; (301) 657-2768; (301) 657-2909 (fax). Annual Subscription Rates: USA Canada Non-USA Individuals $40 $65 $100 Institutions $60 $65 $100 Questions related to subscriptions should be addresssed to: SherryM@ASCLS.org. The cost of single copies is $10. Requests to replace missing issues free of charge are honored up to six months after the date of issue. Send requests to ASCLS headquarters. Annual membership dues of ASCLS are $80, $40 of which is allocated to a subscription of CLS. Periodical postage paid at Bethesda, MD and other additional Contributing Editors Eileen Carreiro-Lewandowski/N Dartmouth MA Deborah Josko/Newark NJ Elaine Kohane/Newark NJ Rebecca Laudicina/Chapel Hill NC Connie Mahon/San Antonio TX Linda Smith/San Antonio TX Michelle Wright-Kanuth/Galveston TX REVIEW BOARD Richard Bamberg/Greenville NC Kathleen Blevins/Oklahoma City OK Dianne Cearlock/DeKalb IL Peter Colaninno/Jamaica NY Jo Ann Fenn/Salt Lake City UT Ellis Frohman/St Louis MO Mildred Fuller/Norfolk VA Abraham Furman/Portland OR Richard Gregory/Indianapolis IN Jesse Guiles/Newark NJ Lester Hardegree/Bluffton SC Denise Harmening/Baltimore MD Daniel Hoefner/Elon, NC Linda Hogan/Wichita KS Virginia Hughes/Montgomery AL Donna Larson/Gresham OR Elizabeth Leinbach-Kenimer/Augusta GA Linda Kasper/Indianapolis IN Nancy Konopka/Gettysburg PA Robin Krefetz/Cherry Hill NJ Linda Laatsch/Milwaukee WI Hal Larsen/Lubbock TX LouAnn Lawrence/New Orleans LA Craig Lehmann/Stony Brook NY Lynn Little/Dallas TX David McGlasson/Lackland AFB TX Sharon Miller/St Charles IL Isaac Montoya/Houston TX Harriette Nadler/King of Prussia PA Joan Prince/Milwaukee WI Margaret Reinhart/Philadelphia PA John Seabolt/Lexington KY Stephen Sodeke/Tuskegee AL P.A.C.E.® Liaison Sharon Miller/St Charles IL ASCLS BOARD OF DIRECTORS 2004-2005 Susan Morris, President Bernie Bekken, President-Elect Barbara Brown, Past President Scott Aikey, Secretary/Treasurer Bobby Lee, Director Region I Mary Ann McLane, Director Region II Lynn Ingram, Director Region III Linda Kasper, Director Region IV Rick Panning, Director Region V John Koenig, Director Region VI Debbie Faubion, Director Region VII Susie Zanto, Director Region VIII Elizabeth L (Heidi) Smith, Director Region IX Marcia Armstrong, Director Region X Robbe Peetz, Student Forum Chair ASCLS Headquarters Executive Staff Elissa Passiment, Executive Director EDITORIAL OFFICE Schwabbauer and Associates 2167 Terra Lane PO Box 5399 Coralville IA 52241-5399 (319) 351-2922; (319) 351-2927 (fax) cls@ia.net www.ascls.org/leadership/cls/index.htm Executive Editor Marian Schwabbauer PhD Managing Editor Ivan Schwabbauer Trends and Technology Editor Mary Jane Gore 6701 Democracy Blvd, Suite 300 Bethesda MD 20814 clstt@aol.com PRODUCTION BB Design Studio 2416 E Avenue NE Cedar Rapids IA 52402 mailing offices. Advertising for CLS is accepted in accordance with the ad- vertising policy of the ASCLS. Contact the CLS advertising representative at (301) 657-2768. Manuscript Submissions: To encourage consistency in style, refer to guidelines in Scientific Style and Format – The Council of Biology Editors Manual for Authors, Editors, and Publishers, 6th ed. Detailed instructions for authors are available on the ASCLS site. Contact the CLS Editorial Office for more information. All articles published represent the opinions of the authors and do not reflect the official policy of ASCLS or the authors’ institutions unless specified. Microfilm and microfiche editions of CLS are available from University Microfilms, 300 N Zeeb Road, Ann Arbor MI 48106. Correspondence related to editorial content should be mailed to: CLS Editorial Office, PO Box 5399, Coralville IA 52241- 5399; (319) 351-2922; (319) 351-2927 (fax). cls@ia.net © Copyright 2005 American Society for Clinical Laboratory Science Inc. All rights reserved. ASCLS Vision Statement The American Society for Clinical Laboratory Science, as the pre- eminent organization for clinical laboratory science practitioners, provides dynamic leadership and vigorously promotes all aspects ofclinicallaboratorysciencepractice,educationandmanagement to ensure excellent, accessible cost-effective laboratory services for the consumers of health care. AMERICAN SOCIETY FOR CLINICAL LABORATORY SCIENCE 6701 Democracy Blvd, Suite 300 Bethesda, Maryland 20817 (301) 657-2768, (301) 657-2909 (fax) www.ascls.org/ ASCLS Mission Statement The mission of the American Society for Clinical Laboratory Science is to promote the profession of clinical laboratory science and provide beneficial services to those who practice it. To enable its members to provide quality services for all consumers, the society is committed to the continuous quest for excellence in all its activities. Inclusion in the journal of product names or author opinions does not constitute endorsement by either Clinical Laboratory Science or ASCLS. Postmaster: Send address changes to Clinical Laboratory Science, 6701 Democracy Blvd, Suite 300, Bethesda MD 20814. Postmaster: Send address changes to Clinical Laboratory Science, 6701 Democracy Blvd, Suite 300, Bethesda MD 20814.
  • 3. CLINICAL LABORATORY SCIENCE DIALOGUE AND DISCUSSION 66 Health Disparities and Public Policy Isaac D Montoya 67 Pay for Performance Kathy Hansen, Don Lavanty CLINICAL PRACTICE 69 Evaluation of Malaria Parasite Screening Procedures Among Sudanese Blood Donors Mohamed Siddig M Ali, Abdul Gader Mohamed Yousif, Mustafa Salih Mustafa, Malik Hassan Ibrahim 74 Clinical Laboratory Educators Conference 2005 Abstracts 79 ASCLS 2005 ANNUAL MEETING PROGRAM RESEARCH AND REPORTS 84 Membranoproliferative Glomerulonephritis Type II in a 10-year-old Girl Martha E Tibbs, Sharon P Andreoli, Carrie L Phillips 90 Children on the Frontline Against E. coli: Typical Hemolytic-Uremic Syndrome Heidi Anderson 100 2003 Workforce Survey of Hospital Clinical Laboratories in New Jersey Elaine M Keohane, Mary Ellen Schaad, Karen Feeney FOCUS: PSYCHOSTIMULANTS 107 Mechanism of Action and Therapeutic Uses of Psychostimulants Kevin F Foley 114 The Use and Abuse of Psychostimulants Susan B Gock, Victor A Skrinska 119 Measurement of 3,4-MDMA and Related Amines in Diagnostic and Forensic Laboratories Victor A Skrinska, Susan B Gock 124 CONTINUING EDUCATION QUESTIONS 127 TRENDS AND TECHNOLOGY SPRING 2005 VOLUME 18/NUMBER 2
  • 4. 66 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE EDITORIAL Health Disparities and Public Policy ISAAC D MONTOYA Health-related disparities are significant differences in the incidence, prevalence, morbidity, mortality, and burden of disease among specific population groups. Medical research has demonstrated glaring disparities for a wide range of health problems and among different groups. It is important that we improve our understanding of what causes health disparities and work to address them. In doing so there is a tendency to discuss health disparities solely in terms of differences among racial and ethnic groups; however it is a myth that only these groups experience disparities. Unfortunately, health dispari- ties occur in all segments of society. Medical research working to achieve the goal of alleviating health disparities in the United States is a goal with broad sup- port that adopts the well-established perspective that various forms of discrimination and poverty are the major contributors to unequal health status. One idea that has been put forth is that genetic research plays a significant role in alleviating this nationalproblem,whichmayoverstatetheimportanceofgenet- ics in explaining health disparities. Over reliance on genetics as a factor in explaining health disparities may lead us to miss the factors that we can control, thus reinforcing stereotyping which contributes to disparities in the first place. Access to care is a primary reason for disparities. For example, disparities due to limited access to coronary artery bypass graft (CABG) surgery are well documented. Evidence shows that even when patients do receive CABG surgery, the poor, people living in rural areas, and racial minorities are more likely to be treated by lower quality providers. In addition, some disparities occur due to the hospital to which patients are admitted and to alesserdegreetobeingtreatedbyalow-volumesurgeon.Efforts to eliminate health disparities should address not only access to care, but also access to high-quality care. Obesity is a major concern nationally that crosses all socio- economic groups. Genetics, and lack of access to dietary/ nutritional information, healthy foods, and needed exercise all contribute to the problem of weight control. A recent study, funded by the National Institutes of Health, analyzed a sample of Americans’ weight. The results show continuing disparities by sex and between racial/ethnic groups in the prevalence of obesity. Adultswithdevelopmentaldisabilitiesexperiencedisparitiesthat areoftennotasobvious.Arecentstudyfoundthispopulationto bemorelikelytoleadsedentarylifestylesandseventimesaslikely to report inadequate emotional support compared to adults without disabilities. Adults with disabilities and developmental disabilities were also more likely to report being in fair or poor health than adults without disabilities. Significant medical care utilization disparities were found for breast and cervical cancer screening as well as for oral healthcare in this group. Mental health and drug abuse, e.g., nicotine, alcohol, and illicit drugproblems,areaclassicexampleofdisparitiesatboththepre- ventionandtreatmentlevel.Thesetypesofbehavioralproblems face stigmas that further contribute to the disparity problem. For example, in a national study the prevalence of smoking during pregnancy ranged from 9.0% to 17.4%. Younger (age <25 years) women, white women, American Indian women, non-Hispanic women, women with a high school education or less, and women with low incomes consistently reported the highest rates of smoking. In the same study the prevalence of alcohol use during pregnancy ranged from 3.4% to 9.9%. In seven states, women age >35 years, non-Hispanic women, women with more than a high school education, and women with higher incomes reported the highest prevalence of alcohol useduringpregnancy.Althoughprevalencedatacannotbeused to identify causes or interventions to improve health outcomes, theydoindicatethemagnitudeofdisparitiesandidentifypopu- lations that should be targeted for intervention. We recognize that a number of groups experience inferior medical care and health status, but may not appreciate the se- riousness of the problem. Each year the United States spends billions of dollars to perfect the ‘technology’ of healthcare, e.g., development of new drugs, new pieces of equipment, and to modernize delivery systems, thereby saving thousands of lives. Correcting known disparities could prevent five times as many deaths. If policymakers adhered to the goal of optimizing population health, greater priority would go to resolving disparities rather than to developing new technol- ogy, but unfortunately reverse priorities prevail. Isaac D Montoya PhD was the Clinical Laboratory Science Research and Reports Editor, 2001-04.
  • 5. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 67 In today’s climate of focus on the healthcare consumer, much attention is being paid to improving patient safety and quality of care. There is heightened interest in distin- guishing healthcare providers who are “good performers”, who provide safe and efficient care from poorer performers whose outcomes may not be as good. Some payers feel that one way to encourage performance improvement is to pay good performers better than other providers. The Center for Medicare and Medicaid Services (CMS) began a Pay for Performance program for hospitals on a pilot basis about two years ago. Hospitals that participate in the pilot project keep statistics on a number of measures in diagnosis groups common in the Medicare population, such as acute myocardial infarction (AMI), coronary artery bypass grafts (CABG), heart failure, pneumonia, and hip and knee replace- ment. If hospitals achieve certain levels of compliance with the goals of the measures, they are paid 1% to 2% more than the usual DRG payment. A few examples of the over thirty pay for performance metrics defined by CMS are: • AMI: aspirin at arrival • AMI: thrombolytic within 30 minutes of arrival • AMI: percutaneous coronary intervention received within 120 minutes of arrival • CABG: post operative hemorrhage or hematoma • Heart failure: smoking cessation advice/counseling provided • Pneumonia: oxygenation assessment within 24 hours • Pneumonia: blood culture collected prior to first anti- biotic assessment • Hip and knee replacement: prophylactic antibiotic re- ceived within one hour prior to surgical incision On January 31, CMS announced that ten large physician groups across the country would participate in a three-year pilot project of pay for performance for physicians. Physicians will continue to be paid on a fee-for-service basis, but will be eligible for performance payments based on how well they improve patient outcomes and avoid costly complications. The quality measures focus on the many of the same chronic illnesses in the Medicare population as do the hospital mea- sures, including congestive heart failure, coronary artery disease, diabetes, and hypertension, as well as preventive services such as screenings for breast and colorectal cancer, and immunization for flu and pneumonia. Another proposal related to pay for performance for physi- cians surfaced in January that would have a very direct im- pact on the laboratory. During hearings on January 12, the Medicare Payment Advisory Commission (MedPAC) made a recommendation that CMS should require laboratories to report test results to CMS on the claim for payment. From a reading of the transcript of the proceedings of the MedPAC, it is not clear how the data would be used to evaluate physi- cian performance, perhaps by measuring the percentage of abnormal results. In addition to using this strategy to evaluate physician performance for pay for performance, MedPAC discussion focused on the requirement as a way to encour- age all providers to use information technology (electronic medical record). The electronic medical record has been a focus of the Bush administration’s health policy. Laboratorians are likely to be skeptical about the effectiveness of laboratory values alone as a measure of physician effective- ness, taken outside the context of the larger medical record. In addition, the American Clinical Laboratory Association (ACLA), the association that represents the larger national reference laboratories, has appeared before the MedPAC to raise a number of concerns about the recommendation: • Considerable cost and effort would be required for labo- ratories and hospitals to reprogram computer systems to transmit test results to the billing systems to appear on claims. In most institutions, results are reported elec- tronically and test charges are billed electronically, but there is no interface between those systems that would link results to test charges. WASHINGTON BEAT Pay for Performance KATHY HANSEN, DON LAVANTY Washington Beat is intended to provide a timely synopsis of activity in the nation’s capitol of importance to clinical laboratory practitioners. This section is coordinated jointly by Kathy Hansen, Chair of the ASCLS Government Affairs Committee, and Don Lavanty, ASCLS Legislative Counsel. Direct all inquiries to ASCLS (301) 657-2768 extension 3022; (301) 657-2909 (fax); or mail to ASCLS, 6701 Democracy Blvd., Suite 300, Bethesda MD 20814, Attention: Washington Beat.
  • 6. 68 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE • The MedPAC suggests that laboratories would standardize nomenclaturefortestsusingLOINCcodes,whicharemore specific that CPT codes. LOINC codes are not commonly used now, and conversion would be a huge effort. • Not all test results are numeric, and not all tests are reported with a reference range. Lengthy narratives ac- company many microbiology and flow cytometry results, for example. • Laboratory values should be interpreted in the light of other information about the patient found in the medical record. • Last but not least, the recommendation would need to be examined in the light of HIPAA’s “minimum necessary” privacy standard. MedPAC discussed whether it might be better to recommend startingtherequirementwithasubsetofspecifictestresults,but in the end stayed with the recommendation that all test results be reported. It did acknowledge that the recommendation rep- resents “…a complex undertaking” and this included a two to three year transition period for implementation. ACLA is now briefing key congressional staff about their concerns about the MedPAC proposal. The College of American Pathologists (CAP), along with other physician groups, opposes the MedPAC’s proposal to set aside 1% to 2% of physician payments to be redistributed on the basis of performance. The MedPAC recommendations have gone to CMS, which will have to decide whether to accept them. If CMS decides to move forward, proposed regulations would be published in the Federal Register for comment. ASCLS will monitor this situation closely and register opinions and submit com- ments whenever appropriate. Regardless of how one views the concept of pay for performance and its potential to improve patient outcomes and patient safety, this particular proposal seems to be unreasonably burdensome for laboratories to implement, and to have limitations in the validity of the con- clusions that could be drawn about physician performance from the raw laboratory data. WASHINGTON BEAT
  • 7. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 69 CLINICAL PRACTICE Evaluation of Malaria Parasite Screening Procedures Among Sudanese Blood Donors MOHAMED SIDDIG M ALI, ABDUL GADER MOHAMED YOUSIF, MUSTAFA SALIH MUSTAFA, MALIK HASSAN IBRAHIM OBJECTIVE: To compare the standard microscopic exami- nation, the polymerase chain reaction (PCR), and the immu- nochromatography test (ICT) to determine the best method for screening blood donors for malaria parasites in Sudan. METHODS: A total of 100 blood donors were screened for malaria parasites by standard microscopic technique, ICT, and PCR.Bloodfilmswereexaminedmicroscopicallyusingstandard Giemsa staining techniques. Qurum (Canadian Company) malaria kits were used to perform the ICT. For performing PCR, DNA was extracted using Chelex method and amplified by the moderately repetitive DNA sequence pBRK-l. RESULTS: Using PCR, a total of 21 blood samples were positive; 8 (38%) of them showed negative blood films and 7 (33%) were negative on ICT. Four blood samples that tested positive by ICT despite a negative PCR and microscopic ex- amination were proved to be false positives. The false negativ- ity of both the microscopic examination and ICT was found to be significant. The sensitivity of microscopy was 61.9% and of ICT was 66.7%, while the specificity of microscopy was 100% and of ICT was 94.9%. When direct microscopy was considered as the standard technique the sensitivity of ICT was 100% and the specificity was 94.3%. CONCLUSION: Although PCR is more sensitive and more specific, it is unaffordable. Microscopy for malaria when com- pared to ICT showed similar sensitivity at low cost. However, all human plasmodium species can be detected using the mi- croscopy while only two species (P. falciparum and P. vivax) can be detected by ICT. The detected false positivity of ICT is not inconsequential since this implies the rejection of a greater proportion of blood donations. Therefore, microscopy is con- sidered more suitable for screening Sudanese blood donors for malaria parasites prior to donation at the present time. RECOMMENDATIONS: To establish a reference malaria diagnosis unit in each blood bank in Sudan as well as to train blood bankers to perform microscopic examinations. ABBREVIATIONS: ICT = immunochromatography test; PCR = polymerase chain reaction. INDEX TERMS: donor testing; malaria testing. Clin Lab Sci 2005;18(2):69 Dr Mohamed Siddig M Ali MSc Haematology is Lecturer and Head, Department of Haematology, AI Neelain University, Khartoum, Sudan. Abdul Gader Mohamed Yousif PhD is Professor of Medicine and Tropical Diseases and Dean, Faculty of Medicine, Uni- versity of Khartoum, Khartoum, Sudan. Mustafa Salih Mustafa MD is General Manager of Planning and Development and Research Directorate, Federal Ministry of Health, Khartoum, Sudan. Dr Malik Hassan Ibrahim MSc Haematology is Lecturer and Head, Department of Haematology, Sudan University of Science and Technology, Khartoum, Sudan. Address for correspondence: Dr Mohamed Siddig Mohamed Ali, Faculty of Medical Laboratory Sciences - AI Neelain Uni- versity, PO Box 12702, Khartoum, Sudan. mohdaru@hotmail. com The medical use of blood and blood derivatives is increasing all over the world, despite the hazards related to transmission of protozoal, spirochaetal, bacterial, and viral diseases. Transmis- sion of malaria by blood transfusion is a significant problem in regionsoftheworldwherethisdiseaseisendemic;P.falciparum intransfusedbloodmayleadtofatality.1 Moreover,transfusion- induced P. falciparum has increased in recent years, probably because it has become increasingly resistant to drugs.2 The peer-reviewed Clinical Practice Section seeks to publish case stud- ies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, includ- ing brief reviews of books, computer programs, audiovisual materials, or other materials of interest to readers. Direct all inquiries to Bernadette Rodak MS CLS(NCA), Clin Lab Sci Clinical Practice Editor, Clinical Laboratory Science Program, Indiana University, Fesler 409, 1120 South Avenue, Indianapolis IN 46202-5113. brodak@iupui.edu.
  • 8. 70 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE Prevention of transfusion-induced malaria depends on the screening of potentially infected blood donors, especially those who provide whole blood or fresh concentrates of erythrocytes, leukocytes, or platelets.3 However, success- ful screening for malaria parasites requires experience in the differential characteristics of the various species of the parasite and should constitute a part of primary health care.2 Microscopic examination of a blood film can be used for the detection of malaria infection in a donor in whom malaria is suspected based on circumstantial evidence. Examina- tion of stained thin blood films can identify the species and give an estimate of the percentage of infected red cells.4-6 Examination of the buffy coat and red cells below can help in detecting parasites when they are few in number (5 to 10 parasites/µL), particularly in the late stages, e.g., trophozoites and gametocytes.5 However, in traditional microscopy, failure to adhere to correct techniques seriously compromises the specificity and sensitivity. Immunochromatography tests (ICT) employ monoclonal antibodies against histidine rich protein (HRP-2), which is produced by the parasite and released into the circulation. Compared to microscopy, it is simple to perform, does not require the use of special equipment, and is faster with low variation between users. Moreover, it can detect asexual parasites and young gametocytes with reasonable sensitivity and specificity (>90%). Serological methods cannot replace the demonstration of parasites in the blood as far as diagnosis of symptomatic patients is concerned.6 In countries where the disease oc- curs, antibody tests are unable to distinguish between active and past infection and they have only a limited value in the clinical diagnosis of malaria.5 During the past few years, the polymerase chain reaction (PCR) has become a major diagnostic and research tech- nique. It is reliable for the detection of parasites present at low concentration in blood or serum samples. Further, the use of PCR could be 100 times more sensitive than the microscopic examination of thick blood films.7 The present study is intended to determine the selection of the best laboratory procedure that can be applied for screen- ing blood donors for malaria parasites in Sudan since such studies have not been conducted previously. MATERIALS AND METHODS The design of the study is descriptive, cross-sectional, and facility based. It was conducted in the Ahmed Gasim Hospi- tal (Khartoum North, Sudan) among a total number of 100 blood donors screened for malaria parasites by ICT, PCR, and standard microscopic technique. Blood samples were col- lected (1 mL from each), processed with EDTA anticoagulant (1.5 mg), and then immediately used to prepare blood films and perform ICT. Three spots of each tested blood sample (50 µL each) to be used for the PCR were stored dry (on #3 filter paper) at -20 °C.8 From the blood collected from each donor, duplicate thick and thin blood films were prepared, stained by Giemsa stain, and examined microscopically as the standard methods.5 The absolute number of parasites (number/µL), was estimat- ed in positive thick blood films by counting the recognized malaria parasites against 200 white blood cells according to the following formula: # of parasites counted x total WBC # of leukocytes counted (200) Qurum (Canadian Company) ICT malaria kit was used. This kit has been designed for the detection of P. falciparum in whole blood. It employs monoclonal antibodies against HRP-2 that is secreted by the parasite. Captured monoclonal antibodies are immobilized on a nitrocellulose membrane strip. When P. falciparum antigen is present in lysed whole blood, it binds antibodies as the blood migrates along the test strip. Colloidal gold particles are coated with these antibodies to form a sandwich resulting in visible red line. Performance of the test and interpretation of results were conducted as directed by manufacturer instructions. For performing PCR, DNA was extracted from the filter paper using Chelex method.8 One of the three blood spots was cut using a sterile blade and put in a 1.5 mL Eppendorf tube containing 1 mL of 0.5% saponine in freshly prepared 1 x phosphate buffer saline (PBS) and then incubated over- night at 4 °C (hemoglobin is released into the wash leaving the DNA of the parasite on the paper). Thetubeswerespunforoneminuteat13x103 rpmandthesu- pernatantfluidwasremoved.OnemLof1xPBSwasaddedand spun again for one minute; the supernatant was also removed. FiftyµLofPCRqualitywaterand50µLof20%Chelexmixture were added to each tube and then boiled for ten minutes. CLINICAL PRACTICE
  • 9. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 71 The tubes were centrifuged for one minute to pellet Chelex and debris. The DNA supernatant was taken off and trans- ferred into a new sterile 0.5 mL Eppendorf tube. DNA amplification was performed using the moderately repetitive DNA sequence pBRKl- 14 forward, 5-CGC TACATATGCTAG TTGCCA GA C-2ʹ and reverse 5ʹ- CGTGTA CCATA CATCCTACCAAC-3ʹ that amplifies a 206 base pair sequence.7 The PCR product was run in a gel electrophoresis tank (1.5% agarose gel) with the addition of ethidium bromide solution (0.5 mg/mL) and DNA molecu- lar weight marker (fraction VI) in a parallel well. DatawereanalyzedbythecomputerusingtheStatisticalPackage forSocialSciences(SPSS).Chi-squareandFisherexacttestswere used for comparison and correlation between proportions. The sensitivity of the test is the proportion of positives, cor- responding to the positive result obtained by the standard test. However, specificity of the test is the proportion of negatives corresponding to the negative result obtained by the standard technique. Sensitivity, specificity, false negative rate, and false positive rate can be calculated using the fol- lowing formulas and Table 1: Sensitivity = (e)/(e + f) Specificity = (h)/(g + h) False negative rate = 1 – sensitivity False positive rate = 1 – specificity RESULTS Out of the 100 randomly selected blood donors, 13 samples (13%) were positive by microscopy as well as by ICT and PCR, with 75 samples (75%) being negative throughout. Twelve blood samples (12%) showed variable results. Comparison between the results of PCR and ICT of the exam- ined blood samples applying PCR as the standard technique is shown in Table 2. Using PCR, a total of 21 blood samples were positive; of them only 14 blood samples tested positive using ICT. Four blood samples tested positive by ICT despite a negative PCR and microscopic examination; therefore, these proved to be false positives. The false positive of the ICT was found to be statistically insignificant (p >0.05). Seven (33%) positive PCR blood samples were negative by ICT. These false negative ICT results were found to be highly significant (p <0.001). Applying PCR as a standard technique, the sensitivity of ICT was 66.7% while the specificity was 94.9%. The false negative rate was 33.3% and the false positive rate was 5.1%. Comparison between the result of PCR and microscopic examination of the donors’ blood samples applying PCR as the standard technique is shown in Table 3. Using PCR as the standard technique, a total of 21 blood samples were positive; 8 (38%) of them showed negative blood films. This false negativity of the microscopic examina- tion was found to be highly significant (p <0.001). CLINICAL PRACTICE Table 1. Sensitivity and specificity table Tested Standard technique technique Positive Negative Total (D+) (D-) Positive (T+) (e) (g) (e + g) Negative (T-) (f) (h) (f + h) Total (e + f) (g + h) Table 2. Comparison between the results of PCR and ICT PCR Total Positive Negative ICT positive 14 4 18 ICT negative 7 75 82 Total 21 79 100 p >0.01 Table 3. Comparison of PCR vs. microscopic examination PCR Total Positive Negative Microscopic examination Positive 13 0 13 Negative 8 79 87 Total 21 79 100 p >0.01
  • 10. 72 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE ApplyingPCRasastandardtechnique,thesensitivityofmicro- scopic examination was 61.9% while the specificity was 100%. The false negative rate was 38.1% and the false positive rate was zero. Comparison between the result of microscopic examina- tion and ICT of the same blood samples applying microscopy as the standard technique is shown in Table 4. Using microscopic examination, a total of 13 blood samples were positive; 5 (38.5%) of them were ICT negative. Only one donor’s blood sample (7.7%) was found positive by ICT and PCR despite a negative blood film. The false positive ICT (the other four positives; 22.2%) which were negative by blood films, representing the dif- ference between microscopy and ICT, were found to be insignificant (p >0.05). Applying microscopic examination as standard technique, the sensitivity of ICT was 100% and the specificity was 94.3%. The false positive and false negative rate was 5.7% and zero respectively. DISCUSSION Transfusion-induced malaria continues its resurgence throughout much of the tropics and subtropics. Success- ful control or eradication measures must include strategies directed to prevent transmitting malaria parasite infected blood to patients. Therefore, it is mandatory to establish an effective technique for screening blood donors for the malaria parasite. Different techniques were compared to determine the best method of screening donors that can be easily and rapidly applied in Sudan. Since PCR is highly specific and highly sensitive for posi- tive and negative results respectively (100 times greater than staining technique), it can be used as a standard technique to avoid false negatives and positives.7 In this study, PCR was adopted as the standard technique. It is worthwhile to men- tion that during this investigation, the difference between PCR and both ICT and direct microscopy was still signifi- cant, although PCR has the drawbacks of being costly and requiring strict laboratory conditions to avoid contamination that can lead to false positive results. In malaria-endemic areas, the use of ICT is known to be rather deceptive when used for investigating symptomatic patients for the presence of malaria parasite in their blood. False positive results can be obtained even 14 days after clearance of the parasites by treatment or by the immune system.5,6, 9-11 This study showed that sensitivity of the ICT versus direct microscopy was 100%, which corresponds to the results obtained by Singh in India, Cavallo in France, and Gaye in Senegal.11-13 However, all of them confirmed the false posi- tivity of ICT within variable periods after infections. These reports are in close agreement with the findings of this study; four false positive tests were detected. The false positive results are important since this implies that a greater proportion of blood donations will be rejected to ensure the prevention of malaria transmission by blood transfusion. Therefore, ICT seems to be not reliable for screening Sudanese blood donors for malaria parasite since whether the parasite is present or not, persistence of HRP-2 will result in false positivity. Fur- ther, only ICT designed for the detection of only two species (P. falciparum and P. vivax) is available, whereas transfusion induced malaria due to the other human plasmodium species (P. malariae and P. ovale) is not uncommon, particularly P. malariae because of its chronicity. The ICT test will be falsely negative when the latter species are present. The standard microscopic examination technique for malaria parasites enables the detection of all four human plasmodium species. It also allows distinction between species and stages of infestation and the capability of determining parasitaemia which may produce some new epidemiological and parasi- tological aspects providing some suggestions for eradication; moreover, it is cheap, relatively rapid, readily available, and easy to perform. When compared with ICT, microscopic examination shows similar sensitivity (no statistical difference; p >0.05). Hence, microscopy of malaria is more suitable for screening blood donors in Sudan. This is in agreement with Wilairat’s findings thatmicroscopicdiagnosisofmalariaisstillmorethanadequate CLINICAL PRACTICE Table 4. Comparison between the result of micro- scopic examination and ICT of the same blood samples applying microscopy as standard technique Microscopic examination Positive Negative Total ICT positive 13 5 18 ICT negative 0 82 82 Total 13 87 100 p =1.20
  • 11. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 73 in the routine investigation of individual cases, in spite of the fact that many authors have not favored the use of this method because of the amount of time and expertise required.14 There is still no international consensus for the exact defini- tions of what constitutes high, intermediate, or low parasi- taemia; undoubtedly, most of the parasite densities that were encountered in our study were relatively low. However, the minimum number of parasites transmitting malaria via blood transfusion may vary among individual recipients; a single parasite can transmit the disease in mice.13 Even if a single parasite can be detected in a thick blood film, which is equiva- lent to 4 μL of blood, more than hundreds of thousands of parasites might escape in a full unit of blood (450 mL). This signifies the importance of the reliable testing of blood donors for malaria parasite to minimize, though never com- pletely eliminate, the risk of malaria transmission by blood transfusion. Moreover, the hazards of transfusion malaria are serious, and justify prior testing of blood donors for malaria even by advanced expensive techniques. These techniques will surely reduce the false negative results and hence minimize the risk of transfusing infected blood. The standard microscopic identification technique of malaria parasites is ideal to be applied at the present time until the development of more feasible application of PCR. The estab- lishment of a reference malaria diagnosis unit in each blood bank as well as trained blood bankers is necessary. CONCLUSION The hazards of transmitting malaria through blood transfu- sion are serious thus justifying the use of expensive techniques for testing blood donors. Transfusion of blood from malaria parasite infected donors to patients will result in transfusion- malaria; screening donors for malaria prior to donation will undoubtedly reduce this risk. Microscopy is much cheaper than both ICT and PCR; in addition, it enables the detec- tion of all human plasmodium species. Therefore, it is the best technique to be adopted for the control of transfusion- induced malaria in the different regions of Sudan until the feasibility of using PCR technology is improved. ACKNOWLEDGEMENT I wish to express my sincere gratitude, thanks, and everlast- ing appreciation to Dr Amna El Subki Khalid, Dr Musa M Kier, Dr A1 Dirdiri Salim, Dr Nawal Taj Elsir, and Dr Mona Abbas Babiker for their invaluable help. REFERENCES 1. Barker RH, Banchongaksorn T, Courval JM, and others. A simple method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction. Am J Trop Med Hyg 1992;46:416-26. 2. Neva FA, Brown HW. Basic clinical parasitology. 6th ed. New York: Appleton and Lange; 1994. p 347. 3. Bruce Chwatt LJ. Transfusion malaria revisited. Trop Dis Bull 1982;79(10):827-40. 4. Sherman IW. Malaria. Washington: ASM: 1998. p 557. 5. Cheesbrough M. Medical laboratory manual for tropical countries. 2nd ed. Oxford: Butterworth; 1987. p 602. 6. Manson Bahr PEC, Bell DR. Mansons’ tropical diseases. 19th ed. London: ELBS; 1987. 7. Vu TT, Tran VB, Phan NT, and others. Screening donor blood for malaria by polymerase chain reaction. Trans R Soc Trop Med Hyg 1995;89:44-7. 8. Tirasophon W, Panyim S. PCR for low level detection of malaria parasite in blood. Protocols in molecular parasitology In: Hyde JE, editor. Methods in molecular biology. 21st ed. Totowa NJ: Humana Press; 1993. p 205-12. 9. Karbwang J, Tasanor O, Kanda T, Wattanagoon Y. ParaSight TM -F test for the detection of treatment failure in multi-drug resistant Plas- modium falciparum. Trans R Soc Trop Med Hyg 1996;90:513-5. 10. Valecha N, Sharma VP, Devi CU. A rapid immunochromatographic test (ICT) for diagnosis of Plasmodium falciparum. Diagn-Microbiol Infect Dis 1998;30(4):257-60. 11. Singh B, Cox-Singh J, Miller AO, and others. Detection of malaria in Malaysia by nested polymerase chain reaction amplification of dried blood spots on filter papers. Trans R Soc Trop Med Hyg 1996;90(5):519-21. 12. Cavallo JD, Hernandez E, Gerome P, and others. Serum HRP-2 antigens and imported Plasmodium falciparum malaria. Comparison of ParaSight-F and ICT malaria P.f. Med Trop 1997;57(4):353-6. 13. Gaye O, Diouf M, Dansokho EF, and others. Diagnosis of Plas- modium falciparum malaria using ParaSight F and ICT malaria P.F. Parasite 1998;5(2):189-92. 14. Wilairat P. DNA probes for malaria parasite detection: current status and future prospects. In: Ko RC, editor. Immunological and molecular basis of pathogenesis in parasitic diseases. Hong Kong: Hong Kong University Press; 1989. p 169-74. CLINICAL PRACTICE
  • 12. 74 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE CLINICAL PRACTICE Clinical Laboratory Educators Conference 2005 Abstracts The peer-reviewed Clinical Practice Section seeks to publish case stud- ies, reports, and articles that are immediately useful, are of a practical nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, includ- ing brief reviews of books, computer programs, audiovisual materials, or other materials of interest to readers. Direct all inquiries to Bernadette Rodak MS CLS(NCA), Clin Lab Sci Clinical Practice Editor, Clinical Laboratory Science Program, Indiana University, Fesler 409, 1120 South Avenue, Indianapolis IN 46202-5113. brodak@iupui.edu. POSTER PRESENTATIONS Authors listed in bold face type will be the presenters. Cold Agglutinins: A Case Study of Patient’s Condition and Its Effects on the Integrity of Laboratory Findings Brenda Bouchard MS, University of Massachusetts Dart- mouth, Dartmouth MA; Lauren Cunha CLS(NCA), Charl- ton Memorial Hospital, Fall River MA. Although frequently seen in clinical practice, the diagnosis of patients who have cold reacting antibodies can be com- plicated. Cold agglutinins affect many areas of laboratory testing. Broad recognition of patient history, symptoms, and laboratory findings can guide one to include the pos- sibility of a cold reacting antibody. A cold agglutinin has optimal reactivity at temperatures below those of normal body temperatures and is almost always IgM subclass. The case to be presented begins with a female patient having a pre-existing anemia and chronic obstructive pulmonary disorder. Laboratory findings include CBC and differential data with abnormally high WBC and platelets, and 90% neutrophils. The patient had a low RBC count, hemoglobin, and hematocrit. Measurement of arterial blood gas levels showed a low pO2 and O2 saturation, with a high pCO2 and HCO3. The patient’s mycology cultures grew out a mold most characteristic of Aspergillus spp., Latex agglutination D-dimer testing revealed a cold agglutinin. Due to the patient’s low hematocrit, the physician ordered two units of packed red blood cells. Further details of laboratory results and treatment followed by how this case may have been misdiagnosed due to the presence of the cold agglutinin are presented. Comparing Academic Performance, Learning Style, and Student Satisfaction in a Pre-CLS Biology Simulation Laboratory Course Sandra M Weiss Ed D CLS (NCA), Tricia McGinnis, Neu- mann College, Aston PA. The purpose of this study was three-fold: 1) to determine if laboratory simulations can replace a traditional laboratory, 2) to determine if there are learning styles more responsive to computer simulations, and 3) to determine if students are more satisfied with computer simulations than traditional wet laboratories. The basic assumption that science labora- tories help students gain a better understanding of concepts described in lectures and textbooks was not challenged. Unfortunately, ideal laboratory teaching conditions are of- ten rare and complex experiments are expensive, hazardous, or time consuming to perform in introductory laboratory courses. Incorporating microcomputer technology enables a variety of teaching techniques that permits the instructor to become a resource person and facilitator of learning rather than the expert who imparts knowledge. Students enrolled in a pre-CLS physiology laboratory course participated in a pilot study July 2004. All wet laboratories were replaced using PhysioExTM 5.0 laboratory simulation in physiology. Students learning styles and personality characteristics were identified using Kolb’s Learning Style and the Meyers-Briggs inventories. Additionally, pre- and post-tests and final grades were compared to determine significant gains in cognition. Students were surveyed twice during the session. The results of the t-test and one-way analysis of variance (Anova) indi- cated that ‘A’ students were more satisfied with the course and knowledge gained (p <0.05) than ‘B’ and ‘C’ students. The results of this study have implications for clinical labo- ratory science education. Laboratory simulations may be an alternative way to introduce CLS students to advanced equipment and complex techniques not routinely performed in the laboratory.
  • 13. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 75 Development and Delivery of an AS to BS Degree Completion Distance Learning Track in CLS Linda J Graeter PhD MT(ASCP), Charity Einhaus Accurso PhD MT(ASCP), Gideon H Labiner MS MT(ASCP) CLS(NCA), Elizabeth C King PhD, University of Cincinnati, Cincinnati OH. The CLS Program at the University of Cincinnati introduced an AS to BS degree completion distance learning track (CLS DL) in June 2004. The track was designed to provide work- ing laboratory professionals with an associate degree the opportunity to earn a bachelor’s degree while continuing full- or part-time employment, and with minimal disruption in their lives. Previous laboratory and life experiences were considered in curriculum development, so students enter the program with advanced standing that includes complete fulfillment of the university’s general education requirements. The curriculum includes upper division science content, didactic courses, and advanced clinical experiences that are completed in each student’s community. The courses are taught using an interactive distance learning course model that includes audio and video PowerPoint presentations, regular discussions between the instructors and the students, and frequent learning assessments. The students matriculate through the curriculum in a learning community of 20 stu- dents that provides a natural support system and study group environment. Fifty-four students were admitted into the first quarter; 94% were retained. One hundred three new students were admitted into the second quarter. Funding, develop- ment, resources, the course model, and the curriculum will be discussed and sample course materials will be presented. Student demographic, assessment, and retention data will be shown. Preliminary data suggest that the distance learning course model and curricular structure utilized in the CLS DL track will successfully provide laboratory professionals who are not able to attend a traditional program with the means to continue their education and advance their careers. Development and Implementation of an Innova- tive MLT/CLT to MT/CLS Articulation Program Using Synchronous and Asynchronous Delivery Formats Tracy S Harrison MS MT(ASCP), Faye E Coleman MS CLS MT(ASCP), C Thomas Somma EdD MT(ASCP) SC, Paul F Magnant MBA, Old Dominion University, Norfolk VA. It is well established that there is a shortage of certified medi- cal technologists/clinical laboratory scientists (MTs/CLSs). The problem identified in this study is that there are limited programs that address the development of a curriculum format that is accessible by working medical laboratory technicians/clinical laboratory technicians (MLTs/CLTs) to earn a bachelor’s degree. In 1996, a new model of curriculum delivery using synchronous and asynchronous formats to deliver the didactic components was developed and imple- mented by Old Dominion University. The program utilizes an interactive, televised, asynchronous weekend format to deliver courses to distant sites. The work sites of MLTs/CLTs provide the clinical component of the program. This study was a retrospective cross-sectional comparison of the scores of traditional and weekend students (n = 97) on the American Society of Clinical Pathology (ASCP) national certification examination over a 4½ year period beginning in 1998. In every comparison there was no difference, i.e., p <0.001, between the scores of traditional and weekend students on the ASCP examination. Furthermore, the scores of traditional and weekend students in four of the six subject areas (hema- tology, blood bank, microbiology, and chemistry) exceeded not only the national average in these subject areas, but also the cumulative overall score. This suggests that the Old Dominion University MLT/CLT to MT/CLS Program, de- livered in this innovative format, is effective. Further research needs to be conducted in order to examine the cost/benefit of this type of program as well as other delivery formats. Educating Medical Students: It’s Not Always “THE LAB’S FAULT!” Smith LA, McKenzie SB, Burns C, Bearden MD, Holton R, Kudolo G, Chumley H, The University of Texas Health Sci- ence Center, San Antonio TX. Clinical laboratory scientists (CLSs) often complain that other healthcare providers, in particular physicians, do not recognize our educational level and underutilize our expertise. In addition, clinicians have little or no knowledge of the ef- fects of pre-analytical errors, especially those resulting from improper specimen collection. As a result, when laboratory values do not match clinical diagnosis – it’s the lab’s fault! This project, directed by the Clinical Laboratory Science Program faculty, was part of a medical school course for students en- tering their junior year. We integrated phlebotomy, clinical data, and laboratory data to expose medical students to the value of laboratory data and the expertise of CLS. The class was divided into five groups, each of which met for a half day – one-half of the time was spent in small work groups, the remainder in phlebotomy. Twelve scenarios were developed covering reflex testing and sources of pre-analytical error in CLINICAL PRACTICE
  • 14. 76 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE all laboratory disciplines. Each student group identified the problem in the scenario and determined which laboratory professional could be consulted regarding questions about test ordering or interpretation. Students then presented the results to other members of the class. CLS faculty moder- ated the sessions. CLS students assisted with venipuncture. Medical students took a pre- and post-test that measured knowledge of laboratory testing. The difference between means of the pre- and post-test was significant. Comparison of the means of the pre- and post-test showed a 30% improve- ment in scores. Session evaluations were favorable with many students suggesting additional time for the activities. Fostering the Development of Expertise in Clini- cal Laboratory Scientists (CLSs) Janet Hudzicki PhD MT(ASCP)SM, Kansas State University, Manhattan KS. The development of expertise is a phenomenon that is little understood. Although there is a body of research that examines the characteristics of experts, and compares experts to novices, the literature on the actual transition process lacks depth. This research describes an investigation of the transition from novice to expert in the clinical laboratory science community of practice using a phenomenological approach. The sample selection process consisted of soliciting names of expert CLSs from the members of the Clinical Laboratory Managers Association. The potential participants were randomly selected from the submitted names and asked to participate in the study. Data were collectedfrom11participantsbysemi-structuredinterviews. The constant comparative method was used to analyze the interview transcripts. Four factors were determined to be essential to the transition process: Self-directedness in learning, storytelling, mentors and mentoring, and reflection. In addition, the transition from novice to expert requires being part of a vital, robust community of practice. Recommendations for helping novices with this transition include establishing a mentoring program for students and new employees, encouraging storytelling among laboratory personnel,andprovidingtoolsthatwillencouragereflection. This research has the potential to impact the education and training of CLSs, the enculturation of novice CLSs into the profession’s community of practice, and the development of expertise in CLSs. Integrating Education of MT/CLS Students and CP Residents in a Single Course Nancy Goodyear PhD CLS(NCA), University ofWashington, Seattle WA. It is unusual for undergraduate MT/CLS students to have the opportunity to interact on an equal basis with clinical pa- thology (CP) residents. Pathology residents in the University of Washington Department of Laboratory Medicine begin their CP training with a three-month structured core course covering all areas of the clinical laboratory. The microbiology portion begins with eight laboratory sessions taught by an experienced clinical technologist or a microbiology post-doc- toral fellow. Following this introduction, the residents join the MT Program clinical microbiology laboratory class for three to four weeks. Following the core, they rotate through the clinical labs, including at least six weeks in microbiology. Many residents have no clinical laboratory or microbiology background; although they don’t need to develop technical expertise, working up the same specimens as the MT/CLS students, from plating to final report, helps them to under- stand the testing performed in microbiology. Interactions between MT/CLS students and residents help both groups recognize the critical role that each plays in healthcare, and appreciate the expertise and limitations of each groups’ training. MT/CLS students assist residents with laboratory procedures and colony morphology. Residents bring human organs from the Department of Pathology teaching organ collection and give demonstrations reviewing normal and abnormal anatomy, especially as it applies to infection. In addition to providing an opportunity for interprofessional interactions, combining MT/CLS students and CP residents in the same microbiology course consolidates teaching workload, improves resource utilization, and provides an opportunity for MT program faculty to contribute to a larger educational mission. Moving from Traditional to Online Delivery: Creating a Hemostasis Course That Promotes Student Participation Margaret Fritsma MA MT(ASCP)SBB, University of Ala- bama at Birmingham, Birmingham AL. University clinical laboratory science programs are mov- ing in the direction of distance learning to make courses more accessible to students. While there are advantages and disadvantages to both classroom-based courses and online courses, most undergraduate students prefer classroom- CLINICAL PRACTICE
  • 15. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 77 based lectures to online delivery. The challenge to educators is to design online courses that engage students, accommodate various learning styles, incorporate a variety of learning activi- ties, and build in accountability and student participation. A hemostasiscourseisdescribedinwhichcoursecontentisplaced online,withclassroomfollow-up.Eachweek’slessonmaterialis placedonlineintheformoflessonobjectives,PowerPointslides with lecture audio, a Microsoft Word handout with slides ac- companied by written text of the lecture, and assignments and supplemental material. There is one classroom meeting each week, which consists of a short quiz on the online material, an interactivediscussion(nolecture)ofthemoredifficultconcepts and implications from the lesson material, a question and an- swer (Q&A) session, and various group activities. Examples of groupactivitiesincludeimpromptugrouppresentationsofshort topics, games, student bowl competition, a Protein C Pathway skit, and case discussions. Each student is assigned one mod- ule to narrate the audio portion of the online lesson from the instructor’sscriptedtext,andtoleadtheclassroomQ&Asession on his/her topic. Student interest and feedback is positive, and will guide future development of the course. Exam scores have shown improvement in grades over the previous course offered in a more traditional format. A Novel Consortium Model for Delivering Clini- cal Laboratory Programs to Rural Regions Karen R Murray PhD CLS(NCA), Tarleton State University, Stephenville TX. Workforce problems including severe shortages of clinical laboratory technicians (CLTs) in surrounding rural regions and shortages of histology technicians (HTs) in both rural and urban regions, coupled with a state mandated initiative (“Closing the Gaps”) to increase student participation and success within higher education institutions in the State of Texas, prompted the Clinical Laboratory Science (CLS) Department at this institution to generate and investigate alternative program and curriculum models in order to ad- dress these problems. The chosen solution was to design a unique consortium model that represents a novel coopera- tion between the two-year and four-year higher education sectors, and is supported by a unique curriculum design that facilitates access and participation. Four key outcomes of the resulting consortium model are that it 1) delivers two high need laboratory science programs (CLT and HT) and thus contributes to the “Closing the Gaps” initiative in three of four key objective areas; 2) utilizes a large community col- lege partnership base with established clinical affiliations to serve a large, predominantly rural geographic region and to maintain sufficient student numbers for program viability; 3) realizes cost efficiencies by capitalizing on an existing infrastructure of resources and expertise already in place to support CLS programs; and 4) facilitates laboratory science student articulation between professional levels. This novel consortium model may be applied to institutions nationwide that are facing similar problems with diminishing state fund- ing, program viability concerns due to low student numbers, workforce shortages, and increasing demands for student access, participation, and success. The Relationship of ProficiencyTest Performance to Personnel Credentials of Laboratory Testing Personnel Maria E Delost MS CLS(NCA), Guang-Hwa Chang PhD, Youngstown State University, Youngstown, OH; W Gregory Miller PhD, William J Korzun PhD DABCC MT(ASCP), Teresa S Nadder PhD CLS(NCA), Virginia Commonwealth University, Richmond VA. Performanceonproficiencytest(PT)surveysprovidesanobjec- tive and consistent evaluation of laboratory quality. The goal of thestudy,aretrospectivereviewofexistingPTresultsperformed at Virginia Commonwealth University Health System labora- tories, was to determine the relationship of PT performance to the personnel credentials of the laboratory testing personnel. Predictorvariablesincludedthepractitioner’smajorareaofstudy, degree, certification, and years of laboratory experience. The studygroupconsistedof185testingpersonnel.Therewere3389 proficiency-testingresultsofwhich3306weregradedacceptable (97.6%),36wereunacceptable(1.1%),and47werenotgraded (1.4%). For those results performed by a single practitioner (n = 3266), the core laboratory performed 3161 (96.8%) of the PT results with 30 unacceptable (0.95%) results. The satellite laboratoriesstaffedbynon-laboratoriansperformed105(3.2%) oftheresultsand6(5.7%)wereunacceptable.Logisticregression analysis of the full model, with all predictors included, showed statistical significance (χ2 = 18.581, p = 0.010, df = 7) for years of experience and level of educational degree. Individuals with less than two years experience were over five times more likely to produce an erroneous result when compared to those with 20 years of clinical experience. Study limitations included the use of a single institution and incomplete demographics for six testing personnel who were responsible for two (5.5%) of the unacceptable PT results. As the laboratory workforce shortage intensifies, the performance of laboratory personnel with lim- ited years of clinical experience or those lacking an educational degree may be important. CLINICAL PRACTICE
  • 16. 78 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE The Use of Games to Review in a Clinical Microbiology Class Linda Jeff MA MT(ASCP), University of Alabama at Bir- mingham, Birmingham AL. A method of reviewing material prior to tests in clinical micro- biologywasdesiredthatdidnotinvolvereteaching.Tomeetthis objective, games were developed such as “You Make Me Sick”, “YouGrowOnMe”,“Jeopardy”,“WhoWantstobeaMicrobi- ologist?”, “Go Streaking”, “MicrobiologyTeam Competition”, “EnterobacteriaceaeSquares”,and“BACT”(bingo).Thegames providedopportunitiesforstudentstoreviewmaterialinanon- threatening, interactive, sometimes competitive, and fun way. The use of games was an effective means of reviewing/reinforc- ing material as indicated by students’ mean scores on the four exams (82.6, 84.7, 87, and 86.1). On an evaluation, 100% of students responded that they enjoyed playing games, the games were a beneficial way to review for tests, the games helped them learn important facts/concepts, and the games were relevant to exam questions. All the students liked team games better than individual games. The games that students felt were most helpful in preparing for exams and learning facts and concepts were“YouGrowonMe”,“YouMakeMeSick”,“Jeopardy”,and “Who Wants to be a Microbiologist?”. Students’ written com- ments about using the games were very positive and included such statements as “The games helped me a lot,” “I really liked the games. They were very helpful in preparing for exams,” and “Thegameswereverygoodreviewtools.”Basedontheseresults Iwillcontinueusingthesegamesandothersinthemicrobiology class as well as in the immunology class that I teach. TECHNOLOGY DEMONSTRATIONS Demonstration of Microsoft Producer for the Development of High Quality Recorded Lectures Based on PowerPoint Presentations Scott Wright MS, Weber State University, Ogden UT. For the past three years, the Clinical Laboratory Sciences De- partment at Weber State University has offered both CLT and CLS degrees online. Presently, one third of the courses offered through the Department deliver course lectures to the online student on a CD. The CD is mailed directly to the student prior to the beginning of the semester, eliminating problems associated with streaming video delivered over the Internet. The lectures are created by first writing a script which is then recorded and synchronized to PowerPoint slides using a program called Microsoft Producer for PowerPoint 2003. This technology demonstration will involve two computers; the first to play various examples of recorded lectures, and the second to demonstrate the relatively simple steps involved in creating high quality recorded lectures using the Microsoft Producer software (available for free at www.microsoft.com). The results of a student survey will be available describing the popularity of the recorded lectures, the ease of use, and convenience for the online student. Integrating Learning Objects into Clinical Mi- crobiology Teaching Materials Jean Brickell EdD, Michelle Kanuth PhD CLS(NCA), Vicki Freeman PhD CLS(NCA), University of Texas Medical Branch, Galveston TX; Sandy Latshaw MA, Carol Larson MSEd CLS(NCA), University of Nebraska Medical Center, Omaha NE. Time to produce educational content is a major concern when planning lessons for CLS/CLT students. Learning objects (LOs) are an approach to producing content in which the instructional material is broken down into ‘bite size’ chunks. These chunks can be independently created, maintained, reused, and pulled apart and then stuck back together into many different forms like Lego toys. LOs are a high quality technical resource for lectures, reviews, or tests that can be used to structure a lesson individually or strung together to create interactive content. LOs may include a combination of pictures, graphics, animation, video, audio, and text components. Because they are visual in nature, they are an asset for the development of lesson structure in both distance learning and computer-assisted learning environments. The use of LOs can reduce the preparation time for lectures, examinations, and remediation materials, freeing instructors to focus on other tasks. The University of Texas Medical Branch Clinical Laboratory Science Pro- gram partnering with the University of Nebraska Medical Center Division of Medical Technology received a Fund for Improvement for Postsecondary Education Grant to create LOs and disseminate them via an online repository. The current focus of this repository is microbiology and provides instructional content on biochemical reactions, organism identification, panel selection, and gram stain quality control. The accompanying technology demonstration will provide the actual LOs and demonstrate the sequencing of LOs to form a cohesive lesson. CLINICAL PRACTICE
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  • 22. 84 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS Membranoproliferative Glomerulonephritis Type II in a 10-year-old Girl MARTHA E TIBBS, SHARON P ANDREOLI, CARRIE L PHILLIPS The peer-reviewed Research and Reports Section seeks to publish reports of original research related to the clinical laboratory or one or more subspecialties, as well as information on important clinical laboratory-related topics such as technological, clinical, and experimental advances and innovations. Literature reviews are also included. Direct all inquiries to David G Fowler PhD CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of Clinical Laboratory Sciences, University of Mississippi Medical Center, 2500 North State St, Jackson MS 39216. (601) 984- 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu The clinical course of a 10-year-old female patient who presented with hematuria, proteinuria, and hypertension is described. Four months after being diagnosed with acute glomerulonephritis, the child was referred to a pediatric nephrologist due to persistent hematuria and unresolved proteinuria. A renal biopsy was performed due to the per- sistent urinary abnormalities and a family history of renal failure. The renal biopsy demonstrated pathological findings characteristic of membranoproliferative glomerulonephritis type II. The child was treated with an antihypertensive agent and steroids. Despite poor prognostic clinical and pathological features, she has minimal urinary abnormali- ties, normal renal function, and normal blood pressure on antihypertensive medication six years after the diagnosis of membranoproliferative glomerulonephritis type II. ABBREVIATIONS: C3 = complement component 3; GBM = glomerular basement membranes; Ig = immunoglobulin; MPGN = membranoproliferative glomerulonephritis. INDEX TERMS: C3 nephritic factor; complement; dense deposit disease; membranoproliferative glomerulonephritis; prednisone. Clin Lab Sci 2005;18(2):84 Martha E Tibbs MT(ASCP) is a Medical Technologist at Indiana University Hospital, Indianapolis IN. Sharon P Andreoli is a pediatric nephrologist at Riley Children’s Hospital, Indianapolis IN. Carrie L Phillips MD is a Nephropathologist at Indiana Uni- versity, Indianapolis IN. Address for correspondence: Martha E Tibbs MT(ASCP), Indiana University Hospital, Pathology/Microbiology, UH3575, 550 N University Blvd, Indianapolis IN 46202. (317) 274- 3896, (317) 278-0049 (fax). metibbs@iupui.edu The nephrotic and nephritic syndromes are clinical conse- quences of structural injury to the renal glomerulus, a vascu- lar filter that clears the blood of waste products. Laboratory testing is required to distinguish the two syndromes. Protein- uria, the major clinical sign of nephrotic syndrome, results from altered permeability of the glomerular filtration barrier due to perturbations in visceral epithelial cells (podocytes). Nephrotic syndrome in children is the clinical manifestation of podocyte injury most commonly due to minimal change disease. Other causes include focal segmental glomeruloscle- rosis, membranoproliferative glomerulonephritis, or mem- branous glomerulopathy. Children with nephritic syndrome usually present with gross hematuria that may be accompa- nied by hypertension and variable degrees of proteinuria. Inflammatory processes that target glomeruli, termed acute glomerulonephritis, alter capillary wall integrity, and allow red blood cells to leak into urine. For a more comprehensive analysis of acute glomerulonephritis, the reader is directed to the review by Vinen and Oliveira.1 Hematuria occurs in IgA nephropathy, hypocomplementemic glomerulonephritis, and hereditary renal disease (Table 1). Children who present with overlapping features of both nephrotic and nephritic syndromes should undergo thorough serological testing to narrow down the differential diagnosis. When hematuria and proteinuria are accompanied by hypocomplementemia (Table 1), a renal biopsy may be required to determine the underlying etiology and to guide therapeutic management. If the acute process is untreated, these patients risk progres- sion to chronic glomerulonephritis and may require renal replacement therapy such as dialysis or transplantation (for an excellent review, see Coppo and Amora 2004).2 Membranoproliferative glomerulonephritis (MPGN), or mesangiocapillary glomerulonephritis, is caused by an ab- normal immune response leading to antibody deposition in
  • 23. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 85 RESEARCH AND REPORTS Table 1. Differential diagnoses of pediatric patients with hematuria and proteinuria Glomerulonephritis Hypocomplementemic glomerulonephritis Postinfectious glomerulonephritis Membranoproliferative glomerulonephritis, types I, II, and III Systemic lupus erythematosus Nephritis of chronic infection IgA related glomerulonephritis Henoch-Schönlein purpura Systemic lupus erythematosus IgA nephropathy Membranous glomerulonephritis Hereditary renal diseases Alport syndrome (hereditary nephritis) Sickle cell nephropathy Autosomal dominant polycystic kidney disease Autosomal recessive kidney disease the kidneys. The membrano- portion refers to the histological observation of glomerular capillary wall thickening due to glomerular basement mem- brane (GBM) alterations. Increased glomerular cellularity suggests there is a proliferative basis to the glomeru- lonephritis. Patients with MPGN typically experience a decline in renal function as a consequence of inflam- mation and structural alterations of the kidney.3,4 MPGN is subtyped into three categories based on the pathway of serum complement activation and altered renal morphology resulting from deposition of immunoglobulin and complement in glomeruli. All three subtypes of MPGN are character- ized by a decreased serum C3 level in 80% to 90% of patients and the low serum complement level is an impor- tant clinical tool in the preliminary diagnosis of glomerulonephritis (Table 1). MPGN type I is characterized by the classical pathway of complement activation and immune deposits along the subendothelial aspect of the GBM. MPGN type II, or dense deposit disease, is characterized by ‘dense’ rib- bon-like immune deposits within the GBM, alternative complement pathway activation, and circulating C3 nephritic factor. MPGN type III is characterized by subendothelial, subepithelial, and mesangial immune deposits with activation of the alterna- tive complement pathway.5 CASE STUDY InSeptember1997,a10-year-oldgirlwas seenbyherfamilyphysicianforaoneweek history of dysuria and brownish-colored urine. Upon physical examination, the patient was afebrile with a blood pressure of124/92mmHg.Theurinedipstickwas positive for protein. Urine microscopy showed numerous white and red blood cells. The child was diagnosed with acute glomerulonephritis and was prescribed a Table 2. Results of serum laboratory tests Test Patient’s results Reference interval Hemoglobin 12.1 10.0-15.5 g/dL Hematocrit 0.351 0.35-0.49 L/L White blood cell count 13.4 4.5-13.5 x 109 /L Sodium 139 138-145 mEq/L Potassium 4.0 3.4-4.7 mEq/L Chloride 105 98-107 mEq/L Carbon dioxide 26 20-28 mEq/L Blood urea nitrogen 11 5-18 mg/dL Serum creatinine 0.6 0.3-0.7 mg/dL Calcium 8.8 8.8-10.8 mg/dL Phosphorus 5.1 4.5-7.0 mg/dL Total protein 5.9 ↓ 6.0-8.0 g/dL Albumin 3.1 ↓ 3.8-5.4 g/dL Cholesterol 243 ↑ 124-201 mg/dL Alkaline phosphatase 177 ↑ 25-125 U/L Anti-nuclear antibody (ANA) <1:40 <1:40 Crithidia negative negative C3 complement 91 83-177 mg/dL C4 complement 29 12-36 mg/dL Strep enzyme screen negative negative
  • 24. 86 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS At the pediatric nephrology clinic, the patient’s only symptom was intermittent abdominal pain. Blood pressure was 130/90 mmHg and the physical exam was otherwise unremarkable. Urinalysis demonstrated persistent hematuria and protein- uria, the serum complement levels were normal, and the 24- hour total urine protein excretion was substantially elevated at 2,420 mg (normal is less than 150 to 250 mg/day). Ad- ditional laboratory results conducted on serum, summarized inTable 2, included decreased albumin, borderline low total protein, and elevated cholesterol and alkaline phosphatase. A renal ultrasound showed bilateral enlargement of the kid- neys. Serology typical of postinfectious glomerulonephritis (Streptozyme® ) or nephritis associated with systemic lupus erythematosus (anti-nuclear antibody) was negative. Her family history was remarkable for two maternal uncles with kidney disease each requiring a renal transplant while her mother’s urinalysis was negative for blood. Her nephrologist suspected the child had a form of glomerulonephritis, likely IgA nephropathy, but was concerned about the possibility of hereditary nephritis. A kidney biopsy was performed to establish a definitive diagnosis. The renal biopsy The renal biopsy specimen contained over 60 glomeruli by light microscopy. Ten percent of the glomeruli were obsolescent or totally scarred. The remaining glomeruli showed lobular accentuation of the glomerular tufts sec- ondary to diffusely increased cellularity, thickened capil- ten-day course of amoxicillin. In January 1998, the child returned toherphysician’sofficewithbronchitis.Atthattimeshehadahis- toryofgrosshematuria,herurinewasbrownincolor,andtheurine dipstick was positive for protein and blood. A ten-day treatment of clarithromycin was prescribed, blood tests were drawn, and the child was referred to a pediatric nephrologist. Figure 1. Light microscopy of glomerulus Hematoxylin and eosin stain demonstrated lobular accentuation of glomerular tufts due to mononuclear hypercellularity and excess accumulation of mesangial matrix (original magnification 400x). ‘Tram-tracks’ or duplication of the GBM was highlighted by the Jones’ silver stain (arrow, inset). Figure 3. Electron microscopy of glomerulus Ultrastructural examination revealed ribbon-like electron dense deposits within the GBM (arrows). U = urinary or Bowman’s space, E = erythrocyte in glomerular capillary lumen, P = podo- cyte, and M = mesangial matrix. Figure 2. Direct immunofluorescence microscopy Strong labeling with fluorescein-conjugated anti-C3 complement antibody was seen along capillary loops of glomeruli (center) and occasional tubular basement membranes (bottom right).
  • 25. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 87 RESEARCH AND REPORTS lary loops, and excess accumulation of mesangial matrix (Figure 1). The glomerular hypercellularity was due to increased mononuclear cells in the mesangium. Jones’ silver stain showed thickened and focally duplicated GBM (‘tram-tracks’) with increased mesangial matrix (Figure 1, inset). Cellular crescents, or extracapillary proliferation in Bowman’s space, were present in 10% of the glomeruli. Interstitial edema was apparent. The tubules and vessels were histologically unremarkable. Direct immunofluorescence examination of the renal biopsy specimen revealed strong immunofluorescence staining for complement C3 along the glomerular capillary loops (3+ to 4+ intensity on a scale of 0 to 4+) accompanied by weaker staining in the mesangium (1+ to 2+) (Figure 2). Staining of capillary loops and mesangium with immunoglobulins IgG, IgA, and IgM was negative to weak (0 to 1+). Staining with complement C1q and fibrinogen was negative. One glomerulus was examined by electron microscopy. Ultra- structural examination revealed numerous dense, elongated ribbon-likedepositsalongthelaminadensaofGBM(Figure3). Podocyte foot processes were effaced and the mesangial matrix was increased. Clinical course The renal biopsy results were consistent with MPGN type II or dense deposit disease. Based on the results of the renal biopsy, the child received six pulses of intravenous meth- ylprednisolone on an every other day schedule. This was followed by oral prednisone, 60 mg every other day. For hypertension, the patient was prescribed an angiotensin- converting enzyme inhibitor, enalapril, 2.5 mg/day. Six months after the kidney biopsy and initiation of therapy, her serum albumin had increased to 3.8 mg/dL, her 24- hour total urine protein excretion had decreased to 760 mg and she had no further episodes of gross hematuria. She was treated with reducing doses of alternate day prednisone for the next three years. During this interval, her serum albumin and creatinine remained normal, her microscopic hematuria resolved and her 24-hour total urine protein excretion decreased to less than 500 mg per 24 hours. Six years after the diagnosis of MPGN type II and nearly three years after discontinuation of prednisone therapy, she continues to have normal renal function and normal blood pressure, no microscopic hematuria, and minimal proteinuria as demonstrated by a urine protein excretion of less than 500 mg per day. MEMBRANOPROLIFERATIVE GLOMERULONEPHRI- TIS TYPE II Clinical findings MPGN is a common childhood glomerulonephritis that usu- ally progresses to chronic renal failure. Of the three subtypes of MPGN, type II is observed less frequently, accounting for about 20% to 30% of the cases of all MPGNs. Adults and children affected by MPGN type II are typically less than 20 years old with a median age of 11.5 years.4 Many children ultimately found to have MPGN originally receive medical attention due to asymptomatic hematuria and proteinuria. MPGN may present as either acute nephritic syndrome or nephrotic syndrome, with or without gross hematuria.3,4,6 Hypertension occurs in some patients due to water and so- dium retention, increased production of renin by the kidney, and other complex mechanisms that regulate blood pressure. In some cases, a patient may present with findings typical of poststreptococcal acute glomerulonephritis; however, if resolution of the symptoms of postinfectious glomerulone- phritis does not occur within six to eight weeks then other glomerulonephritides need to be considered (Table 1).4,5 Since the patient described in this report had persistent uri- nary abnormalities for a period of several months, it became likely that she did not have postinfectious glomerulonephritis and she underwent a kidney biopsy for the determination of a definitive diagnosis. Laboratory findings In a patient with MPGN, the major findings on the uri- nalysis are hematuria and proteinuria. Proteinuria may be pronounced and therefore lead to hypoalbuminemia, hyper- lipidemia, and edema: all features characteristic of nephrotic syndrome. Edema may arise as a result of decreased oncotic pressure resulting in fluid leaking from the intravascular space (within the blood vessel) into the tissues when serum protein levels are decreased due to urinary losses.4,7 At presentation, the patient’s blood urea nitrogen and creatinine levels are usu- ally normal to slightly elevated, unless a rapidly progressive glomerulonephritis is evident. A normocytic, normochromic anemia may also be present.4 In all three subtypes of MPGN, a major laboratory find- ing is hypocomplementemia characterized by low levels of C3, which is important to help characterize and formulate a provisional diagnosis in a child with hematuria and pro- teinuria.3,4,7 Depressed serum C3 is related to increased catabolism and decreased synthesis of complement.5 The C3 nephritic factor, NFa, is present in 30% to 75% of cases.4 C3 nephritic factor is an IgG autoantibody that stabilizes
  • 26. 88 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS the alternative pathway C3 convertase, C3bBb. The presence of C3 nephritic factor causes the formation of C3bBb that becomes resistant to inactivation by factor H and leads to very low C3 levels.7,8,9 Serum C4, C5, and properdin levels are usually normal in MPGN type II.5,9 Pathological findings The kidneys show no gross abnormalities that reflect the pathologic process in MPGN type II, although some patients may have bilateral renal enlargement as is typical in most patients with any form of glomerulonephritis. To separate the types of MPGN, ultrastructural examination is required to demonstrate the glomerular alterations. In MPGN type II, transmission electron microscopy shows sausage-shaped or ribbon-like osmiophilic (electron dense) deposits within the lamina densa of the GBM.3-5,7 Tubular basement mem- branes may be widened by similar deposits. The deposits occupy short segments in the glomerulus that are distributed irregularly in less severe cases. The dense deposits may also occur in the mesangium.10 When examined by brightfield microscopy, the hematoxylin- eosin stain imparts a lobular accentuation to the glomerular architecture due to capillary wall thickening and variable mesangial hypercellularity.3,10 Occasionally crescents may involve a portion of the glomeruli.4,7,10 Mesangial sclerosis, or scarring, is more obvious with disease progression. The source of glomerular capillary wall thickening is revealed by periodic acid-Schiff (PAS) or Jones’ silver stains that densely label duplicated basement membranes or ‘tram-tracks’ and impart weaker staining of the dense deposits.3,7 Thioflavin T stain gives a green coloration to the affected basement membranes when using fluorescence microscopy.5 Similar staining can be observed along Bowman’s capsule and in the mesangia.10 PAS and Jones’ stains highlight the mesangial sclerosis resulting from expansion of extracellular matrix.5,7 Tubular basement membranes may be thickened.3,5 Direct immunofluorescence microscopy shows extensive deposition of C3 within glomerular capillary loops and mesangia. Immunoglobulins are typically scarce; however, if they are present, the immunoglobulins are usually limited to a few glomerular segments.4,10 Pathogenesis The pathogenesis of MPGN type II is unclear but the primary morphology is dense deposits in the GBM. By unknown mechanisms, glomerular inflammation occurs subsequently. The origin and nature of the dense deposits are unknown.3-5 C3 nephritic factor has been implicated in the pathogenesis of MPGN type II due to the hypocomplementemia. How- ever, the disease progression does not appear to be affected by hypocomplementemia nor C3 nephritic factor.3-6 Prognosis The indicators of poor prognosis in MPGN type II are hypertension, impaired renal function at time of diagnosis, nephrotic syndrome, and the presence of crescents.6,7 The higher the percentage of glomeruli with crescents, the more rapidly renal function deteriorates.3,10 Although most patients diagnosed with MPGN type II have no visual complaints, MPGN type II is associated with abnormal retinal function. Dense deposits are observed in the Bruch membrane and the basement membrane of the choriocapillaris. Drusen deposits and retinal pigment epithelial disturbances are characteristic of dense deposit disease retinopathy. These findings are more commonly seen in patients with longstanding MPGN type II.4,11-13 MPGN type II usually progresses slowly to chronic renal failure. Fifty percent of the children will develop chronic renal failure within ten years following diagnosis. Within 20 years of diagnosis, 80% to 90% have chronic renal failure.4 Management Several different therapies have been used, including anti- platelet therapy and immunosuppression.4,6,14 However, to date, there is no universally accepted form of therapy.4,14 Most pediatric nephrologists use alternate-day prednisone therapy.4 Prednisone appears to stabilize renal function and improves disease characteristics; however, it can produce side effects due to drug toxicity.14 These side effects include stunted growth in children, hypertension, weight gain, Cushinoid features, and mood swings/personality changes. Therapy with pulse intravenous methylprednisolone followed by alternate-day oral prednisone has an improved outcome.4,14-17 The alternate- day regimen either suppresses the immune process underlying glomerular inflammation or decreases inflammation itself to inactivate the disease.5 Treatment may vary according to clinical symptoms and labo- ratory evaluation. The goals of the treatment are to reduce symptoms, prevent complications, and slow progression of the disease. Some children may require dietary restrictions on sodium, fluids, and protein to control high blood pres- sure, swelling, and accumulation of waste products in the bloodstream. Antihypertensive and diuretic therapy may be needed for treatment of edema and hypertension.4,7 Therapy with an angiotensin-converting enzyme inhibitor may be used to decrease urinary protein excretion and slow the
  • 27. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 89 RESEARCH AND REPORTS progression of chronic renal failure.To manage chronic renal failure, dialysis or kidney transplantation may eventually be necessary.4 However, in 50% to 100% of kidney transplant recipients, recurrence of dense deposits in the transplanted kidney may develop.18-20 When MPGN type II reoccurs, it leads to graft loss in 10% to 20% of the patients.18,19 SUMMARY The patient described in this case study presented with hema- turia, proteinuria, and elevated blood pressure. Contrary to most patients with MPGN type II, this 10-year-old female had a normal C3 complement level.Without the renal biopsy, this patient would not have been diagnosed with MPGN type II. Although the current therapy has done well in most cases to slow the progression of the disease, there is still a need for the development of a universally effective treatment that does not have significant adverse side effects. ACKNOWLEDGEMENTS This report is in compliance with Indiana University School of Medicine’s institutional review board and the Health Insur- ance Portability and Accountability Act of 1996. REFERENCES 1. Vinen CS, Oliveira DB. Acute glomerulonephritis. Postgrad Med J 2003;79(930):206-13. 2. Coppo R, Amore A. New perspectives in treatment of glomerulone- phritis. Pediatr Nephrol 2004;19(3):256-65. 3. Cameron JS,Turner DR, Heaton J, and others. Idiopathic mesangio- capillary glomerulonephritis: comparison of types I and II in children and adults and long-term prognosis. Am J Med 1983;74:175-92. 4. Andreoli SP. Chronic glomerulonephritis in childhood: membranop- roliferative glomerulonephritis, Henoch-Schönlein purpura nephritis, and IgA nephropathy. Seminars in Nephrol 1995;42(6):1487-1503. 5. West CD. Idiopathic membranoproliferative glomerulonephritis in childhood. Pediatr Nephrol 1992;6:96-103. 6. Schwertz R, de Jong R, Gretz N, and others. Outcome of idiopathic membranoproliferative glomerulonephritis in children. Acta Paediatr 1996;85(3):308-12. 7. Kasinath BS. Sifting the causes of microscopic hematuria. Hosp Pract 1996;31(7):99-106, 109-10. 8. West CD, McAdams AJ. Paramesangial glomerular deposits in membranoproliferative glomerulonephritis type II correlate with hypocomplementemia. Am J of Kidney Dis 1995;25(6):853-61. 9. West CD, McAdams AJ. The alternative pathway C3 convertase and glomerular deposits. Pediatr Nephrol 1999:13:448-53. 10. Churg J. Renal disease classification and atlas of glomerular disease. Tokyo: IGAKU-SHOIN;1982, p 83-109. 11. Kim RY, Faktorovich EG, Kuo CY, and others. Retinal function abnormalities in membranoproliferative glomerulonephritis type II. Am J of Ophthalmol 1997;123(5):619-28. 12. Leys A, Vanrenterghem Y, Van Damme B, and others. Sequential observation of fundus changes in patients with long standing mem- branoproliferative glomerulonephritis type II (MPGN type II). Eur J Ophthalmol 1991;1(1):17-22. 13. O’Brien C, Duvall-Young J, Brown M, and others. Electrophysiol- ogy of type II mesangiocapillary glomerulonephritis with associated fundus abnormalities. Br J Ophthalmol 1993;77(12):778-80. 14. Bergstein JM, Andreoli SP. Response of type I membranoproliferative glomerulonephritis to pulse methylprodnisolone and alternate-day prednisone therapy. Pediatr Nephrol 1995;9:268-71. 15. Tarshish P, Bernstein J, Tobin JN, and others. Treatment of mesan- giocapillary glomerulonephritis with alternate-day prednisone—a report of the international study of kidney disease in children. Pediatr Nephrol 1992;6(2):123-30. 16. McEnery PT, McAdams AJ, West CD. The effect of prednisone in a high-dose, alternate-day regimen on the natural history of idiopathic membranoproliferative glomerulonephritis. Med 1985;64(6):401-24. 17. Faedda R, Satta A, Tanda F, and others. Immunosuppressive treat- ment of membranoproliferative glomerulonephritis. Nephron 1994;67(1):59-65. 18. Shimizu T, Tanabe K, Oshima T, and others. Recurrence of mem- branoproliferative glomerulonephritis in renal allografts. Transplant Proc 1998;30:3910-13. 19. Andresdottir MB, Assmann KJM, Hoitsma AJ, and others. Renal transplantation in patients with dense deposit disease: morphological characteristics of recurrent disease and clinical outcome. Nephrol Dial Transplant 1999;14:1723-31. 20. Turner DR, Cameron JS, Bewick M, and others. Transplantation in mesangiocapillary glomerulonephritis with intramembranous dense “deposits”: recurrence of disease. Kidney Int 1976;9(5):439-48. Congratulations Graduate! Find the answers when you visit www.ascls.org/ jobs/grads/index.asp. Experts in clinical laboratory management, education, and practice have addressed the most frequently asked questions of graduating students just for you! Let ASCLS help you with your career development! To view current job openings, visit the ASCLS Career Center at www.ascls. org/jobs. 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  • 28. 90 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS “Children on the Frontline Against E.coli”: Typical Hemolytic-Uremic Syndrome HEIDI ANDERSEN The peer-reviewed Research and Reports Section seeks to publish reports of original research related to the clinical laboratory or one or more subspecialties, as well as information on important clinical laboratory-related topics such as technological, clinical, and experimental advances and innovations. Literature reviews are also included. Direct all inquiries to David G Fowler PhD CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of Clinical Laboratory Sciences, University of Mississippi Medical Center, 2500 North State St, Jackson MS 39216. (601) 984- 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu A thirteen-month old infant presented with classical hemolytic-uremic syndrome (HUS), but with negative cultures for Escherichia coli (E. coli) 0157:H7. HUS is commonly linked to infection with E. coli 0157:H7; however, traditional culture has demonstrated poor sensitivity. Pathogenesis of the organism in HUS involves the production of a Shiga-like toxin (STX), resulting in a triad of symptoms. An early and accurate differential diagnosis, based on patient presentation with acute renal failure, hemolytic anemia, and thrombocytopenia, is critical for supportive treatment and improved prognosis. Patient prognosis is related to the duration of renal failure and dialysis treatment. Research is aimed at improved detection of E. coli 0157:H7 or the STX produced, and future vaccination to eliminate typical HUS. ABBREVIATIONS: CDC = Centers for Disease Control and Prevention; HUS = hemolytic-uremic syndrome; STX = shiga- like toxin; TTP = thrombotic thrombocytopenic purpura. INDEX TERMS: hemolytic-uremic syndrome. Clin Lab Sci 2005(18(2):90 HeidiAndersenMT(ASCP)works at St John’s Hospital, Ander- son IN and at Indiana University Hospital, Indianapolis IN. Address for correspondence: Heidi Andersen MT(ASCP), 2007 B Parsons Drive, Indianapolis IN 46224. (317) 459- 3023. handerse@iupui.edu This article was written while the author was a student in the Clinical Laboratory Science program at Indiana University, Indianapolis IN. CASE STUDY In January, a thirteen-month-old Caucasian male presented with progressive diarrhea over a period of two weeks. During this time, medical attention was sought, and the infant was diagnosed with a common childhood diarrhea, suspected to be due to a rotavirus. However, the infant continued with progressive diarrhea and began showing signs of pallor, de- hydration, petechiae on his thighs, and decreased appetite. The infant began experiencing episodes of acute abdominal pain with intermittent periods of lethargy. As the diarrhea worsened, one sixteenth of a tablet of Imodium® was given to the infant and he was brought to the local emergency department (ED). The infant presented in the ED with signs of edema in the extremities from oliguria and acute renal failure. He was catheterized, treated with Lasix® to stimulate kidney function, and given nutritional IV support. Vitals revealed hypertension with a blood pressure of 132/55 (normal blood pressure of a 1-year-old Caucasian male is 102/57) and a temperature of 103.5 °F. As a result, the infant was given Hydralazine® and Tylenol® to reduce his elevated blood pressure and temperature respectively. The initial laboratory results indicated a diagnostic triad of thrombocytopenia, hemolytic anemia, and acute renal failure (Table 1). Subsequently, the infant was diagnosed with hemolytic-uremic syndrome (HUS). Also, it is important to note that the infant’s increased white cell count and fever supported the diagnosis of the onset of the inflammatory reaction that occurs in typical HUS, with E. coli 0157:H7 suspected as the cause. QUESTIONS TO BE CONSIDERED • What is the typical presentation of HUS and the patient population at risk? • What is the pathogenesis and pathophysiology of typical HUS? • What is the differential diagnosis of typical HUS?
  • 29. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 91 Table 1. Initial laboratory testing of patient on day 1 of hospitalization Tests Results Reference ranges Albumin 33 30.8 - 46.6 g/L BUN 32 1.8 - 7.1 mmol/L Chloride 102 95 - 105 mmol/L CO2 17 22 - 27 mmol/L Creatinine 336 17.7 - 61.9 mol/L Glucose 5.7 2.2 - 11.1 mmol/L Hematocrit 0.258 0.340 - 0.480 Hemoglobin 85 96 - 156 g/L Platelet # 64 150 - 450 x 109 /L Potassium 5.3 3.5 - 5.5 mmol/L Sodium 131 135 - 145 mmol/L Total bilirubin 11 0 - 10 g/L WBC # 21.5 5.5 - 17.5 x 109 /L RESEARCH AND REPORTS • What is the treatment and long-term prognosis of typical HUS? • What are preventive measures that can be taken to protect children from HUS? INTRODUCTION TO HUS The syndrome now known as HUS, was described in 1955 by Gasser. In 1982, Riley isolated pathogenic Shiga-like toxin producing E. coli (STEC) serotype 0157:H7 from contami- nated hamburger. Then, Karmali linked HUS to E. coli 0157: H7 in 1985. Connection of E. coli 0157:H7 to HUS was a major breakthrough in differentiating the mechanisms of the often-confused conditions of thrombotic thrombocytopenic purpura (TTP) and HUS. HUS is currently accepted as the most common cause of acute renal failure in children in the U.S. and is primarily caused by E. coli 0157:H7. The Centers for Disease Control and Prevention (CDC) estimate that with 73,000 E. coli 0157:H7 infections per year in the U.S., 2% to 7% of E. coli 0157:H7 infections result in HUS with the majority of cases occurring in Caucasians less than five years of age.1 The elderly, adults, and older children have higher mortality rates from HUS than occur in younger children.2,3 Interestingly, HUS caused by infection with E. coli 0157: H7 is less common in African-Americans.4 Overall, E. coli 0157:H7 infections costs about 60 lives and $660 million dollars each year, and is now an endemic cause of HUS in the U.S.1,5,6 Classical HUS is defined as a thrombotic microangiopathy with a diagnostic triad of acute hemolytic anemia with schistocytes, thrombocytopenia, and acute renal failure. However, classical HUS does not always present with a complete diagnostic triad. A major indicator of typical HUS from E. coli 0157:H7 is presentation with a one to eight day acute gastroenteritis prodrome and bloody diarrhea, unless the infection is acquired by a urinary tract infection or a respiratory infection.7,8 The kidney is the major organ target in classical HUS, but other organs such as pancreas, heart, lungs, and brain may also be involved.9-12 Other fac- tors supportive of a diagnosis of HUS due to E. coli 0157: H7 include its predominance in the summer months and its potential to occur in outbreaks. HUS, as noted by its name, is merely a syndrome, and therefore, has many known etiologies. Typical (infectious) HUS is caused by bacterial and viral infections such as E. coli 0157:H7, some non-0157 E. coli strains, Shigella dysenteriae 1, Streptococcus pneumoniae, Salmonella typhi, Campylobacter jejuni, and HIV. Non-infectious, atypical HUS may be sec- ondary to (but not limited to) pregnancy and postpartum, organ transplant, glomerulonephritis, systemic lupus erythe- matosus, or treatment with drug therapies such as tacrolimus (FK506), quinine, and mitomycin. However, it is estimated in the U.S. that 90% of cases of HUS in children are primary infections caused by E. coli 0157:H7 with increasing find- ings of non-0157:H7 E. coli cases, which may cause higher incidences of HUS in other countries.13,14 Infection with E. coli 0157:H7 can be acquired from several sources. Enterohemorrhagic E. coli (EHEC) is carried asymp- tomatically in the intestines of cattle, where Globotriaosylce- ramide (Gb3 ) receptors are found throughout the intestinal tract, but cattle lack Gb3 receptors in the vasculature.15,16 These findings may play a significant role in colonization. However, the reason cattle are asymptomatic carriers of E. coli 0157:H7 is still being studied. During slaughter, the surface of EHEC contaminated meat is ground into and spread throughout the hamburger.Only50to100viableorganismsof E.coli0157:H7 are required to cause infection.7 Other food products, besides ground beef, that can be a reservoir for EHEC after contamina- tionwithcattlefecesinclude:unpasteurizedmilkproducts,apple juice, water, and vegetables. There is seasonality to the shedding of EHEC from cattle, where there is an increased amount of organismshedinthesummermonths,whichcorrelateswithan increase in products with fecal contamination, E. coli 0157:H7 infections, and HUS in the warmer season.17 The infant in the case study, however, presented with HUS in January.
  • 30. 92 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS Although cattle are the major source of E. coli 0157:H7, it has been found in other animals, e.g., sheep, goats, deer, and even a small percentage in cats, dogs, horses, and flies.18,19 E. coli 0157:H7 has been shown to survive ten months or longer in contaminated sources that resulted in human infection.20 Additionally, E. coli 0157:H7 can readily be transmitted from person to person, causing concern to families, nursing homes, daycare facilities, and other crowded living conditions.21,22 After September 11, 2001, E. coli 0157:H7 was considered to be a potential agent of bioterrorism.23 In the case of this thirteen-month old child, no other children or adults with whom he had contact were infected. Pathogenesis and pathophysiology The pathogenesis of typical HUS begins with the hardiness of E. coli 0157:H7. E. coli 0157:H7 can easily live through freezing at –80 °C for as long as nine months. Its acid resis- tance increases survival in acidic foods (conditions that are normally used to preserve food from bacterial contamination) because acidity decreases the nutritional competition with other non-acid-resistant organisms. E. coli 0157:H7 can remain viable in a pH environment less than 2.5.24 Once E. coli 0157:H7 enters the body orally, it survives the pH of the stomach, and colonizes in the mucosa of the colon. The exact mechanism of E. coli 0157:H7 pathogenic- ity is under extensive research. A proposed mechanism is that the lipopolysaccharide and STX from the organism are responsible for activating the phosphatidylinositol cascade, increasing mobilized ionized calcium that allows adherence of the organism to the epithelial cells of the colon and pro- motes lesion formation. Lesions change the permeability of the epithelial cell membrane to water and electrolytes, causing the prodromal diarrhea, and initiate an inflamma- tory response partially responsible for the microangiopathic thrombosis.25 The inflammatory response activates neutro- phils that release cytokines, platelet activating factor (PAF), and tissue necrosis factor alpha. These play a role in increasing activated neutrophils (leukocytosis), platelet aggregation,26 and up-regulating Globotriaosylceramide (Gb3 ) receptors on kidney and brain endothelial cells,12 respectively. Close proximity of the injured epithelial cells to microvasculature results in hemorrhagic colitis (HC) and bloody diarrhea in the typical HUS. STX access to circulation following HC allows for direct and indirect mechanisms of pathogenicity. STX reaches the kid- ney and brain through the circulatory system on the surface of platelets, neutrophils, and monocytes.27-29 STX bound to platelets induces platelet aggregation directly, and STX prevents apoptosis in neutrophils, resulting in leukocytosis and increased inflammation.27,30 The proposed primary target of STX from the circulation is the distal convoluted tubular epithelium in children (Gb3 receptors may not be present in adults).31 The injured renal epithelial cells also initiate an inflammatory response and release endothelin, tissue plasminogen activator inhibitor-1 (PAI-1), and PAF. Endothelin increases production of PAF, white blood cell activation that results in leukocytosis, and may somewhat increase blood pressure early on in typical HUS. PAI-1 inhibits fibrinolysis of microvascular throm- bosis. PAF-activated platelets release thromboxane hence promoting platelet aggregation by vasoconstriction-induced high shear stress. Thromboxane is regulated by prostacyclin released from injured epithelial cells through negative feed- back. However, in typical HUS, platelet activation outnum- bers production of prostacyclin resulting in thrombosis.32 Microvascular endothelial cells that have Gb3 receptors (in children and adults), particularly those of the glomerulus, are extremely sensitive to STX. Injury to the endothelial cells by bound STX is key to the pathogenesis of typical HUS.7,32 STX causes extensive microangiopathic thrombosis, hypoxia, and ischemia in the glomerular endothelial cells, and similar damage can be found in the cerebral endothelial cells of the blood-brain barrier.33 Injury to the endothelial cells is marked by elevated thrombomodulin levels. Lesions from STX in- volve the glomerular capillaries; thickening of the capillary wall near the glomerulus decreases the glomerular filtration rate.34 Both ischemia and thickened capillaries elevate blood pressure. Progressive damage to the kidneys occurs via hy- perfiltration of the functional nephrons that remain after the acute phase.35 Damage to cerebral endothelial cells causes encephalophy, coma, stroke, and cerebral infarcts.33 Toxins are freely permeable to the glomerulus, and the large capillary surface area enhances toxin pathogenicity. The concentration of toxin is increased by countercurrent roles of vasculature and tubules in the kidney.36 STX has one enzymatic subunit A and five receptor-specific B subunits. Subunit A invades and kills the renal endothelial cells by endocytosis and inhibiting translation. Subunit B activates neutrophils, releasing proteases and hydrogen peroxide that irreversibly damage renal cells.37 Oxidative substances and fibrin-platelet aggregations occluding the microvasculature fragment red cells, producing schistocytes on the peripheral blood smear.
  • 31. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 93 Table 2. Differential diagnosis of typical HUS including patient case study results Laboratory and Patient Typical Atypical TTP DIC clinical findings HUS HUS Abnormal kidney tests Y Y Y Y Y/N Abnormal liver tests N N N N Y/N Abnormal PT, PTT, and D-dimer N N N N Y CNS involvement N Y/N Y/N Y Y GI prodrome/ bloody diarrhea Y Y N N N Hemolytic anemia Y Y Y Y Y Hypertension Y Y Y Y Y Leukocytosis Y Y N Y/N Y Positive Coombs test N N N N N Recurrences N N Y/N Y N Renal failure Y Y Y Y/N Y/N Thrombocytopenia Y Y Y Y Y Y = Yes, N = No RESEARCH AND REPORTS Differential diagnosis It is critical to differentiate typical HUS from atypical HUS, thrombotic thrombocytopenic purpura (TTP), and disseminated intravascular coagu- lation (DIC) for both treatment and prognosis (Table 2). For example, typi- cal HUS requires supportive therapy whileTTP mandates plasma exchanges and possibly immunosuppressive treatment with glucocorticoids. Mis- diagnosis could be life threatening; a significant number of deaths from TTP occur within forty-eight hours of presentation.38 Atypical causes of HUS are associated with a high mortality rate and recur- rences. These causes include comple- ment deficiency involvement, neuro- logical involvement, an inborn error in the metabolism of cobalamin, or a factor H deficiency. Most secondary causes of atypical HUS can be ruled out with patient history. Generally, atypical HUS does not present with leukocytosis or GI prodrome and bloody diarrhea, and is seen more fre- quently in adults than young children, perhaps simply because adults are likely to have secondary causes. TTP, a completely separate disease from HUS, is the result of a decreased level of von Willebrand Factor (vWF)-cleaving enzyme (ADAMTS-13) due to a defi- ciency of ADAMTS-13 or antibodies against it.39,40 The classical presentation of TTP includes fever, central nervous system involvement, and the diagnostic triad seen in classical HUS. However, 25% of typical HUS cases have nervous system involvement and many present with fever.41 Unlike typical HUS, TTP does not generally involve gastrointes- tinal inflammation, or leukocytosis.42 TTP is usually systemic with severe thrombocytopenia resulting in platelet counts below 20 x 109 /L, where typical HUSisprimarilylocalizedtothekidney with platelet counts between 30 x 109 /L and 150 x 109 /L. Risk for TTP is not targeted to a specific age group, like typical HUS. In the future, differentiat- ing TTP from HUS may be easier by measuringthefunctionalADAMTS-13 level in the plasma.43 DIC is a systemic activation of both the coagulation cascade and fibrinoly- sis, and is commonly associated with pregnancy-related complications like atypical HUS. The easiest most reliable way to differentiate DIC is with rou- tine coagulation testing. Prothrombin time (PT), partial thromboplastin time (PTT), and D-dimer levels are abnormal in DIC, but are normal in patients with typical and atypical HUS. However, vitamin K deficiency may prolong the PT, and D-dimer levels may be elevated with a high concentration of fibrin in the micro- angiopathic thrombus in patients with typical HUS.44 LABORATORY FINDINGS AND CLINICAL COURSE Upon diagnosis, the thirteen-month- old infant was transferred via am- bulance to a major referral childrens hospital. Albumin, total bilirubin, and hemoglobin from the initial labora- tory tests (Table 1) were near normal supporting the beginning of the acute phase of HUS. Hemoglobin drops as hemolysis increases during the acute
  • 32. 94 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE Table 3. Laboratory evaluation of patient hemolysis and confirmation of typical HUS Tests Patient results Reference ranges AST 176 25 - 45 U/L LDH 10240 425 - 975 U/L Lipase 160 25 - 120 IU/L PT/INR 12.8 sec/1.05 NA/0.9 - 1.1 PTT 32.5 24.7 - 33.4 sec Table 4. Patient urinalysis results on day-2 of hospi- talization Urinalysis and Patient results Reference range microscopic Appearance cloudy clear Bacteria many negative Bilirubin negative negative Color pink colorless-dark yellow Glucose negative negative Hemoglobin 250 Ery/µL negative Ketones 5 mg/dL <75 mg/dL Nitrite negative negative pH 5.0 5.0-8.5 Protein 500 mg/dL negative Red blood cells 663/HPF 0-3/HPF Specific gravity 1.016 1.003-1.030 Urobilinogen negative 0.1-1.0 Ehrlich’s units WBC esterase 100 LEU/µL negative White blood cells 66/HPF 0-5/HPF RESEARCH AND REPORTS phase, and total bilirubin rises as hemoglobin from lysed red cells is broken down, following degradation of the hemoglobin porphyrin ring. A type and screen (T&S) and indirect Coombs were ordered for type-specific supportive transfusions. The indirect Coombs was negative, support- ing a non-immune-mediated cause of hemolysis. Clinically, hemolysis is monitored by the hemoglobin level, but LDH and AST tests supplement evaluation of hemolysis. LDH is more specific to hemolysis; however, extremely elevated LDH also reflects tissue necrosis (Table 3).45 Since there is extensive red cell destruction in HUS, most of the hapto- globin is bound to free hemoglobin, giving lower detectable levels of haptoglobin in the serum. Coagulation studies were found to be normal ruling out DIC in the differential diagnosis (Table 3). Recovery of a small urine sample was obtained from the Foley catheter of the infant for urinalysis (Table 4) which confirmed proteinuria, acidosis, and hemolysis. Red cell casts were not found in this sample, but are commonly found with care- ful examination in patients with typical HUS.46 The infant began peritoneal dialysis on day 3 (Figures 1, 2, and 3), was given nasogastric tube feedings for nutritional support, and was placed in barrier isolation to prevent transmission of suspected E. coli 0157:H7. In addition to the urinalysis, stool cultures were ordered for growth of E. coli 0157:H7, Shigella, Yersinia, Salmonella, and Campylobacter. Cultures are done to confirm a causative agent of HUS and for epidemologic purposes such as track- ing of the contaminated food product and disease control. Sorbitol MacConkey agar is generally the medium of choice for isolating the non-sorbitol-fermenting E. coli 0157:H7. Most non-pathogenic E. coli found in the normal flora of the GI tract ferment sorbitol. After successive negative stool cul- tures for STX-producing organisms, the infant was released from barrier isolation. A negative culture does not rule out a typical HUS diagnosis, and is commonly seen due to the transient shedding of E. coli 0157:H7, culturing too late in the course of the infection, or poor culture sensitivities. The best time to obtain a positive culture for the infectious organism is during the GI prodrome.14 Throughout the infant’s 32-day hospitalization, HUS was monitored with daily basic metabolic panels and cell blood counts. Monitoring hemoglobin, platelets, creatinine, and albumin throughout the acute phase of HUS is indicative of severity and duration of hemolysis, microthrombi, and kidney dysfunction, respectively. An increase in catabolism, during the acute phase of the infection, and the change in distribution of volume to albumin in the serum resulted in hypoalbuminemia, reaching a low of 1.7 mg/dL. Because no platelets were transfused or any other therapy given that would directly affect the platelet count, the rise in platelet count on day-11 marked the initiation of the recovery phase (Figure 2). Day-15 marked the end of the acute phase of the illness with stabilization of the platelet count, hemoglobin, and creatinine (Figures 1-3). After day-15, supportive transfusions were no longer necessary, and on day-20, recovery was confirmed by discontinuation of dialysis with continued stabilization of creatinine levels (Figure 3).
  • 33. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 95 RESEARCH AND REPORTS As commonly seen in classical typical HUS, the WBC differentials showed a left shift with few myelocytes and metamyelocytes along with leuko- cytosis and thrombocytopenia. Cell morphology included moderate hy- pochromic, microcytic red cells with a few schistocytes, nucleated red cells, ovalocytes, polychromasia, and giant platelets. Reticulocyte counts would likely be increased, while the infant at- tempted to compensate for the anemia with increased red cell production. A bone marrow aspirate is certainly not necessary and would be risky given the presence of thrombocytopenia. How- ever, cellular morphology of the bone marrow would be expected to show normal to increased megakaryocytes (depending on platelet consumption), and erythroid hyperplasia. As often occurs with HUS, this infant had several complications. On day- 2, the infant developed tachycardia, cardiac arrythmia, and periorbital edema. Cardiac arrhythmias in HUS are due to cardiac ischemia, which can also result in myocardial infarction in typical HUS.10 On day-4, slight involvement of the pancreas was seen with elevated serum lipase (Table 3). Both lipase and amylase are markers for pancreatitis. Since lipase is not elevated by as many conditions as amylase, it is a more specific indicator for pancreatitis. After the onset of ab- dominal pain, lipase levels rose within 12 hours and rapidly fell to normal range within a few days. In addition, the infant developed peritonitis when the dialysis catheter was compromised, which was reflected by the increasing creatinine levels at day-9 (Figure 3). A dialysate culture revealed coagulase- negative Staphylococcus sp. and Strepto- coccus pyogenes, and a dialysate sample revealed a neutrophil count of 1830 x 109 /L with 75% neutrophils and 23% monocytes with toxic granulation. The infant was treated with vancomycin and serum levels were monitored. On day-25, the infant spiked a fever of 103 °F and was treated with cefotaxime and vancomycin for possible septicemia. Both blood and urine cultures were obtained, and both were found to be negative for growth. After conclusion of the acute phase, a urinalysis still showed significant pro- teinuria along with a few granular casts and renal epithelial cells, but the he- moglobin levels and red cell numbers supported the cessation of hemolysis. The infant was discharged on day-32 with resolution of the fever and the acute phase of typical HUS. The infant continues to be closely monitored for long-term prognosis. TREATMENT The treatment regimen used in the acute phase of typical HUS is primarily supportive and may in- clude medication to control blood pressure, continuous peritoneal dialysis or hemodialysis, packed red cell transfusions, fluid restriction, diuretics, and very careful mainte- nance of electrolytes. In rare cases, a kidney transplant may be required. Untreated typical HUS will lead to coma, cerebral infarcts, pancreatic insufficiency, and death.46 However, Figure 1. Hemoglobin vs. Time Figure 2. Platelet Count vs. Time Figure 3. Creatinine vs. Time
  • 34. 96 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS rigorous management of treatment has decreased the mortality rate of typical HUS from 50% to about 5% to 10% throughout the last five decades.47,48 There are important contraindications to certain treatments when typical HUS is diagnosed. Platelets are generally not transfused, because they may cause more severe microangio- pathic thrombosis. Antibiotics are also not suggested, because killing E. coli 0157:H7 may rupture the organism’s cell wall, thereby increasing the amount of toxin released, and contrib- uting to the severity of the acute phase.49 If complications occur, however, during typical HUS, such as peritonitis or sepsis, antibiotics may be necessary. Anti-motility drugs for diarrhea are not advised, because they may increase the risk of typical HUS by extending the exposure time of STX to the patient’s cells.50 Long-term treatment for patients requires monitoring of blood pressure, creatinine, and urinalysis.51 Patients exhib- iting hypertension and proteinuria are treated to lower the blood pressure with an angiotensinogen-converting enzyme (ACE) inhibitor (blocking the production of renin and consequently, aldosterone) to reduce further damage to the glomerulus, reduce proteinuria, and delay or prevent future chronic renal failure.52 Two years of post-HUS laboratory test- ing on this infant confirmed hypertension and proteinuria. After beginning treatment with an ACE inhibitor, the infant showed a decrease in blood pressure and proteinuria. PROGNOSIS Although prognosis of HUS remains variable, a recent meta- analysis produced significant data. It is estimated that 58% of HUS patients fully recover, 5% die within the acute phase, 11% develop chronic renal failure or die within four years of the acute phase, and 17% experience long-term residual effects such as hypertension, proteinuria, and a decreased glomerular filtration rate.There is often a period of perceived renal recovery before the onset of long-term residual effects. Surprisingly, even some patients who presented with mild HUS (without dialysis or abnormal urine output) developed renal sequelae.47 Renal sequelae may result in renal failure more than twenty years after the acute phase of HUS.53 Thus, it is extremely important that the renal function of any child with a history of HUS be followed for life. The best practical indicator of prognosis is the duration of oliguria or anuria and consequently, the need for dialysis dur- ing the acute phase of HUS.54 The most sensitive indicator is a kidney biopsy, which shows the degree of renal cortical necrosis. This procedure is impractical and contraindicated in the acute phase, but it is used later to assess long-term prognosis.53,55 Other factors that may support poor long-term prognosis include: elevated white cell count >20 x 109 /L, neural involvement, post-HUS proteinuria, and hyperten- sion during the acute phase.35,56-58 In the case history of the thirteen month old infant, the following poor prognostic indicators were found: oliguria/anuria >10 days, leukocytosis >20 x 109 /L, post-HUS proteinuria, and hypertension at onset of HUS. PREVENTION There are three important levels of prevention when dealing with E. coli 0157:H7. On the public level, proper hand- washing and safe preparation of food remain the best prac- tices for the prevention of typical HUS. However, too much responsibility has been placed on the public.59 The state and federal government play critical roles in prevention. They are responsible for keeping the food supply safe and ensuring quality assurance by mandating effective screening of food products for E. coli 0157:H7 and other foodborne pathogens. In the past, only a few states required such screening. With the new rapid STX detection methods available, surveillance of E. coli 0157:H7 in the beef industry is improving.60 The Food and Drug Administration has approved the irradiation of ground beef, which can kill at least 90% of E. coli 0157:H7 contaminants.24 A law mandating irradiation of other poten- tially contaminated food products was approved by the United States Department of Agriculture and is being implemented. Cases involving contaminated water sources have also called attention to enforcing regulations for protecting small water systems that in the past received little attention.61 RESEARCH AND THE FUTURE Current research is aimed at faster and more reliable testing of STX as well as countering the action of STX produced by any organism. The conventional method involving the culture of stool on MacConkey-Sorbitol agar for E. coli 0157:H7 is only 40% sensitive and takes up to three days to complete.62 Further complicating the diagnosis of typical HUS is the usual delay of symptoms for one to two weeks after infection, leaving few organisms in the stool for detec- tion. In addition, more than 100 other shiga toxin-producing E. coli (STEC) serotypes are left undetectable by many clini- cal laboratories because they ferment sorbitol, like normal intestinal strains of E. coli.63 Improved technology is strongly needed to enhance the sensitivity of culture for E. coli 0157:H7 in HUS patients.
  • 35. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 97 RESEARCH AND REPORTS In 1996, research showed a success rate of 90% for E. coli 0157:H7 if an immunomagnetic separation (IMS) technique was used to isolate the 0157 antigen (using beads with the corresponding antibody) after use of a pre-enrichment culture medium. This method proved to have many advantages over new PCR methods for detecting E. coli 0157:H7 including at least a 100-fold increase in sensitivity, and less complex test- ing.64 In 1999, the United States Department of Agriculture began implementing IMS to improve the screening of ground beef. To date, many clinical laboratories have not adopted the improved methods for routine screening of E. coli 0157: H7 in children suspected of HUS.65 If a clinical laboratory is unable to implement new technology for detecting E. coli 0157:H7 and/or STX, the specimen should be referred to an appropriate reference laboratory. Alternative rapid tests for non-0157 STEC are directed to- ward the detection of STX produced instead of the organism. One method uses an EIA technology that requires approxi- mately 18 hours to perform (this includes a 16-hour incuba- tion period for increased sensitivity), while another method is a simple toxin detection tests that only takes three hours to perform. Both methods may significantly shorten the time for diagnosis. In addition, other improved methods include PCR followed by pulse-field gel electrophoresis, impedance technology, and enzyme-linked immunoassays. Vaccination is another potential area of interest. Healthy persons develop an antibody to the antigenic subunit B of STX after exposure.66 When mice were immunized with the B subunit of STX, they were able to produce antibodies that neutralized the potent effects of STX.67 Monkeys have also been successfully immunized and protected by an STX vaccine when challenged with a lethal dosage.68,69 Immuni- zations may be administered for both humans and cattle in order to reduce E. coli 0157:H7 infections and incidence of typical HUS. Treatment of patients with monoclonal antibodies, currently in clinical trials, may neutralize the STX in patients present- ing with early diagnosis of typical HUS. The monoclonal STX antibody binds STX in the intestine and may render the toxin less able gain access to the circulation. Hence, STX is less likely to affect the kidney or other organs.42 One remaining concern with this treatment is the remaining lipopolysaccharide (LPS) of the bacteria that also plays an antigenic role in typical HUS, perhaps warranting the need for treatment with another monoclonal specific antibody or a polyvalent antibody to both STX and LPS.32 CONCLUSION As in the case of this thirteen-month-old infant with HUS, it is estimated that E. coli 0157:H7 causes 73,000 of the 76 mil- lionfood-borneillnesseseachyear.Mostfood-borneillnessesin the U.S. are self-limiting, but those that cause HUS may lead to death or life-long consequences such as renal sequelae and gastrointestinal complications. Progress has been made in the eliminationoffood-bornecausesoftypicalHUSviairradiation offoodproductsandimprovedqualityassuranceandcontrolin the food industry. Current research is focused on vaccination, improved diagnostic testing methods, and intervening treat- ments with early diagnosis. “So HUSH all you children and don’t you cry, together we will beat the bug they call E. coli.”70 REFERENCES 1. Centers for Disease Control and Prevention. Escherichia coli 0157: H7. Available at: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ escherichiacoli_g.htm Accessed April 28, 2004. 2. Carter AO, Borczyk AA, Carlson JA, and others. A severe outbreak of Escherichia coli 0157:H7 - associated hemorrhagic colitis in a nursing home. N Engl J Med 1987;317:01496-500. 3. Melnyk AMS, Solez K, Kjellstrand CM. Adult hemolytic-uremic syndrome: a review of 37 cases. Arch Intern Med. 1997;155:2077-84. 4. Jernigan SM, Waldo FB. Racial incidence of hemolytic uremic syndrome. Pediatr Nephrol 1994;8:545-7. 5. United States Department of Agriculture. Economics of foodborne disease: E. coli. 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Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation. J Clin Invest 1986;78:1456-61. 41. Gerber A, Karch H, Allerberger F, and others. Clinical course and the role of shiga toxin-producing Escherichia coli infection in the hemolytic-uremic syndrome in pediatric patients, 1997- 2000, in Germany and Austria: a prospective study. J Infect Dis 2002;186(4):493-500. 42. Pisoni R, Ruggenenti P, Remuzz G. Thrombotic microangiopathies including hemolytic-uremic syndrome. In: Johnson RJ, Feehally J, editors. Comprehensive Clinical Nephrology, 2nd ed. Philadelphia PA: 2003;413-23. 43. Gerritsen H, Turecek P, Schwarz HP, and others. Assay of von Willebrand factor (vWF)-cleaving protease based on decreased collagen binding affinity of degraded vWF. A tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999;82:1386-9. 44. Chandler WL, Jelacic S, Boster DR. Prothrombotic coagulation abnormalities preceding the hemolytic-uremic syndrome. N Engl J Med 2002;346(1):23-32. 45. Cohen JA, Brecher ME, Bandarenko N. Cellular source of serum lactate dehydrogenase elevation in patients with thrombotic thrombocytopenic purpura. J Clin Apheresis 1998;13:16-9. 46. Corrigan JJ Jr, Boineau FG. Hemolytic-uremic syndrome. Pediatr Rev 2001;22(11):365-9. 47. Garg AX, Suri RS, Barrowman N, and others. Long-term renal prognosis of diarrhea-associated hemolytic uremic syndrome: a systematic review, meta-analysis, and meta-regression. JAMA 2003;290(10):1360-70. 48. Rust RS. Thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Available at: www.emedicine.com/neuro/ topic499.htm Accessed May 2, 2004. 49. Safdar N, Said A, Gangnon RE, and others. Risk of hemolytic uremic syndrome after antibiotic treatment of Escherichia coli 0157: H7 enteritis: a meta-analysis. JAMA 2002;288:996-1001.
  • 37. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 99 RESEARCH AND REPORTS 50. Cimolai N, Carter JE, Morrison BJ. Risk factors for the progression of Escherichia coli 0157:H7 enteritis to hemolytic-uremic syndrome. J Pediatr 1990;116:589-92. 51. Krmar RT, Ferraris JR, Ramirez JA, and others. Ambulatory blood pressure monitoring after recovery from hemolytic uremic syndrome. Pediatr Nephrol 2001;16:812-6. 52. Remuzzi G. The future of reno-protection: frustrations and promises. Presented at: World Congress of Nephrology; June 8-12, 2003; Berlin, Germany. 53. Gagnadoux MF, Habib R, Gubler MC, and others. Long-term (15- 25 years) outcome of childhood hemolytic-uremic syndrome. Clin Nephrol 1996;46(1):39-41. 54. Fitzpatrick MM, Shah V, Trompeter RS, and others. Long term renal outcome of childhood haemolytic uraemic syndrome. BMJ 1991;303:489-92. 55. Nelid GH. Haemolytic-uraemic syndrome in practice. Lancet 1994;343(8894):398-401. 56. Fernandez GC, Rubel C, Dran G, and others. Shiga toxin-2 induces neutrophilia and neutrophil activation in a murine model of hemolytic uremic syndrome. Clin Immunol 2000;95(5):227-34. 57. Siegler RL. Spectrum of extrarenal involvement in postdiarrheal hemolytic-uremic syndrome. J Pediatr 1994;125:511-8. 58. Siegler RL. Postdiarrheal shiga toxin-mediated hemolytic uremic syndrome. JAMA 2003;290(10):1379-81. 59. Safetablesourpriority.Whyarepeoplestilldyingfromcontaminated food? Available at: www.safetables.org/pdf/STOP_Report.pdf. Accessed May 21, 2004. 60 Murano EA. Enhancing and evolving: advancements in 2003 and initiatives to improve food safety in 2004; February 12, 2004; San Antonio TX. 61. Bopp DJ, Sauders BD, Waring AL, and others. Detection, isolation, and molecular subtyping of Escherichia coli 0157:H7 and Campylobacter jejuni associated with a large waterborne outbreak. J Clin Microbiol 2003;41(1):174-80. 62. Elliot EJ, Robins-Browne RM, O’Loughlin EV, and others. Nationwide study of haemolytic uraemic syndrome: clinical, microbiological, and epidemiological features. Arch Dis Child 2001;85:125-31. 63. Tarr PI, Neill MA. Perspective: the problem of non-0157:H7 shiga toxin (verocytotoxin)-producing Escherichia coli. J Infect Dis 1996;174:1136-9. 64. Karch H, Janetzki-Mittmann C, Aleksic S, and others. Isolation of enterohemorrhagic Escherichia coli 0157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods and direct culture. J Clin Microbiol 34:516-9. 65. United States General Accounting Office. Emerging infectious diseases: consensus on needed laboratory capacity could strengthen surveillance. Report to the Chairman, Subcommittee on Public Health, Committee on Health Education, Labor, and Pensions, U.S. Senate. Washington (DC): GAO/HEHS-99-26; 1999. 66. Ludwig K, Sarkim V, Bitzan M, and others. Shiga toxin-producing Escherichia coli infection and antibodies against Stx2 and Stx1 in household contacts of children with enteropathic hemolytic-uremic syndrome. J Clin Microbiol 2002;40(5):1773-82. 67. Byun Y, Ohmura M, Fujihashi K, and others. Nasal immunization with E. coli verotoxin 1(VT)-B subunit and a nontoxic mutant of cholera toxin elicits serum neutralizing antibodies. Vaccine 2001;19:2061-70. 68. Suzaki Y, Ami Y, Nagata N, and others. Protection of monkeys against shiga toxin induced by shiga toxin-liposome conjugates. Int Arch Allergy Immunol 2002;127:294-8. 69. Mukherjee J, Chios K, Fishwild D, and others. Human Stx2-specific monoclonal antibodies prevent systemic complications of Escherichia coli 0157:H7 infection. Infect Immun 2002;70(2):612-9. 70. Haemolytic Uraemic Syndrome Help. H.U.S.H – A Poem. Available at: www.ecoli-uk.com/where.htm. Accessed April 22, 2004. Clinical Laboratory Science Announces 2004 Distinguished Author Award Recipients Recipients of the Clinical Laboratory Science (Clin Lab Sci) Distinguished Author Awards are chosen by Clin Lab Sci readers and editorial board members. Nominations are based upon originality, quality of writing, and relevance and value to the clinical laboratory science profession. The Editorial Board of Clin Lab Sci is pleased to announce the following recipients of the 2004 Distinguished Author Awards: Reports and Reviews Vicky A LeGrys, Katherine Hartmann, and Joan R Walsh for their article The Clinical Consequences and Diagnosis of Hypothyroidism published in the Fall 2004 issue of Clinical Laboratory Science. Research Isaac D Montoya for his article Topography as a Contextual Variable in Infectious Disease Transmission published in the Spring 2004 issue of Clinical Laboratory Science. Focus Section Louann W Lawrence for her article refractory Anemia and the Myelodysplastic Syndromes published in the Summer 2004 issue of Clinical Laboratory Science.
  • 38. 100 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS 2003 Workforce Survey of Hospital Clinical Laboratories in New Jersey ELAINE M KEOHANE, MARY ELLEN SCHAAD, KAREN FEENEY The peer-reviewed Research and Reports Section seeks to publish reports of original research related to the clinical laboratory or one or more subspecialties, as well as information on important clinical laboratory-related topics such as technological, clinical, and experimental advances and innovations. Literature reviews are also included. Direct all inquiries to David G Fowler PhD CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of Clinical Laboratory Sciences, University of Mississippi Medical Center, 2500 North State St, Jackson MS 39216. (601) 984- 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu ABBREVIATIONS: CT = cytotechnologist; HLT = histo- technologist; HT = histotechnician; MLT = medical labora- tory technician; MT = medical technologist. INDEX TERMS: job opportunities; workforce. Clin Lab Sci 2005;18(2):100 Elaine M Keohane PhD CLS(NCA) is Professor, Department of Clinical Laboratory Sciences, University of Medicine and Dentistry of New Jersey, Newark NJ. Mary Ellen Schaad MT(ASCP) isTechnical Manager, Clini- cal Laboratory, Meridian Health System, Neptune NJ. Karen Feeney MPA MT(ASCP)DLM is Administrative Director, Department of Laboratories, Bayshore Community Hospital, Holmdel NJ. Address for correspondence: Elaine M Keohane PhD CLS(NCA), University of Medicine and Dentistry of New Jersey, School of Health Related Professions, 65 Bergen Street, Newark NJ 07107. (973) 972-5510, (973) 972-8527 (fax). keohanem@umdnj.edu The authors are also members of the Coalition for New Jersey Clinical Laboratory Personnel. The clinical laboratory personnel shortage has reached significant proportions in many areas of the country and there is growing concern about its impact on the accessibil- ity and quality of clinical laboratory services. For the years 2002–2012, the U.S. Bureau of Labor Statistics projected a need for 138,000 new clinical laboratory technologists and technicians, or approximately 13,800 per year, due to growth and attrition from the field.1 On the other hand, in 2002, there were only 3,548 clinical laboratory technician/medical laboratory technician (CLT/MLT) and clinical laboratory sci- entist/medical technologist (CLS/MT) graduates in the U.S.2 If the current imbalance between vacancies and graduates continues, the national shortage of clinical laboratory person- nel may grow by more than 10,000 laboratorians per year. In surveys conducted by the American Society for Clinical Pathology (ASCP), vacancy rates in 2000 for medical tech- nologists (MTs) and medical laboratory technicians (MLTs) were 11.1% and 14.3% nationally and 14.9% and 24.5% in the northeast; in 2002 those rates showed a decrease to 7% and 8.6% nationally and 8.3% and 3.5% in the northeast. Although the vacancy rates in the latter study decreased to single digits, the vacancies are nevertheless noteworthy in terms of the actual number of vacant positions, taking into consideration a national workforce estimated at 297,000 clinical laboratory technologists and technicians.1 A Coalition for New Jersey Clinical Laboratory Personnel was formed in April 2002 to study the extent of and ad- dress a perceived shortage of clinical laboratory personnel in New Jersey. This coalition consists of twenty-eight members representing hospital clinical laboratory administrators, su- pervisors, and educators; hospital human resources directors; and representatives from the New Jersey Society for Clinical Laboratory Science, New Jersey Clinical Laboratory Man- agement Association, New Jersey Hospital Association, the New Jersey State Department of Health and Senior Services, and New Jersey Medicaid. One of the goals of the coalition is to document and disseminate data on the supply of and demand for clinical laboratory professionals in the state. An unpublished study conducted by the New Jersey Society for Clinical Laboratory Science showed a 48.5% decrease in MLT and MT graduates since 1998, with only 26 MTs and 21 MLTs graduating in 2003 in the entire state. In addition, during that same time, the state experienced the closure of one MT and three MLT programs. There are no histotech- nologist (HTL) programs, and only one histotechnician (HT) program in the state, but that program recently went
  • 39. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 101 RESEARCH AND REPORTS Table 1. Usable surveys by New Jersey County. Numbers represent surveys received by region and by county North - 31 Central - 9 South - 9 Bergen - 5 Hunterdon - 0 Atlantic - 3 Essex - 9 Mercer - 3 Burlington - 1 Hudson - 6 Middlesex - 1 Camden - 3 Morris - 0 Monmouth - 5 Cape May - 0 Passaic - 6 Somerset - 0 Cumberland - 0 Sussex - 1 Gloucester - 1 Union - 2 Ocean - 1 Warren - 2 Salem - 0 Figure 1. Percent distribution of all categories of NJ laboratory personnel by age (n = 2,004). Note that 49.1% are older than 45 years, and 13.8% are older than 55 years into inactive status. There is also only one cytotechnologist (CT) program. In 2003, these programs produced only seven HT and five CT graduates. Although there was some anecdotal information from New Jersey laboratory managers about difficulties in hiring quali- fied laboratory practitioners, there was insufficient data on vacancies and shortages for these practitioners in the state’s workforce. Therefore, the Coalition conducted a survey of hospital clinical laboratory managers to determine the extent of the clinical laboratory personnel shortage in NJ, and to begin a data collection process to project workforce needs into the future. METHODS In January 2003, a one-page survey was mailed to the clinical laboratory managers of the 95 hospitals in NJ. Surveys were codedfortrackingpurposes.AsecondsurveywassentinMarch 2003 to the non-responders, followed by phone contact. The survey requested data on county, hospital size, total number of billable tests, total current budgeted FTEs, the number and age of clinical laboratory employees in six cat- egories, the number of current vacancies, and the average time it took to fill vacancies. The six personnel categories included MT staff, MT supervisor, MLT, HTL, HT, and CT. In addition, managers were asked to indicate if they had difficulties hiring or recruiting for a particular position, department, or shift, and if they had incentives in place to hire laboratory personnel. All survey responses were received between February and April 2003, and were reviewed, tabu- lated, and summarized. RESULTS A total of 55 surveys were received for a response rate of 57.9%. Forty-nine (51.6%) of the surveys contained data that were usable in the analysis, and represented data from hospitals in fifteen of the twenty-one NJ counties (Table 1). A majority of the surveys (31) were received from the Northern NJ counties. Forty-seven percent of the usable responses were from hos- pitals with greater than 300 beds, while 53% had less than 300 beds. A total of 33,094,905 annual billable laboratory tests were reported by 39 hospitals, ranging from 97,800 to 3,822,755 per hospital. A total of 2,697 total budgeted FTEs were reported by 49 hospitals. Figure 1 depicts the breakdown of all categories of NJ clinical laboratory personnel by age. A breakdown of personnel by category and age is depicted in Figure 2 and Table 2. The largest category reported was MT staff with 1,455 employees in 49 hospitals. In the MT staff category, 50.8% of the employees were over 45 years, and 14% were over 55 years, while 61.5% of the MT supervisors were over 45 years, with 21.6% over 55 years. The MLT population was somewhat younger with only 38.3% over 45 years. In the MLT, HTL, HT, and CT categories, 10% of the employees were over 55 years. Table 3 lists the number of vacancies by category and region. The highest number occurred in the MT category with 49 full time and 52 part time vacancies among the
  • 40. 102 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS Table 2. Breakdown of clinical laboratory personnel by category and age Category Total MT staff MT Supervisor MLT HTL HT CT Current total # 2331 1455 251 428 34 116 47 (n = 49 hospitals) Current total # 2004 1247 213 381 28 98 37 reported by age (n = 42 hospitals) Under 35 yrs 315 177 15 90 4 21 8 15.7% 14.2% 7.0% 23.6% 14.3% 21.4% 21.6% 36 – 45 yrs 706 436 67 145 9 36 13 35.2% 35.0% 31.5% 38.1% 32.1% 36.8% 35.2% 46 – 55 yrs 708 460 85 108 12 31 12 35.3% 36.8% 39.9% 28.3% 42.9% 31.6% 32.4% Over 55 yrs 275 174 46 38 3 10 4 13.8% 14.0% 21.6% 10.0% 10.7% 10.2% 10.8% 45 or younger 1021 613 82 235 13 57 21 50.9% 49.2% 38.5% 61.7% 46.4% 58.2% 56.8% 46 or older 983 634 131 146 15 41 16 49.1% 50.8% 61.5% 38.3% 53.6% 41.8% 43.2% 49 hospitals. In the MT supervisor category, there were 11 full time and 4 part time vacancies, and in the MLT category, there were 6 full time and 20 part time vacan- cies. There were seven vacancies for full time HT and one vacancy for a full time CT. Figure 2. Percent distribution of categories of laboratory personnel by age
  • 41. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 103 RESEARCH AND REPORTS Table 3. Number of vacancies by category and region (n = 49 hospitals) MT Staff MT Sup MLT HTL HT CT Tot N C S Tot N C S Tot N C S Tot N C S Tot N C S Tot N C S FT 49 26 18 5 11 8 3 0 6 2 3 1 0 0 0 0 7 5 2 0 1 1 0 0 PT 52 36 12 4 4 4 0 0 20 5 4 11 1 0 0 1 1 1 0 0 1 1 0 0 FT = full time; PT = part time; Sup = supervisor; Tot = total number vacancies; N = vacancies in northern NJ; C = vacancies in central NJ; S = vacancies in southern NJ Figure 3. Percentage of laboratory managers reporting hiring difficulties by category (n = 49) Table 4 summarizes the responses for the average time to fill vacancies in the various categories. The time ranged from an average of 10.6 and 11 weeks for MLTs and MTs, respectively, to 104 weeks for HTs. A majority of laboratory managers reported difficulties in hiring generalist MTs (71.4%), as well as night shift (46.9%), evening shift (28.6%), HT (24.5%), and blood bank (20.4%) positions. Other positions in which difficul- ties in hiring were reported included part time, weekend, CT, and general supervisory positions. Figure 3 presents a summary of the data. The miscellaneous category includes one to two responses each for microbiology technologist, phlebotomist, evening supervisor, blood bank supervisor, and histology supervisor. Laboratory managers reported using the following incentives for hiring laboratory personnel: 42.9% of laboratory man- agers reported no incentives, 30.6% had shift differentials, 26.5% had tuition reimbursement, 24.5% had a sign-on bonus, and 20.4% made market adjustments in salaries. The incentives are summarized in Figure 4. In addition, some laboratory managers had comments related to their hiring difficulties. These are summarized in Table 5. DISCUSSION The data reflect responses from over half of the hospital clinical laboratories in NJ. The largest number of surveys being received from the northern region of the state re- flects the larger number of hospitals in that region. The responding hospitals were almost evenly divided between those that are greater than 300 beds and those that are less than 300 beds. A total of 33,094,905 annual billable tests were reported by 39 hospitals. Projecting that figure across 95 hospitals at an average cost of $14 per test, hospital laboratories represent a billion dollar industry in the state.
  • 42. 104 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS Table 4. Average time to fill vacancies MT Staff MT Sup* MLT HTL HT CT Average number of weeks 11.0 14.8 10.6 22.6 25.6 25.4 Range in weeks 1 to 52 2 to >52 2 to 52 8 to >52 8 to104 12 to 78 Number of hospitals n = 39 n = 32 n = 27 n = 8 n = 21 n = 10 * Sup = supervisor Figure 4. Percentage of hospitals offering hiring incentives for laboratory personnel (n = 49) Nearly half of the practitioners employed in the responding NJ hospital clinical laboratories are over the age of 45, which is only slightly lower than the median age of 47 years reported in 2002 for clinical laboratory practitioners nationwide. In NJ, 13.8% of the clinical laboratory professionals in the 49 responding hospitals were over the age of 55; projecting that number over the 95 hospitals in the State, it is likely that over 500 laboratorians in the current workforce will be over 65 within the next 10 years. There were a total number of 2,697 budgeted FTEs in 49 hospitals, with 2,331 current clinical laboratory employees. Using these figures, an estimate of the overall vacancy rate for hospital laboratory personnel is 13.6%. During the 3-month survey period, there were a total of 49 full time and 52 part time vacancies for staff MTs, and 11 full time and 4 part time vacancies for supervisory MTs in the 49 responding hospitals. Projecting those figures to the 95 hospitals in the state, the number of actual vacancies could be as high as 116 full time and 109 part time MT positions. Given that only 26 MTs graduated in 2003, and that not all of that cohort will choose to work in hospital laboratories, there was a significant shortfall of MTs with approximately eight vacant MT positions per MT graduate. Over 70% of the laboratory managers reported difficulty in filling vacant MT generalist positions, while difficulty in filling night, evening, and blood bank positions were reported by 46.9%, 28.6%, and 20.4% of the managers, respectively. As indicated in a comment in Table 5, an unknown, but prob- ably significant, number of MTs work more than one job to supplement their salaries. If this did not occur, (assuming the second job is in another hospital laboratory) the shortage would be even greater. During this same period, there were 4 full time and 20 part time MLT vacancies. Again, projecting these figures to the 95 hospitals in the state, the amount of actual vacancies could be as high as 8 full time and 39 part time positions. Given that only 21 MLTs graduated in 2003, and that not
  • 43. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 105 RESEARCH AND REPORTS Table 5. Comments from laboratory managers about hiring difficulties “Most employees are working more than one job to earn a living. This makes it very difficult to staff the lab.” “Shift differential increases are desperately needed but being delayed at least 6 months due to reim- bursement problems.” “Much difficulty in recruiting supervisors due to lack of qualified outside applicants and lack of interest in qualified in-house staff due to salary compression with increased responsibilities.” “We gave up trying to fill a part time MLT day job and converted it to a lab aide.” “Blood bank position vacant since 3/02; histologist position vacant since 8/02.” “Could not find a CT, so closed cytology.” “Unable to find qualified candidate for blood bank supervisor. Three blood bank techs went to industry. Histotech asking salary was above our approved range.” “We are lucky enough to have a MT school; CTs and HTs are extremely hard to find.” all of that cohort will work in hospital laboratories, there was a shortage of MLTs to fill the open part time positions. Some laboratory managers indicated that they would hire a MLT to fill a vacant MT position if a qualified MT was not available, prompting them to reevaluate their staffing mix of MLTs vs. MTs. If this staffing shift should occur, there will likely be more vacant MLT positions, thus exacerbating the MLT shortage. Although there were only two vacancies for CTs, there is only one program in the state producing five to eight graduates per year, many of whom are employed, often at higher salaries, by reference laboratories. This situation creates difficulties in filling vacancies at some hospitals, requiring as long as 78 weeks to fill a CT vacancy (Table 4). In fact, one hospital reported closing their cytology department since they were unable to fill a vacant CT position (See Table 5). There are no HTL programs in the state, and only the one HT Program in the southern region of the state. That one HT Program, however, recently went inactive. It was pro- ducing five to ten graduates per year; however, the graduates traditionally preferred employment in their home region, resulting in unfilled positions in the northern and central regions of the state. Nearly 25% of the laboratory managers reported difficulty in filling HT positions, with up to 104 weeks required to fill a vacant position (Table 4). With no active HT or HTL programs in the state, the personnel shortages in histology will become even greater. The U.S. Bureau of Labor Statistics projected that between 2002–2012 the clinical laboratory field would experience a combination of growth and attrition requiring 13,800 new clinical laboratory technologists and technicians per year. Although the simplification and automation of tests will result in some loss of positions, a net increase in need for new practitioners was predicted due to the population growth, an increase in the elderly population, and the introduction of new types of tests that will spur the utilization of more laboratory services. The general staffing pattern was 3.4 MTs to 1 MLT in NJ hospital laboratories. Utilization of MLTs to fill some of the vacant MT positions may help to alleviate the shortage and spur an increase in enrollments in MLT Programs. Supply and demand in the state’s clinical laboratory work- force will continue to be monitored by surveying the edu- cational programs and laboratory managers every two years, and developing a prediction model for future needs. It is also recommended to continue to study and address work- ing conditions, incentives, and salaries to make the clinical laboratory profession more attractive and competitive with other fields that require comparable education, and to begin to study the impact of the shortage on the delivery and ac- cessibility of quality laboratory services. This information is critical for strategic planning for both laboratory managers and educators. The Coalition launched a Website in May 2003 (www.lab- science.org) to inform potential students and science teachers about the clinical laboratory professions. The Coalition will
  • 44. 106 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE RESEARCH AND REPORTS continue to address the shortage through efforts to promote awareness of the professions to potential students and the public, to assist in recruitment of students into the state’s educational programs, to encourage hospitals, colleges, and universities to maintain their educational programs, and to share information about successful retention programs to keep our highly skilled professionals in hospital settings. Nationwide, and statewide, clinical laboratory practitioners are not being produced in sufficient quantity to meet the cur- rent and future demand. Nationwide, it has been estimated that the shortage is growing by over 10,000 laboratorians per year. In NJ, even if the five existing MT programs operate at full capacity (an estimated 52 students per year), there will still be insufficient graduates to meet the current and the projected demand for MTs in the future. In addition, due to the location of a large number of pharmaceutical compa- nies and commercial laboratories in NJ, the state’s hospitals must compete for skilled laboratory personnel with those industries that recruit professionals out of the hospital setting with higher wages and/or better working conditions, e.g., no weekends, holidays, etc. MT programs either need to expand or new programs need to be established, and perhaps the hospital laboratory industry in the state should re-examine the utilization of MLTs vs. MTs in the workplace. REFERENCES 1. Hecker DE. Occupational employment projections to 2012. Monthly Labor Review. February 2004: 80-105. 2. American Medical Association, Health Professions: Career and Education Data Book 2003-2004. Chicago: AMA;2003. 3. Ward-Cook K, Chapman S: 2002 wage and vacancy survey of medical laboratories. Lab Med 2003,34:702-7. 4. Ward-Cook, K, Tanner S. 2000 wage and vacancy survey of medical laboratories. Lab Med 2001,32:124-38.
  • 45. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 107 FOCUS: PSYCHOSTIMULANTS Mechanism of Action and Therapeutic Uses of Psychostimulants KEVIN F FOLEY ABBREVIATIONS: ADHD = attention-deficit hyper- activity disorder; CNS = central nervous system; MDA = 3,4-methylenedioxyamphetamine; MDMA = 3,4-methyl- enedioxymethamphetamine; NC = not controlled; OTC = over the counter. INDEX TERMS: psychostimulants. Clin Lab Sci 2005;18(2):107 Kevin F Foley PhD MT(ASCP) is with the Department of Bio- medical Technologies, University of Vermont, Burlington VT. Address for correspondence: Kevin F Foley PhD MT (ASCP), Assistant Professor, University ofVermont, Department of Medi- cal Laboratory and Radiation Sciences, 302 Rowell Building, 106 Carrigan Drive, BurlingtonVT 05405. (802) 656-2506. kfoley@uvm.edu. Victor A Skrinska PhD DABCC is the Focus: Psychostimu- lants guest editor. Focus Continuing Education Credits: see pages 124 to 126 for learning objectives, test questions, and application form. The name stimulant can be given to any drug that increases the rateofaphysiologicfunction.Thetermpsychostimulantismore specific, referring to compounds that have direct neurological effects,typically:heightenedalertness,increasedenergy,appetite suppression,andsometimeseuphoria.Useofpsychostimulants is widespread and occurs in both recreational and clinical set- tings. Therapeutic monitoring and screening for use and abuse ofthesedrugsiscommoninclinicallaboratories.Understanding the physiological effects and uses of stimulants will be of value to clinical and forensic laboratory scientists. EXAMPLES OF PSYCHOSTIMULANTS Table 1 lists several psychostimulants that are commonly prescribed, have widespread illicit use, or are well-known by the general public. Table 1 also identifies the control sched- ule of each compound. Many psychostimulants are listed as controlled substances under the Controlled Substances Act, Title II of the Comprehensive Drug Abuse Prevention and Control Act of 1970. Schedules I through IV are assigned to compounds based on their abuse potential and their medical utility. Schedules II through V contain drugs which have known medical uses whereas Schedule I compounds have no current, sanctioned medical use. THERAPEUTIC USES OF PSYCHOSTIMULANTS Amphetamine has long been known to be a mental stimulant. Because of its psychostimulant properties, amphetamine has beenusedsuccessfullybyU.S.fighterpilotsbecauseitenhances cockpit performance by reducing the effects of fatigue.1 Since amphetamine heightens alertness it has found use in the treat- ment of narcolepsy and in attention-deficit hyperactivity disor- der.2 The drug Adderall® for example, is used widely in cases of ADHDandismerelyamixtureofamphetamine’sstereoisomers: dextro- and levoamphetamine. Amphetamine also has appetite suppressant effects and thus is used in the treatment of obesity, although it is not FDA approved for this use. Although amphetamine can be consid- ered the prototype psychostimulant, many psychostimulants have been identified or created that resemble amphetamine in their chemical structures. These compounds vary in their effects and utility as well as their abuse potential. Meth- amphetamine, like amphetamine has a history of illicit use and is known to be considerably more potent in vivo than the unmethylated amphetamine.3,4 Methamphetamine, like amphetamine, has been used with some success in ADHD patients. It is also approved for use in treating obesity.5 Pseudoephedrine, the name given to the 1R2R enantio- mer, and ephedrine, the 1R2S enantiomer, are common medications used primarily as nasal decongestants. Unlike amphetamine and methamphetamine they have little abuse potential allowing them to be obtained OTC.6 Ephedrine is also approved for treatment of bronchospasm, enuresis, hypotension, and narcolepsy.5 Some psychostimulants have been found to have uses that are not typical for other drugs in the same class. Although bupropion is a phenylalkylamine, the structural dissimilar- ity with amphetamine is significant enough to produce considerably different physiologic effects. Bupropion is an
  • 46. 108 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE example of a weak psychostimulant that has found use as an antidepressant (Wellbutrin® ) and is also marketed as a smok- ing cessation aid (Zyban® ). Although still a mild psycho- stimulant, the behavioral effects of bupropion are reported to be unlike amphetamine when used in humans. The abuse potential is also considerably less than with amphetamine.7 Off-label uses of bupropion include treatment of ADHD, diabetic neuropathy, and neuropathic pain.5 Another clini- cally-useful β-ketone psychostimulant is diethylpropion. Diethylpropion is currently used only in the treatment of obesity. This psychostimulant is commonly prescribed as an appetite suppressant under the name Tenuate® . Methcathinone is a Schedule I psychostimulant that is eas- ily synthesized from ephedrine. Methcathinone is the N- methylated version of cathinone, a natural psychostimulant obtained from the Catha edulis plant and known as “qat” or “khat”.8 Methcathinone has no sanctioned medical use and is only used recreationally. Its effects are those of a classic psychostimulant, causing ‘flying euphoria’ followed by a five-to-eight hour period of feeling tough, invincible with increased libido, and desire to be physical.8,9 The next two compounds listed in Table 1 are also Schedule I compounds. 3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedioxymethamphetamine (MDMA) are popular recreational drugs known respectively as ‘the love (or hug) drug’ and ‘ecstasy’. MDMA use is growing rapidly and in some European countries is the second most fre- quently used illegal drug after marijuana.10 Interestingly, both MDMA and MDA were used therapeutically before being classified Schedule I in 1985. These compounds were used by patients in constructive psychotherapy sessions.8 MDMA has received much negative attention in recent years as claims of its neurotoxicity mounted. However data concerning the neurotoxicity of MDMA have been inconsistent and one major study was even retracted after it was found that the toxicity in the study was brought about by methamphetamine rather than MDMA.11,12 The widespread use and popularity of MDMA coupled with the lack of definitive reports on neurotoxicity have encouraged some to reconsider the util- ity of this drug.13-15 Because of MDMA’s unique effects, a new study investigating the therapeutic use of MDMA has recently earned IRB and FDA approval. This research will assess the use of MDMA in the treatment of post-traumatic stress disorder.16,17 Perhaps the most well-known psychostimulant after amphet- amineismethylphenidate,madepopularunderthetradename Ritalin® . Methylphenidate has pharmacological effects similar FOCUS: PSYCHOSTIMULANTS Table 1. Some common psychostimulants Compound Trade or common name Schedule Amphetamine Dexedrine® , Adderall® C-II Methamphetamine Desoxyn® , Methampex® ‘speed’, ‘crystal meth’ C-II Ephedrine Herbal sources: ma huang, ephedra, ephedra sinica, epitonin, Pretz-D® OTC Bupropion Wellbutrin® , Zyban® , Buproban™ NC Diethylpropion Tenuate® , Dipro™ Durad™ Radtue™ C-IV Methcathinone ‘cat’ C-I 3,4-Methylenedioxyamphetamine MDA, ‘love drug’ C-I 3,4-Methylenedioxymethamphetamine MDMA, ecstasy, XTC C-I Methylphenidate Concerta™, Metadate® , Methylin™, Ritalin® C-II Phentermine Adipex-P® , Fastin® , Obenix® , Phentamine® , Supramine™ C-IV Fenfluramine Pondimin® (discontinued, removed from the U.S. market) C-IV Cocaine Methyl benzoylecgonine, coke, crack C-II C-I = Schedule I: Compounds with high potential for abuse having no currently accepted medical use in treatment in the United States. C-II = Schedule II: Compounds with high potential for abuse having currently accepted medical use(s) in treatment in the United States or a currently accepted medical use with severe restrictions. C-IV = Schedule IV: Compounds having a low potential for abuse relative to drugs scheduled as C-III. NC = not controlled (unscheduled). OTC = over the counter.
  • 47. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 109 Figure 1. Structure of amphetamine and catecholamine neurotransmitters to amphetamine; however, its abuse po- tentialissomewhatlower,althoughthere are many conflicting reports regarding methylphenidate abuse potential (see reference 18 for review). Methylpheni- date has become the drug of choice for treating ADHD but has also found off- label use as an antidepressant.5 Two other drugs worth mentioning as psychostimulants are phentermine and fenfluramine. Both of these drugs have been used as appetite suppres- sants and both have amphetamine-like sympathomimetic effects. Fenflu- ramine contains a chiral center and fenfluramine is the name given to the racemic mixture of D- and L-isomers. The D-isomer (dexfenfluramine) was purportedly responsible for the anorectic actions of fenfluramine and was also associated with fewer side ef- fects than the racemic fenfluramine. Dexfenfluramine was marketed as Redux® .19 By 1997 both fenfluramine and dexfenfluramine were removed from the U.S. market at the request of the FDA when cases of cardiac valvulopathy were reported.5,20,21 Fen- fluramine is perhaps best known by its off-label use with phentermine, known as “Fen-phen”. Fen-phen was widely used for the long-term management of obesity. The fenfluramine-phenter- mine combination was also associated with valvulopathies and a bulletin was issued by the FDA to cease off-label use of the Fen-Phen combination.5,21 Of the two drugs, only phentermine is still used as an appetite suppressant although tolerance to the anorexiant effects of phentermine usually develops a few weeks after starting therapy. When considering the clinically-used drugs listed in Table 1, cocaine is the only one that is not used for its psycho- stimulant effects. Cocaine is used clini- cally only as a local anesthetic, usually in mucosal or ophthalmic procedures. MECHANISM OF ACTION It is easy to see the resemblance between the chemical structures of common psychostimulants and endog- enous monoamine neurotransmitters (Figure 1). The prototype psychostim- ulant amphetamine closely resembles the catecholamine neurotransmitters norepinephrine, epinephrine, and do- pamine. Since many psychostimulants share the features of a phenyl ring, a nitrogen group, and carbon side chains of varying lengths, many stimulants fall into the category of phenylalkylamines. With the possible exception of cocaine, all the compounds listed inTable 1 can be classified as phenylalkylamines. Be- FOCUS: PSYCHOSTIMULANTS cause amphetamine is considered the prototype stimulant, other compounds that have similar chemical structures and similar physiologic effects are often termed ‘amphetamines’ (Figure 2). Given their structural similarity to en- dogenous neurotransmitters, it is not surprisingthatmanyphenylalkylamines have autonomic nervous system activ- ity, i.e., sympathomimetics, as well as mood-altering effects. Amphetamine and other closely related phenylal- kylamines can activate receptors that normally bind catecholamines or serotonin. In addition, amphetamine and related compounds can cause the release of catecholamines and serotonin from nerve endings.22-26 Once released, the endogenous neurotransmitters are free to act on their extracellular recep- tors. When catecholamine receptors in the brain are activated, myriad effects can result. Neuropsychological effects of catecholamine receptor activation or potentiation by psychostimulants can include: increased alertness, in- somnia, euphoria, decreased appetite, and at higher doses, psychosis. Indeed, amphetamine can correctly be called a “psychotomimetic” since high doses can bring about a psychosis very similar to that seen in schizophrenic patients.27,28 In the periphery, activation or poten- tiation of catecholamine receptors by psychostimulants can result in: vaso- constriction and subsequent hyperten- sion, mydriasis, tachycardia, and other general sympathomimetic effects. The mechanism of action of amphetamine and amphetamine-like psychostimu- lants involves four major effects: 1. Binding to extracellular catechol- amine receptors 2. Inhibition of monoamine neu- rotransmitter uptake 3. Release of catecholamines from neurons 4. Inhibition of monoamine oxidase
  • 48. 110 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE Psychostimulants alter neurotransmitter release. Psycho- stimulants can effectively alter the amount or the rate of neurotransmitter released by a monoaminergic neuron. The resulting change in mood or behavior is the summation of these neuromodulations and is quite complex. At the cellular level this neuromodulation is due to one or more of the ef- fects listed above. Amphetamine-like compounds have a wide range of affinities to catecholamine and serotonin receptors. The overall pattern of receptor binding for a psychostimulant is unique for a given stimulant and contributes to the distinc- tive behavioral effects of a given compound. In addition to having direct receptor-binding effects, many psychostimulants inhibit monoamine transporters. Mono- amine transporter proteins serve to recycle neurotransmitters after they are released from the neuron and in so doing, ter- minate the neurotransmitter signal. These same monoamine transporters are also the targets for antidepressant medica- tions including the popular drugs fluoxetine (Prozac® ), paroxetine (Paxil® ) and sertraline (Zoloft® ). Inhibitors of monoamine transporters block the uptake, or ‘reuptake’, of neurotransmitters by neurons (Figure 3). This blockade effec- tively increases the concentration of neurotransmitter in the synapse, resulting in increased binding of neurotransmitters to their receptors. FOCUS: PSYCHOSTIMULANTS Figure 2. Structures of some common psychostimulants Structural similarity can be seen among many of the compounds containing the phenylalkylamine structure (compounds shown are from Table 1).
  • 49. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 111 Psychostimulants can also elevate the concentration of neurotransmitter in the synapse by evoking release of neu- rotransmitters. The release of stored neurotransmitters can be triggered directly when psychostimulants bind receptors present on neurons or can release neurotransmitters indirectly via an exchange mechanism occurring through monoamine transporter proteins. The exchange mechanism or efflux process which can occur via monoamine transporters has been observed with many amphetamine derivatives.29,30 This efflux is thought to be a type of ‘reverse-transport’ mediated by monoamine transporters.31,32 Finally, amphetamine and amphetamine-like psychostimulants often act as competitive inhibitors of the enzyme monoamine oxidase (MAO).33,34 MAO is a mitochondrial enzyme that breaks down mono- amine neurotransmitters. Products of catecholamine break- down include 3-methoxy-4-hydroxymandelic acid (VMA), homovanillic acid (HVA), and dihydroxyphenylacetic acid (DOPAC). These compounds are commonly-measured me- tabolites of catecholamines whose formation is due in part or in full to MAO. Inhibition of MAO would thus bring about an expected increase in monoamine neurotransmitters since their breakdown would be impeded. In summary, psychostimulants can affect neurotransmitter release in at least four ways; by binding extracellular receptors, by blocking monoamine transporters, by evoking neurotrans- FOCUS: PSYCHOSTIMULANTS Figure 3. Psychostimulant mechanisms of action MAO = Monoamine oxidose. Psychostimulants can have one or more of the following actions: 1) activation of neurotransmitter receptors; 2) inhibition of monoamine transporters; 3) stimulation of neurotransmitter release; or 4) inhibition of monoamine oxidase.
  • 50. 112 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE mitter release, and by antagonizing the enzyme MAO (Figure 3). A given psychostimulant can have multiple mechanisms of actions. Indeed, the actions of some psychostimulants, such as MDMA and bupropion have not yet been fully explained but it is clear that all or some of the mechanisms discussed play a role in the unique actions of these drugs. PHYSIOLOGIC EFFECTS OF PSYCHOSTIMULANTS Having already considered the cellular actions of psycho- stimulants it is worth mentioning the overall physiologic effects of these drugs. Because of the complex circuitry in the brain and the overlap of monoamine neurotransmitter systems, understanding how subtle differences in chemical structure bring about significantly different psychological effects can be difficult. Although all differ in their overall physiological effects, the psychostimulants listed in Table 1 do share the following adverse side effects: agitation/anxi- ety, dizziness, restlessness, psychosis, and seizures. Many of these compounds can also cause hypertension, insomnia, hyperthermia, tachycardia, and euphoria.5 A discussion of the psychopharmacological mechanisms of action of each individual psychostimulant is beyond the scope of this article (see reference 35 for excellent reviews). In general, the physiologic effects of psychostimulants can be represented by the prototype psychostimulant, amphetamine. In the periphery amphetamine acts as a sympathomimetic. Mydriasis, hypertension, and tachycardia would be expected from a compound with close structural similarity to endog- enous catecholamines. Amphetamine releases norepinephrine from sympathetic nerve endings which causes increases in systolic and diastolic blood pressure, often causing a reflex- decrease in heart rate. At higher doses or overdoses tachycardia or cardiac arrhythmias can occur.5 Because of these cardio- vascular effects, amphetamine is contraindicated in patients with arteriosclerosis, coronary artery disease, hypertension, and closed-angle glaucoma.5 Interestingly, the commonly used drugs ephedrine and pseudoephedrine are utilized for their vascular effects. By acting directly on alpha-adrenergic recep- tors in the mucosa of the respiratory tract, pseudoephedrine produces vasoconstriction leading to nasal decongestion. In the CNS, amphetamine modulates monoamine neu- rotransmitter release and kinetics. The overall effect of these actions is consistent with known roles of monoamines in the brain. The anorectic effects of amphetamine involve release of norepinephrine in the paraventricular nucleus of the hypothalamus, a brain area central to feeding behavior.36,37 Serotonergic systems also play a role in appetite and satiety and these systems are also affected by psychostimulants like amphetamine.38,39 In addition to appetite control, amphet- amine is also used for its ability to focus attention. The basis for this effect is not well understood. The seemingly para- doxical fact that psychostimulants can act as calming agents in humans seems enigmatic. The effects of amphetamine on attention and alertness are ultimately due to the modulation of normal patterns of central activity. These modulations are brought about by modulating serotonergic, dopaminergic, and noradrenergic pathways but the precise mechanism of action of amphetamine in ADHD is unknown (see references 2 and 40 for reviews). Euphoria, of course, is an important, high-dose effect of amphetamine. The euphoric effects of amphetamine and methamphetamine are very similar to cocaine however un- like cocaine, amphetamine and methamphetamine are taken orally and are metabolized more slowly than cocaine, making it the psychostimulant of choice for many drug users. Cen- trally, amphetamine and cocaine cause an acute dopamine release in addition to inhibiting neuron dopamine uptake.41-43 This release of dopamine in the nucleus accumbens and prefrontal cortex is thought to cause the prominent euphoric and reinforcing effects associated with amphetamine and cocaine.44 These psychostimulants can produce a significant euphoria, locomotor stimulation, reduced fatigue, sexual stimulation, increased mental attention, and increased social- ity. Higher doses can produce tremors and vomiting, as well as tonic-clonic convulsions. CONCLUSION Psychostimulants are important pharmacological agents used in a variety of conditions including the treatment of obesity, ADHD, narcolepsy, and as decongestants. Psychostimulants typically contain a phenylalkylamine structure that closely resembles that of endogenous monoamines. The actions of psychostimulants are mediated through their ability to bind monoaminereceptorsand/ormodulatemonoaminetransmitter release. Although psychostimulants have a history of abuse and misuse their therapeutic uses are numerous and significant. REFERENCES 1. Emonson DL, Vanderbeek RD. The use of amphetamines in U.S. Air Force tactical operations during Desert Shield and Storm. Aviat Space Environ Med 1995;66:260-3. 2. Gainetdinov RR, Wetsel WC, Jones SR, and others. Role of serotonin in the paradoxical calming effect of psychostimulants on hyperactiv- ity. Science 1999;283:397-401. 3. Glennon RA, Yousif M, Naiman N, Kalix P. Methcathinone: a new and potent amphetamine-like agent. Pharmacol Biochem Behav 1987;26:547-51. FOCUS: PSYCHOSTIMULANTS
  • 51. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 113 4. Glennon RA. Discriminative stimulus properties of phenylisopro- pylamine derivatives. Drug Alcohol Depend 1986;17:119-34. 5. Clinical Pharmacology 2000 Database. 2004. Gold Standard Medical Media, http://cpip.gsm.com/ Accessed January 7, 2005. 6. Wee S, Ordway GA, Woolverton WL. Reinforcing effect of pseudo- ephedrine isomers and the mechanism of action. Eur J Pharmacol 2004;493:117-25. 7. Griffith JD, Carranza J, Griffith C, Miller LL. Bupropion: clini- cal assay for amphetamine-like abuse potential. J Clin Psychiatry 1983;44:206-8. 8. Perrine DM. The chemistry of mind-altering drugs. Washington DC: American Chemical Society;1996. 9. EmersonTS,CisekJE.Methcathinone:aRussiandesigneramphetamine infiltrates the rural midwest. Ann Emerg Med 1993;22:1897-903. 10. Landry MJ. MDMA: a review of epidemiologic data. J Psychoactive Drugs 2002;34:163-9. 11. Ricaurte GA, Yuan J, Hatzidimitriou G, and others. Severe dopami- nergic neurotoxicity in primates after a common recreational dose regimen of MDMA (‘ecstasy’). Science 2002;297:2260-3. 12. Ricaurte GA, Yuan J, Hatzidimitriou G, and others. Retraction. Science 2003;301:1479. 13. Lyles J, Cadet JL. Methylenedioxymethamphetamine (MDMA, ecstasy) neurotoxicity: cellular and molecular mechanisms. Brain Res Brain Res Rev 2003;42:155-68. 14. Kish SJ. How strong is the evidence that brain serotonin neurons are damaged in human users of ecstasy? Pharmacol Biochem Behav 2002;71:845-55. 15. PentneyAR.Anexplorationofthehistoryandcontroversiessurrounding MDMA and MDA. J Psychoactive Drugs 2001;33:213-21. 16. Michael C. Mithoefer MD, Mark T, Wagner PD. Experimental proposal: a human phase II study –safety and efficacy of 3,4-methy- lenedioxymethamphetamine (MDMA)-assisted psychotherapy in the treatment of chronic posttraumatic stress disorder (PTSD). Charleston SC: Medical University of South Carolina, 2002. 17. Weiss R. DEA approves trial use of ecstasy in trauma cases. The Washington Post March 2, 2004:02. 18. Kollins SH, MacDonald EK, Rush CR. Assessing the abuse potential of methylphenidate in nonhuman and human subjects: a review. Pharmacol Biochem Behav 2001;68:611-27. 19. Bremer JM, Scott RS, Lintott CJ. Dexfenfluramine reduces cardiovas- cular risk factors. Int J Obes Relat Metab Disord 1994;18:199-205. 20. Shively BK, Roldan CA, Gill EA, and others. Prevalence and deter- minants of valvulopathy in patients treated with dexfenfluramine. Circ 1999;100:2161-7. 21. Connolly HM, Crary JL, McGoon MD, and others. Valvular heart disease associated with fenfluramine-phentermine. N Engl J Med 1997;337:581-8. 22. Rubin RP, Jaanus SD. A study of the release of catecholamines from the adrenalmedullabyindirectlyactingsympathomimeticamines.Naunyn Schmiedebergs Arch Pharmakol Exp Pathol 1966;254:125-37. 23. Magyar K, Knoll J. Para-substituted amphetamines and brain sero- tonin. Pol J Pharmacol Pharm 1975;27:139-43. 24. Paton DM. Structure—activity relations for the acceleration of efflux of noradrenaline from adrenergic nerves in rabbit atria by sympatho- mimetic amines. Can J Physiol Pharmacol 1975;53:822-9. 25. Carr LA, Moore KE. Release of norepinephrine and normetaneph- rine from cat brain by central nervous system stimulants. Biochem Pharmacol 1970;19:2671-5. 26. Fitzgerald JL, Reid JJ. Effects of methylenedioxymethamphetamine on the release of monoamines from rat brain slices. Eur J Pharmacol 1990;191:217-20. 27. Snyder SH. The dopamine hypothesis of schizophrenia: focus on the dopamine receptor. Am J Psychiatry 1976;133:197-202. 28. Angrist B, Sathananthan G, Wilk S, Gershon S. Amphetamine psychosis: behavioral and biochemical aspects. J Psychiatr Res 1974;11:13-23. 29. Rudnick G, Wall SC. p-Chloroamphetamine induces serotonin release through serotonin transporters. Biochem 1992;31:6710-8. 30. Wall SC, Gu H, Rudnick G. Biogenic amine flux mediated by cloned transportersstablyexpressedinculturedcelllines:amphetaminespecific- ity for inhibition and efflux. Mol Pharmacol 1995;47:544-50. 31. Sasa M. Function of monoamine neurotransmitter transporters. Nippon Rinsho 2001;59:1457-64. 32. Scholze P, Norregaard L, Singer EA, and others. The role of zinc ions in reverse transport mediated by monoamine transporters. J Biol Chem 2002;277:21505-13. 33. Weyler W, Salach JI. Purification and properties of mitochondrial monoamine oxidase type A from human placenta. J Biol Chem 1985;260:13199-207. 34. Pearce LB, Roth JA. Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods. Biochem 1985;24:1821-6. 35. Neuropsychopharmacology, The fifth generation of progress. Phila- delphia: Lippincott, Williams and Wilkins, 2002. 36. Frankish HM, Dryden S, Hopkins D, and others. Neuropeptide Y, the hypothalamus, and diabetes: insights into the central control of metabolism. Peptides 1995;16:757-71. 37. Samanin R, Garattini S. Neurochemical mechanism of action of anorectic drugs. Pharmacol Toxicol 1993;73:63-8. 38. Blundell JE, Leshem MB. The effect of 5-hydroxytryptophan on food intake and on the anorexic action of amphetamine and fenfluramine. J Pharm Pharmacol 1975;27:31-7. 39. Halford JC, Blundell JE. Separate systems for serotonin and leptin in appetite control. Ann Med 2000;32:222-32. 40. Ohno M. The dopaminergic system in attention deficit/hyperactivity disorder. Congenit Anom (Kyoto) 2003;43:114-22. 41. Karoum F, Chrapusta SJ, Brinjak R, and others. Regional effects of amphetamine, cocaine, nomifensine, and GBR 12909 on the dynamics of dopamine release and metabolism in the rat brain. Br J Pharmacol 1994;113:1391-9. 42. Reid MS, Hsu K, Jr., Berger SP. Cocaine and amphetamine prefer- entially stimulate glutamate release in the limbic system: studies on the involvement of dopamine. Synapse 1997;27:95-105. 43. Jacocks HM 3rd, Cox BM. Serotonin-stimulated release of [3H]dopamine via reversal of the dopamine transporter in rat striatum and nucleus accumbens: a comparison with release elicited by potassium, N-methyl-D-aspartic acid, glutamic acid and D-am- phetamine. J Pharmacol Exp Ther 1992;262:356-64. 44. Moghaddam B, Bunney BS. Differential effect of cocaine on extra- cellular dopamine levels in rat medial prefrontal cortex and nucleus accumbens: comparison to amphetamine. Synapse 1989;4:156-61. FOCUS: PSYCHOSTIMULANTS
  • 52. 114 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE FOCUS: PSYCHOSTIMULANTS The Use and Abuse of Psychostimulants SUSAN B GOCK, VICTOR A SKRINSKA ABBREVIATIONS: DAWN = Drug Abuse Warning Network; ED = emergency department; MDA = methyl- enedioxyamphetamine; MDMA = methylenedioxymetham- phetamine; OTC = over the counter. INDEX TERMS: drug abuse; psychostimulants. Clin Lab Sci 2005;18(2):114 Susan B Gock MS MT(ASCP) is Technical Director, Mil- waukee County Medical Examiner’s Toxicology Laboratory, Milwaukee WI. Victor A Skrinska PhD DABCC is Professor and Chair, Uni- versity of Alabama at Birmingham, Birmingham AL. Address for correspondence: Susan B Gock MS MT(ASCP), Forensic Laboratory Technical Director, Milwaukee County Medical Examiner’s Office, 933West Highland Ave, Milwaukee WI 53233. (414)-223-1228. sgock@milwcnty.com. Victor A Skrinska PhD DABCC is the Focus: Psychostimu- lants guest editor. Focus Continuing Education Credit: see pages 124 to 126 for learning objectives, test questions, and application form. Drugusehasbecomeasignificantmedicalandsocialproblemin theUnitedStates.Toxicologicalanalysisofbiologicalspecimens from individuals is generally accepted to be the most objective method for determining drug use and abuse. As a science, forensic toxicology deals with the medico-legal implications of drug use, misuse, and abuse. This may include the following: criminal penalties imposed for the distribution, possession, and use of illicit drugs; assessment of drug impairment in human performance (behavioral) toxicology; assessment of drug toxic- ity as a contributing factor in the cause and manner of death in postmortem forensic toxicology cases; or detection of drug use in workplace drug testing programs. Psychostimulants include a diverse class of drugs exhibiting central nervous system stimulant properties, and have a high abuse potential. Drugs in this class include illicit drugs, pre- scription medications, over the counter (OTC) preparations, and dietary supplements. Clinical indications for therapeutic use include treatment of narcolepsy, attention deficit disorder, and as an appetite suppressant in the treatment of obesity. Pharmacological effects of psychostimulant drugs include the ability to increase alertness, relieve fatigue, decrease appetite, elevate mood, increase confidence, and produce euphoria. Abuse of psychostimulant drugs may lead to tolerance that is exhibited by the need of higher doses of the drug to pro- duce the same desired effects. Consequently, users may try to intensify the drug’s positive effects by increasing the drug dosage, taking it more frequently, or changing the route of administration leading to the possibility of drug abuse, mis- use, or toxicity. The most common psychostimulant drugs of forensic interest with documented abuse potential are listed in Table 1. These drugs are characterized as either sympa- thomimetic or hallucinogenic amines with psychostimulant effects. Phentermine, fenfluramine, and diethylproprion are related prescription medications used as appetite suppres- sants. There is little epidemiological evidence to support the abuse of these three psychostimulant drugs. However, their use is not recommended for individuals with current or past drug abuse problems. TRENDS IN PSYCHOSTIMULANT ABUSE Statistics released by the Drug Abuse Warning Network (DAWN) reflect the reporting of specific illicit, prescrip- tion, and OTC drugs that are linked to drug abuse in visits to hospital emergency departments (EDs). The data are presented in two issues of The Dawn Report.1,2 In 2002, there were an estimated 670,307 ED visits related to drug abuse in the continental U.S. This translates to 0.7% of all visits. Over the past nine years, reports of drug-related ED visits associated with drugs grew at roughly twice the rate of total ED visits. From 1994 to 2002, ED visits related to drug abuse rose 29%, while total ED visits rose only 15%. In 2002, about half (54%) of all visits related to drug abuse involved multiple drugs. Four illicit drugs including cocaine, marijuana, heroin, and methamphetamine accounted for 36% of the drugs involved in these visits. Cocaine continues to be the most frequently mentioned illicit substance reported to DAWN by hospital EDs nationwide.2
  • 53. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 115 Illicit use of psychostimulant drugs reported in 2002 included 199,198 cases for cocaine; 17,696 cases for methamphetamine; and 4,026 cases for methylenedioxymethamphetamine (MDMA). From 1995 to 2002, the rate of cocaine-related ED visits increased 33% whereas the rate of methamphetamine-related visits re- mained stable. Visits involving cocaine exceeded the number of visits for any other illicit drug in 18 of the 21 met- ropolitan areas monitored by DAWN. Western metropolitan areas including San Francisco, Seattle, San Diego, Los Angeles, and Phoenix had the highest rates of methamphetamine ED visits. According to the National Drug Threat Assessment 2003, prepared by the National Drug Intelligence Center, cocaine is the primary drug threat in the U.S. because of its high demand and availability, expanding distribu- tion to new markets, high rate of associated toxicity issues, and relation to violence.3 According to 2002 estimates from DAWN, there were almost 39,000 ED visits related to drug abuse involving amphetamines and methamphet- amine. In more than half of these visits the age of the patient was 18 to 34.1,4 In the report, the category of amphetamines included dextro- amphetamine, methcatinone, and methylenedioxyamphetamine (MDA). Visits involving the amphetamines and methamphetamine increased 54% between 1995 and 2002.4 Data also show that methamphetamine abuse, which was predominately located on the west coast of the U.S. may be spreading eastward. This was dem- onstrated by an increase in ED visits for amphetamines and methamphet- amine in several metropolitan areas in the Midwest, South, and Northeast. However, even though there was an increase, the overall numbers of related visits in these areas remained low. More than 60% of the amphet- amine and methamphetamine visits also involved other drugs. Marijuana, alcohol, cocaine, benzodiazepines, opi- oid pain relievers, and heroin were the most frequently reported substances in combination with amphetamines and methamphetamine. The term ‘club drugs’ is used to de- scribe illicit recreational drugs that have gained popularity in recent years at nightclubs or large dance parties called ‘raves’.5,6 Club or rave drugs include stimulants, depressants, and hallucinogenic substances frequently taken for their psychedelic and/or euphoric effects to enhance dancing, auditory, and visual perceptions. Drugs in this class include the following: lysergic acid diethylamide (LSD), FOCUS: PSYCHOSTIMULANTS Table 1. Common psychostimulant drugs of forensic interest Drug Type Amphetamine Sympathomimetic stimulant Cocaine Sympathomimetic stimulant Ephedrine Sympathomimetic stimulant Methamphetamine Sympathomimetic stimulant Methylenedioxyamphetamine (MDA) Hallucinogen Methylenedioxymethamphetamine (MDMA) Hallucinogen Methylphenidate Sympathomimetic stimulant gamma-hydroxybutyrate (GHB), ketamine, flunitrazepam (Rohypnol), and MDMA or ecstasy. For the year 2002, club or rave drugs were impli- cated in approximately 1.2% of all ED visits related to drug abuse that were reported to DAWN.6 Some of these visits involved multiple club drugs, that were frequently used in combina- tion with alcohol, marijuana, cocaine, and heroin. The incidence of ED visits involving MDMA is increasing based on data provided to DAWN.1,6 In 1994 MDMA was mentioned 253 times. In 2001 the number of times MDMA was mentioned increased to 5,542 and then subsequently declined to 4,026 in 2002. In 2002, MDMA was the most common club drug de- tected in drug related ED visits, with 75% of these visits involving patients 26 years or younger. Methylphenidate (Ritalin® ), a sched- ule II substance, is associated with patterns of abuse similar to other psychostimulants.7,8 Since it produces many of the same pharmacological ef- fects as cocaine or the amphetamines, a high potential for abuse of this drug also exists. The primary legitimate medicinal use of methylphenidate is for the treatment of attention deficit hyperactivity disorder (ADHD) in children. The increased therapeutic use of this drug for treatment of ADHD is paralleled by an increase in the abuse of the drug among adolescents and young adults due to its increased avail- ability. A recent survey of Wisconsin schools found that most schools did not control how Ritalin was stored or dispensed on school property, making it easy to steal, give away, or sell the drug.9 Approximately 16% of the stu- dents surveyed reported that they had been asked to sell, give, or trade their Ritalin to other students. This pattern of abuse is characterized by increasing
  • 54. 116 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE doses, frequent episodes of binge use followed by depres- sion, and the desire to continue the use of the drug despite medical and social consequences. The abuser may change the route of administration of the drug from oral to snorting or intravenous injection to intensify the effects. Recent years have seen an increase in the use of dietary supplements, not only to promote good health, but also as a means of obtaining a ‘natural’ legal high.10 Stimulants are the most commonly abused dietary supplements used for this purpose. Manufacturers of stimulant dietary supplements market them as natural and safe alternatives for enhanced mental alertness, weight loss, bodybuilding, and athletic performance enhancement. See the table in reference 10 for information on common dietary supplements. Most stimu- lant dietary supplements contain one or a combination of the following ingredients: ephedrine, pseudoephedrine, syneph- rine, caffeine, or yohimbine. Herbal Ecstacy® , Metabolife® , and Xenadrine® are examples of OTC dietary supplements containing ephedrine as the selected stimulant. Even though these products are probably safe in most cases when used as directed, a potential for abuse and toxicity still exists as with the other illicit, prescription, and OTC psychostimulants. According to the Toxic Exposure Surveillance System of the American Association of Poison Control Centers, ephedrine products accounted for 77% of the cases of abused or misused dietary supplements in 2002.10 Due to the increased abuse of stimulant dietary supplements with potential risk of serious adverse events including arrhythmias, seizures, heart attacks, and strokes, the FDA prohibited the sale of all dietary supple- ments containing ephedrine as of April 12, 2004. PHARMACOKINETICS OF ILLICIT PSYCHO- STIMULANTS Cocaine, an alkaloid, is a naturally occurring central ner- vous system stimulant found in the South American plant Erythroxylon coca. For medicinal use as a local anesthetic, cocaine is administered topically as a hydrochloride in 10% to 20% solutions for the membranes of the throat and nose or in 1% to 4% solutions for ophthalmologic procedures.11 Street forms of cocaine are sold as either hydrochloride salt or crack. Crack is cocaine that has been processed from cocaine hydrochloride to a free base for smoking. Common routes of administration include intranasal, intravenous, or smoked. Oral is not the preferred route of administration because, when taken orally, the first-pass effects result in low drug bioavailability and reduced euphoric effects due to inefficient delivery to the brain.12 The intravenous route of administration produces 100% drug bioavailability whereas bioavailability by the intranasal and smoked routes can be quite variable. Due to the convenience of administration, and the rapid, intense onset of effects from the smoked route, intranasal and smoked routes are most commonly used for cocaine self-administration. Cocaine is metabolized to benzoylecgonine and ecgonine methyl ester, the two major metabolites, by different mechanisms.12,13 Cocaine is metabolized in the blood to benzoylecgonine via spontaneous hydrolysis at a physiological and alkaline pH and metabolized to ecgonine methyl ester via enzymatic hydrolysis by pseudocholinesterase and liver esterases with the reaction rate being dependent on drug con- centration. Both benzoylecgonine and ecgonine methyl ester are further metabolized to ecgonine. When cocaine is used in combination with ethyl alcohol, a pharmacologically active metabolite, cocaethylene (ethylcocaine), is produced by the transesterification of cocaine with ethyl alcohol. Therefore, in cases of simultaneous alcohol and cocaine use, cocaethylene concentrations should also be considered when interpreting results for assessment of toxicity. Anhydroecgonine methyl ester has been identified as a unique metabolite in the post- mortem blood and urine specimens of persons after smoking cocaine.14 Benzoylecgonine can be detected in blood within 15 to 30 minutes after intravenous, intranasal, and smoking routes of administration. Detection of benzoylecgonine in the urine can provide a means of estimating an approximate window of time of the use of cocaine. Benzoylecgonine can be detected in urine for two to four days after use of cocaine depending on dose, frequency of use, urine pH, and clear- ance. Detection has been reported seven to sixteen days after chronic compulsive cocaine use.12,13 Ecstasy or MDMA is the most prominent member of the methylenedioxy-substituted amphetamines (hallucinogenic amines) to gain popularity for illicit recreational drug use.5,6 At normal doses, the effects include mild to moderate cen- tral nervous system stimulating effects as well as enhanced feelings of empathy, closeness, and response to intimate touch, which is why it is also classified as an empathogen- entactogen.13,15 Currently, there is no legitimate approved therapeutic use for MDMA in the U.S. The manufacture of MDMA is typically in clandestine laboratories, primarily in Western Europe where it is easier to obtain the precursor chemicals. It is estimated that 80% of the MDMA consumed worldwide is produced in Belgium, Luxembourg, and the Netherlands. The drug is typically supplied in tablet form with an embossed logo, usually white but also available in a variety of colors. Most frequently, MDMA is administered orally in tablet or capsule form in doses of 100 mg to 150 FOCUS: PSYCHOSTIMULANTS
  • 55. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 117 mg. A popular variation on oral ingestion is ‘parachuting’, in which a tablet is crushed, wrapped in tissue paper, and swallowed for more rapid absorption.15 This is sometimes supplemented with an uncrushed tablet to achieve both rapid onset as well as a sustained effect. The onset of action following oral administration is 30 to 60 minutes.5 MDMA has a half-life of approximately seven hours andundergoesN-demethylationtotheactivemetaboliteMDA, which is the metabolite usually detected in blood. Illicitly pro- duced MDMA is a racemic mixture. The enantiomers exhibit differences in pharmacological activity as well as affinity for the enzymeresponsiblefortheirmetabolism.15 MDMAalsoexhibits nonlinear pharmacokinetics suggesting that beyond a certain threshold, small increases in dose may result in larger increases in blood concentrations with greater risk of toxicity.15 About 65% of the dose is eliminated in the urine as parent drug and 7% as MDA.11 Mono- and di-hydroxy metabolites are excreted in the urine as conjugates. Amphetamine and methamphetamine have limited le- gitimate pharmacologic use. Methamphetamine is the more potent of these two drugs, producing greater central nervous system effects and having a longer duration of action, most likely due to its greater ability to penetrate the central nervous system (CNS). Current therapeutic uses of these drugs are for the treatment of narcolepsy, obesity, and ADHD. Am- phetamine can also be detected as a metabolite of other drugs including fenethylline, fenproporex, and methamphetamine. Illicit preparations of amphetamine usually contain a racemic mixture.11 Clandestine laboratories use phenylpropanolamine as a precursor to amphetamine manufacture. Because of its ease of manufacture and ready availability, methamphetamine has become the sympathomimetic amine of choice among stimulant abusers. In the U.S., methamphetamine is the most frequently encountered clandestinely produced con- trolled substance.13 Methamphetamine is easily synthesized from ephedrine. In the U.S., l-methamphetamine (under the label l-deoxyephedrine) is the active constituent of the OTC decongestant Vicks Inhaler containing approximately 50 mg of drug.11, 16 This isomer is reported to have less CNS activity and greater peripheral sympathomimetic activity than the d-enantiomer. l-methamphetamine can also be detected as a metabolite of selegiline. d-methamphetamine is a legal prescription drug (Desoxyn® ) and a prominent form in many illicit methamphetamine drug preparations available as a water soluble, white, crystalline powder (methamphetamine hydrochloride). Determination of the enantiometric ratio can be useful in determining drug source as illicit, diverted, or licit.13,16 Common routes of administration include oral, intranasal, and intravenous. Smoking remains a minor route of ad- ministration compared to the others.16 Methamphetamine users feel a short yet intense ‘rush’ when the drug is initially administered. In abuse, there is a progression following the start of use, from oral or intranasal routes to intravenous use of the drug. At higher doses, particularly following in- travenous use, users typically report intense exhilaration and euphoria, elevated self-esteem, extreme wakefulness, rapid flow of ideas, and increased physical and mental capacity. These effects are perceived as positive and generally encourage repeated administration and produce a binge-type pattern of use.16 In the binge-type pattern of use, the initial positive effects may be followed by restlessness, irritability, and pos- sibly paranoid psychoses that reinforce the continued use of the drug to maintain the ‘high’ which eventually will lead to tolerance and psychological dependence. Methamphetamine undergoes phase I metabolism by N- demethylation to amphetamine, its major active metabolite via the cytochrome P450 2D6 isoenzyme system. Amphet- amine is metabolized to a variety of metabolites, including norephedrine and p-hydroxyamphetamine, both of which are pharmacologically active and may contribute to the effects of the drug, especially during chronic usage. Accumulated hydroxylated metabolites have been implicated in the devel- opment of amphetamine psychosis.13 Under normal condi- tions, up to 43% of a dose of methamphetamine is eliminated unchanged in the 24-hour urine, with about 4% to 7% as amphetamine.11 Elimination of the sympathomimetic amines is highly pH dependent. Urinary acidification to a pH less than 5.6 decreases the plasma half-life from 11 to 12 hours to 7 to 8 hours whereas alkalinization increases the half-life to 18 to 34 hours. PSYCHOSTIMULANT TOXICITY In toxic doses, the psychostimulants begin to produce unpleasant CNS symptoms including anxiety, agitation, hallucinations, delirium, seizures, and death.13 High-dose, long-term use of stimulants can induce an acute psychotic state in previously healthy individuals. CNS-induced ab- normalities, seizures, or muscular hyperactivity may induce hyperthermia. Secondary rhabdomyolysis may also be seen. Cardiovascular manifestations include hypertension, tachy- cardia, arrhythmias, and myocardial ischemia. Cerebrovas- cular accidents are precipitated by elevated blood pressure or drug-induced vasospasms. FOCUS: PSYCHOSTIMULANTS
  • 56. 118 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE The clinical picture of stimulant intoxication also includes a wide array of psychiatric symptoms including schizophrenic symptoms, manic-like states, psychoses, depressions (espe- cially during withdrawal), and various types of anxiety con- ditions including panic states.8 Psychotic symptoms usually arise with chronic abuse but may also appear acutely with large doses of stimulants. With high doses of stimulants, symptoms of extreme anger in conjunction with aggressive behavior can also be a catalyst for both violence and murder and is especially seen in cases of methamphetamine and cocaine intoxication.12,16 Many factors must be considered when interpreting psy- chostimulant concentrations in blood for an assessment of toxicity since clinical and postmortem studies clearly show that therapeutic, toxic, and lethal concentrations overlap. Tolerance, sensitization, and specimen stability problems are factors complicating the correlation of blood concentrations with assessment of toxicity. Many pathological conditions may predispose an individual to toxicity and possibly death at lower than expected blood concentrations.17 Sudden death that is attributed to the complications of methamphetamine and cocaine abuse is most commonly associated with cardio- vascular effects (disturbances in heart rhythm, heart attacks), but may also be associated with respiratory failure, and neu- rological effects (strokes, seizure). Seizures are often associated with stimulant abuse but tend to occur only at higher doses. Convulsions are a common sequel to long-term and high dose use that is associated with typical abuse patterns. Cocaine-induced excited or agitated delirium is an example of a toxic response to cocaine that is frequently associated with death. This syndrome is associated with severe hyper- thermia, extreme agitation and delirium, respiratory arrest, and sudden death. These individuals exhibit extreme strength in combination with bizarre and/or violent behavior.12 Use of methamphetamine can cause damage to the brain that is detectable months after the use of the drug. The damage to the brain is similar to damage caused by Alzheimer’s disease, stroke, or epilepsy.18 Rave party attendees who ingest MDMA are at risk of de- hydration, hyperthermia, and heart or kidney failure. These risks are due to a combination of the drug’s stimulant effect that allows the user to dance for long periods of time in the hot and crowded environment of rave parties. The combina- tion of crowded all-night dance parties and MDMA use has been reported to cause fatalities.5,15 REFERENCES 1. The DAWN report: trends in drug-related emergency department visits, 1994-2004 at a glance. Drug Abuse Warning Network, Office of Applied Statistics, Substance Abuse and Mental Health Services Administration. 2003. 2. The DAWN report: major drugs of abuse in emergency department visits, 2002 update. Drug Abuse Warning Network, Office of Applied Statistics, Substance Abuse and Mental Health Services Administra- tion. 2004. 3. National drug threat assessment, 2003. Publication # 2003-Q0317- 001. National Drug Intelligence Center. 2003. 4. The DAWN report: amphetamine and methamphetamine ED visits, 1995-2002.DrugAbuseWarningNetwork,OfficeofAppliedStatistics, Substance Abuse and Mental Health Services Administration. 2004. 5. Valentine JL, Kerrigan S. “Club” or “rave” drugs offer challenges to laboratories. Clin Forens Tox News 2001;September:1-8. 6. The DAWN report: club drugs, 2002 Update. Drug Abuse Warning Network, Office of Applied Statistics, Substance Abuse and Mental Health Services Administration. 2004. 7. Evans C, Blackburn D, Butt P, and others. Use and abuse of meth- ylphenidate in attention-deficit hyperactivity disorder. CPJ/RPC 2004;137:30-3. 8. Morton WA, Stockton GG. Methylphenidate abuse and psychiatric side effects. J Clin Psychiatry 2000;2:159-64. 9. Musser CJ, Ahmann PA, Theye FW, and others. Stimulant use and the potential for abuse in Wisconsin as reported by school admin- istrators and longitudinally followed children. J Dev Behav Pediatr 1998;19:187-92. 10. Simone KE. Abuse of dietary supplements is common and dangerous. Clin Forens Tox News 2004;September:2-6. 11. Baselt RC. Disposition of toxic drugs and chemicals in man. 6th ed. Foster City CA: Chemical Toxicology Institute, 2002. 12. Isenschmid DS. Cocaine: effects on human performance and behav- ior. Forens Sci Rev 2002;14:62-100. 13. Levine B. Principles of forensic toxicology. 2nd ed. Washington DC: AACC Press; 2003. 14. Jenkins AJ, Goldberger BA. Identification of unique cocaine me- tabolites and smoking by-products in postmortem blood and urine specimens. J Forensic Sci 1997;42:824-7. 15. Logan BK, Couper FJ. 3,4-Methylenedioxymethamphetamine: effects on human performance and behavior. Forens Sci Rev 2003;15:11-28. 16. Logan BK. Methamphetamine: effects on human performance and behavior. Forens Sci Rev 2002;14:133-51. 17. Karch S. Pathology of drug abuse. 2nd ed. Philadelphia PA: CRC Press;1993. 18. Methamphetamine abuse linked to long-term damage to brain cells. NIDA News Release, March 27, 2000. FOCUS: PSYCHOSTIMULANTS
  • 57. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 119 The Focus section seeks to publish relevant and timely continuing education for clinical laboratory practitioners. Section editors, topics, and authors are selected in advance to cover current areas of interest in each discipline. Readers can obtain continuing education credit (CE) through P.A.C.E.® by completing the tearout form/examination questions included in each issue of Clin Lab Sci and mailing it with the appropriate fee to the address designated on the form. Suggestions for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to George Fritsma MS MT(ASCP), Department of Pathology, 619 South 19th Street, West Pavilion, University of Alabama at Birmingham, Birmingham AL 35233, (205)821-5641, gfritsma@path.uab.edu. FOCUS: PSYCHOSTIMULANTS Measurement of 3,4-MDMA and Related Amines in Diagnostic and Forensic Laboratories VICTOR A SKRINSKA, SUSAN B GOCK The phenylalkylamine derivatives, 3,4-methylenedioxymeth- amphetamine (MDMA, ecstasy, XTC, Adam), 3,4-methy- lenedioxyethamphetamine (MDEA, MDE, Eve), and 3,4- methylenedioxyamphetamine (MDA), are psychostimulants with hallucinogenic properties. MDA is also a metabolite of both MDMA and MDEA. These drugs are ring-substituted amphetamine derivatives that produce hallucinogenic, en- tactogenic (‘love drug’), and stimulating effects.1-3 MDMA was initially developed as an appetite suppressant, however, its use as a therapeutic drug has been very limited.4 Because of its effects as a hallucinogenic psychostimulant with rela- tively low toxicity, it has emerged over the last two decades as a common recreational psychostimulant or ‘club drug’ at ‘raves’.5 MDMA, MDEA, and MDA are often referred to as ‘rave’ or ‘designer’ drugs. They are produced in clandestine laboratories and have an increasing presence on the illicit drug market worldwide. Significant adverse health effects have been reported that include: serotonin neurotoxicity, severe psychiatric disorders, renal failure, malignant hyperthermia, hepatitis, rhabdomyolysis, and disseminated intravascular coagulation.6-8 A number of fatal outcomes associated with severe MDMA intoxication have been reported.9-12 ABBREVIATIONS: Adam = 3,4-methylenedioxymetham- phetamine; ecstasy = 3,4-methylenedioxymethamphetamine; Eve = 3,4-methylenedioxyethamphetamine; GC = capillary gas chromatography; GC/MS = gas chromatography/mass spectrometry; HPLC = high performance liquid chro- matography; MDA = 3,4-methylenedioxyamphetamine; MDE = 3,4-methylenedioxyethamphetamine; MDEA = 3,4-methylenedioxyethamphetamine; MDMA = 3,4-methy- lenedioxymethamphetamine; XTC = 3,4-methylenedioxy- methamphetamine. INDEX TERMS: drug testing; hallucinogenic drugs. Clin Lab Sci 2005;18(2):119 Victor A Skrinska PhD DABCC is Professor and Chair, Uni- versity of Alabama at Birmingham, Department of Diagnostic and Therapeutic Sciences, Birmingham AL. Susan B Gock MS MT(ASCP) is Forensic Laboratory Techni- cal Director, Milwaukee County Medical Examiner’s Office, Milwaukee WI. Address for correspondence: Victor A Skrinska PhD DABCC, Professor and Chair, University of Alabama at Birmingham, Department of Diagnostic and Therapeutic Sciences, 1530 3rd Ave S, Birmingham AL 35294-1212. (205) 934-9124. skrinska@uab.edu Victor A Skrinska PhD DABCC is the Focus: Psychostimu- lants guest editor. Focus Continuing Education Credit: see pages 124 to 126 For learning objectives, test questions, and application form. The analysis of MDMA, MDEA, and MDA can be broken down into several categories. The first is the need to identify the presence of the drugs in tablets that are seized and suspect- ed to contain illicit drugs. The second is the need to detect ‘rave’ drugs onsite with the intent to determine recent use of the drugs. The third category is the typical laboratory drug screen used to determine either recent or chronic exposure to the drugs. And finally, the fourth category is forensic analysis of postmortem specimens for the presence of the drugs. The specimens, methodology, and instrumentation vary with each of the categories. Table 1 summarizes the methods that have been developed and reported for these categories. ANALYSIS OF TABLETS Tablets containing MDMA and other psychostimulants are prepared in clandestine laboratories worldwide. The tablets
  • 58. 120 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE vary in size and typically have logos such as a pitbull, spar- row, butterfly, ‘e’, or ‘X-files’ imprinted on the tablets.13 The concentration of the active ingredients varies widely even among tablets from same origin.14 The excipients or inert ingredients found in tablets include glucose, sorbitol, and cellulose.15 Despite variation in concentration of the active ingredients, analysis of the tablets is helpful in identification of the clandestine laboratory that manufactured them. A number of analytical techniques have been applied to the characterization of the seized tablets. Raman spectroscopy of the active components and the excipients in tablets has been successfully used to identify tablets from the same source based on the state of hydration and the drug/excipi- ent ratio.15-18 Another approach is analysis of impurities and byproducts of synthesis by gas chromatography/mass spec- trometry (GC/MS), capillary gas chromatography (GC), or high performance liquid chromatography (HPLC).19,20 Isotopic analysis of the tablets for the ratios of deuterium, carbon 13, and nitrogen 15 in the active ingredients has been reported as a characteristic that is unique to the site of manufacture and may be a reliable method of fingerprinting the tablets.21,22 Capillary zone electrophoresis with ultraviolet detection is a rapid method suitable for routine analysis of MDMA content in tablets.23 ONSITE DETECTION OF PSYCHOSTIMULANTS When individuals at the scene of a ‘rave’ party, accident, or crime are taken into custody, the need arises for a rapid onsite detection method for MDMA and related drugs. Some immunoassays that have been developed for detec- tion of methamphetamine have high cross reactivity with MDMA and MDA, which make the assays potentially suit- able for onsite screens where abuse of psychostimulants is suspected.24 Procedures have been reported for onsite analysis of saliva and sweat.24-27 The concentrations of MDMA and MDA in saliva have pharmacokinetic parameters that are similar to plasma, thus demonstrating that saliva is a useful and less invasive alternative to analysis of plasma.28 Studies have shown that individuals taking a single 100 mg dose of MDMA consistently have detectable levels of MDMA in both sweat and saliva after 1.5 hours. After six hours, most individuals remain positive; however, the number of false negatives begins to increase significantly to almost 20%.25 Drugwipe has been successfully applied to onsite screens of both saliva wiped from the tongue and sweat collected from armpits.26,27 The drug may be quantified and cutoff limits established with a hand photometer, Drugread .26 DETECTION OF ILLICIT PSYCHOSTIMULANTS IN DRUG SCREENS Laboratory drug screens for detection of MDMA, MDEA, and MDA typically measure the presence of these substances in plasma, urine, saliva, or hair.29-31 The methods used for de- tection range from relatively rapid basic immunoassays to the Table 1. Summary of methods of analysis cited for MDMA and related amines Specimen Immunoassay HPLC Electrophoresis GC/MS LC/MS Other Tablet — 19 23 19 — 15-18, 20-22 Onsite testing Saliva 26 — — — — — Sweat 24, 25, 27 — — — — — Laboratory screening Hair 36 36, 43 31 – 33 35 – 42 — 44 Plasma — 34 — — 29 — Saliva — — — 28 29 — Urine 30 34 — 30 — — Forensic analysis Hair — — — 47 — — Plasma — 45, 46 — — — — Vitreous humor — 45, 46 — — — — F O C U S : PSYCHOSTIMULANTS
  • 59. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 121 more sophisticated and more labor intensive methods such as liquid chromatography with tandem mass spectrometry (LC/MS/MS).29,30 Plasma and saliva concentrations indicate recent drug abuse less than 24 hours, whereas urine concen- trations reflect drug intake within the previous 48 hours.27 Analysis of hair provides a historical perspective that suggests chronic abuse of the drugs.31 After a single 100 mg dose of MDMA, the concentration of the drug peaks at 1.5 hours after intake with concentrations ranging from 135 to 233 ng/ mL in plasma and from 1729 to 6510 ng/mL in saliva. After 24 hours, the MDMA levels in plasma and saliva decrease to mean concentrations of 14 ng/mL and 126 ng/mL, respec- tively.28 In urine, significant concentrations of MDMA are detectable up to 48 hours yielding a positive screen in urine while the results for plasma and saliva are negative.26,27 Immunoassays for amphetamine and methamphetamine gen- erally have high cross reactivity with related drugs and have been successfully applied to urine screens for the detection of MDMA and MDA.30 The assays have sufficient sensitivity for reliable detection of the drugs at the established cutoff of 500 ng/mL and agree well with results confirmed by GC/MS. The high cross reactivity of immunoassays with structures similar to amphetamine and methamphetamine may result in false positives for MDMA when other substances such as ephedrine or pseudoephedrine are present.30 Capillary electrophoresis with electrochemical and fluores- cence detection has been successfully applied to the analysis of MDMA in urine with detection limits of 4 ng/mL with an electrochemical detector and 50 ng/mL with a fluorescence detector.32,33 Chromatographic techniques such as HPLC with fluorescence detection have been used for measurement of MDMA in plasma and urine with a detection limit of 25 ng/mL.34 Mass spectrometry methods including GC/MS and LC/MS/MS have been reported for the analysis of plasma and saliva for MDMA and its metabolites with detection limits of 6 ng/mL and 2 ng/mL, respectively.28,29 Hair analysis has been studied as a specimen that may be useful for determination of past chronic exposure to illicit drugs.31 Hair is a complex structure that grows approxi- mately one cm/month. During its growth, hair is exposed to substances present in the capillary blood circulation near the follicle and substances excreted in sweat at the base of the follicle. Drugs in contact with hair penetrate and embed in the core of the hair stock and remain in the stock for an extended period of time. In the hair stock, drugs are rela- tively protected from the environment; however, extensive washing of the hair will cause some loss of the embedded drugs. Also, external contact with drugs in powder or liquid form will cause penetration of the hair stock.31 Nevertheless, it has been shown to be a useful indicator of chronic drug use and is widely accepted as a suitable specimen for drug screens including the detection of MDMA and MDA.35 All methods for hair analysis require digestion of the hair sample followed by extraction of the drugs. The accepted cutoff for a positive hair sample is 0.1 ng/mg.36 Most methods developed for the analysis of drugs in hair use GC/MS instrumentation to obtain the necessary sensitivity.37-42 Hair samples range in weight from 10 to 50 mg. Other methods that have been successfully applied to hair analysis for MDMA include capillary electrophoresis, radioimmunoassay, HPLC, and ion mobility spectrometry.31,36,43,44 FORENSIC ANALYSIS OF PSYCHOSTIMULANTS Analysis of postmortem specimens for the presence of psychostimulants such as MDMA, MDEA, and MDA typi- cally involves extraction of the drugs from tissues including liver, muscle, and brain as well as from urine, central blood, peripheral blood, and vitreous humor.45 Varying degrees of putrefaction and postmortem redistribution of drugs further complicate the analysis. While hair and urine are suitable forensic specimens to determine the presence of the drugs, peripheral blood and vitreous humor are reported to provide the best estimate of the blood concentration at the time of death.45-48 There are various analytical techniques available for initial screening, confirmation, and quantification of forensic speci- mens such as thin-layer chromatography (TLC), HPLC, and GC/MS.45-47 TLC is a common initial screening technique when a method capable of detecting a broad-spectrum of drugs in urine specimens is required. Identification is based on Rf value and the color characteristics following exposure to specific staining reagents. The Toxi-Lab A® system for the detection of basic and neutral drugs in urine specimens is able to differentiate sympathomimetic amines such as ephedrine, pseudoephedrine, and phenylpropanolamine from illicit dugs such as amphetamine, methamphetamine, MDMA, and MDA. Sensitivity for most of the drugs in this class using this procedure is approximately 500 ng/mL. Immunoassays may also be applied to forensic drug screens for the presence of MDMA and related metabolites.30 However, as mentioned earlier, immunoassay techniques for the detection of amphetamine and methamphetamine have variable amounts of antibody cross reactivity to other F O C U S : PSYCHOSTIMULANTS
  • 60. 122 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE structurally related sympathomimetic amines including pseudoephedrine and ephedrine.30 Antibody cross reactivity is variable and dependent on both the concentration of the structurally related analyte present in the specimen as well as the source of the antibodies used for detection. Higher levels of antibody cross reactivity occur with polyclonal an- tibody assays in comparison to monoclonal antibody assays. Monoclonal antibody assays are more specific and exhibit less cross-reactivity to structurally related compounds and should be used when high selectivity is desired. Application of immunoassay techniques for the analysis of postmortem specimens poses a problem due to decomposition occurring during the postmortem interval. This may result in the pro- duction of biogenic amines such as beta-phenethylamine or tyramine that have the potential to produce a false positive with amphetamine immunoassays due to the cross reactivity with these analytes. Due to the lack of specificity associated with immunoassays for identification of the specific psycho- stimulants present in the sample, confirmation of positive immunoassay results should be made using an alternate analytical methodology. GC/MS analysis with selective ion monitoring is the analytical methodology routinely utilized for drug confirmation and quantification. Derivatization of the drugs with heptafluorobutyric anhydride, pentafluoro- propionic anhydride, or trifluoroacetic anhydride prior to analysis improves chromatographic behavior and reduces fragmentation so that higher mass fragments can be used for GC/MS selective ion monitoring which allows a more definitive identification and confirmation of the drugs. CONCLUSION A wide range of analytical methods have been developed for analysis of MDMA and related psychostimulants. Analysis by GC/MS is the technique that has been reported the most often and has been applied to the widest range of specimen types. No doubt this is due to the high specificity combined with high sensitivity that is found in GC/MS applications. However, there are a number of alternate methods using technologies such as immunoassay, HPLC, and electrophoresis that have sufficient sensitivity and specificity for detection of MDMA in routine screening applications in the laboratory. Furthermore, these alternative methods are more easily automated and more suitable for high volume applications. REFERENCES 1. Nichols DE. Differences between the mechanism of action of MDMA, MBDB, and the classic hallucinogens. Identification of a new therapeutic class: entactogens. J Psychoactive Drugs 1986;18:305-13. 2. Steele TD, McCann UD, Ricaurte GA. 3,4-methylenedioxymeth- amphetamine (MDMA, ‘ecstasy’): pharmacology and toxicology in animals and humans. Addiction 1994;89:539-51. 3. Hermle L, Spitzer M, Borchardt D, and others. Psychological effects of MDE in normal subjects. Are entactogens a new class of psychoac- tive agents? Neuropsychopharmacology 1993;8(2):171-6. 4. Climko RP, Roehrich H, Sweeney DR, and others. Ecstacy: a review of MDMA and MDA. Int J Psychiatry Med 1986;16:359-72. 5. Arria AM, Yacoubian GS Jr, Fost E, and others. The pediatric forum: ecstasy use among club rave attendees. Arch Pediatr Adolesc Med 2002;156:295-6. 6. Parrott AC. Recreational ecstasy/MDMA, the serotonin syn- drome, and serotonergic neurotoxicity. Pharmacol Biochem Behav 2002;71:837-44. 7. McCann UD, Eligulashvili V, Ricaurte GA. (+/-)3,4-methylene- dioxymethamphetamine (‘ecstasy’)-induced serotonin neurotoxicity: clinical studies. Neuropsychobiology 2000;42:11-6. 8. McCann UD, Slate SO, Ricaurte GA. Adverse reactions with 3,4- methylenedioxymethamphetamine (MDMA; ‘ecstasy’). Drug Saf 1996;15:107-15. 9. FineschiV, Masti A. Fatal poisoning by MDMA (ecstasy) and MDEA: a case report. Int J Legal Med 1996;108:272-5. 10. Iwersen S, Schmoldt A.Two very different fatal cases associated with the use of methylenedioxyethylamphetamine (MDEA): Eve as deadly as Adam. Clin Toxicol 1996;34:241-4. 11. Randall T. Ecstasy-fueled ‘Rave’ parties become dances of death for English youths. JAMA 1992;268:1505-6. 12. Henry JA, Jeffreys KJ, Dawling S.Toxicity and deaths from 3,4-methy- lenedioxymethamphetamine (‘ecstasy’). Lancet 1992;340:384-7. 13. Schneider RC, Kovar KA. Analysis of ecstasy tablets: comparison of reflectance and transmittance near infrared spectroscopy. Forensic Sci Int 2003;134:187-95. 14. Sherlock K, Wolff K, Hay AW, and others. Analysis of illicit ecstasy tablets: implications for clinical management in the accident and emergency department. J Accid Emerg Med 1999;16:194-7. 15. Bell SE, Burns DT, Dennis AC, and others. Composition pro- filing of seized ecstasy tablets by Raman spectroscopy. Analyst 2000;125:1811-5. 16. Bell SE, Burns DT, Dennis AC, and others. Rapid analysis of ecstasy and related phenethylamines in seized tablets by Raman spectroscopy. Analyst 2000;125:541-4. 17. Bell SE, Barrett LJ, Burns DT, and others. Tracking the distribution of ‘ecstasy’ tablets by Raman composition profiling: a large scale feasibility study. Analyst 2003;128:1331-5. 18. Ryder AG. Classification of narcotics in solid mixtures using prin- cipal component analysis and Raman spectroscopy. J Forensic Sci 2002;47:275-84. 19. Cheng WC, Poon NL, Chan MF. Chemical profiling of 3,4-methyl- enedioxymethamphetamine (MDMA) tablets seized in Hong Kong. J Forensic Sci 2003;48:1249-59. 20. Palhol F, Boyer S, Naulet N, and others. Impurity profiling of seized MDMA tablets by capillary gas chromatography. Anal Bioanal Chem 2002;374:274-81. 21. Carter JF,Titterton EL, Murray M, and others. Isotopic characterisa- tion of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy- methylamphetamine (ecstasy). Analyst 2002;127:830-3. F O C U S : PSYCHOSTIMULANTS
  • 61. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 123 22. PalholF,LamoureuxC,NauletN.15Nisotopicanalyses:apowerfultool toestablishlinksbetweenseized3,4-methylenedioxymethamphetamine (MDMA) tablets. Anal Bioanal Chem 2003;376:486-90. 23. Frost M, Kohler H, Blaschke G. Analysis of ‘ecstasy’ by capillary electrophoresis. Int J Legal Med 1996;109:53-7. 24. Fay J, Fogerson R, Schoendorfer D, and others. Detection of methamphetamine in sweat by EIA and GC-MS. J Anal Toxicol 1996;20:398-403. 25. Pichini S, Navarro M, Pacifici R, and others. Usefulness of sweat testing for the detection of MDMA after a single-dose administra- tion. J Anal Toxicol 2003;27:294-303. 26. Pichini S, Navarro M, Farre M, and others. On-site testing of 3,4- methylenedioxymethamphetamine (ecstasy) in saliva with Drugwipe and Drugread: a controlled study in recreational users. Clin Chem 2002;48:174-6. 27. Pacifici R, Farre M, Pichini S, and others. Sweat testing of MDMA with the Drugwipe analytical device: a controlled study with two volunteers. J Anal Toxicol 2001;25:144-6. 28. Navarro M, Pichini S, Farre M, and others. Usefulness of saliva for measurement of 3,4-methylenedioxymethamphetamine and its metabolites: correlation with plasma drug concentrations and effect of salivary pH. Clin Chem 2001;47:1788-95. 29. Wood M, De Boeck G, Samyn N, and others. Development of a rapid and sensitive method for the quantitation of amphetamines in human plasma and oral fluid by LC-MS-MS. J Anal Toxicol 2003;27:78-87. 30. Stout PR, Klette KL, Wiegand R. Comparison and evaluation of DRI methamphetamine, DRI ecstasy, Abuscreen ONLINE amphet- amine, and a modified Abuscreen ONLINE amphetamine screening immunoassays for the detection of amphetamine (AMP), metham- phetamine (MTH), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in human urine. J Anal Toxicol 2003;27:265-9. 31. Tagliaro F, Manetto G, Crivellente F, and others. Hair analysis for abused drugs by capillary zone electrophoresis with field-amplified sample stacking. Forensic Sci Int 1998;92:201-11. 32. Backofen U, Matysik FM, Hoffmann W, and others. Analysis of illicit drugs by nonaqueous capillary electrophoresis and electrochemical detection. Fresenius J Anal Chem 2000;367:359-63. 33. Fang C, Chung YL, Liu JT, and others. Rapid analysis of 3,4-methy- lenedioxymethamphetamine: a comparison of nonaqueous capillary electrophoresis/fluorescence detection with GC/MS. Forensic Sci Int 2002;125:142-8. 34. Herraez-Hernandez R, Campins-Falco P, Verdu-Andres J. Sensitive determination of methylenedioxylated amphetamines by liquid chromatography. Analyst 2001;126:581-6. 35. Kintz P, Cirimele V. Interlaboratory comparison of quantitative de- termination of amphetamine and related compounds in hair samples. Forensic Sci Int 1997;84:151-6. 36. Tagliaro F, Valentini R, Manetto G, and others. Hair analysis by using radioimmunoassay, high-performance liquid chromatography and capillary electrophoresis to investigate chronic exposure to heroin, cocaine and/or ecstasy in applicants for driving licenses. Forensic Sci Int 2000;107:121-8. 37. Pujadas M, Pichini S, Poudevida S, and others. Development and validation of a gas chromatography-mass spectrometry assay for hair analysis of amphetamine, methamphetamine, and methylene- dioxy derivatives. J Chromatogr B Analyt Technol Biomed Life Sci 2003;798:249-55. 38. Kintz P, Cirimele V,Tracqui A, and others. Simultaneous determina- tion of amphetamine, methamphetamine, 3,4-methylenedioxyam- phetamine, and 3,4-methylenedioxymethamphetamine in human hair by gas chromatography-mass spectrometry. J Chromatogr B Biomed Appl 1995;670:162-6. 39. Allen DL, Oliver JS. The use of supercritical fluid extraction for the determination of amphetamines in hair. Forensic Sci Int 2000;107:191-9. 40. Cooper GA, Allen DL, Scott KS, and others. Hair analysis: self-re- ported use of ‘speed’ and ‘ecstasy’ compared with laboratory findings. J Forensic Sci 2000;45:400-6. 41. Uhl M. Determination of drugs in hair using GC/MS/MS. Forensic Sci Int 1997;84:281-94. 42. Rothe M, Pragst F, Spiegel K, and others. Hair concentrations and self-reported abuse history of 20 amphetamine and ecstasy users. Forensic Sci Int 1997;89:111-28. 43. Tagliaro F, De Battisti Z, Groppi A, and others. High sensitivity simultaneous determination in hair of the major constituents of ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylene- dioxyamphetamine, and 3,4-methylene-dioxyethylamphetamine) by high-performance liquid chromatography with direct fluorescence detection. J Chromatogr B Biomed Sci Appl 1999;723:195-202. 44. Keller T. Miki A. Regenscheit P, and others. Detection of designer drugs in human hair by ion mobility spectrometry (IMS). Forensic Sci Int 1998;94:55-63. 45. De Letter EA, Bouche MP, Van Bocxlaer JF, and others. Interpreta- tion of a 3,4-methylenedioxymethamphetamine (MDMA) blood level: discussion by means of a distribution study in two fatalities. Forensic Sci Int 2004;141:85-90. 46. Clauwaert KM, Van Bocxlaer JF, De Letter EA, and others. Determi- nation of the designer drugs 3, 4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxyam- phetamine with HPLC and fluorescence detection in whole blood, serum, vitreous humor, and urine. Clin Chem 2000;46:1968-77. 47. Uhl M. Tandem mass spectrometry: a helpful tool in hair analysis for the forensic expert. Forensic Sci Int 2000;107:169-79. 48. De Letter EA, De Paepe P, Clauwaert KM, and others. Is vitreous humour useful for the interpretation of 3,4-methylenedioxymeth- amphetamine (MDMA) blood levels? Experimental approach with rabbits. Int J Legal Med 2000;114:29-35. FOCUS
  • 62. 124 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE FOCUS: PSYCHOSTIMULANTS Continuing Education Questions SPRING 2005 To receive three contact hours of basic level P.A.C.E. credit for the Focus: Psychostimulants questions, insert your an- swers in the appropriate spots on the immediately following page; then complete and mail the form as directed. NOTE: There may be more answer spaces on the answer sheet than needed. If so, leave them blank. Make sure the number of the answer space being filled matches the number of the questions being answered. LEARNING OBJECTIVES After reading the three Focus: Psychostimulants articles in this issue, the reader will be able to: 1. Describe the physiologic actions for amphetamine. 2. Describe the behavioral effects of some common psychostimulants. 3. Identify several therapeutic uses for psychostimulants. 4. Describe the mechanisms of action of amphetamine. 5. List symptoms associated with high doses of amphetamine. 6. List the common psychostimulants of forensic interest. 7. List the illicit psychostimulants that show an increasing trend of abuse in the United States. 8. Describe the composition of crack. 9. List the common routes of administration of cocaine. 10. Identify the major metabolites of cocaine, MDMA, and methamphetamine. 11. Describe symptoms related to toxic doses of cocaine, MDMA, and methamphetamine. 12. List the drugs that are commonly referred to as ‘designer’ or ‘rave’ drugs. 13. Describe the adverse effects of MDMA and related drugs caused by chronic intoxication. 14. Describe specimen collection for on site screening for MDMA. 15. List suitable analytical methods for analysis of MDMA and related amines in tablets, saliva, sweat, plasma, urine, hair, and vitreous humor. CONTINUING EDUCATION QUESTIONS 1. Which of the following physiologic actions is NOT attributed to amphetamine? a. Euphoria b. Appetite suppression c. Hypotension d. Tachycardia 2. Which of the following behavioral effects is NOT as- sociated with common psychostimulants? a. Locomotor suppression b. Sexual stimulation c. Increased sociality d. Increased mental attention 3. Amphetamine is used in the treatment of all of the fol- lowing disorders EXCEPT: a. obesity. b. narcolepsy. c. attention-deficit hyperactivity disorder. d. nasal congestion. 4. The mechanisms of action of amphetamine include all of the following EXCEPT: a. inhibition of monoamine neurotransmitter uptake. b. binding to intracellular catecholamine receptors. c. inhibition of monoamine oxidase. d. release of catecholamines from neurons. 5. An individual exhibiting symptoms of tremors, vomiting, and tonic-clonic convulsions is possibly experiencing: a. withdrawal from amphetamine. b. high dose amphetamine. c. chronic tolerance to amphetamine. d. gastrointestinalcomplicationassociatedwithamphetamine. 6. Allofthefollowingdrugsarelikelytobeincludedinadrug screen protocol in a forensic laboratory EXCEPT: a. cocaine. b. methamphetamine. c. ethylenedioxyamphetamine. d. methylenedioxymethamphetamine. 7. The most frequently mentioned illicit drug reported in emergency room visits over the last several years is: a. cocaine. b. methamphetamine. c. methylenedioxymethamphetamine. d. amphetamine.
  • 63. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 125 FOCUS: PSYCHOSTIMULANTS 8. Crack is composed of: a. cocaine hydrochloride. b. methamphetamine free base. c. methamphetamine hydrochloride. d. cocaine free base. 9. The routes of self administration of cocaine include all of the following EXCEPT: a. smoked. b. oral. c. intranasal. d. intravenous. 10. Analysis of a blood sample following cocaine admin- istration will include all of the following metabolites EXCEPT: a. benzoyl methyl ester. b. ecgonine methyl ester. c. benzoylecgonine. d. ecgonine. 11. At toxic levels, both cocaine and MDMA cause: a. fatigue. b. nausea. c. hyperthermia. d. hypothermia. 12. The group of hallucinogenic psychostimulants that are often found at ‘raves’ includes all of following drugs EXCEPT: a. MDMA. b. MDEA. c. MDA. d. MDME. 13. The adverse effects associated with chronic use of ‘rave’ drugs include all of the following EXCEPT: a. renal failure. b. pancreatitis. c. malignant hyperthermia. d. rhabdomyolysis. 14. Severaltechniqueshavebeendevelopedforonsitescreening of ‘rave’ drugs. The onsite screens require collection of: a. urine. b. hair. c. blood. d. saliva. 15. The analytical technique most commonly used for de- termination of MDMA levels in hair is: a. GC/MS. b. HPLC. c. LC/MS. d. TLC. 16. The chemical structures of most common psychostimu- lants share the features of: a. phenyl ring, alkyl side chain, amine. b. phenol ring, unconjugated side chain, amine. c. phenyl ring, alkyl side chain, carboxylic acid. d. phenol ring, alkyl side chain, ketone. 17. An individual is taken to the emergency room where a blood sample is collected and tested for possible cocaine abuse. The toxicology screen shows the presence of ben- zoylecgonine and cocaethylene. The conclusion is the: a. sample is a false positive. b. chronic use of cocaine. c. use of cocaine and ethanol. d. use of cocaine and amphetamine. 18. Inrecentyears,thepercentageofemergencyroomvisitsas- sociated with abuse of multiple drugs is approximately: a. one third. b. two thirds. c. three quarters. d. one half. 19. The best estimate of a blood concentration of a psycho- stimulant at the time of death is provided by analysis of a postmortem sample of: a. hair. b. central blood. c. peripheral blood. d. urine. 20. An individual is taken into custody at a rave. After 36 hours, a drug screen is ordered. Which specimen would give the best indication of abuse of MDMA at the rave? a. Plasma b. Saliva c. Sweat d. Urine
  • 64. 126 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE To earn continuing education (P.A.C.E.®) credit, (1) complete the form below, (2) record your answers, and (3) tear out and mail this form with a check or money order ($18 for ASCLS members, $28 for non-members for all articles) to: American Society for Clinical Laboratory Science P.O. Box 79154, Baltimore MD 21279-0154 A certificate and credit will be awarded to participants who achieve a passing grade of 70% or better. Participants should allow eight weeks for notification of scores and receipt of certificates. Focus: Psychostimulants carries 3.0 hours of basic level P.A.C.E.® credit. This form can be submitted for credit for up to one year from the date of issue. Print or type carefully. (01) NAME ______________________________________________________________________________________________ Last First Middle ASCLS membership number __________________________________ Licensure number ______________________________ (02) ADDRESS __________________________________________________________________________________________ (03) CITY________________________(04) STATE/COUNTRY _____________(05) ZIP/POSTAL CODE_________________ (06) DAYTIME PHONE ( ______ )__________________________(07) E-MAIL:______________________________________ (08) CREDIT CARD # _____________________________ TYPE (CIRCLE) AE MC VIS EXP. DATE_____________ Continuing Education Registration Form Check all that apply q I am an ASCLS member q I am not an ASCLS member q I would like to receive ASCLS membership information q I have previously participated in Focus q I would like information on other continuing education sources Answers Circle correct answer (questions are on previous two pages). 1. a b c d e 8. a b c d e 15. a b c d e 22. a b c d e 2. a b c d e 9. a b c d e 16. a b c d e 23. a b c d e 3. a b c d e 10. a b c d e 17. a b c d e 24. a b c d e 4. a b c d e 11. a b c d e 18. a b c d e 25. a b c d e 5. a b c d e 12. a b c d e 19. a b c d e 26. a b c d e 6. a b c d e 13. a b c d e 20. a b c d e 27. a b c d e 7. a b c d e 14. a b c d e 21. a b c d e 28. a b c d e Participant Information Please circle the most appropriate answers. 1. Is this program used to meet your CE requirements for: (a) state license (b) NCA (c) employment (d) other 2. Specialty: (a) biochemistry/urinalysis (b) microbiology (c) lab administration (d) hematology/hemostasis (e) education (f)immunology (g) immunohematology 3. Workplace: (a) hospital over 500 beds (b) hospital 200–499 beds (c) hospital 100–199 beds (d) hospital under 100 beds (e)private lab (f ) community blood bank (g) group practice (h) private physician (i) clinic (j) other 4. Salary range: (a) under $10,000 (b) $10,000 to $20,000 (c) $20,000 to $30,000 (d) $30,000 to $40,000 (e) over $40,000 5. Did these articles achieve their stated objectives? (a) yes (b) no 6. How much of these articles can you apply in practice? (a) all (b) some (c) very little (d) none 7. Employment status: (a) full time (b) part time (c) student (d) not employed (e) retired 8. How long did it take you to complete both the reading and the quiz? ___________minutes 9. What subjects would you like to see addressed in future Focus articles?
  • 65. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 127 TRENDS AND TECHNOLOGY Trends and Technology: Spring 2005 MARY JANE GORE The Trends and Technology section seeks to publish product and technology information (including black-and-white glossy photo- graphs), news items (such as FDA approvals), and information about laboratory resources of all kinds. The intent of this section is to provide a cutting-edge, one-stop shop tailored to the current practical needs and concerns of clinical laboratory practitioners. Let us hear from you with suggestions on how to improve this section. Direct inquiries and information to Mary Jane Gore, CLSTrends and Technology Editor, c/o ASCLS, 6701 Democracy Blvd., Suite 300, Bethesda, MD 20814, clstt@aol.com. Please send all materi- als clearly marked NEW PRODUCTS. ONLINE With this issue ofTrends andTechnol- ogy, I decided to break with tradition – the tradition of not reviewing man- ufacturers’ Web sites. I did not want to create the illusion of favoritism or persuasion by featuring a clinical di- agnostics Web site, but after so many years of reviewing other types of sites – governmental agencies, personal lab-related Web sites, some associa- tion sites of interest, quality-related sites – I began to wonder: why not? As always, I welcome reader input about sites that may be helpful – or not so helpful – to review for other readers (send your ideas to clstt@aol. com). This time, I visited the site of Ortho-Clinical Diagnostics (www. orthoclincal.com), a subdivision of Johnson & Johnson, and found that when I answered the Web survey, I got a timely response to my query, which led to me look at the site from the standpoint of a laboratory scientist. I have to say that helpfulness is a hall- mark of the Ortho-Clinical site. All of its major analyzers and other product lines are clearly labeled as links to the left on the homepage. The information under “Contact Us” is extensive and inviting, and not just an email address that is often found online. If you want to contact these folks, they make it easy. The homepage also has links to technical support (including holiday photos of these people, putting faces to the voices on the phone) and techni- cal documentation, which brought up 123 “Instructions for Use” documents, down to the last calibrator kit. The most impressive fact of this Web site, for me, is that it is all available with a glance at the homepage, including headline news about new products. Ortho appears to be a company dedicated to customer service and response and its site is in- deed user friendly. NEW PRODUCTS The VITROS 350 Chemistry System offers easy operation, maintenance, and training. Whether you use it as a primary, STAT, or back-up to the pow- erful VITROS 5,1 FS, the VITROS 350 Chemistry System can perform the work. The VITROS 350 comes complete with enhanced throughput, a broad accessible menu, and new ergo- nomic design. “Load-and-go” reagent preparation, MicroSlide™ Technology and up to six months’ calibration sta- bility mean labor can be redeployed for value-added tasks. Minimal in- strument maintenance assists with productivity and reduces costs with the system that delivers the right results the first time. Ortho-Clinical Diagnostics studies indicate that the VITROS 350 Chemistry System with enhanced software improves throughput 10% to 25% and time-to-first-result up to 12% when compared to the VITROS 250 System. Visit www.orthoclinical. com for more information on this and other products. Ortho-Clinical VITROS 350 Maxell Corporation of America, leader in advanced recordable media products, has announced new ultra- durable and ultra-reliable DVD media designed specifically for the medical market. Maxell’s new medical-grade media incorporates the innovative MAXPRO™ Hardcoat technology to produce enhanced DVD-R media that delivers the highest level of data protection for up to twice the archival shelf life. Maxell Medical DVD-R is HIPAA- and DICOM-compliant, and with its superior scratch, dust and smudge resistance and extended archival life, is ideal for critical medical images, patient records, backup, and fixed content storage. Maxell’s 700 MB, 8X speed Medical DVD-R media will be available in March as a single disc in a jewel case and in 50-pack spindles with printable white surfaces
  • 66. 128 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE TRENDS AND TECHNOLOGY for either thermal or inkjet printers. Pricing will be affordable compared to other backup media. Contact www.maxell.com. Healthcare organizations now have continuous access to the Joint Com- mission on Accreditation of Healthcare Organizations’ Periodic Performance Review (PPR) on the Joint Com- mission’s secure extranet, “JAYCO.” The PPR is an integral component of the Joint Commission’s accreditation process that promotes continuous standards compliance through ongo- ing, internal monitoring. Before mak- ing the PPR continuously available, organizations had received access to the PPR tool 15 months after its last triennial survey and had three months to complete it. Those timeframes for access have now been eliminated. The schedule for completing the PPR re- mains unchanged until January 2006, when organizations will be expected to update the PPR annually. For 2005, the PPR process requires each accredit- ed organization to conduct a mid-cycle self-assessment against applicable Joint Commission standards, develop a Plan of Action to address identified areas of non-compliance and identify Measures of Success for validating resolution of the identified problem areas. Under the usual PPR process, organizations will be expected to share this information with the Joint Commission at the mid- cycle point. Contact Charlene Hill at Charlene D. Hill 630-792-5175 or email chill@jcaho.org. Tecan introduced several new labo- ratory automation products at Lab Automation 2005, the pre-eminent industry meeting, held at the San Jose McEnery Convention Center. Tecan’s interactive trade show booth included the latest unique robotic solutions.Te- can’s participation at Lab Automation 2005 demonstrated robotic solutions that deliver speed, efficiency, and reli- ability for laboratories focused on clini- cal diagnostics, genomics, proteomics and drug discovery. Contact Greg Porter, Ph.D., at 919-361-5200. AxSYM Anti-HCV (hepatitis C virus) is a Microparticle Enzyme Immu- noassay (MEIA) for the qualitative detection of anti-HCV IgG to HCV recombinant proteins in human se- rum or plasma containing potassium EDTA, sodium EDTA, sodium hepa- rin, lithium heparin, sodium citrate, or potassium oxalate. Assay results, in conjunction with other laboratory re- sults and clinical information, may be used to provide presumptive evidence of infection with HCV virus (state of infection or associated disease not determined) in persons with signs or symptoms of hepatitis and in persons at risk for hepatitis C infection. Visit www.abbottdiagnostics.com for the 2005 product releases and listings. FDA APPROVALS Bayer Diagnostics has received U.S. Food and Drug Administration (FDA) approval for its automated assay for the hepatitis C virus on the ADVIA Cen- taur Immunoassay System. Contact Susan Hager at 781-551-7916. Bio-Rad Laboratories has received marketing clearance from the FDA for its new BioPlex 2200 system, a revo- lutionary new immunoassay platform that employs multiplexing technology to analyze for multiple disease states from single patient samples. It is the first clinical diagnostics platform to offer multiplexing technology on a fully-automated, fully-integrated random access platform. The system was unveiled last July at the American Association of Clinical Chemistry meeting and will soon be available in the U.S. The BioPlex 2200 system is a bead-based multiplexing immu- noassay platform that can deliver up to 2200 results per hour. The system will initially include a panel of assays targeting autoimmune diagnostics. Future assays in development are in the areas of serology, infectious disease, cardiac, and toxicology. Contact Gary Mantha at 510-741-4637 or email Gary_Mantha@bio-rad.com. BioPlex 2200 Bio-Rad Laboratories recently received approvalfromtheFDAforitsnewMul- tispot HIV-1/HIV-2 Rapid Test. This highly sensitive test kit this year became availableintheU.S.andwillsignificantly aid in the diagnosis of HIV-1/HIV-2 (human immunodeficiency virus, types 1 and 2), the virus that causes AIDS. ContactSusanBergat510-741-6063or email susan_berg@bio-rad.com. Roche announced that its first microar- ray-based test, the AmpliChip CYP450 Test, has been cleared by the FDA for di- agnosticuseintheU.S.Thistest,whichis powered by Affymetrix microarray tech- nology, analyses a patient’s cytochrome P450, 2D6, and 2C19 genotypes from genomic DNA extracted from a blood sample. Test results will allow physicians to consider unique genetic information from patients in selecting medications and doses of medications for a wide vari- etyofcommonconditionssuchascardiac diseases, pain, and cancer. This new test allows physicians access to information that could help to prevent harmful drug interactions and to assure drugs are used optimally. Adverse drug reactions cause
  • 67. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 129 TRENDS AND TECHNOLOGY a huge number of hospitalizations in the U.S. The new test also will, in some cases,enablepatientstoavoidsuboptimal or even harmful treatment choices. For patientsitisextremelyimportanttoknow whether pain killers or anesthetics might work differently or not at all for them. More information is available at www. roche-diagnostics.com. Dade Behring (NASDAQ: DADE) has received clearance from the FDA for the use of its Advanced D-Dimer as- say as an aid in the diagnosis of venous thromboembolism (VTE), [deep vein thrombosis (DVT) or pulmonary em- bolism (PE)]. The clearance included performance data with a defined cutoff value for the Dade Behring BCS System and Sysmex CA-1500 Sys- tem. The assay is also for use on Dade Behring’sBCT System, and Sysmex CA-7000 and CA-560 Systems. ARRANGEMENTS VWR International announces the recent acquisition of Alpha-Omega Calibrations, LLC. This acquisition expandsVWR’sserviceportfoliotonow include instrument calibration services and repairs for customers nationwide. Alpha-Omega Calibrations’ proven process includes a quality manual, well- documented standard operating proce- dures, a world-class training program for all their technicians, state-of-the-art facilities and equipment, and decades of gravimetric and metrology experience. Service advantages include fast turn- around times, competitive pricing, full traceability to NIST, and fulfillment of ISO and all other compliance record- keeping requirements. Contact Robin Gervasoni at 610-430-7258 or email robin_gervasoni@vwr.com. Infotrieve Inc, a provider of content software technology and information services, has announced its acquisi- tion of GenSys Software, Inc, pro- vider of the GenSys/ELN™ electronic laboratory notebook for life sciences, chemistry, and other research-intensive industries. The GenSys/ELN will serve as the anchor for Infotrieve’s life sciences electronic research platform, which has been designed to collectively increase the value of organizational content and improve scientists’ existing work- flow by enabling links from electronic laboratory notebooks to discovery tools, literature and scientific data, laboratory product information, and integrated retrieval capabilities for literature and laboratory products. Contact Infotrieve Marketing Manager Ian Palmer by phone at 310-445-3038 or via e-mail at ipalmer@infotrieve.com. International Technidyne has struck a deal with Medical Automation Sys- tems for the MAS RALS to integrate ITC’s IRMA TRUpoint™ analyzer for point-of-care blood gas monitoring with the widely used RALS -Plus data management system, marketed by MAS. Contact Check Weber at 847-705-1802. Abbott and Nihon Kohden Corpora- tion announced that they have entered into an agreement for the commer- cialization of automated hematology diagnostic instruments for use in hos- pital laboratories and physician offices. Under terms of the agreement, two 5-part differential hematology instru- ments, which are designed to offer red and white blood cell analysis, will be manufactured by Nihon Kohden and distributed by Abbott under the CELL-DYN Pearl™ brand name. Ab- bott obtains exclusive distribution rights for the two instruments in the United States and Canada and non- exclusive distribution rights for the instruments in other countries with the exception of China and Japan. Contact Amy Woodworth at 847-935-4755. Instructions to Authors Detailed Instructions to Authors can be found on the ASCLS Website (www.ascls.org) by fol- lowing the Publications links or going directly to http://www.ascls.org/leadership/cls/index.htm, or obtained by contacting the Clin Lab Sci Editorial Office, PO Box 5399, Coralville, IA 52241. (319) 351-2922. cls@ia.net Questions may be addressed to Ivan Schwabbauer, Managing Editor.
  • 68. ASCLS 73rd Annual Meeting, July 26-30, 2005 AACC/ASCLS Clinical Lab Expo, July 26-28, 2005 Orlando, Florida Roundtables Include: POCT Issues * Pre-Analytical Effects with Heparin Monitoring * Patient ID and Risk Management * MRSA * Fun in the Classroom * Meeting Planning * Planning a State Legislative Day * and More! Opening & Closing Keynotes: Leadership - Disney Style! Discover the business behind the magic. It’s a Jungle Out There! Real world solutions to acheive goals. Scientific Sessions Include: Connectivity to the Electronic Health Record * Six Sigma and LEAN * Laboratory Design * Body Fluid Cellular Morphology * Molecular Detection of Hematologic Malignancies * Technical Advances in Diagnostic Microbiology * Cardiac Markers * Emergency Preparedness Training for the Lab * Neonatal Screening for Meta- bolic Diseases * Improved POCT Outcomes * Thrombotic Microangiopathies * Automating Transfusion Services * and More! Visit www.ascls.org/conferences/2005AM/ for details and online registration. Hilton in the Walt Disney World Resort Orange County Convention Center