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................................ CLINICAL LABORATORY SCIENCE ................................ Focus: Psychostimulants A S C L S JOURNAL OF THE AMERICAN SOCIETY FOR CLINICAL LABORATORY SCIENCE

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CLINICAL SPRING 2005 LABORATORY VOLUME 18/NUMBER 2 SCIENCE DIALOGUE AND DISCUS S I O N 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 REPORT S 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: PSYCHOSTIMUL A N T S 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 EDUCATIO N Q U E S T I O N S 127 TRENDS AND TECHNOLO G Y

EDITORIAL Health Disparities and Public Policy ISAAC D MONTOYA Health-related disparities are significant differences in the Adults with developmental disabilities experience disparities that incidence, prevalence, morbidity, mortality, and burden of are often not as obvious. A recent study found this population to disease among specific population groups. Medical research be more likely to lead sedentary lifestyles and seven times as likely has demonstrated glaring disparities for a wide range of health to report inadequate emotional support compared to adults problems and among different groups. It is important that we without disabilities. Adults with disabilities and developmental improve our understanding of what causes health disparities disabilities were also more likely to report being in fair or poor and work to address them. In doing so there is a tendency to health than adults without disabilities. Significant medical care discuss health disparities solely in terms of differences among utilization disparities were found for breast and cervical cancer racial and ethnic groups; however it is a myth that only these screening as well as for oral healthcare in this group. groups experience disparities. Unfortunately, health disparities occur in all segments of society. Mental health and drug abuse, e.g., nicotine, alcohol, and illicit drug problems, are a classic example of disparities at both the pre- Medical research working to achieve the goal of alleviating vention and treatment level. These types of behavioral problems health disparities in the United States is a goal with broad sup- face stigmas that further contribute to the disparity problem. port that adopts the well-established perspective that various For example, in a national study the prevalence of smoking forms of discrimination and poverty are the major contributors during pregnancy ranged from 9.0% to 17.4%. Younger (age to unequal health status. One idea that has been put forth is <25 years) women, white women, American Indian women, that genetic research plays a significant role in alleviating this non-Hispanic women, women with a high school education national problem, which may overstate the importance of genet- or less, and women with low incomes consistently reported ics in explaining health disparities. Over reliance on genetics as the highest rates of smoking. In the same study the prevalence a factor in explaining health disparities may lead us to miss the of alcohol use during pregnancy ranged from 3.4% to 9.9%. factors that we can control, thus reinforcing stereotyping which In seven states, women age >35 years, non-Hispanic women, contributes to disparities in the first place. women with more than a high school education, and women with higher incomes reported the highest prevalence of alcohol Access to care is a primary reason for disparities. For example, use during pregnancy. Although prevalence data cannot be used disparities due to limited access to coronary artery bypass graft to identify causes or interventions to improve health outcomes, (CABG) surgery are well documented. Evidence shows that they do indicate the magnitude of disparities and identify popu- even when patients do receive CABG surgery, the poor, people lations that should be targeted for intervention. living in rural areas, and racial minorities are more likely to be treated by lower quality providers. In addition, some disparities We recognize that a number of groups experience inferior occur due to the hospital to which patients are admitted and to medical care and health status, but may not appreciate the se- a lesser degree to being treated by a low-volume surgeon. Efforts riousness of the problem. Each year the United States spends to eliminate health disparities should address not only access to billions of dollars to perfect the ‘technology’ of healthcare, care, but also access to high-quality care. e.g., development of new drugs, new pieces of equipment, and to modernize delivery systems, thereby saving thousands Obesity is a major concern nationally that crosses all socio- of lives. Correcting known disparities could prevent five economic groups. Genetics, and lack of access to dietary/ times as many deaths. If policymakers adhered to the goal of nutritional information, healthy foods, and needed exercise optimizing population health, greater priority would go to all contribute to the problem of weight control. A recent resolving disparities rather than to developing new technol- study, funded by the National Institutes of Health, analyzed ogy, but unfortunately reverse priorities prevail. a sample of Americans’ weight. The results show continuing disparities by sex and between racial/ethnic groups in the Isaac D Montoya PhD was the Clinical Laboratory Science prevalence of obesity. Research and Reports Editor, 2001-04. 66 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

WASHINGTON BEAT Pay for Performance KATHY HANSEN, DON LAVANTY In today’s climate of focus on the healthcare consumer, On January 31, CMS announced that ten large physician much attention is being paid to improving patient safety groups across the country would participate in a three-year and quality of care. There is heightened interest in distin- pilot project of pay for performance for physicians. Physicians guishing healthcare providers who are “good performers”, will continue to be paid on a fee-for-service basis, but will be who provide safe and efficient care from poorer performers eligible for performance payments based on how well they whose outcomes may not be as good. Some payers feel that improve patient outcomes and avoid costly complications. one way to encourage performance improvement is to pay The quality measures focus on the many of the same chronic good performers better than other providers. illnesses in the Medicare population as do the hospital measures, including congestive heart failure, coronary artery The Center for Medicare and Medicaid Services (CMS) began disease, diabetes, and hypertension, as well as preventive a Pay for Performance program for hospitals on a pilot basis services such as screenings for breast and colorectal cancer, about two years ago. Hospitals that participate in the pilot and immunization for flu and pneumonia. project keep statistics on a number of measures in diagnosis groups common in the Medicare population, such as acute Another proposal related to pay for performance for physi- myocardial infarction (AMI), coronary artery bypass grafts cians surfaced in January that would have a very direct im- (CABG), heart failure, pneumonia, and hip and knee replace- pact on the laboratory. During hearings on January 12, the ment. If hospitals achieve certain levels of compliance with Medicare Payment Advisory Commission (MedPAC) made the goals of the measures, they are paid 1% to 2% more than a recommendation that CMS should require laboratories to the usual DRG payment. report test results to CMS on the claim for payment. From a reading of the transcript of the proceedings of the MedPAC, A few examples of the over thirty pay for performance metrics it is not clear how the data would be used to evaluate physi- defined by CMS are: cian performance, perhaps by measuring the percentage of • AMI: aspirin at arrival abnormal results. In addition to using this strategy to evaluate • AMI: thrombolytic within 30 minutes of arrival physician performance for pay for performance, MedPAC • AMI: percutaneous coronary intervention received discussion focused on the requirement as a way to encour- within 120 minutes of arrival age all providers to use information technology (electronic • CABG: post operative hemorrhage or hematoma medical record). The electronic medical record has been a • Heart failure: smoking cessation advice/counseling focus of the Bush administration’s health policy. provided • Pneumonia: oxygenation assessment within 24 hours Laboratorians are likely to be skeptical about the effectiveness • Pneumonia: blood culture collected prior to first anti- of laboratory values alone as a measure of physician effective- biotic assessment ness, taken outside the context of the larger medical record. • Hip and knee replacement: prophylactic antibiotic re- In addition, the American Clinical Laboratory Association ceived within one hour prior to surgical incision (ACLA), the association that represents the larger national reference laboratories, has appeared before the MedPAC to raise a number of concerns about the recommendation: Washington Beat is intended to provide a timely synopsis of activity in the nation’s capitol of importance to clinical laboratory practitioners. • Considerable cost and effort would be required for labo- This section is coordinated jointly by Kathy Hansen, Chair of the ratories and hospitals to reprogram computer systems to ASCLS Government Affairs Committee, and Don Lavanty, ASCLS transmit test results to the billing systems to appear on Legislative Counsel. Direct all inquiries to ASCLS (301) 657-2768 claims. In most institutions, results are reported elec- extension 3022; (301) 657-2909 (fax); or mail to ASCLS, 6701 tronically and test charges are billed electronically, but Democracy Blvd., Suite 300, Bethesda MD 20814, Attention: there is no interface between those systems that would Washington Beat. link results to test charges. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 67

WASHINGTON BEAT • The MedPAC suggests that laboratories would standardize ACLA is now briefing key congressional staff about their nomenclature for tests using LOINC codes, which are more concerns about the MedPAC proposal. The College of specific that CPT codes. LOINC codes are not commonly American Pathologists (CAP), along with other physician used now, and conversion would be a huge effort. groups, opposes the MedPAC’s proposal to set aside 1% to • Not all test results are numeric, and not all tests are 2% of physician payments to be redistributed on the basis reported with a reference range. Lengthy narratives ac- of performance. company many microbiology and flow cytometry results, for example. The MedPAC recommendations have gone to CMS, which • Laboratory values should be interpreted in the light will have to decide whether to accept them. If CMS decides of other information about the patient found in the to move forward, proposed regulations would be published medical record. in the Federal Register for comment. ASCLS will monitor • Last but not least, the recommendation would need to be this situation closely and register opinions and submit com- examined in the light of HIPAA’s “minimum necessary” ments whenever appropriate. Regardless of how one views the privacy standard. concept of pay for performance and its potential to improve patient outcomes and patient safety, this particular proposal MedPAC discussed whether it might be better to recommend seems to be unreasonably burdensome for laboratories to starting the requirement with a subset of specific test results, but implement, and to have limitations in the validity of the con- in the end stayed with the recommendation that all test results clusions that could be drawn about physician performance be reported. It did acknowledge that the recommendation rep- from the raw laboratory data. resents “…a complex undertaking” and this included a two to three year transition period for implementation. 68 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

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- sidered more suitable for screening Sudanese blood donors for nation, the polymerase chain reaction (PCR), and the immu- malaria parasites prior to donation at the present time. nochromatography test (ICT) to determine the best method for screening blood donors for malaria parasites in Sudan. RECOMMENDATIONS: To establish a reference malaria diagnosis unit in each blood bank in Sudan as well as to train METHODS: A total of 100 blood donors were screened for blood bankers to perform microscopic examinations. malaria parasites by standard microscopic technique, ICT, and PCR. Blood films were examined microscopically using standard ABBREVIATIONS: ICT = immunochromatography test; Giemsa staining techniques. Qurum (Canadian Company) PCR = polymerase chain reaction. malaria kits were used to perform the ICT. For performing PCR, DNA was extracted using Chelex method and amplified INDEX TERMS: donor testing; malaria testing. by the moderately repetitive DNA sequence pBRK-l. Clin Lab Sci 2005;18(2):69 RESULTS: Using PCR, a total of 21 blood samples were positive; 8 (38%) of them showed negative blood films and 7 Dr Mohamed Siddig M Ali MSc Haematology is Lecturer (33%) were negative on ICT. Four blood samples that tested and Head, Department of Haematology, AI Neelain University, positive by ICT despite a negative PCR and microscopic ex- Khartoum, Sudan. amination were proved to be false positives. The false negativity of both the microscopic examination and ICT was found Abdul Gader Mohamed Yousif PhD is Professor of Medicine to be significant. The sensitivity of microscopy was 61.9% and Tropical Diseases and Dean, Faculty of Medicine, Uni- and of ICT was 66.7%, while the specificity of microscopy versity of Khartoum, Khartoum, Sudan. was 100% and of ICT was 94.9%. When direct microscopy was considered as the standard technique the sensitivity of Mustafa Salih Mustafa MD is General Manager of Planning ICT was 100% and the specificity was 94.3%. and Development and Research Directorate, Federal Ministry of Health, Khartoum, Sudan. CONCLUSION: Although PCR is more sensitive and more specific, it is unaffordable. Microscopy for malaria when com- Dr Malik Hassan Ibrahim MSc Haematology is Lecturer pared to ICT showed similar sensitivity at low cost. However, and Head, Department of Haematology, Sudan University of all human plasmodium species can be detected using the mi- Science and Technology, Khartoum, Sudan. croscopy while only two species (P. falciparum and P. vivax) can be detected by ICT. The detected false positivity of ICT is Address for correspondence: Dr Mohamed Siddig Mohamed not inconsequential since this implies the rejection of a greater Ali, Faculty of Medical Laboratory Sciences - AI Neelain Uni- proportion of blood donations. Therefore, microscopy is con- versity, PO Box 12702, Khartoum, Sudan. mohdaru@hotmail. com The peer-reviewed Clinical Practice Section seeks to publish case stud- The medical use of blood and blood derivatives is increasing all ies, reports, and articles that are immediately useful, are of a practical over the world, despite the hazards related to transmission of nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, includ- protozoal, spirochaetal, bacterial, and viral diseases. Transmis- ing brief reviews of books, computer programs, audiovisual materials, or sion of malaria by blood transfusion is a significant problem in other materials of interest to readers. Direct all inquiries to Bernadette regions of the world where this disease is endemic; P. falciparum Rodak MS CLS(NCA), Clin Lab Sci Clinical Practice Editor, Clinical in transfused blood may lead to fatality.1 Moreover, transfusion- Laboratory Science Program, Indiana University, Fesler 409, 1120 induced P. falciparum has increased in recent years, probably South Avenue, Indianapolis IN 46202-5113. brodak@iupui.edu. because it has become increasingly resistant to drugs.2 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 69

C LINICAL PRACTICE Prevention of transfusion-induced malaria depends on the MATERIALS AND METHODS screening of potentially infected blood donors, especially The design of the study is descriptive, cross-sectional, and those who provide whole blood or fresh concentrates of facility based. It was conducted in the Ahmed Gasim Hospi- erythrocytes, leukocytes, or platelets.3 However, success- tal (Khartoum North, Sudan) among a total number of 100 ful screening for malaria parasites requires experience in blood donors screened for malaria parasites by ICT, PCR, the differential characteristics of the various species of the and standard microscopic technique. Blood samples were col- parasite and should constitute a part of primary health care.2 lected (1 mL from each), processed with EDTA anticoagulant Microscopic examination of a blood film can be used for the (1.5 mg), and then immediately used to prepare blood films detection of malaria infection in a donor in whom malaria and perform ICT. Three spots of each tested blood sample is suspected based on circumstantial evidence. Examina- (50 µL each) to be used for the PCR were stored dry (on #3 tion of stained thin blood films can identify the species and filter paper) at -20 °C.8 give an estimate of the percentage of infected red cells.4-6 Examination of the buffy coat and red cells below can help From the blood collected from each donor, duplicate thick in detecting parasites when they are few in number (5 to 10 and thin blood films were prepared, stained by Giemsa stain, parasites/µL), particularly in the late stages, e.g., trophozoites and examined microscopically as the standard methods.5 and gametocytes.5 However, in traditional microscopy, failure to adhere to correct techniques seriously compromises the The absolute number of parasites (number/µL), was estimat- specificity and sensitivity. ed in positive thick blood films by counting the recognized malaria parasites against 200 white blood cells according to Immunochromatography tests (ICT) employ monoclonal the following formula: antibodies against histidine rich protein (HRP-2), which is produced by the parasite and released into the circulation. # of parasites counted x total WBC Compared to microscopy, it is simple to perform, does not # of leukocytes counted (200) require the use of special equipment, and is faster with low variation between users. Moreover, it can detect asexual Qurum (Canadian Company) ICT malaria kit was used. parasites and young gametocytes with reasonable sensitivity This kit has been designed for the detection of P. falciparum and specificity (>90%). in whole blood. It employs monoclonal antibodies against HRP-2 that is secreted by the parasite. Captured monoclonal Serological methods cannot replace the demonstration of antibodies are immobilized on a nitrocellulose membrane parasites in the blood as far as diagnosis of symptomatic strip. When P. falciparum antigen is present in lysed whole patients is concerned.6 In countries where the disease oc- blood, it binds antibodies as the blood migrates along the test curs, antibody tests are unable to distinguish between active strip. Colloidal gold particles are coated with these antibodies and past infection and they have only a limited value in the to form a sandwich resulting in visible red line. Performance clinical diagnosis of malaria.5 of the test and interpretation of results were conducted as directed by manufacturer instructions. During the past few years, the polymerase chain reaction (PCR) has become a major diagnostic and research tech- For performing PCR, DNA was extracted from the filter nique. It is reliable for the detection of parasites present paper using Chelex method.8 One of the three blood spots at low concentration in blood or serum samples. Further, was cut using a sterile blade and put in a 1.5 mL Eppendorf the use of PCR could be 100 times more sensitive than the tube containing 1 mL of 0.5% saponine in freshly prepared microscopic examination of thick blood films.7 1 x phosphate buffer saline (PBS) and then incubated over- night at 4 °C (hemoglobin is released into the wash leaving The present study is intended to determine the selection of the DNA of the parasite on the paper). the best laboratory procedure that can be applied for screening blood donors for malaria parasites in Sudan since such The tubes were spun for one minute at 13 x 103 rpm and the su- studies have not been conducted previously. pernatant fluid was removed. One mL of 1x PBS was added and spun again for one minute; the supernatant was also removed. Fifty µL of PCR quality water and 50 µL of 20% Chelex mixture were added to each tube and then boiled for ten minutes. 70 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

C L INICAL PRACTICE The tubes were centrifuged for one minute to pellet Chelex Comparison between the results of PCR and ICT of the exam- and debris. The DNA supernatant was taken off and trans- ined blood samples applying PCR as the standard technique ferred into a new sterile 0.5 mL Eppendorf tube. is shown in Table 2. Using PCR, a total of 21 blood samples were positive; of them only 14 blood samples tested positive DNA amplification was performed using the moderately using ICT. Four blood samples tested positive by ICT despite repetitive DNA sequence pBRKl- 14 forward, 5-CGC a negative PCR and microscopic examination; therefore, these TACATATGCTAG TTGCCA GA C-2ʹ and reverse 5ʹ- proved to be false positives. The false positive of the ICT was CGTGTA CCATA CATCCTACCAAC-3ʹ that amplifies found to be statistically insignificant (p >0.05). a 206 base pair sequence.7 The PCR product was run in a gel electrophoresis tank (1.5% agarose gel) with the addition of Seven (33%) positive PCR blood samples were negative by ethidium bromide solution (0.5 mg/mL) and DNA molecu- ICT. These false negative ICT results were found to be highly lar weight marker (fraction VI) in a parallel well. significant (p <0.001). Data were analyzed by the computer using the Statistical Package Applying PCR as a standard technique, the sensitivity of for Social Sciences (SPSS). Chi-square and Fisher exact tests were ICT was 66.7% while the specificity was 94.9%. The false used for comparison and correlation between proportions. negative rate was 33.3% and the false positive rate was 5.1%. Comparison between the result of PCR and microscopic The sensitivity of the test is the proportion of positives, cor- examination of the donors’ blood samples applying PCR as responding to the positive result obtained by the standard the standard technique is shown in Table 3. test. However, specificity of the test is the proportion of negatives corresponding to the negative result obtained by Using PCR as the standard technique, a total of 21 blood the standard technique. Sensitivity, specificity, false negative samples were positive; 8 (38%) of them showed negative rate, and false positive rate can be calculated using the fol- blood films. This false negativity of the microscopic examina- lowing formulas and Table 1: tion was found to be highly significant (p <0.001). Sensitivity = (e)/(e + f) Specificity = (h)/(g + h) False negative rate = 1 – sensitivity False positive rate = 1 – specificity Table 2. Comparison between the results of PCR and ICT RESULTS PCR Total Out of the 100 randomly selected blood donors, 13 samples Positive Negative (13%) were positive by microscopy as well as by ICT and ICT positive 14 4 18 PCR, with 75 samples (75%) being negative throughout. ICT negative 7 75 82 Twelve blood samples (12%) showed variable results. Total 21 79 100 p >0.01 Table 1. Sensitivity and specificity table Table 3. Comparison of PCR vs. microscopic examination Tested Standard technique technique PCR Total Positive Negative Total Positive Negative (D+) (D-) Microscopic examination Positive 13 0 13 Positive (T+) (e) (g) (e + g) Negative 8 79 87 Negative (T-) (f ) (h) (f + h) Total 21 79 100 Total (e + f ) (g + h) p >0.01 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 71

C LINICAL PRACTICE Applying PCR as a standard technique, the sensitivity of micro- avoid false negatives and positives.7 In this study, PCR was scopic examination was 61.9% while the specificity was 100%. adopted as the standard technique. It is worthwhile to men- The false negative rate was 38.1% and the false positive rate was tion that during this investigation, the difference between zero. Comparison between the result of microscopic examina- PCR and both ICT and direct microscopy was still signifi- tion and ICT of the same blood samples applying microscopy cant, although PCR has the drawbacks of being costly and as the standard technique is shown in Table 4. requiring strict laboratory conditions to avoid contamination that can lead to false positive results. Using microscopic examination, a total of 13 blood samples were positive; 5 (38.5%) of them were ICT negative. Only In malaria-endemic areas, the use of ICT is known to be one donor’s blood sample (7.7%) was found positive by ICT rather deceptive when used for investigating symptomatic and PCR despite a negative blood film. patients for the presence of malaria parasite in their blood. False positive results can be obtained even 14 days after The false positive ICT (the other four positives; 22.2%) clearance of the parasites by treatment or by the immune which were negative by blood films, representing the dif- system.5,6, 9-11 ference between microscopy and ICT, were found to be insignificant (p >0.05). This study showed that sensitivity of the ICT versus direct microscopy was 100%, which corresponds to the results Applying microscopic examination as standard technique, obtained by Singh in India, Cavallo in France, and Gaye in the sensitivity of ICT was 100% and the specificity was Senegal.11-13 However, all of them confirmed the false posi- 94.3%. The false positive and false negative rate was 5.7% tivity of ICT within variable periods after infections. These and zero respectively. reports are in close agreement with the findings of this study; four false positive tests were detected. The false positive results DISCUSSION are important since this implies that a greater proportion of Transfusion-induced malaria continues its resurgence blood donations will be rejected to ensure the prevention of throughout much of the tropics and subtropics. Success- malaria transmission by blood transfusion. Therefore, ICT ful control or eradication measures must include strategies seems to be not reliable for screening Sudanese blood donors directed to prevent transmitting malaria parasite infected for malaria parasite since whether the parasite is present or blood to patients. Therefore, it is mandatory to establish an not, persistence of HRP-2 will result in false positivity. Fur- effective technique for screening blood donors for the malaria ther, only ICT designed for the detection of only two species parasite. Different techniques were compared to determine (P. falciparum and P. vivax) is available, whereas transfusion the best method of screening donors that can be easily and induced malaria due to the other human plasmodium species rapidly applied in Sudan. (P. malariae and P. ovale) is not uncommon, particularly P. malariae because of its chronicity. The ICT test will be falsely Since PCR is highly specific and highly sensitive for posi- negative when the latter species are present. tive and negative results respectively (100 times greater than staining technique), it can be used as a standard technique to 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 Table 4. Comparison between the result of micro- of infestation and the capability of determining parasitaemia scopic examination and ICT of the same blood which may produce some new epidemiological and parasi- samples applying microscopy as standard technique tological aspects providing some suggestions for eradication; moreover, it is cheap, relatively rapid, readily available, and Microscopic examination easy to perform. Positive Negative Total ICT positive 13 5 18 When compared with ICT, microscopic examination shows ICT negative 0 82 82 similar sensitivity (no statistical difference; p >0.05). Hence, Total 13 87 100 microscopy of malaria is more suitable for screening blood p =1.20 donors in Sudan. This is in agreement with Wilairat’s findings that microscopic diagnosis of malaria is still more than adequate 72 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

C L INICAL PRACTICE in the routine investigation of individual cases, in spite of the ACKNOWLEDGEMENT fact that many authors have not favored the use of this method I wish to express my sincere gratitude, thanks, and everlast- because of the amount of time and expertise required.14 ing appreciation to Dr Amna El Subki Khalid, Dr Musa M Kier, Dr A1 Dirdiri Salim, Dr Nawal Taj Elsir, and Dr There is still no international consensus for the exact defini- Mona Abbas Babiker for their invaluable help. tions of what constitutes high, intermediate, or low parasitaemia; undoubtedly, most of the parasite densities that were REFERENCES encountered in our study were relatively low. However, the 1. Barker RH, Banchongaksorn T, Courval JM, and others. A simple minimum number of parasites transmitting malaria via blood method to detect Plasmodium falciparum directly from blood samples using the polymerase chain reaction. Am J Trop Med Hyg transfusion may vary among individual recipients; a single 1992;46:416-26. parasite can transmit the disease in mice.13 Even if a single 2. Neva FA, Brown HW. Basic clinical parasitology. 6th ed. New York: parasite can be detected in a thick blood film, which is equiva- Appleton and Lange; 1994. p 347. lent to 4 μL of blood, more than hundreds of thousands of 3. Bruce Chwatt LJ. Transfusion malaria revisited. Trop Dis Bull parasites might escape in a full unit of blood (450 mL). 1982;79(10):827-40. 4. Sherman IW. Malaria. Washington: ASM: 1998. p 557. 5. Cheesbrough M. Medical laboratory manual for tropical countries. This signifies the importance of the reliable testing of blood 2nd ed. Oxford: Butterworth; 1987. p 602. donors for malaria parasite to minimize, though never com- 6. Manson Bahr PEC, Bell DR. Mansons’ tropical diseases. 19th ed. pletely eliminate, the risk of malaria transmission by blood London: ELBS; 1987. transfusion. Moreover, the hazards of transfusion malaria are 7. Vu TT, Tran VB, Phan NT, and others. Screening donor blood for serious, and justify prior testing of blood donors for malaria malaria by polymerase chain reaction. Trans R Soc Trop Med Hyg 1995;89:44-7. even by advanced expensive techniques. These techniques will 8. Tirasophon W, Panyim S. PCR for low level detection of malaria surely reduce the false negative results and hence minimize parasite in blood. Protocols in molecular parasitology In: Hyde JE, the risk of transfusing infected blood. editor. Methods in molecular biology. 21st ed. Totowa NJ: Humana Press; 1993. p 205-12. The standard microscopic identification technique of malaria 9. Karbwang J, Tasanor O, Kanda T, Wattanagoon Y. ParaSight TM-F test for the detection of treatment failure in multi-drug resistant Plas- parasites is ideal to be applied at the present time until the modium falciparum. Trans R Soc Trop Med Hyg 1996;90:513-5. development of more feasible application of PCR. The estab- 10. Valecha N, Sharma VP, Devi CU. A rapid immunochromatographic lishment of a reference malaria diagnosis unit in each blood test (ICT) for diagnosis of Plasmodium falciparum. Diagn-Microbiol bank as well as trained blood bankers is necessary. Infect Dis 1998;30(4):257-60. 11. Singh B, Cox-Singh J, Miller AO, and others. Detection of malaria CONCLUSION in Malaysia by nested polymerase chain reaction amplification of dried blood spots on filter papers. Trans R Soc Trop Med Hyg The hazards of transmitting malaria through blood transfu- 1996;90(5):519-21. sion are serious thus justifying the use of expensive techniques 12. Cavallo JD, Hernandez E, Gerome P, and others. Serum HRP-2 for testing blood donors. Transfusion of blood from malaria antigens and imported Plasmodium falciparum malaria. Comparison parasite infected donors to patients will result in transfusion- of ParaSight-F and ICT malaria P.f. Med Trop 1997;57(4):353-6. malaria; screening donors for malaria prior to donation will 13. Gaye O, Diouf M, Dansokho EF, and others. Diagnosis of Plas- modium falciparum malaria using ParaSight F and ICT malaria P.F. undoubtedly reduce this risk. Microscopy is much cheaper Parasite 1998;5(2):189-92. than both ICT and PCR; in addition, it enables the detec- 14. Wilairat P. DNA probes for malaria parasite detection: current tion of all human plasmodium species. Therefore, it is the status and future prospects. In: Ko RC, editor. Immunological and best technique to be adopted for the control of transfusion- molecular basis of pathogenesis in parasitic diseases. Hong Kong: induced malaria in the different regions of Sudan until the Hong Kong University Press; 1989. p 169-74. feasibility of using PCR technology is improved. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 73

CLINICAL PRACTICE Clinical Laboratory Educators Conference 2005 Abstracts POSTER PRESENTATIONS Comparing Academic Performance, Learning Authors listed in bold face type will be the presenters. Style, and Student Satisfaction in a Pre-CLS Biology Simulation Laboratory Course Cold Agglutinins: A Case Study of Patient’s Sandra M Weiss Ed D CLS (NCA), Tricia McGinnis, Neu- Condition and Its Effects on the Integrity of mann College, Aston PA. Laboratory Findings Brenda Bouchard MS, University of Massachusetts Dart- The purpose of this study was three-fold: 1) to determine if mouth, Dartmouth MA; Lauren Cunha CLS(NCA), Charl- laboratory simulations can replace a traditional laboratory, ton Memorial Hospital, Fall River MA. 2) to determine if there are learning styles more responsive to computer simulations, and 3) to determine if students are Although frequently seen in clinical practice, the diagnosis more satisfied with computer simulations than traditional of patients who have cold reacting antibodies can be com- wet laboratories. The basic assumption that science labora- plicated. Cold agglutinins affect many areas of laboratory tories help students gain a better understanding of concepts testing. Broad recognition of patient history, symptoms, described in lectures and textbooks was not challenged. and laboratory findings can guide one to include the pos- Unfortunately, ideal laboratory teaching conditions are of- sibility of a cold reacting antibody. A cold agglutinin has ten rare and complex experiments are expensive, hazardous, optimal reactivity at temperatures below those of normal or time consuming to perform in introductory laboratory body temperatures and is almost always IgM subclass. The courses. Incorporating microcomputer technology enables a case to be presented begins with a female patient having variety of teaching techniques that permits the instructor to a pre-existing anemia and chronic obstructive pulmonary become a resource person and facilitator of learning rather disorder. Laboratory findings include CBC and differential than the expert who imparts knowledge. Students enrolled data with abnormally high WBC and platelets, and 90% in a pre-CLS physiology laboratory course participated in neutrophils. The patient had a low RBC count, hemoglobin, a pilot study July 2004. All wet laboratories were replaced and hematocrit. Measurement of arterial blood gas levels using PhysioExTM 5.0 laboratory simulation in physiology. showed a low pO2 and O2 saturation, with a high pCO2 and Students learning styles and personality characteristics were HCO3. The patient’s mycology cultures grew out a mold most identified using Kolb’s Learning Style and the Meyers-Briggs characteristic of Aspergillus spp., Latex agglutination D-dimer inventories. Additionally, pre- and post-tests and final grades testing revealed a cold agglutinin. Due to the patient’s low were compared to determine significant gains in cognition. hematocrit, the physician ordered two units of packed red Students were surveyed twice during the session. The results blood cells. Further details of laboratory results and treatment of the t-test and one-way analysis of variance (Anova) indi- followed by how this case may have been misdiagnosed due cated that ‘A’ students were more satisfied with the course to the presence of the cold agglutinin are presented. and knowledge gained (p <0.05) than ‘B’ and ‘C’ students. The results of this study have implications for clinical laboratory science education. Laboratory simulations may be an alternative way to introduce CLS students to advanced The peer-reviewed Clinical Practice Section seeks to publish case stud- equipment and complex techniques not routinely performed ies, reports, and articles that are immediately useful, are of a practical in the laboratory. nature, or contain information that could lead to improvement in the quality of the clinical laboratory’s contribution to patient care, including 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. 74 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

C LINICAL PRACTICE Development and Delivery of an AS to BS Degree The problem identified in this study is that there are limited Completion Distance Learning Track in CLS programs that address the development of a curriculum Linda J Graeter PhD MT(ASCP), Charity Einhaus format that is accessible by working medical laboratory Accurso PhD MT(ASCP), Gideon H Labiner MS MT(ASCP) technicians/clinical laboratory technicians (MLTs/CLTs) to CLS(NCA), Elizabeth C King PhD, University of Cincinnati, earn a bachelor’s degree. In 1996, a new model of curriculum Cincinnati OH. delivery using synchronous and asynchronous formats to deliver the didactic components was developed and imple- The CLS Program at the University of Cincinnati introduced mented by Old Dominion University. The program utilizes an AS to BS degree completion distance learning track (CLS an interactive, televised, asynchronous weekend format to DL) in June 2004. The track was designed to provide work- deliver courses to distant sites. The work sites of MLTs/CLTs ing laboratory professionals with an associate degree the provide the clinical component of the program. This study opportunity to earn a bachelor’s degree while continuing was a retrospective cross-sectional comparison of the scores of full- or part-time employment, and with minimal disruption traditional and weekend students (n = 97) on the American in their lives. Previous laboratory and life experiences were Society of Clinical Pathology (ASCP) national certification considered in curriculum development, so students enter examination over a 4½ year period beginning in 1998. In the program with advanced standing that includes complete every comparison there was no difference, i.e., p <0.001, fulfillment of the university’s general education requirements. between the scores of traditional and weekend students on The curriculum includes upper division science content, the ASCP examination. Furthermore, the scores of traditional didactic courses, and advanced clinical experiences that are and weekend students in four of the six subject areas (hema- completed in each student’s community. The courses are tology, blood bank, microbiology, and chemistry) exceeded taught using an interactive distance learning course model not only the national average in these subject areas, but that includes audio and video PowerPoint presentations, also the cumulative overall score. This suggests that the Old regular discussions between the instructors and the students, Dominion University MLT/CLT to MT/CLS Program, de- and frequent learning assessments. The students matriculate livered in this innovative format, is effective. Further research through the curriculum in a learning community of 20 stu- needs to be conducted in order to examine the cost/benefit dents that provides a natural support system and study group of this type of program as well as other delivery formats. environment. Fifty-four students were admitted into the first quarter; 94% were retained. One hundred three new students Educating Medical Students: It’s Not Always were admitted into the second quarter. Funding, develop- “THE LAB’S FAULT!” ment, resources, the course model, and the curriculum will Smith LA, McKenzie SB, Burns C, Bearden MD, Holton R, be discussed and sample course materials will be presented. Kudolo G, Chumley H, The University of Texas Health Sci- Student demographic, assessment, and retention data will be ence Center, San Antonio TX. shown. Preliminary data suggest that the distance learning course model and curricular structure utilized in the CLS DL Clinical laboratory scientists (CLSs) often complain that track will successfully provide laboratory professionals who other healthcare providers, in particular physicians, do not are not able to attend a traditional program with the means recognize our educational level and underutilize our expertise. to continue their education and advance their careers. In addition, clinicians have little or no knowledge of the effects of pre-analytical errors, especially those resulting from Development and Implementation of an Innova- improper specimen collection. As a result, when laboratory tive MLT/CLT to MT/CLS Articulation Program values do not match clinical diagnosis – it’s the lab’s fault! This Using Synchronous and Asynchronous Delivery project, directed by the Clinical Laboratory Science Program faculty, was part of a medical school course for students en- Formats tering their junior year. We integrated phlebotomy, clinical Tracy S Harrison MS MT(ASCP), Faye E Coleman MS CLS data, and laboratory data to expose medical students to the MT(ASCP), C Thomas Somma EdD MT(ASCP) SC, Paul F value of laboratory data and the expertise of CLS. The class Magnant MBA, Old Dominion University, Norfolk VA. 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 It is well established that there is a shortage of certified medi- remainder in phlebotomy. Twelve scenarios were developed cal technologists/clinical laboratory scientists (MTs/CLSs). covering reflex testing and sources of pre-analytical error in VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 75

C LINICAL PRACTICE all laboratory disciplines. Each student group identified the Integrating Education of MT/CLS Students and problem in the scenario and determined which laboratory CP Residents in a Single Course professional could be consulted regarding questions about Nancy Goodyear PhD CLS(NCA), University of Washington, test ordering or interpretation. Students then presented the Seattle WA. results to other members of the class. CLS faculty moder- ated the sessions. CLS students assisted with venipuncture. It is unusual for undergraduate MT/CLS students to have Medical students took a pre- and post-test that measured the opportunity to interact on an equal basis with clinical pa- knowledge of laboratory testing. The difference between thology (CP) residents. Pathology residents in the University means of the pre- and post-test was significant. Comparison of Washington Department of Laboratory Medicine begin of the means of the pre- and post-test showed a 30% improve- their CP training with a three-month structured core course ment in scores. Session evaluations were favorable with many covering all areas of the clinical laboratory. The microbiology students suggesting additional time for the activities. portion begins with eight laboratory sessions taught by an experienced clinical technologist or a microbiology post-doc- Fostering the Development of Expertise in Clini- toral fellow. Following this introduction, the residents join cal Laboratory Scientists (CLSs) the MT Program clinical microbiology laboratory class for Janet Hudzicki PhD MT(ASCP)SM, Kansas State University, three to four weeks. Following the core, they rotate through Manhattan KS. the clinical labs, including at least six weeks in microbiology. Many residents have no clinical laboratory or microbiology The development of expertise is a phenomenon that is background; although they don’t need to develop technical little understood. Although there is a body of research that expertise, working up the same specimens as the MT/CLS examines the characteristics of experts, and compares experts students, from plating to final report, helps them to under- to novices, the literature on the actual transition process stand the testing performed in microbiology. Interactions lacks depth. This research describes an investigation of the between MT/CLS students and residents help both groups transition from novice to expert in the clinical laboratory recognize the critical role that each plays in healthcare, and science community of practice using a phenomenological appreciate the expertise and limitations of each groups’ approach. The sample selection process consisted of training. MT/CLS students assist residents with laboratory soliciting names of expert CLSs from the members of the procedures and colony morphology. Residents bring human Clinical Laboratory Managers Association. The potential organs from the Department of Pathology teaching organ participants were randomly selected from the submitted collection and give demonstrations reviewing normal and names and asked to participate in the study. Data were abnormal anatomy, especially as it applies to infection. In collected from 11 participants by semi-structured interviews. addition to providing an opportunity for interprofessional The constant comparative method was used to analyze interactions, combining MT/CLS students and CP residents the interview transcripts. Four factors were determined in the same microbiology course consolidates teaching to be essential to the transition process: Self-directedness workload, improves resource utilization, and provides an in learning, storytelling, mentors and mentoring, and opportunity for MT program faculty to contribute to a larger reflection. In addition, the transition from novice to expert educational mission. requires being part of a vital, robust community of practice. Recommendations for helping novices with this transition Moving from Traditional to Online Delivery: include establishing a mentoring program for students and Creating a Hemostasis Course That Promotes new employees, encouraging storytelling among laboratory Student Participation personnel, and providing tools that will encourage reflection. Margaret Fritsma MA MT(ASCP)SBB, University of Ala- This research has the potential to impact the education and bama at Birmingham, Birmingham AL. training of CLSs, the enculturation of novice CLSs into the profession’s community of practice, and the development of University clinical laboratory science programs are mov- expertise in CLSs. 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- 76 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

C LINICAL PRACTICE based lectures to online delivery. The challenge to educators maintain sufficient student numbers for program viability; is to design online courses that engage students, accommodate 3) realizes cost efficiencies by capitalizing on an existing various learning styles, incorporate a variety of learning activi- infrastructure of resources and expertise already in place to ties, and build in accountability and student participation. A support CLS programs; and 4) facilitates laboratory science hemostasis course is described in which course content is placed student articulation between professional levels. This novel online, with classroom follow-up. Each week’s lesson material is consortium model may be applied to institutions nationwide placed online in the form of lesson objectives, PowerPoint slides that are facing similar problems with diminishing state fund- with lecture audio, a Microsoft Word handout with slides ac- ing, program viability concerns due to low student numbers, companied by written text of the lecture, and assignments and workforce shortages, and increasing demands for student supplemental material. There is one classroom meeting each access, participation, and success. week, which consists of a short quiz on the online material, an interactive discussion (no lecture) of the more difficult concepts and implications from the lesson material, a question and an- The Relationship of Proficiency Test Performance swer (Q&A) session, and various group activities. Examples of to Personnel Credentials of Laboratory Testing group activities include impromptu group presentations of short Personnel topics, games, student bowl competition, a Protein C Pathway Maria E Delost MS CLS(NCA), Guang-Hwa Chang PhD, skit, and case discussions. Each student is assigned one mod- Youngstown State University, Youngstown, OH; W Gregory ule to narrate the audio portion of the online lesson from the Miller PhD, William J Korzun PhD DABCC MT(ASCP), instructor’s scripted text, and to lead the classroom Q&A session Teresa S Nadder PhD CLS(NCA), Virginia Commonwealth on his/her topic. Student interest and feedback is positive, and University, Richmond VA. will guide future development of the course. Exam scores have shown improvement in grades over the previous course offered Performance on proficiency test (PT) surveys provides an objec- in a more traditional format. tive and consistent evaluation of laboratory quality. The goal of the study, a retrospective review of existing PT results performed A Novel Consortium Model for Delivering Clini- at Virginia Commonwealth University Health System labora- cal Laboratory Programs to Rural Regions tories, was to determine the relationship of PT performance to Karen R Murray PhD CLS(NCA), Tarleton State University, the personnel credentials of the laboratory testing personnel. Stephenville TX. Predictor variables included the practitioner’s major area of study, degree, certification, and years of laboratory experience. The Workforce problems including severe shortages of clinical study group consisted of 185 testing personnel. There were 3389 laboratory technicians (CLTs) in surrounding rural regions proficiency-testing results of which 3306 were graded acceptable and shortages of histology technicians (HTs) in both rural (97.6%), 36 were unacceptable (1.1%), and 47 were not graded and urban regions, coupled with a state mandated initiative (1.4%). For those results performed by a single practitioner (n (“Closing the Gaps”) to increase student participation and = 3266), the core laboratory performed 3161 (96.8%) of the success within higher education institutions in the State of PT results with 30 unacceptable (0.95%) results. The satellite Texas, prompted the Clinical Laboratory Science (CLS) laboratories staffed by non-laboratorians performed 105 (3.2%) Department at this institution to generate and investigate of the results and 6 (5.7%) were unacceptable. Logistic regression alternative program and curriculum models in order to ad- analysis of the full model, with all predictors included, showed dress these problems. The chosen solution was to design a statistical significance (χ2 = 18.581, p = 0.010, df = 7) for years unique consortium model that represents a novel coopera- of experience and level of educational degree. Individuals with tion between the two-year and four-year higher education less than two years experience were over five times more likely sectors, and is supported by a unique curriculum design that to produce an erroneous result when compared to those with facilitates access and participation. Four key outcomes of the 20 years of clinical experience. Study limitations included the resulting consortium model are that it 1) delivers two high use of a single institution and incomplete demographics for six need laboratory science programs (CLT and HT) and thus testing personnel who were responsible for two (5.5%) of the contributes to the “Closing the Gaps” initiative in three of unacceptable PT results. As the laboratory workforce shortage four key objective areas; 2) utilizes a large community col- intensifies, the performance of laboratory personnel with lim- lege partnership base with established clinical affiliations to ited years of clinical experience or those lacking an educational serve a large, predominantly rural geographic region and to degree may be important. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 77

C LINICAL PRACTICE The Use of Games to Review in a Clinical The lectures are created by first writing a script which is Microbiology Class then recorded and synchronized to PowerPoint slides using Linda Jeff MA MT(ASCP), University of Alabama at Bir- a program called Microsoft Producer for PowerPoint 2003. mingham, Birmingham AL. This technology demonstration will involve two computers; the first to play various examples of recorded lectures, and the A method of reviewing material prior to tests in clinical micro- second to demonstrate the relatively simple steps involved in biology was desired that did not involve reteaching. To meet this creating high quality recorded lectures using the Microsoft objective, games were developed such as “You Make Me Sick”, Producer software (available for free at www.microsoft.com). “You Grow On Me”, “Jeopardy”, “Who Wants to be a Microbi- The results of a student survey will be available describing ologist?”, “Go Streaking”, “Microbiology Team Competition”, the popularity of the recorded lectures, the ease of use, and “Enterobacteriaceae Squares”, and “BACT” (bingo). The games convenience for the online student. provided opportunities for students to review material in a non- threatening, interactive, sometimes competitive, and fun way. Integrating Learning Objects into Clinical Mi- The use of games was an effective means of reviewing/reinforc- crobiology Teaching Materials ing material as indicated by students’ mean scores on the four Jean Brickell EdD, Michelle Kanuth PhD CLS(NCA), Vicki exams (82.6, 84.7, 87, and 86.1). On an evaluation, 100% of Freeman PhD CLS(NCA), University of Texas Medical students responded that they enjoyed playing games, the games Branch, Galveston TX; Sandy Latshaw MA, Carol Larson were a beneficial way to review for tests, the games helped them MSEd CLS(NCA), University of Nebraska Medical Center, learn important facts/concepts, and the games were relevant Omaha NE. to exam questions. All the students liked team games better than individual games. The games that students felt were most Time to produce educational content is a major concern helpful in preparing for exams and learning facts and concepts when planning lessons for CLS/CLT students. Learning were “You Grow on Me”, “You Make Me Sick”, “Jeopardy”, and objects (LOs) are an approach to producing content in “Who Wants to be a Microbiologist?”. Students’ written com- which the instructional material is broken down into ‘bite ments about using the games were very positive and included size’ chunks. These chunks can be independently created, such statements as “The games helped me a lot,” “I really liked maintained, reused, and pulled apart and then stuck back the games. They were very helpful in preparing for exams,” and together into many different forms like Lego toys. LOs are a “The games were very good review tools.” Based on these results high quality technical resource for lectures, reviews, or tests I will continue using these games and others in the microbiology that can be used to structure a lesson individually or strung class as well as in the immunology class that I teach. 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 TECHNOLOGY DEMONSTRATIONS in both distance learning and computer-assisted learning environments. The use of LOs can reduce the preparation Demonstration of Microsoft Producer for the time for lectures, examinations, and remediation materials, Development of High Quality Recorded Lectures freeing instructors to focus on other tasks. The University Based on PowerPoint Presentations of Texas Medical Branch Clinical Laboratory Science Pro- Scott Wright MS, Weber State University, Ogden UT. gram partnering with the University of Nebraska Medical Center Division of Medical Technology received a Fund for For the past three years, the Clinical Laboratory Sciences De- Improvement for Postsecondary Education Grant to create partment at Weber State University has offered both CLT and LOs and disseminate them via an online repository. The CLS degrees online. Presently, one third of the courses offered current focus of this repository is microbiology and provides through the Department deliver course lectures to the online instructional content on biochemical reactions, organism student on a CD. The CD is mailed directly to the student identification, panel selection, and gram stain quality control. prior to the beginning of the semester, eliminating problems The accompanying technology demonstration will provide associated with streaming video delivered over the Internet. the actual LOs and demonstrate the sequencing of LOs to form a cohesive lesson. 78 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

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RESEARCH AND REPOR TS Membranoproliferative Glomerulonephritis Type II in a 10-year-old Girl MARTHA E TIBBS, SHARON P ANDREOLI, CARRIE L PHILLIPS The clinical course of a 10-year-old female patient who Carrie L Phillips MD is a Nephropathologist at Indiana Uni- presented with hematuria, proteinuria, and hypertension versity, Indianapolis IN. is described. Four months after being diagnosed with acute glomerulonephritis, the child was referred to a pediatric Address for correspondence: Martha E Tibbs MT(ASCP), nephrologist due to persistent hematuria and unresolved Indiana University Hospital, Pathology/Microbiology, UH3575, proteinuria. A renal biopsy was performed due to the per- 550 N University Blvd, Indianapolis IN 46202. (317) 274- sistent urinary abnormalities and a family history of renal 3896, (317) 278-0049 (fax). metibbs@iupui.edu failure. The renal biopsy demonstrated pathological findings characteristic of membranoproliferative glomerulonephritis The nephrotic and nephritic syndromes are clinical conse- type II. The child was treated with an antihypertensive quences of structural injury to the renal glomerulus, a vascu- agent and steroids. Despite poor prognostic clinical and lar filter that clears the blood of waste products. Laboratory pathological features, she has minimal urinary abnormali- testing is required to distinguish the two syndromes. Protein- ties, normal renal function, and normal blood pressure on uria, the major clinical sign of nephrotic syndrome, results antihypertensive medication six years after the diagnosis of from altered permeability of the glomerular filtration barrier membranoproliferative glomerulonephritis type II. due to perturbations in visceral epithelial cells (podocytes). Nephrotic syndrome in children is the clinical manifestation ABBREVIATIONS: C3 = complement component 3; GBM of podocyte injury most commonly due to minimal change = glomerular basement membranes; Ig = immunoglobulin; disease. Other causes include focal segmental glomeruloscle- MPGN = membranoproliferative glomerulonephritis. rosis, membranoproliferative glomerulonephritis, or membranous glomerulopathy. Children with nephritic syndrome INDEX TERMS: C3 nephritic factor; complement; dense usually present with gross hematuria that may be accompa- deposit disease; membranoproliferative glomerulonephritis; nied by hypertension and variable degrees of proteinuria. prednisone. Inflammatory processes that target glomeruli, termed acute glomerulonephritis, alter capillary wall integrity, and allow Clin Lab Sci 2005;18(2):84 red blood cells to leak into urine. For a more comprehensive analysis of acute glomerulonephritis, the reader is directed Martha E Tibbs MT(ASCP) is a Medical Technologist at to the review by Vinen and Oliveira.1 Hematuria occurs in Indiana University Hospital, Indianapolis IN. IgA nephropathy, hypocomplementemic glomerulonephritis, and hereditary renal disease (Table 1). Children who present Sharon P Andreoli is a pediatric nephrologist at Riley Children’s with overlapping features of both nephrotic and nephritic Hospital, Indianapolis IN. 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 The peer-reviewed Research and Reports Section seeks to publish underlying etiology and to guide therapeutic management. reports of original research related to the clinical laboratory or If the acute process is untreated, these patients risk progres- one or more subspecialties, as well as information on important sion to chronic glomerulonephritis and may require renal clinical laboratory-related topics such as technological, clinical, replacement therapy such as dialysis or transplantation (for and experimental advances and innovations. Literature reviews an excellent review, see Coppo and Amora 2004).2 are also included. Direct all inquiries to David G Fowler PhD CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of Membranoproliferative glomerulonephritis (MPGN), or Clinical Laboratory Sciences, University of Mississippi Medical mesangiocapillary glomerulonephritis, is caused by an ab- Center, 2500 North State St, Jackson MS 39216. (601) 984- normal immune response leading to antibody deposition in 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu 84 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS the kidneys. The membrano- portion Table 1. Diﬀerential diagnoses of pediatric patients with hematuria and refers to the histological observation proteinuria of glomerular capillary wall thickening due to glomerular basement mem- Glomerulonephritis brane (GBM) alterations. Increased Hypocomplementemic glomerulonephritis glomerular cellularity suggests there Postinfectious glomerulonephritis is a proliferative basis to the glomeru- Membranoproliferative glomerulonephritis, types I, II, and III lonephritis. Patients with MPGN Systemic lupus erythematosus typically experience a decline in renal Nephritis of chronic infection function as a consequence of inﬂam- IgA related glomerulonephritis mation and structural alterations of Henoch-Schönlein purpura the kidney.3,4 MPGN is subtyped into Systemic lupus erythematosus three categories based on the pathway IgA nephropathy of serum complement activation and altered renal morphology resulting Membranous glomerulonephritis from deposition of immunoglobulin Hereditary renal diseases and complement in glomeruli. All Alport syndrome (hereditary nephritis) three subtypes of MPGN are character- Sickle cell nephropathy ized by a decreased serum C3 level in Autosomal dominant polycystic kidney disease 80% to 90% of patients and the low Autosomal recessive kidney disease serum complement level is an important clinical tool in the preliminary diagnosis of glomerulonephritis (Table 1). MPGN type I is characterized by Table 2. Results of serum laboratory tests the classical pathway of complement activation and immune deposits along Test Patient’s results Reference interval the subendothelial aspect of the GBM. Hemoglobin 12.1 10.0-15.5 g/dL MPGN type II, or dense deposit Hematocrit 0.351 0.35-0.49 L/L disease, is characterized by ‘dense’ rib- White blood cell count 13.4 4.5-13.5 x 109/L bon-like immune deposits within Sodium 139 138-145 mEq/L the GBM, alternative complement Potassium 4.0 3.4-4.7 mEq/L pathway activation, and circulating Chloride 105 98-107 mEq/L C3 nephritic factor. MPGN type III Carbon dioxide 26 20-28 mEq/L is characterized by subendothelial, Blood urea nitrogen 11 5-18 mg/dL subepithelial, and mesangial immune Serum creatinine 0.6 0.3-0.7 mg/dL deposits with activation of the alterna- Calcium 8.8 8.8-10.8 mg/dL tive complement pathway.5 Phosphorus 5.1 4.5-7.0 mg/dL CASE STUDY Total protein 5.9 ↓ 6.0-8.0 g/dL In September 1997, a 10-year-old girl was Albumin 3.1 ↓ 3.8-5.4 g/dL seen by her family physician for a one week Cholesterol 243 ↑ 124-201 mg/dL history of dysuria and brownish-colored Alkaline phosphatase 177 ↑ 25-125 U/L urine. Upon physical examination, the Anti-nuclear antibody (ANA) <1:40 <1:40 patient was afebrile with a blood pressure Crithidia negative negative of 124/92 mmHg. The urine dipstick was C3 complement 91 83-177 mg/dL positive for protein. Urine microscopy C4 complement 29 12-36 mg/dL showed numerous white and red blood Strep enzyme screen negative negative cells. The child was diagnosed with acute glomerulonephritis and was prescribed a VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 85

R E S EARCH AND REPORTS ten-day course of amoxicillin. In January 1998, the child returned At the pediatric nephrology clinic, the patient’s only symptom to her physician’s office with bronchitis. At that time she had a his- was intermittent abdominal pain. Blood pressure was 130/90 tory of gross hematuria, her urine was brown in color, and the urine mmHg and the physical exam was otherwise unremarkable. dipstick was positive for protein and blood. A ten-day treatment Urinalysis demonstrated persistent hematuria and protein- of clarithromycin was prescribed, blood tests were drawn, and the uria, the serum complement levels were normal, and the 24- child was referred to a pediatric nephrologist. 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 Figure 1. Light microscopy of glomerulus in Table 2, included decreased albumin, borderline low total protein, and elevated cholesterol and alkaline phosphatase. A renal ultrasound showed bilateral enlargement of the kidneys. 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 Hematoxylin and eosin stain demonstrated lobular accentuation The renal biopsy specimen contained over 60 glomeruli of glomerular tufts due to mononuclear hypercellularity and by light microscopy. Ten percent of the glomeruli were excess accumulation of mesangial matrix (original magnification obsolescent or totally scarred. The remaining glomeruli 400x). ‘Tram-tracks’ or duplication of the GBM was highlighted by the Jones’ silver stain (arrow, inset). showed lobular accentuation of the glomerular tufts secondary to diffusely increased cellularity, thickened capil- Figure 2. Direct immunofluorescence microscopy Figure 3. Electron microscopy of glomerulus Ultrastructural examination revealed ribbon-like electron dense Strong labeling with fluorescein-conjugated anti-C3 complement deposits within the GBM (arrows). U = urinary or Bowman’s antibody was seen along capillary loops of glomeruli (center) and space, E = erythrocyte in glomerular capillary lumen, P = podo- occasional tubular basement membranes (bottom right). cyte, and M = mesangial matrix. 86 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS lary loops, and excess accumulation of mesangial matrix MEMBRANOPROLIFERATIVE GLOMERULONEPHRI- (Figure 1). The glomerular hypercellularity was due to TIS TYPE II increased mononuclear cells in the mesangium. Jones’ Clinical findings silver stain showed thickened and focally duplicated GBM MPGN is a common childhood glomerulonephritis that usu- (‘tram-tracks’) with increased mesangial matrix (Figure 1, ally progresses to chronic renal failure. Of the three subtypes inset). Cellular crescents, or extracapillary proliferation in of MPGN, type II is observed less frequently, accounting for Bowman’s space, were present in 10% of the glomeruli. about 20% to 30% of the cases of all MPGNs. Adults and Interstitial edema was apparent. The tubules and vessels children affected by MPGN type II are typically less than 20 were histologically unremarkable. years old with a median age of 11.5 years.4 Many children ultimately found to have MPGN originally receive medical Direct immunofluorescence examination of the renal biopsy attention due to asymptomatic hematuria and proteinuria. specimen revealed strong immunofluorescence staining for MPGN may present as either acute nephritic syndrome or complement C3 along the glomerular capillary loops (3+ to nephrotic syndrome, with or without gross hematuria.3,4,6 4+ intensity on a scale of 0 to 4+) accompanied by weaker Hypertension occurs in some patients due to water and so- staining in the mesangium (1+ to 2+) (Figure 2). Staining of dium retention, increased production of renin by the kidney, capillary loops and mesangium with immunoglobulins IgG, and other complex mechanisms that regulate blood pressure. IgA, and IgM was negative to weak (0 to 1+). Staining with In some cases, a patient may present with findings typical complement C1q and fibrinogen was negative. of poststreptococcal acute glomerulonephritis; however, if resolution of the symptoms of postinfectious glomerulone- One glomerulus was examined by electron microscopy. Ultra- phritis does not occur within six to eight weeks then other structural examination revealed numerous dense, elongated glomerulonephritides need to be considered (Table 1).4,5 ribbon-like deposits along the lamina densa of GBM (Figure 3). Since the patient described in this report had persistent uri- Podocyte foot processes were effaced and the mesangial matrix nary abnormalities for a period of several months, it became was increased. likely that she did not have postinfectious glomerulonephritis and she underwent a kidney biopsy for the determination of Clinical course a definitive diagnosis. The renal biopsy results were consistent with MPGN type II or dense deposit disease. Based on the results of the renal Laboratory findings biopsy, the child received six pulses of intravenous meth- In a patient with MPGN, the major findings on the uri- ylprednisolone on an every other day schedule. This was nalysis are hematuria and proteinuria. Proteinuria may be followed by oral prednisone, 60 mg every other day. For pronounced and therefore lead to hypoalbuminemia, hyper- hypertension, the patient was prescribed an angiotensin- lipidemia, and edema: all features characteristic of nephrotic converting enzyme inhibitor, enalapril, 2.5 mg/day. Six syndrome. Edema may arise as a result of decreased oncotic months after the kidney biopsy and initiation of therapy, pressure resulting in fluid leaking from the intravascular space her serum albumin had increased to 3.8 mg/dL, her 24- (within the blood vessel) into the tissues when serum protein hour total urine protein excretion had decreased to 760 levels are decreased due to urinary losses.4,7 At presentation, mg and she had no further episodes of gross hematuria. the patient’s blood urea nitrogen and creatinine levels are usu- She was treated with reducing doses of alternate day ally normal to slightly elevated, unless a rapidly progressive prednisone for the next three years. During this interval, glomerulonephritis is evident. A normocytic, normochromic her serum albumin and creatinine remained normal, her anemia may also be present.4 microscopic hematuria resolved and her 24-hour total urine protein excretion decreased to less than 500 mg per In all three subtypes of MPGN, a major laboratory find- 24 hours. Six years after the diagnosis of MPGN type II ing is hypocomplementemia characterized by low levels of and nearly three years after discontinuation of prednisone C3, which is important to help characterize and formulate therapy, she continues to have normal renal function and a provisional diagnosis in a child with hematuria and pro- normal blood pressure, no microscopic hematuria, and teinuria.3,4,7 Depressed serum C3 is related to increased minimal proteinuria as demonstrated by a urine protein catabolism and decreased synthesis of complement.5 The C3 excretion of less than 500 mg per day. nephritic factor, NFa, is present in 30% to 75% of cases.4 C3 nephritic factor is an IgG autoantibody that stabilizes VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 87

R E S EARCH AND REPORTS the alternative pathway C3 convertase, C3bBb. The presence C3 nephritic factor has been implicated in the pathogenesis of C3 nephritic factor causes the formation of C3bBb that of MPGN type II due to the hypocomplementemia. How- becomes resistant to inactivation by factor H and leads to ever, the disease progression does not appear to be affected very low C3 levels.7,8,9 Serum C4, C5, and properdin levels by hypocomplementemia nor C3 nephritic factor.3-6 are usually normal in MPGN type II.5,9 Prognosis Pathological findings The indicators of poor prognosis in MPGN type II are The kidneys show no gross abnormalities that reflect the hypertension, impaired renal function at time of diagnosis, pathologic process in MPGN type II, although some patients nephrotic syndrome, and the presence of crescents.6,7 The may have bilateral renal enlargement as is typical in most higher the percentage of glomeruli with crescents, the more patients with any form of glomerulonephritis. To separate rapidly renal function deteriorates.3,10 Although most patients the types of MPGN, ultrastructural examination is required diagnosed with MPGN type II have no visual complaints, to demonstrate the glomerular alterations. In MPGN type MPGN type II is associated with abnormal retinal function. II, transmission electron microscopy shows sausage-shaped Dense deposits are observed in the Bruch membrane and the or ribbon-like osmiophilic (electron dense) deposits within basement membrane of the choriocapillaris. Drusen deposits the lamina densa of the GBM.3-5,7 Tubular basement mem- and retinal pigment epithelial disturbances are characteristic branes may be widened by similar deposits. The deposits of dense deposit disease retinopathy. These findings are more occupy short segments in the glomerulus that are distributed commonly seen in patients with longstanding MPGN type irregularly in less severe cases. The dense deposits may also II.4,11-13 MPGN type II usually progresses slowly to chronic occur in the mesangium.10 renal failure. Fifty percent of the children will develop chronic renal failure within ten years following diagnosis. Within 20 When examined by brightfield microscopy, the hematoxylin- years of diagnosis, 80% to 90% have chronic renal failure.4 eosin stain imparts a lobular accentuation to the glomerular architecture due to capillary wall thickening and variable Management mesangial hypercellularity.3,10 Occasionally crescents may Several different therapies have been used, including anti- involve a portion of the glomeruli.4,7,10 Mesangial sclerosis, platelet therapy and immunosuppression.4,6,14 However, to or scarring, is more obvious with disease progression. The date, there is no universally accepted form of therapy.4,14 Most source of glomerular capillary wall thickening is revealed by pediatric nephrologists use alternate-day prednisone therapy.4 periodic acid-Schiff (PAS) or Jones’ silver stains that densely Prednisone appears to stabilize renal function and improves label duplicated basement membranes or ‘tram-tracks’ and disease characteristics; however, it can produce side effects due impart weaker staining of the dense deposits.3,7 Thioflavin to drug toxicity.14 These side effects include stunted growth T stain gives a green coloration to the affected basement in children, hypertension, weight gain, Cushinoid features, membranes when using fluorescence microscopy.5 Similar and mood swings/personality changes. Therapy with pulse staining can be observed along Bowman’s capsule and in the intravenous methylprednisolone followed by alternate-day mesangia.10 PAS and Jones’ stains highlight the mesangial oral prednisone has an improved outcome.4,14-17 The alternate- sclerosis resulting from expansion of extracellular matrix.5,7 day regimen either suppresses the immune process underlying Tubular basement membranes may be thickened.3,5 glomerular inflammation or decreases inflammation itself to inactivate the disease.5 Direct immunofluorescence microscopy shows extensive deposition of C3 within glomerular capillary loops and Treatment may vary according to clinical symptoms and labo- mesangia. Immunoglobulins are typically scarce; however, ratory evaluation. The goals of the treatment are to reduce if they are present, the immunoglobulins are usually limited symptoms, prevent complications, and slow progression of to a few glomerular segments.4,10 the disease. Some children may require dietary restrictions on sodium, fluids, and protein to control high blood pres- Pathogenesis sure, swelling, and accumulation of waste products in the The pathogenesis of MPGN type II is unclear but the primary bloodstream. Antihypertensive and diuretic therapy may be morphology is dense deposits in the GBM. By unknown needed for treatment of edema and hypertension.4,7 Therapy mechanisms, glomerular inflammation occurs subsequently. with an angiotensin-converting enzyme inhibitor may be The origin and nature of the dense deposits are unknown.3-5 used to decrease urinary protein excretion and slow the 88 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS progression of chronic renal failure. To manage chronic renal J Ophthalmol 1991;1(1):17-22. failure, dialysis or kidney transplantation may eventually be 13. O’Brien C, Duvall-Young J, Brown M, and others. Electrophysiol- ogy of type II mesangiocapillary glomerulonephritis with associated necessary.4 However, in 50% to 100% of kidney transplant fundus abnormalities. Br J Ophthalmol 1993;77(12):778-80. recipients, recurrence of dense deposits in the transplanted 14. Bergstein JM, Andreoli SP. Response of type I membranoproliferative kidney may develop.18-20 When MPGN type II reoccurs, it glomerulonephritis to pulse methylprodnisolone and alternate-day leads to graft loss in 10% to 20% of the patients.18,19 prednisone therapy. Pediatr Nephrol 1995;9:268-71. 15. Tarshish P, Bernstein J, Tobin JN, and others. Treatment of mesan- SUMMARY giocapillary glomerulonephritis with alternate-day prednisone—a report of the international study of kidney disease in children. Pediatr The patient described in this case study presented with hema- Nephrol 1992;6(2):123-30. turia, proteinuria, and elevated blood pressure. Contrary to 16. McEnery PT, McAdams AJ, West CD. The effect of prednisone in a most patients with MPGN type II, this 10-year-old female high-dose, alternate-day regimen on the natural history of idiopathic had a normal C3 complement level. Without the renal biopsy, membranoproliferative glomerulonephritis. Med 1985;64(6):401-24. this patient would not have been diagnosed with MPGN type 17. Faedda R, Satta A, Tanda F, and others. Immunosuppressive treatment of membranoproliferative glomerulonephritis. Nephron II. Although the current therapy has done well in most cases 1994;67(1):59-65. to slow the progression of the disease, there is still a need for 18. Shimizu T, Tanabe K, Oshima T, and others. Recurrence of mem- the development of a universally effective treatment that does branoproliferative glomerulonephritis in renal allografts. Transplant not have significant adverse side effects. Proc 1998;30:3910-13. 19. Andresdottir MB, Assmann KJM, Hoitsma AJ, and others. Renal ACKNOWLEDGEMENTS transplantation in patients with dense deposit disease: morphological characteristics of recurrent disease and clinical outcome. Nephrol This report is in compliance with Indiana University School Dial Transplant 1999;14:1723-31. of Medicine’s institutional review board and the Health Insur- 20. Turner DR, Cameron JS, Bewick M, and others. Transplantation in ance Portability and Accountability Act of 1996. mesangiocapillary glomerulonephritis with intramembranous dense “deposits”: recurrence of disease. Kidney Int 1976;9(5):439-48. 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 glomerulonephritis. Pediatr Nephrol 2004;19(3):256-65. 3. Cameron JS, Turner DR, Heaton J, and others. Idiopathic mesangio- Congratulations Graduate! capillary glomerulonephritis: comparison of types I and II in children and adults and long-term prognosis. Am J Med 1983;74:175-92. Do you have 4. Andreoli SP. Chronic glomerulonephritis in childhood: membranop- questions about roliferative glomerulonephritis, Henoch-Schönlein purpura nephritis, employment opportunities, and IgA nephropathy. Seminars in Nephrol 1995;42(6):1487-1503. salary expectations, 5. West CD. Idiopathic membranoproliferative glomerulonephritis in benefit packages, childhood. Pediatr Nephrol 1992;6:96-103. performance appraisals, 6. Schwertz R, de Jong R, Gretz N, and others. Outcome of idiopathic membranoproliferative glomerulonephritis in children. Acta Paediatr personnel licensure 1996;85(3):308-12. requirements, 7. Kasinath BS. Sifting the causes of microscopic hematuria. Hosp Pract or other topics? 1996;31(7):99-106, 109-10. 8. West CD, McAdams AJ. Paramesangial glomerular deposits in Find the answers when you visit www.ascls.org/ membranoproliferative glomerulonephritis type II correlate with jobs/grads/index.asp. hypocomplementemia. Am J of Kidney Dis 1995;25(6):853-61. Experts in clinical laboratory management, education, 9. West CD, McAdams AJ. The alternative pathway C3 convertase and and practice have addressed the most frequently asked glomerular deposits. Pediatr Nephrol 1999:13:448-53. questions of graduating students just for you! 10. Churg J. Renal disease classification and atlas of glomerular disease. Tokyo: IGAKU-SHOIN;1982, p 83-109. Let ASCLS help you with your 11. Kim RY, Faktorovich EG, Kuo CY, and others. Retinal function career development! abnormalities in membranoproliferative glomerulonephritis type II. Am J of Ophthalmol 1997;123(5):619-28. To view current job openings, 12. Leys A, Vanrenterghem Y, Van Damme B, and others. Sequential visit the ASCLS Career Center at www.ascls. observation of fundus changes in patients with long standing mem- org/jobs. branoproliferative glomerulonephritis type II (MPGN type II). Eur VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 89

RESEARCH AND REPOR TS “Children on the Frontline Against E.coli ”: Typical Hemolytic-Uremic Syndrome HEIDI ANDERSEN A thirteen-month old infant presented with classical This article was written while the author was a student in the hemolytic-uremic syndrome (HUS), but with negative Clinical Laboratory Science program at Indiana University, cultures for Escherichia coli (E. coli) 0157:H7. HUS is Indianapolis IN. commonly linked to infection with E. coli 0157:H7; however, traditional culture has demonstrated poor CASE STUDY sensitivity. Pathogenesis of the organism in HUS involves In January, a thirteen-month-old Caucasian male presented the production of a Shiga-like toxin (STX), resulting in with progressive diarrhea over a period of two weeks. During a triad of symptoms. An early and accurate differential this time, medical attention was sought, and the infant was diagnosis, based on patient presentation with acute renal diagnosed with a common childhood diarrhea, suspected to failure, hemolytic anemia, and thrombocytopenia, is be due to a rotavirus. However, the infant continued with critical for supportive treatment and improved prognosis. progressive diarrhea and began showing signs of pallor, de- Patient prognosis is related to the duration of renal failure hydration, petechiae on his thighs, and decreased appetite. and dialysis treatment. Research is aimed at improved The infant began experiencing episodes of acute abdominal detection of E. coli 0157:H7 or the STX produced, and pain with intermittent periods of lethargy. As the diarrhea future vaccination to eliminate typical HUS. worsened, one sixteenth of a tablet of Imodium® was given to the infant and he was brought to the local emergency ABBREVIATIONS: CDC = Centers for Disease Control and department (ED). Prevention; HUS = hemolytic-uremic syndrome; STX = shiga- like toxin; TTP = thrombotic thrombocytopenic purpura. The infant presented in the ED with signs of edema in the extremities from oliguria and acute renal failure. He INDEX TERMS: hemolytic-uremic syndrome. was catheterized, treated with Lasix® to stimulate kidney function, and given nutritional IV support. Vitals revealed Clin Lab Sci 2005(18(2):90 hypertension with a blood pressure of 132/55 (normal blood pressure of a 1-year-old Caucasian male is 102/57) Heidi Andersen MT(ASCP) works at St John’s Hospital, Ander- and a temperature of 103.5 °F. As a result, the infant was son IN and at Indiana University Hospital, Indianapolis IN. given Hydralazine® and Tylenol® to reduce his elevated blood pressure and temperature respectively. Address for correspondence: Heidi Andersen MT(ASCP), 2007 B Parsons Drive, Indianapolis IN 46224. (317) 459- The initial laboratory results indicated a diagnostic triad 3023. handerse@iupui.edu 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 The peer-reviewed Research and Reports Section seeks to publish inflammatory reaction that occurs in typical HUS, with E. reports of original research related to the clinical laboratory or coli 0157:H7 suspected as the cause. one or more subspecialties, as well as information on important clinical laboratory-related topics such as technological, clinical, QUESTIONS TO BE CONSIDERED and experimental advances and innovations. Literature reviews • What is the typical presentation of HUS and the patient are also included. Direct all inquiries to David G Fowler PhD population at risk? CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of • What is the pathogenesis and pathophysiology of typical Clinical Laboratory Sciences, University of Mississippi Medical HUS? Center, 2500 North State St, Jackson MS 39216. (601) 984- • What is the differential diagnosis of typical HUS? 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu 90 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS • What is the treatment and long-term prognosis of typical Classical HUS is defined as a thrombotic microangiopathy HUS? with a diagnostic triad of acute hemolytic anemia with • What are preventive measures that can be taken to protect schistocytes, thrombocytopenia, and acute renal failure. children from HUS? However, classical HUS does not always present with a complete diagnostic triad. A major indicator of typical HUS INTRODUCTION TO HUS from E. coli 0157:H7 is presentation with a one to eight The syndrome now known as HUS, was described in 1955 day acute gastroenteritis prodrome and bloody diarrhea, by Gasser. In 1982, Riley isolated pathogenic Shiga-like toxin unless the infection is acquired by a urinary tract infection producing E. coli (STEC) serotype 0157:H7 from contami- or a respiratory infection.7,8 The kidney is the major organ nated hamburger. Then, Karmali linked HUS to E. coli 0157: target in classical HUS, but other organs such as pancreas, H7 in 1985. Connection of E. coli 0157:H7 to HUS was a heart, lungs, and brain may also be involved.9-12 Other fac- major breakthrough in differentiating the mechanisms of the tors supportive of a diagnosis of HUS due to E. coli 0157: often-confused conditions of thrombotic thrombocytopenic H7 include its predominance in the summer months and its purpura (TTP) and HUS. HUS is currently accepted as the potential to occur in outbreaks. most common cause of acute renal failure in children in the U.S. and is primarily caused by E. coli 0157:H7. The Centers HUS, as noted by its name, is merely a syndrome, and for Disease Control and Prevention (CDC) estimate that with therefore, has many known etiologies. Typical (infectious) 73,000 E. coli 0157:H7 infections per year in the U.S., 2% HUS is caused by bacterial and viral infections such as E. coli to 7% of E. coli 0157:H7 infections result in HUS with the 0157:H7, some non-0157 E. coli strains, Shigella dysenteriae majority of cases occurring in Caucasians less than five years 1, Streptococcus pneumoniae, Salmonella typhi, Campylobacter of age.1 The elderly, adults, and older children have higher jejuni, and HIV. Non-infectious, atypical HUS may be sec- mortality rates from HUS than occur in younger children.2,3 ondary to (but not limited to) pregnancy and postpartum, Interestingly, HUS caused by infection with E. coli 0157: organ transplant, glomerulonephritis, systemic lupus erythe- H7 is less common in African-Americans.4 Overall, E. coli matosus, or treatment with drug therapies such as tacrolimus 0157:H7 infections costs about 60 lives and $660 million (FK506), quinine, and mitomycin. However, it is estimated dollars each year, and is now an endemic cause of HUS in in the U.S. that 90% of cases of HUS in children are primary the U.S.1,5,6 infections caused by E. coli 0157:H7 with increasing findings 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 Table 1. Initial laboratory testing of patient on day 1 sources. Enterohemorrhagic E. coli (EHEC) is carried asymp- of hospitalization tomatically in the intestines of cattle, where Globotriaosylce- ramide (Gb3) receptors are found throughout the intestinal Tests Results Reference ranges tract, but cattle lack Gb3 receptors in the vasculature.15,16 These findings may play a significant role in colonization. However, Albumin 33 30.8 - 46.6 g/L the reason cattle are asymptomatic carriers of E. coli 0157:H7 BUN 32 1.8 - 7.1 mmol/L is still being studied. During slaughter, the surface of EHEC Chloride 102 95 - 105 mmol/L contaminated meat is ground into and spread throughout the CO2 17 22 - 27 mmol/L hamburger. Only 50 to 100 viable organisms of E. coli 0157:H7 Creatinine 336 17.7 - 61.9 mol/L are required to cause infection.7 Other food products, besides Glucose 5.7 2.2 - 11.1 mmol/L ground beef, that can be a reservoir for EHEC after contamina- Hematocrit 0.258 0.340 - 0.480 tion with cattle feces include: unpasteurized milk products, apple Hemoglobin 85 96 - 156 g/L juice, water, and vegetables. There is seasonality to the shedding Platelet # 64 150 - 450 x 109/L of EHEC from cattle, where there is an increased amount of Potassium 5.3 3.5 - 5.5 mmol/L organism shed in the summer months, which correlates with an Sodium 131 135 - 145 mmol/L increase in products with fecal contamination, E. coli 0157:H7 Total bilirubin 11 0 - 10 g/L infections, and HUS in the warmer season.17 The infant in the WBC # 21.5 5.5 - 17.5 x 109/L case study, however, presented with HUS in January. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 91

R E S EARCH AND REPORTS Although cattle are the major source of E. coli 0157:H7, it to platelets induces platelet aggregation directly, and STX has been found in other animals, e.g., sheep, goats, deer, and prevents apoptosis in neutrophils, resulting in leukocytosis even a small percentage in cats, dogs, horses, and flies.18,19 E. and increased inflammation.27,30 coli 0157:H7 has been shown to survive ten months or longer in contaminated sources that resulted in human infection.20 The proposed primary target of STX from the circulation Additionally, E. coli 0157:H7 can readily be transmitted from is the distal convoluted tubular epithelium in children (Gb3 person to person, causing concern to families, nursing homes, receptors may not be present in adults).31 The injured renal daycare facilities, and other crowded living conditions.21,22 epithelial cells also initiate an inflammatory response and After September 11, 2001, E. coli 0157:H7 was considered release endothelin, tissue plasminogen activator inhibitor-1 to be a potential agent of bioterrorism.23 In the case of this (PAI-1), and PAF. Endothelin increases production of PAF, thirteen-month old child, no other children or adults with white blood cell activation that results in leukocytosis, and whom he had contact were infected. may somewhat increase blood pressure early on in typical HUS. PAI-1 inhibits fibrinolysis of microvascular throm- Pathogenesis and pathophysiology bosis. PAF-activated platelets release thromboxane hence The pathogenesis of typical HUS begins with the hardiness promoting platelet aggregation by vasoconstriction-induced of E. coli 0157:H7. E. coli 0157:H7 can easily live through high shear stress. Thromboxane is regulated by prostacyclin freezing at –80 °C for as long as nine months. Its acid resis- released from injured epithelial cells through negative feed- tance increases survival in acidic foods (conditions that are back. However, in typical HUS, platelet activation outnum- normally used to preserve food from bacterial contamination) bers production of prostacyclin resulting in thrombosis.32 because acidity decreases the nutritional competition with other non-acid-resistant organisms. E. coli 0157:H7 can Microvascular endothelial cells that have Gb3 receptors (in remain viable in a pH environment less than 2.5.24 children and adults), particularly those of the glomerulus, are extremely sensitive to STX. Injury to the endothelial cells Once E. coli 0157:H7 enters the body orally, it survives by bound STX is key to the pathogenesis of typical HUS.7,32 the pH of the stomach, and colonizes in the mucosa of the STX causes extensive microangiopathic thrombosis, hypoxia, colon. The exact mechanism of E. coli 0157:H7 pathogenic- and ischemia in the glomerular endothelial cells, and similar ity is under extensive research. A proposed mechanism is damage can be found in the cerebral endothelial cells of the that the lipopolysaccharide and STX from the organism are blood-brain barrier.33 Injury to the endothelial cells is marked responsible for activating the phosphatidylinositol cascade, by elevated thrombomodulin levels. Lesions from STX in- increasing mobilized ionized calcium that allows adherence volve the glomerular capillaries; thickening of the capillary of the organism to the epithelial cells of the colon and pro- wall near the glomerulus decreases the glomerular filtration motes lesion formation. Lesions change the permeability rate.34 Both ischemia and thickened capillaries elevate blood of the epithelial cell membrane to water and electrolytes, pressure. Progressive damage to the kidneys occurs via hy- causing the prodromal diarrhea, and initiate an inflamma- perfiltration of the functional nephrons that remain after the tory response partially responsible for the microangiopathic acute phase.35 Damage to cerebral endothelial cells causes thrombosis.25 The inflammatory response activates neutro- encephalophy, coma, stroke, and cerebral infarcts.33 phils that release cytokines, platelet activating factor (PAF), and tissue necrosis factor alpha. These play a role in increasing Toxins are freely permeable to the glomerulus, and the large activated neutrophils (leukocytosis), platelet aggregation,26 capillary surface area enhances toxin pathogenicity. The and up-regulating Globotriaosylceramide (Gb3) receptors concentration of toxin is increased by countercurrent roles on kidney and brain endothelial cells,12 respectively. Close of vasculature and tubules in the kidney.36 STX has one proximity of the injured epithelial cells to microvasculature enzymatic subunit A and five receptor-specific B subunits. results in hemorrhagic colitis (HC) and bloody diarrhea in Subunit A invades and kills the renal endothelial cells by the typical HUS. endocytosis and inhibiting translation. Subunit B activates neutrophils, releasing proteases and hydrogen peroxide that STX access to circulation following HC allows for direct and irreversibly damage renal cells.37 Oxidative substances and indirect mechanisms of pathogenicity. STX reaches the kid- fibrin-platelet aggregations occluding the microvasculature ney and brain through the circulatory system on the surface fragment red cells, producing schistocytes on the peripheral of platelets, neutrophils, and monocytes.27-29 STX bound blood smear. 92 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS Differential diagnosis ment deficiency involvement, neuro- of TTP includes fever, central nervous It is critical to differentiate typical logical involvement, an inborn error system involvement, and the diagnostic HUS from atypical HUS, thrombotic in the metabolism of cobalamin, or a triad seen in classical HUS. However, thrombocytopenic purpura (TTP), factor H deficiency. Most secondary 25% of typical HUS cases have nervous and disseminated intravascular coagu- causes of atypical HUS can be ruled system involvement and many present lation (DIC) for both treatment and out with patient history. Generally, with fever.41 Unlike typical HUS, TTP prognosis (Table 2). For example, typi- atypical HUS does not present with does not generally involve gastrointes- cal HUS requires supportive therapy leukocytosis or GI prodrome and tinal inflammation, or leukocytosis.42 while TTP mandates plasma exchanges bloody diarrhea, and is seen more fre- TTP is usually systemic with severe and possibly immunosuppressive quently in adults than young children, thrombocytopenia resulting in platelet treatment with glucocorticoids. Mis- perhaps simply because adults are counts below 20 x 109/L, where typical diagnosis could be life threatening; likely to have secondary causes. HUS is primarily localized to the kidney a significant number of deaths from with platelet counts between 30 x 109/L TTP occur within forty-eight hours TTP, a completely separate disease from and 150 x 109/L. Risk for TTP is not of presentation.38 HUS, is the result of a decreased level of targeted to a specific age group, like von Willebrand Factor (vWF)-cleaving typical HUS. In the future, differentiat- Atypical causes of HUS are associated enzyme (ADAMTS-13) due to a defi- ing TTP from HUS may be easier by with a high mortality rate and recur- ciency of ADAMTS-13 or antibodies measuring the functional ADAMTS-13 rences. These causes include comple- against it.39,40 The classical presentation level in the plasma.43 DIC is a systemic activation of both the coagulation cascade and fibrinoly- Table 2. Differential diagnosis of typical HUS including patient case sis, and is commonly associated with study results pregnancy-related complications like atypical HUS. The easiest most reliable Laboratory and Patient Typical Atypical TTP DIC way to differentiate DIC is with rou- clinical findings HUS HUS tine coagulation testing. Prothrombin time (PT), partial thromboplastin Abnormal kidney tests Y Y Y Y Y/N time (PTT), and D-dimer levels are Abnormal liver tests N N N N Y/N abnormal in DIC, but are normal in patients with typical and atypical Abnormal PT, PTT, HUS. However, vitamin K deficiency and D-dimer N N N N Y may prolong the PT, and D-dimer CNS involvement N Y/N Y/N Y Y levels may be elevated with a high concentration of fibrin in the micro- GI prodrome/ angiopathic thrombus in patients with bloody diarrhea Y Y N N N typical HUS.44 Hemolytic anemia Y Y Y Y Y LABORATORY FINDINGS AND Hypertension Y Y Y Y Y CLINICAL COURSE Leukocytosis Y Y N Y/N Y Upon diagnosis, the thirteen-month- old infant was transferred via am- Positive Coombs test N N N N N bulance to a major referral childrens Recurrences N N Y/N Y N hospital. Albumin, total bilirubin, and Renal failure Y Y Y Y/N Y/N hemoglobin from the initial laboratory tests (Table 1) were near normal Thrombocytopenia Y Y Y Y Y supporting the beginning of the acute Y = Yes, N = No phase of HUS. Hemoglobin drops as hemolysis increases during the acute VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 93

R E S EARCH AND REPORTS phase, and total bilirubin rises as hemoglobin from lysed from barrier isolation. A negative culture does not rule out red cells is broken down, following degradation of the a typical HUS diagnosis, and is commonly seen due to the hemoglobin porphyrin ring. A type and screen (T&S) and transient shedding of E. coli 0157:H7, culturing too late indirect Coombs were ordered for type-specific supportive in the course of the infection, or poor culture sensitivities. transfusions. The indirect Coombs was negative, support- The best time to obtain a positive culture for the infectious ing a non-immune-mediated cause of hemolysis. Clinically, organism is during the GI prodrome.14 hemolysis is monitored by the hemoglobin level, but LDH and AST tests supplement evaluation of hemolysis. LDH Throughout the infant’s 32-day hospitalization, HUS was is more specific to hemolysis; however, extremely elevated monitored with daily basic metabolic panels and cell blood LDH also reflects tissue necrosis (Table 3).45 Since there is counts. Monitoring hemoglobin, platelets, creatinine, and extensive red cell destruction in HUS, most of the hapto- albumin throughout the acute phase of HUS is indicative globin is bound to free hemoglobin, giving lower detectable of severity and duration of hemolysis, microthrombi, and levels of haptoglobin in the serum. Coagulation studies kidney dysfunction, respectively. An increase in catabolism, were found to be normal ruling out DIC in the differential during the acute phase of the infection, and the change in diagnosis (Table 3). distribution of volume to albumin in the serum resulted in hypoalbuminemia, reaching a low of 1.7 mg/dL. Because Recovery of a small urine sample was obtained from the Foley no platelets were transfused or any other therapy given that catheter of the infant for urinalysis (Table 4) which confirmed would directly affect the platelet count, the rise in platelet proteinuria, acidosis, and hemolysis. Red cell casts were not count on day-11 marked the initiation of the recovery phase found in this sample, but are commonly found with care- (Figure 2). Day-15 marked the end of the acute phase of the ful examination in patients with typical HUS.46 The infant illness with stabilization of the platelet count, hemoglobin, began peritoneal dialysis on day 3 (Figures 1, 2, and 3), was and creatinine (Figures 1-3). After day-15, supportive given nasogastric tube feedings for nutritional support, and transfusions were no longer necessary, and on day-20, was placed in barrier isolation to prevent transmission of recovery was confirmed by discontinuation of dialysis with suspected E. coli 0157:H7. continued stabilization of creatinine levels (Figure 3). In addition to the urinalysis, stool cultures were ordered for growth of E. coli 0157:H7, Shigella, Yersinia, Salmonella, Table 4. Patient urinalysis results on day-2 of hospi- and Campylobacter. Cultures are done to confirm a causative talization agent of HUS and for epidemologic purposes such as tracking of the contaminated food product and disease control. Urinalysis and Patient results Reference range Sorbitol MacConkey agar is generally the medium of choice microscopic for isolating the non-sorbitol-fermenting E. coli 0157:H7. Most non-pathogenic E. coli found in the normal flora of the Appearance cloudy clear GI tract ferment sorbitol. After successive negative stool cul- Bacteria many negative tures for STX-producing organisms, the infant was released Bilirubin negative negative Color pink colorless-dark yellow Glucose negative negative Table 3. Laboratory evaluation of patient hemolysis Hemoglobin 250 Ery/µL negative and confirmation of typical HUS Ketones 5 mg/dL <75 mg/dL Nitrite negative negative Tests Patient results Reference ranges pH 5.0 5.0-8.5 Protein 500 mg/dL negative AST 176 25 - 45 U/L Red blood cells 663/HPF 0-3/HPF LDH 10240 425 - 975 U/L Specific gravity 1.016 1.003-1.030 Lipase 160 25 - 120 IU/L Urobilinogen negative 0.1-1.0 Ehrlich’s units PT/INR 12.8 sec/1.05 NA/0.9 - 1.1 WBC esterase 100 LEU/µL negative PTT 32.5 24.7 - 33.4 sec White blood cells 66/HPF 0-5/HPF 94 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS As commonly seen in classical typical normal to increased megakaryocytes for pancreatitis. After the onset of ab- HUS, the WBC differentials showed (depending on platelet consumption), dominal pain, lipase levels rose within a left shift with few myelocytes and and erythroid hyperplasia. 12 hours and rapidly fell to normal metamyelocytes along with leuko- range within a few days. In addition, cytosis and thrombocytopenia. Cell As often occurs with HUS, this infant the infant developed peritonitis when morphology included moderate hy- had several complications. On day- the dialysis catheter was compromised, pochromic, microcytic red cells with 2, the infant developed tachycardia, which was reflected by the increasing a few schistocytes, nucleated red cells, cardiac arrythmia, and periorbital creatinine levels at day-9 (Figure 3). A ovalocytes, polychromasia, and giant edema. Cardiac arrhythmias in HUS dialysate culture revealed coagulase- platelets. Reticulocyte counts would are due to cardiac ischemia, which can negative Staphylococcus sp. and Strepto- likely be increased, while the infant at- also result in myocardial infarction coccus pyogenes, and a dialysate sample tempted to compensate for the anemia in typical HUS.10 On day-4, slight revealed a neutrophil count of 1830 x with increased red cell production. A involvement of the pancreas was seen 109/L with 75% neutrophils and 23% bone marrow aspirate is certainly not with elevated serum lipase (Table 3). monocytes with toxic granulation. The necessary and would be risky given the Both lipase and amylase are markers infant was treated with vancomycin presence of thrombocytopenia. How- for pancreatitis. Since lipase is not and serum levels were monitored. On ever, cellular morphology of the bone elevated by as many conditions as day-25, the infant spiked a fever of 103 marrow would be expected to show amylase, it is a more specific indicator °F and was treated with cefotaxime and vancomycin for possible septicemia. Both blood and urine cultures were obtained, and both were found to be Figure 1. Hemoglobin vs. Time negative for growth. After conclusion of the acute phase, a urinalysis still showed significant proteinuria along with a few granular casts and renal epithelial cells, but the hemoglobin levels and red cell numbers supported the cessation of hemolysis. The infant was discharged on day-32 with resolution of the fever and the Figure 2. Platelet Count vs. Time 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 include medication to control blood pressure, continuous peritoneal Figure 3. Creatinine vs. Time dialysis or hemodialysis, packed red cell transfusions, fluid restriction, diuretics, and very careful maintenance 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, VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 95

R E S EARCH AND REPORTS rigorous management of treatment has decreased the necrosis. This procedure is impractical and contraindicated mortality rate of typical HUS from 50% to about 5% to in the acute phase, but it is used later to assess long-term 10% throughout the last five decades.47,48 prognosis.53,55 Other factors that may support poor long-term prognosis include: elevated white cell count >20 x 109/L, There are important contraindications to certain treatments neural involvement, post-HUS proteinuria, and hyperten- when typical HUS is diagnosed. Platelets are generally not sion during the acute phase.35,56-58 In the case history of the transfused, because they may cause more severe microangio- thirteen month old infant, the following poor prognostic pathic thrombosis. Antibiotics are also not suggested, because indicators were found: oliguria/anuria >10 days, leukocytosis killing E. coli 0157:H7 may rupture the organism’s cell wall, >20 x 109/L, post-HUS proteinuria, and hypertension at thereby increasing the amount of toxin released, and contrib- onset of HUS. uting to the severity of the acute phase.49 If complications occur, however, during typical HUS, such as peritonitis or PREVENTION sepsis, antibiotics may be necessary. Anti-motility drugs for There are three important levels of prevention when dealing diarrhea are not advised, because they may increase the risk with E. coli 0157:H7. On the public level, proper hand- of typical HUS by extending the exposure time of STX to washing and safe preparation of food remain the best prac- the patient’s cells.50 tices for the prevention of typical HUS. However, too much responsibility has been placed on the public.59 The state and Long-term treatment for patients requires monitoring of federal government play critical roles in prevention. They are blood pressure, creatinine, and urinalysis.51 Patients exhib- responsible for keeping the food supply safe and ensuring iting hypertension and proteinuria are treated to lower the quality assurance by mandating effective screening of food blood pressure with an angiotensinogen-converting enzyme products for E. coli 0157:H7 and other foodborne pathogens. (ACE) inhibitor (blocking the production of renin and In the past, only a few states required such screening. With consequently, aldosterone) to reduce further damage to the the new rapid STX detection methods available, surveillance glomerulus, reduce proteinuria, and delay or prevent future of E. coli 0157:H7 in the beef industry is improving.60 The chronic renal failure.52 Two years of post-HUS laboratory test- Food and Drug Administration has approved the irradiation ing on this infant confirmed hypertension and proteinuria. of ground beef, which can kill at least 90% of E. coli 0157:H7 After beginning treatment with an ACE inhibitor, the infant contaminants.24 A law mandating irradiation of other poten- showed a decrease in blood pressure and proteinuria. tially contaminated food products was approved by the United States Department of Agriculture and is being implemented. PROGNOSIS Cases involving contaminated water sources have also called Although prognosis of HUS remains variable, a recent meta- attention to enforcing regulations for protecting small water analysis produced significant data. It is estimated that 58% of systems that in the past received little attention.61 HUS patients fully recover, 5% die within the acute phase, 11% develop chronic renal failure or die within four years RESEARCH AND THE FUTURE of the acute phase, and 17% experience long-term residual Current research is aimed at faster and more reliable testing effects such as hypertension, proteinuria, and a decreased of STX as well as countering the action of STX produced glomerular filtration rate. There is often a period of perceived by any organism. The conventional method involving the renal recovery before the onset of long-term residual effects. culture of stool on MacConkey-Sorbitol agar for E. coli Surprisingly, even some patients who presented with mild 0157:H7 is only 40% sensitive and takes up to three days HUS (without dialysis or abnormal urine output) developed to complete.62 Further complicating the diagnosis of typical renal sequelae.47 Renal sequelae may result in renal failure HUS is the usual delay of symptoms for one to two weeks more than twenty years after the acute phase of HUS.53 Thus, after infection, leaving few organisms in the stool for detec- it is extremely important that the renal function of any child tion. In addition, more than 100 other shiga toxin-producing with a history of HUS be followed for life. E. coli (STEC) serotypes are left undetectable by many clinical laboratories because they ferment sorbitol, like normal The best practical indicator of prognosis is the duration of intestinal strains of E. coli.63 oliguria or anuria and consequently, the need for dialysis during the acute phase of HUS.54 The most sensitive indicator Improved technology is strongly needed to enhance the is a kidney biopsy, which shows the degree of renal cortical sensitivity of culture for E. coli 0157:H7 in HUS patients. 96 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS In 1996, research showed a success rate of 90% for E. coli CONCLUSION 0157:H7 if an immunomagnetic separation (IMS) technique As in the case of this thirteen-month-old infant with HUS, it was used to isolate the 0157 antigen (using beads with the is estimated that E. coli 0157:H7 causes 73,000 of the 76 mil- corresponding antibody) after use of a pre-enrichment culture lion food-borne illnesses each year. Most food-borne illnesses in medium. This method proved to have many advantages over the U.S. are self-limiting, but those that cause HUS may lead new PCR methods for detecting E. coli 0157:H7 including at to death or life-long consequences such as renal sequelae and least a 100-fold increase in sensitivity, and less complex test- gastrointestinal complications. Progress has been made in the ing.64 In 1999, the United States Department of Agriculture elimination of food-borne causes of typical HUS via irradiation began implementing IMS to improve the screening of ground of food products and improved quality assurance and control in beef. To date, many clinical laboratories have not adopted the food industry. Current research is focused on vaccination, the improved methods for routine screening of E. coli 0157: improved diagnostic testing methods, and intervening treat- H7 in children suspected of HUS.65 If a clinical laboratory ments with early diagnosis. “So HUSH all you children and is unable to implement new technology for detecting E. coli don’t you cry, together we will beat the bug they call E. coli.”70 0157:H7 and/or STX, the specimen should be referred to an appropriate reference laboratory. REFERENCES 1. Centers for Disease Control and Prevention. Escherichia coli 0157: Alternative rapid tests for non-0157 STEC are directed to- H7. Available at: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ ward the detection of STX produced instead of the organism. escherichiacoli_g.htm Accessed April 28, 2004. 2. Carter AO, Borczyk AA, Carlson JA, and others. A severe outbreak One method uses an EIA technology that requires approxi- of Escherichia coli 0157:H7 - associated hemorrhagic colitis in a mately 18 hours to perform (this includes a 16-hour incuba- nursing home. N Engl J Med 1987;317:01496-500. tion period for increased sensitivity), while another method 3. Melnyk AMS, Solez K, Kjellstrand CM. Adult hemolytic-uremic is a simple toxin detection tests that only takes three hours syndrome: a review of 37 cases. Arch Intern Med. 1997;155:2077-84. to perform. Both methods may significantly shorten the time 4. Jernigan SM, Waldo FB. Racial incidence of hemolytic uremic syndrome. Pediatr Nephrol 1994;8:545-7. for diagnosis. In addition, other improved methods include 5. United States Department of Agriculture. Economics of PCR followed by pulse-field gel electrophoresis, impedance foodborne disease: E. coli. Available at: www.ers.usda.gov/briefing/ technology, and enzyme-linked immunoassays. FoodborneDisease/ecoli/index.htm Accessed May 17, 2004. 6. Sears, CL. Update on shiga toxin-producing E. coli infections. Adv Vaccination is another potential area of interest. Healthy Stud Med 2003;3(5):259-64. persons develop an antibody to the antigenic subunit B of 7. Kaplan BS, Meyers KE, Schulman SL. The pathogenesis and treatment of hemolytic uremic syndrome. J Am Soc Nephrol STX after exposure.66 When mice were immunized with 1998;9:1126-33. the B subunit of STX, they were able to produce antibodies 8. Tarr PI, Fouser LS, Stapleton AE, and others. Hemolytic-uremic that neutralized the potent effects of STX.67 Monkeys have syndrome in a six-year old girl after a urinary tract infection with also been successfully immunized and protected by an STX shiga-toxin-producing Escherichia coli 0103:H2. N Engl J Med. vaccine when challenged with a lethal dosage.68,69 Immuni- 1996;335(9):635-8. 9. Robitaille P Gonthier M, Grignon A, and others. Pancreatic injury in , zations may be administered for both humans and cattle in the hemolytic-uremic syndrome. Pediatr Nephrol 1997;11(5):631-2. order to reduce E. coli 0157:H7 infections and incidence of 10. Thayu M, Chandler WL, Jelacic S, and others. Cardiac typical HUS. ischemia during hemolytic uremic syndrome. Pediatr Nephrol 2003;18(3):286-9. Treatment of patients with monoclonal antibodies, currently 11. Butani L, Polinsky MS, Kaiser BA, and others. Pleural effusion in clinical trials, may neutralize the STX in patients present- complicating acute peritoneal dialysis in hemolytic uremic syndrome. Pediatr Nephrol 1998;12(9):772-4. ing with early diagnosis of typical HUS. The monoclonal 12. Eisenhauer PB, Chaturvedi P, Fine RE, and others. Tumor necrosis STX antibody binds STX in the intestine and may render factor alpha increases human cerebral endothelial cell Gb3 and the toxin less able gain access to the circulation. Hence, sensitivity to shiga toxin. Infect Immun 2001;69(3):1889-94. STX is less likely to affect the kidney or other organs.42 One 13. Griffin PM, Tauxe RV: The epidemiology of infections caused by remaining concern with this treatment is the remaining Escherichia coli 0157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epid Reviews 1991;13:60-98. lipopolysaccharide (LPS) of the bacteria that also plays an 14. Banatvala N, Griffin PM, Greene KD, and others. The United antigenic role in typical HUS, perhaps warranting the need States national prospective hemolytic uremic syndrome study: for treatment with another monoclonal specific antibody or microbiologic, serologic, clinical, and epidemiologic findings. a polyvalent antibody to both STX and LPS.32 J Infect Dis 2001;183(7):1063-70. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 97

R E S EARCH AND REPORTS 15. Pruimboom-Brees IM, Morgan TW, Ackermann MR, and others. 32. Siegler RL. The hemolytic uremic syndrome. Pediatr Clin North Cattle lack vascular receptors for Escherichia coli 0157:H7 shiga Am 1995;42(6)1505-29. toxins. Proc Natl Acad Sci USA 2000;97(19):10325-9. 33. Hutchison JS, Stanimirovic D, Shapiro A, and others. Shiga toxin 16. Hoey DE, Currie C, Else RW, and others. Expression of receptors (verotoxin) toxicity in human cerebral endothelial cells. In: Kaper JB, for verotoxin 1 from Escherichia coli 0157:H7 on bovine intestinal O’Brien AD, editors. Escherichia coli 0157:H7 and other shiga toxin- epithelium. J Med Microbiol 2002;51(2):143-9. producing E. coli strains. Washington DC: ASM Press, 1998; 323-8. 17. Griffin PM. Epidemiology of shiga toxin-producing Escherichia coli 34. Buckalew VM. Pathophysiology of progressive renal failure. South infections in humans in the United States. In: Kaper JB, and O’Brien Med 1994;87:1028-33. AD, editors. 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Karpman D, Papadopoulou D, Nilsson K, and others. Platelet activation J Med 2002;346(1):23-32. by shiga toxin and circulatory factors as a pathogenetic mechanism in 45. Cohen JA, Brecher ME, Bandarenko N. Cellular source of serum the hemolytic uremic syndrome. Blood 2001; 97(10):3100-8. lactate dehydrogenase elevation in patients with thrombotic 28. Te Loo DMWM, van Hinsbergh VWM, van den Heuvel LPWJ, thrombocytopenic purpura. J Clin Apheresis 1998;13:16-9. and others. Detection of verocytotoxin bound to circulating 46. Corrigan JJ Jr, Boineau FG. Hemolytic-uremic syndrome. Pediatr polymorphonuclear leukocytes of patients with hemolytic uremic Rev 2001;22(11):365-9. syndrome. J Am Soc Nephrol 2001;12(4):800-6. 47. Garg AX, Suri RS, Barrowman N, and others. Long-term renal 29. van Setten PA, Monnens LA, Verstraten RG, and others. 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R E S EARCH AND REPORTS 50. Cimolai N, Carter JE, Morrison BJ. Risk factors for the progression 62. Elliot EJ, Robins-Browne RM, O’Loughlin EV, and others. of Escherichia coli 0157:H7 enteritis to hemolytic-uremic syndrome. Nationwide study of haemolytic uraemic syndrome: clinical, J Pediatr 1990;116:589-92. microbiological, and epidemiological features. Arch Dis Child 51. Krmar RT, Ferraris JR, Ramirez JA, and others. Ambulatory 2001;85:125-31. blood pressure monitoring after recovery from hemolytic uremic 63. Tarr PI, Neill MA. Perspective: the problem of non-0157:H7 syndrome. Pediatr Nephrol 2001;16:812-6. shiga toxin (verocytotoxin)-producing Escherichia coli. J Infect Dis 52. Remuzzi G. The future of reno-protection: frustrations and 1996;174:1136-9. promises. Presented at: World Congress of Nephrology; June 8-12, 64. Karch H, Janetzki-Mittmann C, Aleksic S, and others. Isolation of 2003; Berlin, Germany. enterohemorrhagic Escherichia coli 0157 strains from patients with 53. Gagnadoux MF, Habib R, Gubler MC, and others. Long-term (15- hemolytic-uremic syndrome by using immunomagnetic separation, 25 years) outcome of childhood hemolytic-uremic syndrome. Clin DNA-based methods and direct culture. J Clin Microbiol 34:516-9. Nephrol 1996;46(1):39-41. 65. United States General Accounting Oﬃce. Emerging infectious 54. Fitzpatrick MM, Shah V, Trompeter RS, and others. Long term diseases: consensus on needed laboratory capacity could strengthen renal outcome of childhood haemolytic uraemic syndrome. BMJ surveillance. Report to the Chairman, Subcommittee on Public 1991;303:489-92. Health, Committee on Health Education, Labor, and Pensions, 55. Nelid GH. Haemolytic-uraemic syndrome in practice. Lancet U.S. Senate. Washington (DC): GAO/HEHS-99-26; 1999. 1994;343(8894):398-401. 66. Ludwig K, Sarkim V, Bitzan M, and others. Shiga toxin-producing 56. Fernandez GC, Rubel C, Dran G, and others. Shiga toxin-2 induces Escherichia coli infection and antibodies against Stx2 and Stx1 in neutrophilia and neutrophil activation in a murine model of hemolytic household contacts of children with enteropathic hemolytic-uremic uremic syndrome. Clin Immunol 2000;95(5):227-34. syndrome. J Clin Microbiol 2002;40(5):1773-82. 57. Siegler RL. Spectrum of extrarenal involvement in postdiarrheal 67. Byun Y, Ohmura M, Fujihashi K, and others. Nasal immunization hemolytic-uremic syndrome. J Pediatr 1994;125:511-8. with E. coli verotoxin 1(VT)-B subunit and a nontoxic mutant 58. Siegler RL. Postdiarrheal shiga toxin-mediated hemolytic uremic of cholera toxin elicits serum neutralizing antibodies. Vaccine syndrome. JAMA 2003;290(10):1379-81. 2001;19:2061-70. 59. Safe tables our priority. Why are people still dying from contaminated 68. Suzaki Y, Ami Y, Nagata N, and others. Protection of monkeys food? Available at: www.safetables.org/pdf/STOP_Report.pdf. against shiga toxin induced by shiga toxin-liposome conjugates. Int Accessed May 21, 2004. Arch Allergy Immunol 2002;127:294-8. 60 Murano EA. Enhancing and evolving: advancements in 2003 and 69. Mukherjee J, Chios K, Fishwild D, and others. Human Stx2-speciﬁc initiatives to improve food safety in 2004; February 12, 2004; San monoclonal antibodies prevent systemic complications of Escherichia Antonio TX. coli 0157:H7 infection. Infect Immun 2002;70(2):612-9. 61. Bopp DJ, Sauders BD, Waring AL, and others. Detection, 70. Haemolytic Uraemic Syndrome Help. H.U.S.H – A Poem. Available isolation, and molecular subtyping of Escherichia coli 0157:H7 and at: www.ecoli-uk.com/where.htm. Accessed April 22, 2004. Campylobacter jejuni associated with a large waterborne outbreak. J Clin Microbiol 2003;41(1):174-80. 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. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 99

RESEARCH AND REPOR TS 2003 Workforce Survey of Hospital Clinical Laboratories in New Jersey ELAINE M KEOHANE, MARY ELLEN SCHAAD, KAREN FEENEY ABBREVIATIONS: CT = cytotechnologist; HLT = histo- 2002–2012, the U.S. Bureau of Labor Statistics projected a technologist; HT = histotechnician; MLT = medical labora- need for 138,000 new clinical laboratory technologists and tory technician; MT = medical technologist. technicians, or approximately 13,800 per year, due to growth and attrition from the ﬁeld.1 On the other hand, in 2002, INDEX TERMS: job opportunities; workforce. there were only 3,548 clinical laboratory technician/medical laboratory technician (CLT/MLT) and clinical laboratory sci- Clin Lab Sci 2005;18(2):100 entist/medical technologist (CLS/MT) graduates in the U.S.2 If the current imbalance between vacancies and graduates Elaine M Keohane PhD CLS(NCA) is Professor, Department continues, the national shortage of clinical laboratory person- of Clinical Laboratory Sciences, University of Medicine and nel may grow by more than 10,000 laboratorians per year. Dentistry of New Jersey, Newark NJ. In surveys conducted by the American Society for Clinical Pathology (ASCP), vacancy rates in 2000 for medical tech- Mary Ellen Schaad MT(ASCP) is Technical Manager, Clini- nologists (MTs) and medical laboratory technicians (MLTs) cal Laboratory, Meridian Health System, Neptune NJ. 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% Karen Feeney MPA MT(ASCP)DLM is Administrative and 8.6% nationally and 8.3% and 3.5% in the northeast. Director, Department of Laboratories, Bayshore Community Although the vacancy rates in the latter study decreased to Hospital, Holmdel NJ. single digits, the vacancies are nevertheless noteworthy in terms of the actual number of vacant positions, taking into Address for correspondence: Elaine M Keohane PhD consideration a national workforce estimated at 297,000 CLS(NCA), University of Medicine and Dentistry of New clinical laboratory technologists and technicians.1 Jersey, School of Health Related Professions, 65 Bergen Street, Newark NJ 07107. (973) 972-5510, (973) 972-8527 (fax). A Coalition for New Jersey Clinical Laboratory Personnel keohanem@umdnj.edu was formed in April 2002 to study the extent of and address a perceived shortage of clinical laboratory personnel in The authors are also members of the Coalition for New Jersey New Jersey. This coalition consists of twenty-eight members Clinical Laboratory Personnel. representing hospital clinical laboratory administrators, supervisors, and educators; hospital human resources directors; The clinical laboratory personnel shortage has reached and representatives from the New Jersey Society for Clinical signiﬁcant proportions in many areas of the country and Laboratory Science, New Jersey Clinical Laboratory Man- there is growing concern about its impact on the accessibil- agement Association, New Jersey Hospital Association, the ity and quality of clinical laboratory services. For the years New Jersey State Department of Health and Senior Services, and New Jersey Medicaid. One of the goals of the coalition The peer-reviewed Research and Reports Section seeks to publish is to document and disseminate data on the supply of and reports of original research related to the clinical laboratory or demand for clinical laboratory professionals in the state. An one or more subspecialties, as well as information on important unpublished study conducted by the New Jersey Society for clinical laboratory-related topics such as technological, clinical, Clinical Laboratory Science showed a 48.5% decrease in and experimental advances and innovations. Literature reviews MLT and MT graduates since 1998, with only 26 MTs and are also included. Direct all inquiries to David G Fowler PhD 21 MLTs graduating in 2003 in the entire state. In addition, CLS(NCA), Clin Lab Sci Research and Reports Editor, Dept of during that same time, the state experienced the closure of Clinical Laboratory Sciences, University of Mississippi Medical one MT and three MLT programs. There are no histotech- Center, 2500 North State St, Jackson MS 39216. (601) 984- nologist (HTL) programs, and only one histotechnician 6309, (601) 815-1717 (fax). dfowler@shrp.umsmed.edu (HT) program in the state, but that program recently went 100 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS into inactive status. There is also only one cytotechnologist RESULTS (CT) program. In 2003, these programs produced only seven A total of 55 surveys were received for a response rate of HT and five CT graduates. 57.9%. Forty-nine (51.6%) of the surveys contained data that were usable in the analysis, and represented data from Although there was some anecdotal information from New hospitals in fifteen of the twenty-one NJ counties (Table Jersey laboratory managers about difficulties in hiring quali- 1). A majority of the surveys (31) were received from the fied laboratory practitioners, there was insufficient data on Northern NJ counties. vacancies and shortages for these practitioners in the state’s workforce. Therefore, the Coalition conducted a survey of Forty-seven percent of the usable responses were from hos- hospital clinical laboratory managers to determine the extent pitals with greater than 300 beds, while 53% had less than of the clinical laboratory personnel shortage in NJ, and to 300 beds. A total of 33,094,905 annual billable laboratory begin a data collection process to project workforce needs tests were reported by 39 hospitals, ranging from 97,800 to into the future. 3,822,755 per hospital. A total of 2,697 total budgeted FTEs were reported by 49 hospitals. METHODS In January 2003, a one-page survey was mailed to the clinical Figure 1 depicts the breakdown of all categories of NJ clinical laboratory managers of the 95 hospitals in NJ. Surveys were laboratory personnel by age. A breakdown of personnel by coded for tracking purposes. A second survey was sent in March category and age is depicted in Figure 2 and Table 2. 2003 to the non-responders, followed by phone contact. The largest category reported was MT staff with 1,455 The survey requested data on county, hospital size, total employees in 49 hospitals. In the MT staff category, 50.8% number of billable tests, total current budgeted FTEs, the of the employees were over 45 years, and 14% were over number and age of clinical laboratory employees in six cat- 55 years, while 61.5% of the MT supervisors were over 45 egories, the number of current vacancies, and the average years, with 21.6% over 55 years. The MLT population was time it took to fill vacancies. The six personnel categories somewhat younger with only 38.3% over 45 years. In the included MT staff, MT supervisor, MLT, HTL, HT, and MLT, HTL, HT, and CT categories, 10% of the employees CT. In addition, managers were asked to indicate if they were over 55 years. had difficulties hiring or recruiting for a particular position, department, or shift, and if they had incentives in place to Table 3 lists the number of vacancies by category and hire laboratory personnel. All survey responses were received region. The highest number occurred in the MT category between February and April 2003, and were reviewed, tabu- with 49 full time and 52 part time vacancies among the lated, and summarized. Figure 1. Percent distribution of all categories of NJ laboratory personnel by age (n = 2,004). Note Table 1. Usable surveys by New Jersey County. that 49.1% are older than 45 years, and 13.8% are Numbers represent surveys received by region and older than 55 years 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 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 101

R E S EARCH AND REPORTS 49 hospitals. In the MT supervisor category, there were cies. There were seven vacancies for full time HT and one 11 full time and 4 part time vacancies, and in the MLT vacancy for a full time CT. category, there were 6 full time and 20 part time vacan- 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% Figure 2. Percent distribution of categories of laboratory personnel by age 102 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS Table 4 summarizes the responses for the average time to fill 26.5% had tuition reimbursement, 24.5% had a sign-on vacancies in the various categories. The time ranged from an bonus, and 20.4% made market adjustments in salaries. The average of 10.6 and 11 weeks for MLTs and MTs, respectively, incentives are summarized in Figure 4. In addition, some to 104 weeks for HTs. laboratory managers had comments related to their hiring difficulties. These are summarized in Table 5. A majority of laboratory managers reported difficulties in hiring generalist MTs (71.4%), as well as night shift DISCUSSION (46.9%), evening shift (28.6%), HT (24.5%), and blood The data reflect responses from over half of the hospital bank (20.4%) positions. Other positions in which difficul- clinical laboratories in NJ. The largest number of surveys ties in hiring were reported included part time, weekend, being received from the northern region of the state re- CT, and general supervisory positions. Figure 3 presents a flects the larger number of hospitals in that region. The summary of the data. The miscellaneous category includes responding hospitals were almost evenly divided between one to two responses each for microbiology technologist, those that are greater than 300 beds and those that are phlebotomist, evening supervisor, blood bank supervisor, less than 300 beds. and histology supervisor. A total of 33,094,905 annual billable tests were reported by Laboratory managers reported using the following incentives 39 hospitals. Projecting that figure across 95 hospitals at an for hiring laboratory personnel: 42.9% of laboratory man- average cost of $14 per test, hospital laboratories represent agers reported no incentives, 30.6% had shift differentials, a billion dollar industry in the state. 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) VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 103

R E S EARCH AND REPORTS Nearly half of the practitioners employed in the responding be as high as 116 full time and 109 part time MT positions. NJ hospital clinical laboratories are over the age of 45, which Given that only 26 MTs graduated in 2003, and that not all is only slightly lower than the median age of 47 years reported of that cohort will choose to work in hospital laboratories, in 2002 for clinical laboratory practitioners nationwide. In there was a significant shortfall of MTs with approximately NJ, 13.8% of the clinical laboratory professionals in the 49 eight vacant MT positions per MT graduate. Over 70% responding hospitals were over the age of 55; projecting that of the laboratory managers reported difficulty in filling number over the 95 hospitals in the State, it is likely that vacant MT generalist positions, while difficulty in filling over 500 laboratorians in the current workforce will be over night, evening, and blood bank positions were reported by 65 within the next 10 years. 46.9%, 28.6%, and 20.4% of the managers, respectively. As indicated in a comment in Table 5, an unknown, but prob- There were a total number of 2,697 budgeted FTEs in 49 ably significant, number of MTs work more than one job to hospitals, with 2,331 current clinical laboratory employees. supplement their salaries. If this did not occur, (assuming Using these figures, an estimate of the overall vacancy rate the second job is in another hospital laboratory) the shortage for hospital laboratory personnel is 13.6%. would be even greater. During the 3-month survey period, there were a total of 49 During this same period, there were 4 full time and 20 part full time and 52 part time vacancies for staff MTs, and 11 time MLT vacancies. Again, projecting these figures to the full time and 4 part time vacancies for supervisory MTs in 95 hospitals in the state, the amount of actual vacancies the 49 responding hospitals. Projecting those figures to the could be as high as 8 full time and 39 part time positions. 95 hospitals in the state, the number of actual vacancies could Given that only 21 MLTs graduated in 2003, and that not 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) 104 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

R E S EARCH AND REPORTS all of that cohort will work in hospital laboratories, there weeks to fill a CT vacancy (Table 4). In fact, one hospital was a shortage of MLTs to fill the open part time positions. reported closing their cytology department since they were Some laboratory managers indicated that they would hire a unable to fill a vacant CT position (See Table 5). MLT to fill a vacant MT position if a qualified MT was not available, prompting them to reevaluate their staffing mix of There are no HTL programs in the state, and only the one MLTs vs. MTs. If this staffing shift should occur, there will HT Program in the southern region of the state. That one likely be more vacant MLT positions, thus exacerbating the HT Program, however, recently went inactive. It was pro- MLT shortage. ducing five to ten graduates per year; however, the graduates traditionally preferred employment in their home region, Although there were only two vacancies for CTs, there is only resulting in unfilled positions in the northern and central one program in the state producing five to eight graduates per regions of the state. Nearly 25% of the laboratory managers year, many of whom are employed, often at higher salaries, reported difficulty in filling HT positions, with up to 104 by reference laboratories. This situation creates difficulties in weeks required to fill a vacant position (Table 4). With no filling vacancies at some hospitals, requiring as long as 78 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 Table 5. Comments from laboratory managers 2002–2012 the clinical laboratory field would experience a about hiring difficulties combination of growth and attrition requiring 13,800 new clinical laboratory technologists and technicians per year. “Most employees are working more than one job to Although the simplification and automation of tests will earn a living. This makes it very difficult to staff the result in some loss of positions, a net increase in need for new lab.” practitioners was predicted due to the population growth, an increase in the elderly population, and the introduction “Shift differential increases are desperately needed of new types of tests that will spur the utilization of more but being delayed at least 6 months due to reim- laboratory services. bursement problems.” The general staffing pattern was 3.4 MTs to 1 MLT in NJ “Much difficulty in recruiting supervisors due to hospital laboratories. Utilization of MLTs to fill some of the lack of qualified outside applicants and lack of vacant MT positions may help to alleviate the shortage and interest in qualified in-house staff due to salary spur an increase in enrollments in MLT Programs. compression with increased responsibilities.” Supply and demand in the state’s clinical laboratory work- “We gave up trying to fill a part time MLT day job force will continue to be monitored by surveying the edu- and converted it to a lab aide.” cational programs and laboratory managers every two years, and developing a prediction model for future needs. It is “Blood bank position vacant since 3/02; histologist also recommended to continue to study and address work- position vacant since 8/02.” ing conditions, incentives, and salaries to make the clinical laboratory profession more attractive and competitive with “Could not find a CT, so closed cytology.” other fields that require comparable education, and to begin to study the impact of the shortage on the delivery and ac- “Unable to find qualified candidate for blood cessibility of quality laboratory services. This information is bank supervisor. Three blood bank techs went to critical for strategic planning for both laboratory managers industry. Histotech asking salary was above our and educators. approved range.” The Coalition launched a Website in May 2003 (www.lab- “We are lucky enough to have a MT school; CTs science.org) to inform potential students and science teachers and HTs are extremely hard to find.” about the clinical laboratory professions. The Coalition will VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 105

R E S EARCH AND REPORTS continue to address the shortage through efforts to promote nies and commercial laboratories in NJ, the state’s hospitals awareness of the professions to potential students and the must compete for skilled laboratory personnel with those public, to assist in recruitment of students into the state’s industries that recruit professionals out of the hospital setting educational programs, to encourage hospitals, colleges, and with higher wages and/or better working conditions, e.g., no universities to maintain their educational programs, and to weekends, holidays, etc. MT programs either need to expand share information about successful retention programs to or new programs need to be established, and perhaps the keep our highly skilled professionals in hospital settings. hospital laboratory industry in the state should re-examine the utilization of MLTs vs. MTs in the workplace. Nationwide, and statewide, clinical laboratory practitioners are not being produced in sufficient quantity to meet the cur- REFERENCES rent and future demand. Nationwide, it has been estimated 1. Hecker DE. Occupational employment projections to 2012. Monthly Labor Review. February 2004: 80-105. that the shortage is growing by over 10,000 laboratorians per 2. American Medical Association, Health Professions: Career and year. In NJ, even if the five existing MT programs operate at Education Data Book 2003-2004. Chicago: AMA;2003. full capacity (an estimated 52 students per year), there will 3. Ward-Cook K, Chapman S: 2002 wage and vacancy survey of medical still be insufficient graduates to meet the current and the laboratories. Lab Med 2003,34:702-7. projected demand for MTs in the future. In addition, due 4. Ward-Cook, K, Tanner S. 2000 wage and vacancy survey of medical laboratories. Lab Med 2001,32:124-38. to the location of a large number of pharmaceutical compa- 106 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

FOCUS: PSYCHOSTIMULANTS Mechanism of Action and Therapeutic Uses of Psychostimulants KEVIN F FOLEY ABBREVIATIONS: ADHD = attention-deficit hyper- Control Act of 1970. Schedules I through IV are assigned to activity disorder; CNS = central nervous system; MDA = compounds based on their abuse potential and their medical 3,4-methylenedioxyamphetamine; MDMA = 3,4-methyl- utility. Schedules II through V contain drugs which have enedioxymethamphetamine; NC = not controlled; OTC = known medical uses whereas Schedule I compounds have over the counter. no current, sanctioned medical use. INDEX TERMS: psychostimulants. THERAPEUTIC USES OF PSYCHOSTIMULANTS Amphetamine has long been known to be a mental stimulant. Clin Lab Sci 2005;18(2):107 Because of its psychostimulant properties, amphetamine has been used successfully by U.S. fighter pilots because it enhances Kevin F Foley PhD MT(ASCP) is with the Department of Bio- cockpit performance by reducing the effects of fatigue.1 Since medical Technologies, University of Vermont, Burlington VT. amphetamine heightens alertness it has found use in the treatment of narcolepsy and in attention-deficit hyperactivity disor- Address for correspondence: Kevin F Foley PhD MT (ASCP), der.2 The drug Adderall® for example, is used widely in cases of Assistant Professor, University of Vermont, Department of Medi- ADHD and is merely a mixture of amphetamine’s stereoisomers: cal Laboratory and Radiation Sciences, 302 Rowell Building, dextro- and levoamphetamine. 106 Carrigan Drive, Burlington VT 05405. (802) 656-2506. kfoley@uvm.edu. Amphetamine also has appetite suppressant effects and thus is used in the treatment of obesity, although it is not FDA Victor A Skrinska PhD DABCC is the Focus: Psychostimu- approved for this use. Although amphetamine can be consid- lants guest editor. ered the prototype psychostimulant, many psychostimulants have been identified or created that resemble amphetamine Focus Continuing Education Credits: see pages 124 to 126 for in their chemical structures. These compounds vary in their learning objectives, test questions, and application form. effects and utility as well as their abuse potential. Meth- amphetamine, like amphetamine has a history of illicit use The name stimulant can be given to any drug that increases the and is known to be considerably more potent in vivo than rate of a physiologic function. The term psychostimulant is more the unmethylated amphetamine.3,4 Methamphetamine, like specific, referring to compounds that have direct neurological amphetamine, has been used with some success in ADHD effects, typically: heightened alertness, increased energy, appetite patients. It is also approved for use in treating obesity.5 suppression, and sometimes euphoria. Use of psychostimulants is widespread and occurs in both recreational and clinical set- Pseudoephedrine, the name given to the 1R2R enantio- tings. Therapeutic monitoring and screening for use and abuse mer, and ephedrine, the 1R2S enantiomer, are common of these drugs is common in clinical laboratories. Understanding medications used primarily as nasal decongestants. Unlike the physiological effects and uses of stimulants will be of value amphetamine and methamphetamine they have little abuse to clinical and forensic laboratory scientists. potential allowing them to be obtained OTC.6 Ephedrine is also approved for treatment of bronchospasm, enuresis, EXAMPLES OF PSYCHOSTIMULANTS hypotension, and narcolepsy.5 Table 1 lists several psychostimulants that are commonly prescribed, have widespread illicit use, or are well-known by Some psychostimulants have been found to have uses that the general public. Table 1 also identifies the control sched- are not typical for other drugs in the same class. Although ule of each compound. Many psychostimulants are listed as bupropion is a phenylalkylamine, the structural dissimilar- controlled substances under the Controlled Substances Act, ity with amphetamine is significant enough to produce Title II of the Comprehensive Drug Abuse Prevention and considerably different physiologic effects. Bupropion is an VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 107

F O C U S: PSYCHOSTIMULANTS example of a weak psychostimulant that has found use as an and 3,4-methylenedioxymethamphetamine (MDMA) are antidepressant (Wellbutrin®) and is also marketed as a smok- popular recreational drugs known respectively as ‘the love ing cessation aid (Zyban®). Although still a mild psycho- (or hug) drug’ and ‘ecstasy’. MDMA use is growing rapidly stimulant, the behavioral effects of bupropion are reported and in some European countries is the second most fre- to be unlike amphetamine when used in humans. The abuse quently used illegal drug after marijuana.10 Interestingly, both potential is also considerably less than with amphetamine.7 MDMA and MDA were used therapeutically before being Off-label uses of bupropion include treatment of ADHD, classified Schedule I in 1985. These compounds were used diabetic neuropathy, and neuropathic pain.5 Another clini- by patients in constructive psychotherapy sessions.8 MDMA cally-useful β-ketone psychostimulant is diethylpropion. has received much negative attention in recent years as claims Diethylpropion is currently used only in the treatment of of its neurotoxicity mounted. However data concerning the obesity. This psychostimulant is commonly prescribed as an neurotoxicity of MDMA have been inconsistent and one appetite suppressant under the name Tenuate®. major study was even retracted after it was found that the toxicity in the study was brought about by methamphetamine Methcathinone is a Schedule I psychostimulant that is eas- rather than MDMA.11,12 The widespread use and popularity ily synthesized from ephedrine. Methcathinone is the N- of MDMA coupled with the lack of definitive reports on methylated version of cathinone, a natural psychostimulant neurotoxicity have encouraged some to reconsider the util- obtained from the Catha edulis plant and known as “qat” or ity of this drug.13-15 Because of MDMA’s unique effects, a “khat”.8 Methcathinone has no sanctioned medical use and new study investigating the therapeutic use of MDMA has is only used recreationally. Its effects are those of a classic recently earned IRB and FDA approval. This research will psychostimulant, causing ‘flying euphoria’ followed by a assess the use of MDMA in the treatment of post-traumatic five-to-eight hour period of feeling tough, invincible with stress disorder.16,17 increased libido, and desire to be physical.8,9 Perhaps the most well-known psychostimulant after amphet- The next two compounds listed in Table 1 are also Schedule amine is methylphenidate, made popular under the trade name I compounds. 3,4-methylenedioxyamphetamine (MDA) Ritalin®. Methylphenidate has pharmacological effects similar 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. 108 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S: PSYCHOSTIMULANTS to amphetamine; however, its abuse po- mine combination was also associated cause amphetamine is considered the tential is somewhat lower, although there with valvulopathies and a bulletin was prototype stimulant, other compounds are many conflicting reports regarding issued by the FDA to cease off-label that have similar chemical structures methylphenidate abuse potential (see use of the Fen-Phen combination.5,21 and similar physiologic effects are often reference 18 for review). Methylpheni- Of the two drugs, only phentermine termed ‘amphetamines’ (Figure 2). date has become the drug of choice for is still used as an appetite suppressant treating ADHD but has also found off- although tolerance to the anorexiant Given their structural similarity to en- label use as an antidepressant.5 effects of phentermine usually develops dogenous neurotransmitters, it is not a few weeks after starting therapy. surprising that many phenylalkylamines Two other drugs worth mentioning have autonomic nervous system activ- as psychostimulants are phentermine When considering the clinically-used ity, i.e., sympathomimetics, as well as and fenfluramine. Both of these drugs drugs listed in Table 1, cocaine is the mood-altering effects. Amphetamine have been used as appetite suppres- only one that is not used for its psycho- and other closely related phenylal- sants and both have amphetamine-like stimulant effects. Cocaine is used clini- kylamines can activate receptors that sympathomimetic effects. Fenflu- cally only as a local anesthetic, usually in normally bind catecholamines or ramine contains a chiral center and mucosal or ophthalmic procedures. serotonin. In addition, amphetamine fenfluramine is the name given to the and related compounds can cause the racemic mixture of D- and L-isomers. MECHANISM OF ACTION release of catecholamines and serotonin The D-isomer (dexfenfluramine) It is easy to see the resemblance from nerve endings.22-26 Once released, was purportedly responsible for the between the chemical structures of the endogenous neurotransmitters are anorectic actions of fenfluramine and common psychostimulants and endog- free to act on their extracellular recep- was also associated with fewer side ef- enous monoamine neurotransmitters tors. When catecholamine receptors in fects than the racemic fenfluramine. (Figure 1). The prototype psychostim- the brain are activated, myriad effects Dexfenfluramine was marketed as ulant amphetamine closely resembles can result. Neuropsychological effects Redux®.19 By 1997 both fenfluramine the catecholamine neurotransmitters of catecholamine receptor activation and dexfenfluramine were removed norepinephrine, epinephrine, and do- or potentiation by psychostimulants from the U.S. market at the request pamine. Since many psychostimulants can include: increased alertness, in- of the FDA when cases of cardiac share the features of a phenyl ring, a somnia, euphoria, decreased appetite, valvulopathy were reported.5,20,21 Fen- nitrogen group, and carbon side chains and at higher doses, psychosis. Indeed, fluramine is perhaps best known by its of varying lengths, many stimulants fall amphetamine can correctly be called a off-label use with phentermine, known into the category of phenylalkylamines. “psychotomimetic” since high doses can as “Fen-phen”. Fen-phen was widely With the possible exception of cocaine, bring about a psychosis very similar to used for the long-term management all the compounds listed in Table 1 can that seen in schizophrenic patients.27,28 of obesity. The fenfluramine-phenter- be classified as phenylalkylamines. Be- In the periphery, activation or potentiation of catecholamine receptors by psychostimulants can result in: vaso- Figure 1. Structure of amphetamine and catecholamine neurotransmitters constriction and subsequent hypertension, mydriasis, tachycardia, and other general sympathomimetic effects. The mechanism of action of amphetamine and amphetamine-like psychostimulants involves four major effects: 1. Binding to extracellular catecholamine receptors 2. Inhibition of monoamine neurotransmitter uptake 3. Release of catecholamines from neurons 4. Inhibition of monoamine oxidase VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 109

F O C U S: PSYCHOSTIMULANTS Psychostimulants alter neurotransmitter release. Psycho- In addition to having direct receptor-binding effects, many stimulants can effectively alter the amount or the rate of psychostimulants inhibit monoamine transporters. Mono- neurotransmitter released by a monoaminergic neuron. The amine transporter proteins serve to recycle neurotransmitters resulting change in mood or behavior is the summation of after they are released from the neuron and in so doing, ter- these neuromodulations and is quite complex. At the cellular minate the neurotransmitter signal. These same monoamine level this neuromodulation is due to one or more of the ef- transporters are also the targets for antidepressant medica- fects listed above. Amphetamine-like compounds have a wide tions including the popular drugs fluoxetine (Prozac ®), range of affinities to catecholamine and serotonin receptors. paroxetine (Paxil®) and sertraline (Zoloft®). Inhibitors of The overall pattern of receptor binding for a psychostimulant monoamine transporters block the uptake, or ‘reuptake’, of is unique for a given stimulant and contributes to the distinc- neurotransmitters by neurons (Figure 3). This blockade effective behavioral effects of a given compound. tively increases the concentration of neurotransmitter in the synapse, resulting in increased binding of neurotransmitters to their receptors. 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). 110 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S: PSYCHOSTIMULANTS Psychostimulants can also elevate the concentration of MAO is a mitochondrial enzyme that breaks down mono- neurotransmitter in the synapse by evoking release of neu- amine neurotransmitters. Products of catecholamine break- rotransmitters. The release of stored neurotransmitters can down include 3-methoxy-4-hydroxymandelic acid (VMA), be triggered directly when psychostimulants bind receptors homovanillic acid (HVA), and dihydroxyphenylacetic acid present on neurons or can release neurotransmitters indirectly (DOPAC). These compounds are commonly-measured me- via an exchange mechanism occurring through monoamine tabolites of catecholamines whose formation is due in part or transporter proteins. The exchange mechanism or efflux in full to MAO. Inhibition of MAO would thus bring about process which can occur via monoamine transporters has an expected increase in monoamine neurotransmitters since been observed with many amphetamine derivatives.29,30 This their breakdown would be impeded. efflux is thought to be a type of ‘reverse-transport’ mediated by monoamine transporters.31,32 Finally, amphetamine and In summary, psychostimulants can affect neurotransmitter amphetamine-like psychostimulants often act as competitive release in at least four ways; by binding extracellular receptors, inhibitors of the enzyme monoamine oxidase (MAO).33,34 by blocking monoamine transporters, by evoking neurotrans- 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. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 111

F O C U S: PSYCHOSTIMULANTS mitter release, and by antagonizing the enzyme MAO (Figure and these systems are also affected by psychostimulants like 3). A given psychostimulant can have multiple mechanisms of amphetamine.38,39 In addition to appetite control, amphet- actions. Indeed, the actions of some psychostimulants, such amine is also used for its ability to focus attention. The basis as MDMA and bupropion have not yet been fully explained for this effect is not well understood. The seemingly para- but it is clear that all or some of the mechanisms discussed doxical fact that psychostimulants can act as calming agents play a role in the unique actions of these drugs. in humans seems enigmatic. The effects of amphetamine on attention and alertness are ultimately due to the modulation PHYSIOLOGIC EFFECTS OF PSYCHOSTIMULANTS of normal patterns of central activity. These modulations are Having already considered the cellular actions of psycho- brought about by modulating serotonergic, dopaminergic, stimulants it is worth mentioning the overall physiologic and noradrenergic pathways but the precise mechanism of effects of these drugs. Because of the complex circuitry in action of amphetamine in ADHD is unknown (see references the brain and the overlap of monoamine neurotransmitter 2 and 40 for reviews). systems, understanding how subtle differences in chemical structure bring about significantly different psychological Euphoria, of course, is an important, high-dose effect of effects can be difficult. Although all differ in their overall amphetamine. The euphoric effects of amphetamine and physiological effects, the psychostimulants listed in Table 1 methamphetamine are very similar to cocaine however un- do share the following adverse side effects: agitation/anxi- like cocaine, amphetamine and methamphetamine are taken ety, dizziness, restlessness, psychosis, and seizures. Many of orally and are metabolized more slowly than cocaine, making these compounds can also cause hypertension, insomnia, it the psychostimulant of choice for many drug users. Cen- hyperthermia, tachycardia, and euphoria.5 A discussion of trally, amphetamine and cocaine cause an acute dopamine the psychopharmacological mechanisms of action of each release in addition to inhibiting neuron dopamine uptake.41-43 individual psychostimulant is beyond the scope of this article This release of dopamine in the nucleus accumbens and (see reference 35 for excellent reviews). prefrontal cortex is thought to cause the prominent euphoric and reinforcing effects associated with amphetamine and In general, the physiologic effects of psychostimulants can be cocaine.44 These psychostimulants can produce a significant represented by the prototype psychostimulant, amphetamine. euphoria, locomotor stimulation, reduced fatigue, sexual In the periphery amphetamine acts as a sympathomimetic. stimulation, increased mental attention, and increased social- Mydriasis, hypertension, and tachycardia would be expected ity. Higher doses can produce tremors and vomiting, as well from a compound with close structural similarity to endog- as tonic-clonic convulsions. enous catecholamines. Amphetamine releases norepinephrine from sympathetic nerve endings which causes increases in CONCLUSION systolic and diastolic blood pressure, often causing a reflex- Psychostimulants are important pharmacological agents used decrease in heart rate. At higher doses or overdoses tachycardia in a variety of conditions including the treatment of obesity, or cardiac arrhythmias can occur.5 Because of these cardio- ADHD, narcolepsy, and as decongestants. Psychostimulants vascular effects, amphetamine is contraindicated in patients typically contain a phenylalkylamine structure that closely with arteriosclerosis, coronary artery disease, hypertension, resembles that of endogenous monoamines. The actions of and closed-angle glaucoma.5 Interestingly, the commonly used psychostimulants are mediated through their ability to bind drugs ephedrine and pseudoephedrine are utilized for their monoamine receptors and/or modulate monoamine transmitter vascular effects. By acting directly on alpha-adrenergic recep- release. Although psychostimulants have a history of abuse and tors in the mucosa of the respiratory tract, pseudoephedrine misuse their therapeutic uses are numerous and significant. produces vasoconstriction leading to nasal decongestion. REFERENCES In the CNS, amphetamine modulates monoamine neu- 1. Emonson DL, Vanderbeek RD. The use of amphetamines in U.S. rotransmitter release and kinetics. The overall effect of these Air Force tactical operations during Desert Shield and Storm. Aviat Space Environ Med 1995;66:260-3. actions is consistent with known roles of monoamines in the 2. Gainetdinov RR, Wetsel WC, Jones SR, and others. Role of serotonin brain. 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Pharmacol Biochem Behav mechanism of deamination as probed by steady-state methods. 2002;71:845-55. Biochem 1985;24:1821-6. 15. Pentney AR. An exploration of the history and controversies surrounding 35. Neuropsychopharmacology, The fifth generation of progress. Phila- MDMA and MDA. J Psychoactive Drugs 2001;33:213-21. delphia: Lippincott, Williams and Wilkins, 2002. 16. Michael C. Mithoefer MD, Mark T, Wagner PD. Experimental 36. Frankish HM, Dryden S, Hopkins D, and others. Neuropeptide Y, proposal: a human phase II study –safety and efficacy of 3,4-methy- the hypothalamus, and diabetes: insights into the central control of lenedioxymethamphetamine (MDMA)-assisted psychotherapy in metabolism. Peptides 1995;16:757-71. the treatment of chronic posttraumatic stress disorder (PTSD). 37. Samanin R, Garattini S. Neurochemical mechanism of action of Charleston SC: Medical University of South Carolina, 2002. anorectic drugs. Pharmacol Toxicol 1993;73:63-8. 17. Weiss R. DEA approves trial use of ecstasy in trauma cases. The 38. Blundell JE, Leshem MB. The effect of 5-hydroxytryptophan on food Washington Post March 2, 2004:02. intake and on the anorexic action of amphetamine and fenfluramine. 18. Kollins SH, MacDonald EK, Rush CR. Assessing the abuse potential J Pharm Pharmacol 1975;27:31-7. of methylphenidate in nonhuman and human subjects: a review. 39. Halford JC, Blundell JE. Separate systems for serotonin and leptin Pharmacol Biochem Behav 2001;68:611-27. in appetite control. Ann Med 2000;32:222-32. 19. Bremer JM, Scott RS, Lintott CJ. Dexfenfluramine reduces cardiovas- 40. Ohno M. The dopaminergic system in attention deficit/hyperactivity cular risk factors. Int J Obes Relat Metab Disord 1994;18:199-205. disorder. Congenit Anom (Kyoto) 2003;43:114-22. 20. Shively BK, Roldan CA, Gill EA, and others. Prevalence and deter- 41. Karoum F, Chrapusta SJ, Brinjak R, and others. Regional effects minants of valvulopathy in patients treated with dexfenfluramine. of amphetamine, cocaine, nomifensine, and GBR 12909 on the Circ 1999;100:2161-7. dynamics of dopamine release and metabolism in the rat brain. Br J 21. Connolly HM, Crary JL, McGoon MD, and others. Valvular heart Pharmacol 1994;113:1391-9. disease associated with fenfluramine-phentermine. N Engl J Med 42. Reid MS, Hsu K, Jr., Berger SP. Cocaine and amphetamine prefer- 1997;337:581-8. entially stimulate glutamate release in the limbic system: studies on 22. Rubin RP, Jaanus SD. A study of the release of catecholamines from the the involvement of dopamine. Synapse 1997;27:95-105. adrenal medulla by indirectly acting sympathomimetic amines. Naunyn 43. Jacocks HM 3rd, Cox BM. Serotonin-stimulated release of Schmiedebergs Arch Pharmakol Exp Pathol 1966;254:125-37. [3H]dopamine via reversal of the dopamine transporter in rat 23. Magyar K, Knoll J. Para-substituted amphetamines and brain sero- striatum and nucleus accumbens: a comparison with release elicited tonin. Pol J Pharmacol Pharm 1975;27:139-43. by potassium, N-methyl-D-aspartic acid, glutamic acid and D-am- 24. Paton DM. Structure—activity relations for the acceleration of efflux phetamine. J Pharmacol Exp Ther 1992;262:356-64. of noradrenaline from adrenergic nerves in rabbit atria by sympatho- 44. Moghaddam B, Bunney BS. Differential effect of cocaine on extra- mimetic amines. Can J Physiol Pharmacol 1975;53:822-9. cellular dopamine levels in rat medial prefrontal cortex and nucleus accumbens: comparison to amphetamine. Synapse 1989;4:156-61. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 113

FOCUS: PSYCHOSTIMULANTS The Use and Abuse of Psychostimulants SUSAN B GOCK, VICTOR A SKRINSKA ABBREVIATIONS: DAWN = Drug Abuse Warning scription medications, over the counter (OTC) preparations, Network; ED = emergency department; MDA = methyl- and dietary supplements. Clinical indications for therapeutic enedioxyamphetamine; MDMA = methylenedioxymetham- use include treatment of narcolepsy, attention deficit disorder, phetamine; OTC = over the counter. and as an appetite suppressant in the treatment of obesity. Pharmacological effects of psychostimulant drugs include the INDEX TERMS: drug abuse; psychostimulants. ability to increase alertness, relieve fatigue, decrease appetite, elevate mood, increase confidence, and produce euphoria. Clin Lab Sci 2005;18(2):114 Abuse of psychostimulant drugs may lead to tolerance that is exhibited by the need of higher doses of the drug to pro- Susan B Gock MS MT(ASCP) is Technical Director, Mil- duce the same desired effects. Consequently, users may try waukee County Medical Examiner’s Toxicology Laboratory, to intensify the drug’s positive effects by increasing the drug Milwaukee WI. dosage, taking it more frequently, or changing the route of administration leading to the possibility of drug abuse, mis- Victor A Skrinska PhD DABCC is Professor and Chair, Uni- use, or toxicity. The most common psychostimulant drugs of versity of Alabama at Birmingham, Birmingham AL. forensic interest with documented abuse potential are listed in Table 1. These drugs are characterized as either sympa- Address for correspondence: Susan B Gock MS MT(ASCP), thomimetic or hallucinogenic amines with psychostimulant Forensic Laboratory Technical Director, Milwaukee County effects. Phentermine, fenfluramine, and diethylproprion are Medical Examiner’s Office, 933 West Highland Ave, Milwaukee related prescription medications used as appetite suppres- WI 53233. (414)-223-1228. sgock@milwcnty.com. sants. There is little epidemiological evidence to support the abuse of these three psychostimulant drugs. However, their Victor A Skrinska PhD DABCC is the Focus: Psychostimu- use is not recommended for individuals with current or past lants guest editor. drug abuse problems. Focus Continuing Education Credit: see pages 124 to 126 for TRENDS IN PSYCHOSTIMULANT ABUSE learning objectives, test questions, and application form. Statistics released by the Drug Abuse Warning Network (DAWN) reflect the reporting of specific illicit, prescrip- Drug use has become a significant medical and social problem in tion, and OTC drugs that are linked to drug abuse in visits the United States. Toxicological analysis of biological specimens to hospital emergency departments (EDs). The data are from individuals is generally accepted to be the most objective presented in two issues of The Dawn Report.1,2 In 2002, method for determining drug use and abuse. As a science, there were an estimated 670,307 ED visits related to drug forensic toxicology deals with the medico-legal implications of abuse in the continental U.S. This translates to 0.7% of all drug use, misuse, and abuse. This may include the following: visits. Over the past nine years, reports of drug-related ED criminal penalties imposed for the distribution, possession, and visits associated with drugs grew at roughly twice the rate use of illicit drugs; assessment of drug impairment in human of total ED visits. From 1994 to 2002, ED visits related to performance (behavioral) toxicology; assessment of drug toxic- drug abuse rose 29%, while total ED visits rose only 15%. ity as a contributing factor in the cause and manner of death in In 2002, about half (54%) of all visits related to drug abuse postmortem forensic toxicology cases; or detection of drug use involved multiple drugs. Four illicit drugs including cocaine, in workplace drug testing programs. marijuana, heroin, and methamphetamine accounted for 36% of the drugs involved in these visits. Psychostimulants include a diverse class of drugs exhibiting central nervous system stimulant properties, and have a high Cocaine continues to be the most frequently mentioned illicit abuse potential. Drugs in this class include illicit drugs, pre- substance reported to DAWN by hospital EDs nationwide.2 114 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S: PSYCHOSTIMULANTS Illicit use of psychostimulant drugs methylenedioxyamphetamine (MDA). gamma-hydroxybutyrate (GHB), reported in 2002 included 199,198 Visits involving the amphetamines and ketamine, flunitrazepam (Rohypnol), cases for cocaine; 17,696 cases for methamphetamine increased 54% and MDMA or ecstasy. For the year methamphetamine; and 4,026 cases between 1995 and 2002.4 Data also 2002, club or rave drugs were impli- for methylenedioxymethamphetamine show that methamphetamine abuse, cated in approximately 1.2% of all ED (MDMA). From 1995 to 2002, which was predominately located on visits related to drug abuse that were the rate of cocaine-related ED visits the west coast of the U.S. may be reported to DAWN.6 Some of these increased 33% whereas the rate of spreading eastward. This was dem- visits involved multiple club drugs, methamphetamine-related visits re- onstrated by an increase in ED visits that were frequently used in combina- mained stable. Visits involving cocaine for amphetamines and methamphet- tion with alcohol, marijuana, cocaine, exceeded the number of visits for any amine in several metropolitan areas in and heroin. The incidence of ED other illicit drug in 18 of the 21 met- the Midwest, South, and Northeast. visits involving MDMA is increasing ropolitan areas monitored by DAWN. However, even though there was based on data provided to DAWN.1,6 Western metropolitan areas including an increase, the overall numbers of In 1994 MDMA was mentioned 253 San Francisco, Seattle, San Diego, Los related visits in these areas remained times. In 2001 the number of times Angeles, and Phoenix had the highest low. More than 60% of the amphet- MDMA was mentioned increased to rates of methamphetamine ED visits. amine and methamphetamine visits 5,542 and then subsequently declined According to the National Drug Threat also involved other drugs. Marijuana, to 4,026 in 2002. In 2002, MDMA Assessment 2003, prepared by the alcohol, cocaine, benzodiazepines, opi- was the most common club drug de- National Drug Intelligence Center, oid pain relievers, and heroin were the tected in drug related ED visits, with cocaine is the primary drug threat in most frequently reported substances 75% of these visits involving patients the U.S. because of its high demand in combination with amphetamines 26 years or younger. and availability, expanding distribu- and methamphetamine. tion to new markets, high rate of Methylphenidate (Ritalin®), a sched- associated toxicity issues, and relation The term ‘club drugs’ is used to de- ule II substance, is associated with to violence.3 scribe illicit recreational drugs that patterns of abuse similar to other have gained popularity in recent years psychostimulants.7,8 Since it produces According to 2002 estimates from at nightclubs or large dance parties many of the same pharmacological ef- DAWN, there were almost 39,000 ED called ‘raves’.5,6 Club or rave drugs fects as cocaine or the amphetamines, visits related to drug abuse involving include stimulants, depressants, and a high potential for abuse of this drug amphetamines and methamphet- hallucinogenic substances frequently also exists. The primary legitimate amine. In more than half of these taken for their psychedelic and/or medicinal use of methylphenidate is visits the age of the patient was 18 euphoric effects to enhance dancing, for the treatment of attention deficit to 34.1,4 In the report, the category auditory, and visual perceptions. Drugs hyperactivity disorder (ADHD) in of amphetamines included dextro- in this class include the following: children. The increased therapeutic use amphetamine, methcatinone, and lysergic acid diethylamide (LSD), of this drug for treatment of ADHD is paralleled by an increase in the abuse of the drug among adolescents and Table 1. Common psychostimulant drugs of forensic interest young adults due to its increased availability. A recent survey of Wisconsin Drug Type schools found that most schools did Amphetamine Sympathomimetic stimulant not control how Ritalin was stored or Cocaine Sympathomimetic stimulant dispensed on school property, making Ephedrine Sympathomimetic stimulant it easy to steal, give away, or sell the Methamphetamine Sympathomimetic stimulant drug.9 Approximately 16% of the stu- Methylenedioxyamphetamine (MDA) Hallucinogen dents surveyed reported that they had Methylenedioxymethamphetamine (MDMA) Hallucinogen been asked to sell, give, or trade their Methylphenidate Sympathomimetic stimulant Ritalin to other students. This pattern of abuse is characterized by increasing VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 115

F O C U S: PSYCHOSTIMULANTS doses, frequent episodes of binge use followed by depres- bioavailability by the intranasal and smoked routes can be sion, and the desire to continue the use of the drug despite quite variable. Due to the convenience of administration, and medical and social consequences. The abuser may change the the rapid, intense onset of effects from the smoked route, route of administration of the drug from oral to snorting or intranasal and smoked routes are most commonly used for intravenous injection to intensify the effects. cocaine self-administration. Recent years have seen an increase in the use of dietary Cocaine is metabolized to benzoylecgonine and ecgonine supplements, not only to promote good health, but also as methyl ester, the two major metabolites, by different a means of obtaining a ‘natural’ legal high.10 Stimulants are mechanisms.12,13 Cocaine is metabolized in the blood to the most commonly abused dietary supplements used for this benzoylecgonine via spontaneous hydrolysis at a physiological purpose. Manufacturers of stimulant dietary supplements and alkaline pH and metabolized to ecgonine methyl ester market them as natural and safe alternatives for enhanced via enzymatic hydrolysis by pseudocholinesterase and liver mental alertness, weight loss, bodybuilding, and athletic esterases with the reaction rate being dependent on drug con- performance enhancement. See the table in reference 10 for centration. Both benzoylecgonine and ecgonine methyl ester information on common dietary supplements. Most stimu- are further metabolized to ecgonine. When cocaine is used in lant dietary supplements contain one or a combination of the combination with ethyl alcohol, a pharmacologically active following ingredients: ephedrine, pseudoephedrine, syneph- metabolite, cocaethylene (ethylcocaine), is produced by the rine, caffeine, or yohimbine. Herbal Ecstacy®, Metabolife®, transesterification of cocaine with ethyl alcohol. Therefore, in and Xenadrine® are examples of OTC dietary supplements cases of simultaneous alcohol and cocaine use, cocaethylene containing ephedrine as the selected stimulant. Even though concentrations should also be considered when interpreting these products are probably safe in most cases when used as results for assessment of toxicity. Anhydroecgonine methyl directed, a potential for abuse and toxicity still exists as with ester has been identified as a unique metabolite in the post- the other illicit, prescription, and OTC psychostimulants. mortem blood and urine specimens of persons after smoking According to the Toxic Exposure Surveillance System of the cocaine.14 Benzoylecgonine can be detected in blood within American Association of Poison Control Centers, ephedrine 15 to 30 minutes after intravenous, intranasal, and smoking products accounted for 77% of the cases of abused or misused routes of administration. Detection of benzoylecgonine in dietary supplements in 2002.10 Due to the increased abuse of the urine can provide a means of estimating an approximate stimulant dietary supplements with potential risk of serious window of time of the use of cocaine. Benzoylecgonine can adverse events including arrhythmias, seizures, heart attacks, be detected in urine for two to four days after use of cocaine and strokes, the FDA prohibited the sale of all dietary supple- depending on dose, frequency of use, urine pH, and clear- ments containing ephedrine as of April 12, 2004. ance. Detection has been reported seven to sixteen days after chronic compulsive cocaine use.12,13 PHARMACOKINETICS OF ILLICIT PSYCHO- STIMULANTS Ecstasy or MDMA is the most prominent member of the Cocaine, an alkaloid, is a naturally occurring central ner- methylenedioxy-substituted amphetamines (hallucinogenic vous system stimulant found in the South American plant amines) to gain popularity for illicit recreational drug use.5,6 Erythroxylon coca. For medicinal use as a local anesthetic, At normal doses, the effects include mild to moderate cen- cocaine is administered topically as a hydrochloride in 10% tral nervous system stimulating effects as well as enhanced to 20% solutions for the membranes of the throat and nose feelings of empathy, closeness, and response to intimate or in 1% to 4% solutions for ophthalmologic procedures.11 touch, which is why it is also classified as an empathogen- Street forms of cocaine are sold as either hydrochloride salt entactogen.13,15 Currently, there is no legitimate approved or crack. Crack is cocaine that has been processed from therapeutic use for MDMA in the U.S. The manufacture cocaine hydrochloride to a free base for smoking. Common of MDMA is typically in clandestine laboratories, primarily routes of administration include intranasal, intravenous, or in Western Europe where it is easier to obtain the precursor smoked. Oral is not the preferred route of administration chemicals. It is estimated that 80% of the MDMA consumed because, when taken orally, the first-pass effects result in worldwide is produced in Belgium, Luxembourg, and the low drug bioavailability and reduced euphoric effects due to Netherlands. The drug is typically supplied in tablet form inefficient delivery to the brain.12 The intravenous route of with an embossed logo, usually white but also available in a administration produces 100% drug bioavailability whereas variety of colors. Most frequently, MDMA is administered orally in tablet or capsule form in doses of 100 mg to 150 116 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S: PSYCHOSTIMULANTS mg. A popular variation on oral ingestion is ‘parachuting’, in which a tablet is crushed, wrapped in tissue paper, and Common routes of administration include oral, intranasal, swallowed for more rapid absorption.15 This is sometimes and intravenous. Smoking remains a minor route of ad- supplemented with an uncrushed tablet to achieve both rapid ministration compared to the others.16 Methamphetamine onset as well as a sustained effect. users feel a short yet intense ‘rush’ when the drug is initially administered. In abuse, there is a progression following the The onset of action following oral administration is 30 to 60 start of use, from oral or intranasal routes to intravenous minutes.5 MDMA has a half-life of approximately seven hours use of the drug. At higher doses, particularly following in- and undergoes N-demethylation to the active metabolite MDA, travenous use, users typically report intense exhilaration and which is the metabolite usually detected in blood. Illicitly pro- euphoria, elevated self-esteem, extreme wakefulness, rapid duced MDMA is a racemic mixture. The enantiomers exhibit flow of ideas, and increased physical and mental capacity. differences in pharmacological activity as well as affinity for the These effects are perceived as positive and generally encourage enzyme responsible for their metabolism.15 MDMA also exhibits repeated administration and produce a binge-type pattern nonlinear pharmacokinetics suggesting that beyond a certain of use.16 In the binge-type pattern of use, the initial positive threshold, small increases in dose may result in larger increases effects may be followed by restlessness, irritability, and pos- in blood concentrations with greater risk of toxicity.15 About sibly paranoid psychoses that reinforce the continued use of 65% of the dose is eliminated in the urine as parent drug and the drug to maintain the ‘high’ which eventually will lead to 7% as MDA.11 Mono- and di-hydroxy metabolites are excreted tolerance and psychological dependence. in the urine as conjugates. Methamphetamine undergoes phase I metabolism by N- Amphetamine and methamphetamine have limited le- demethylation to amphetamine, its major active metabolite gitimate pharmacologic use. Methamphetamine is the more via the cytochrome P450 2D6 isoenzyme system. Amphet- potent of these two drugs, producing greater central nervous amine is metabolized to a variety of metabolites, including system effects and having a longer duration of action, most norephedrine and p-hydroxyamphetamine, both of which are likely due to its greater ability to penetrate the central nervous pharmacologically active and may contribute to the effects system (CNS). Current therapeutic uses of these drugs are of the drug, especially during chronic usage. Accumulated for the treatment of narcolepsy, obesity, and ADHD. Am- hydroxylated metabolites have been implicated in the devel- phetamine can also be detected as a metabolite of other drugs opment of amphetamine psychosis.13 Under normal condi- including fenethylline, fenproporex, and methamphetamine. tions, up to 43% of a dose of methamphetamine is eliminated Illicit preparations of amphetamine usually contain a racemic unchanged in the 24-hour urine, with about 4% to 7% as mixture.11 Clandestine laboratories use phenylpropanolamine amphetamine.11 Elimination of the sympathomimetic amines as a precursor to amphetamine manufacture. Because of its is highly pH dependent. Urinary acidification to a pH less ease of manufacture and ready availability, methamphetamine than 5.6 decreases the plasma half-life from 11 to 12 hours has become the sympathomimetic amine of choice among to 7 to 8 hours whereas alkalinization increases the half-life stimulant abusers. In the U.S., methamphetamine is the to 18 to 34 hours. most frequently encountered clandestinely produced controlled substance.13 Methamphetamine is easily synthesized PSYCHOSTIMULANT TOXICITY from ephedrine. In the U.S., l-methamphetamine (under In toxic doses, the psychostimulants begin to produce the label l-deoxyephedrine) is the active constituent of the unpleasant CNS symptoms including anxiety, agitation, OTC decongestant Vicks Inhaler containing approximately hallucinations, delirium, seizures, and death.13 High-dose, 50 mg of drug.11, 16 This isomer is reported to have less CNS long-term use of stimulants can induce an acute psychotic activity and greater peripheral sympathomimetic activity than state in previously healthy individuals. CNS-induced ab- the d-enantiomer. l-methamphetamine can also be detected normalities, seizures, or muscular hyperactivity may induce as a metabolite of selegiline. d-methamphetamine is a legal hyperthermia. Secondary rhabdomyolysis may also be seen. prescription drug (Desoxyn®) and a prominent form in many Cardiovascular manifestations include hypertension, tachy- illicit methamphetamine drug preparations available as a cardia, arrhythmias, and myocardial ischemia. Cerebrovas- water soluble, white, crystalline powder (methamphetamine cular accidents are precipitated by elevated blood pressure hydrochloride). Determination of the enantiometric ratio or drug-induced vasospasms. can be useful in determining drug source as illicit, diverted, or licit.13,16 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 117

F O C U S: PSYCHOSTIMULANTS The clinical picture of stimulant intoxication also includes a REFERENCES wide array of psychiatric symptoms including schizophrenic 1. The DAWN report: trends in drug-related emergency department symptoms, manic-like states, psychoses, depressions (espe- visits, 1994-2004 at a glance. Drug Abuse Warning Network, Office cially during withdrawal), and various types of anxiety con- of Applied Statistics, Substance Abuse and Mental Health Services Administration. 2003. ditions including panic states.8 Psychotic symptoms usually 2. The DAWN report: major drugs of abuse in emergency department arise with chronic abuse but may also appear acutely with visits, 2002 update. Drug Abuse Warning Network, Office of Applied large doses of stimulants. With high doses of stimulants, Statistics, Substance Abuse and Mental Health Services Administra- symptoms of extreme anger in conjunction with aggressive tion. 2004. behavior can also be a catalyst for both violence and murder 3. National drug threat assessment, 2003. Publication # 2003-Q0317- 001. National Drug Intelligence Center. 2003. and is especially seen in cases of methamphetamine and 4. The DAWN report: amphetamine and methamphetamine ED visits, cocaine intoxication.12,16 1995-2002. Drug Abuse Warning Network, Office of Applied Statistics, Substance Abuse and Mental Health Services Administration. 2004. Many factors must be considered when interpreting psy- 5. Valentine JL, Kerrigan S. “Club” or “rave” drugs offer challenges to chostimulant concentrations in blood for an assessment of laboratories. Clin Forens Tox News 2001;September:1-8. toxicity since clinical and postmortem studies clearly show 6. The DAWN report: club drugs, 2002 Update. Drug Abuse Warning Network, Office of Applied Statistics, Substance Abuse and Mental that therapeutic, toxic, and lethal concentrations overlap. Health Services Administration. 2004. Tolerance, sensitization, and specimen stability problems are 7. Evans C, Blackburn D, Butt P, and others. Use and abuse of meth- factors complicating the correlation of blood concentrations ylphenidate in attention-deficit hyperactivity disorder. CPJ/RPC with assessment of toxicity. Many pathological conditions 2004;137:30-3. may predispose an individual to toxicity and possibly death 8. Morton WA, Stockton GG. Methylphenidate abuse and psychiatric side effects. J Clin Psychiatry 2000;2:159-64. at lower than expected blood concentrations.17 Sudden death 9. Musser CJ, Ahmann PA, Theye FW, and others. Stimulant use and that is attributed to the complications of methamphetamine the potential for abuse in Wisconsin as reported by school admin- and cocaine abuse is most commonly associated with cardio- istrators and longitudinally followed children. J Dev Behav Pediatr vascular effects (disturbances in heart rhythm, heart attacks), 1998;19:187-92. but may also be associated with respiratory failure, and neu- 10. Simone KE. Abuse of dietary supplements is common and dangerous. rological effects (strokes, seizure). Seizures are often associated Clin Forens Tox News 2004;September:2-6. 11. Baselt RC. Disposition of toxic drugs and chemicals in man. 6th ed. with stimulant abuse but tend to occur only at higher doses. Foster City CA: Chemical Toxicology Institute, 2002. Convulsions are a common sequel to long-term and high dose 12. Isenschmid DS. Cocaine: effects on human performance and behav- use that is associated with typical abuse patterns. ior. Forens Sci Rev 2002;14:62-100. 13. Levine B. Principles of forensic toxicology. 2nd ed. Washington DC: Cocaine-induced excited or agitated delirium is an example AACC Press; 2003. 14. Jenkins AJ, Goldberger BA. Identification of unique cocaine me- of a toxic response to cocaine that is frequently associated tabolites and smoking by-products in postmortem blood and urine with death. This syndrome is associated with severe hyper- specimens. J Forensic Sci 1997;42:824-7. thermia, extreme agitation and delirium, respiratory arrest, 15. Logan BK, Couper FJ. 3,4-Methylenedioxymethamphetamine: and sudden death. These individuals exhibit extreme strength effects on human performance and behavior. Forens Sci Rev in combination with bizarre and/or violent behavior.12 2003;15:11-28. 16. Logan BK. Methamphetamine: effects on human performance and behavior. Forens Sci Rev 2002;14:133-51. Use of methamphetamine can cause damage to the brain that 17. Karch S. Pathology of drug abuse. 2nd ed. Philadelphia PA: CRC is detectable months after the use of the drug. The damage to Press;1993. the brain is similar to damage caused by Alzheimer’s disease, 18. Methamphetamine abuse linked to long-term damage to brain cells. stroke, or epilepsy.18 NIDA News Release, March 27, 2000. 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 combination of crowded all-night dance parties and MDMA use has been reported to cause fatalities.5,15 118 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

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- lenedioxymethamphetamine; XTC = 3,4-methylenedioxyamphetamine (MDMA, ecstasy, XTC, Adam), 3,4-methy- methamphetamine. lenedioxyethamphetamine (MDEA, MDE, Eve), and 3,4- methylenedioxyamphetamine (MDA), are psychostimulants INDEX TERMS: drug testing; hallucinogenic drugs. with hallucinogenic properties. MDA is also a metabolite of both MDMA and MDEA. These drugs are ring-substituted Clin Lab Sci 2005;18(2):119 amphetamine derivatives that produce hallucinogenic, en- tactogenic (‘love drug’), and stimulating effects.1-3 MDMA Victor A Skrinska PhD DABCC is Professor and Chair, Uni- was initially developed as an appetite suppressant, however, versity of Alabama at Birmingham, Department of Diagnostic its use as a therapeutic drug has been very limited.4 Because and Therapeutic Sciences, Birmingham AL. of its effects as a hallucinogenic psychostimulant with relatively low toxicity, it has emerged over the last two decades Susan B Gock MS MT(ASCP) is Forensic Laboratory Techni- as a common recreational psychostimulant or ‘club drug’ at cal Director, Milwaukee County Medical Examiner’s Office, ‘raves’.5 MDMA, MDEA, and MDA are often referred to as Milwaukee WI. ‘rave’ or ‘designer’ drugs. They are produced in clandestine laboratories and have an increasing presence on the illicit drug Address for correspondence: Victor A Skrinska PhD DABCC, market worldwide. Significant adverse health effects have Professor and Chair, University of Alabama at Birmingham, been reported that include: serotonin neurotoxicity, severe Department of Diagnostic and Therapeutic Sciences, 1530 psychiatric disorders, renal failure, malignant hyperthermia, 3rd Ave S, Birmingham AL 35294-1212. (205) 934-9124. hepatitis, rhabdomyolysis, and disseminated intravascular skrinska@uab.edu coagulation.6-8 A number of fatal outcomes associated with severe MDMA intoxication have been reported.9-12 Victor A Skrinska PhD DABCC is the Focus: Psychostimu- lants guest editor. ABBREVIATIONS: Adam = 3,4-methylenedioxymethamphetamine; ecstasy = 3,4-methylenedioxymethamphetamine; Focus Continuing Education Credit: see pages 124 to 126 For Eve = 3,4-methylenedioxyethamphetamine; GC = capillary learning objectives, test questions, and application form. gas chromatography; GC/MS = gas chromatography/mass spectrometry; HPLC = high performance liquid chro- The analysis of MDMA, MDEA, and MDA can be broken matography; MDA = 3,4-methylenedioxyamphetamine; down into several categories. The first is the need to identify MDE = 3,4-methylenedioxyethamphetamine; MDEA = the presence of the drugs in tablets that are seized and suspect- 3,4-methylenedioxyethamphetamine; MDMA = 3,4-methy- ed to contain illicit drugs. The second is the need to detect ‘rave’ drugs onsite with the intent to determine recent use of The Focus section seeks to publish relevant and timely continuing the drugs. The third category is the typical laboratory drug education for clinical laboratory practitioners. Section editors, topics, screen used to determine either recent or chronic exposure to and authors are selected in advance to cover current areas of interest the drugs. And finally, the fourth category is forensic analysis in each discipline. Readers can obtain continuing education credit of postmortem specimens for the presence of the drugs. The (CE) through P.A.C.E.® by completing the tearout form/examination specimens, methodology, and instrumentation vary with each questions included in each issue of Clin Lab Sci and mailing it with of the categories. Table 1 summarizes the methods that have the appropriate fee to the address designated on the form. Suggestions been developed and reported for these categories. for future Focus topics and authors, and manuscripts appropriate for CE credit are encouraged. Direct all inquiries to George Fritsma MS ANALYSIS OF TABLETS MT(ASCP), Department of Pathology, 619 South 19th Street, West Tablets containing MDMA and other psychostimulants are Pavilion, University of Alabama at Birmingham, Birmingham AL prepared in clandestine laboratories worldwide. The tablets 35233, (205)821-5641, gfritsma@path.uab.edu. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 119

F O C U S : PSYCHOSTIMULANTS vary in size and typically have logos such as a pitbull, spar- or crime are taken into custody, the need arises for a rapid row, butterfly, ‘e’, or ‘X-files’ imprinted on the tablets.13 The onsite detection method for MDMA and related drugs. concentration of the active ingredients varies widely even Some immunoassays that have been developed for detec- among tablets from same origin.14 The excipients or inert tion of methamphetamine have high cross reactivity with ingredients found in tablets include glucose, sorbitol, and MDMA and MDA, which make the assays potentially suit- cellulose.15 Despite variation in concentration of the active able for onsite screens where abuse of psychostimulants is ingredients, analysis of the tablets is helpful in identification suspected.24 Procedures have been reported for onsite analysis of the clandestine laboratory that manufactured them. A of saliva and sweat.24-27 The concentrations of MDMA and number of analytical techniques have been applied to the MDA in saliva have pharmacokinetic parameters that are characterization of the seized tablets. Raman spectroscopy similar to plasma, thus demonstrating that saliva is a useful of the active components and the excipients in tablets has and less invasive alternative to analysis of plasma.28 Studies been successfully used to identify tablets from the same have shown that individuals taking a single 100 mg dose of source based on the state of hydration and the drug/excipi- MDMA consistently have detectable levels of MDMA in ent ratio.15-18 Another approach is analysis of impurities and both sweat and saliva after 1.5 hours. After six hours, most byproducts of synthesis by gas chromatography/mass spec- individuals remain positive; however, the number of false trometry (GC/MS), capillary gas chromatography (GC), negatives begins to increase significantly to almost 20%.25 or high performance liquid chromatography (HPLC).19,20 Drugwipe has been successfully applied to onsite screens of Isotopic analysis of the tablets for the ratios of deuterium, both saliva wiped from the tongue and sweat collected from carbon 13, and nitrogen 15 in the active ingredients has armpits.26,27 The drug may be quantified and cutoff limits been reported as a characteristic that is unique to the site of established with a hand photometer, Drugread.26 manufacture and may be a reliable method of fingerprinting the tablets.21,22 Capillary zone electrophoresis with ultraviolet DETECTION OF ILLICIT PSYCHOSTIMULANTS IN detection is a rapid method suitable for routine analysis of DRUG SCREENS MDMA content in tablets.23 Laboratory drug screens for detection of MDMA, MDEA, and MDA typically measure the presence of these substances ONSITE DETECTION OF PSYCHOSTIMULANTS in plasma, urine, saliva, or hair.29-31 The methods used for de- When individuals at the scene of a ‘rave’ party, accident, 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 — — — — 120 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S : PSYCHOSTIMULANTS more sophisticated and more labor intensive methods such washing of the hair will cause some loss of the embedded as liquid chromatography with tandem mass spectrometry drugs. Also, external contact with drugs in powder or liquid (LC/MS/MS).29,30 Plasma and saliva concentrations indicate form will cause penetration of the hair stock.31 Nevertheless, recent drug abuse less than 24 hours, whereas urine concen- it has been shown to be a useful indicator of chronic drug trations reflect drug intake within the previous 48 hours.27 use and is widely accepted as a suitable specimen for drug Analysis of hair provides a historical perspective that suggests screens including the detection of MDMA and MDA.35 All chronic abuse of the drugs.31 After a single 100 mg dose of methods for hair analysis require digestion of the hair sample MDMA, the concentration of the drug peaks at 1.5 hours followed by extraction of the drugs. The accepted cutoff for a after intake with concentrations ranging from 135 to 233 ng/ positive hair sample is 0.1 ng/mg.36 Most methods developed mL in plasma and from 1729 to 6510 ng/mL in saliva. After for the analysis of drugs in hair use GC/MS instrumentation 24 hours, the MDMA levels in plasma and saliva decrease to to obtain the necessary sensitivity.37-42 Hair samples range in mean concentrations of 14 ng/mL and 126 ng/mL, respec- weight from 10 to 50 mg. Other methods that have been tively.28 In urine, significant concentrations of MDMA are successfully applied to hair analysis for MDMA include detectable up to 48 hours yielding a positive screen in urine capillary electrophoresis, radioimmunoassay, HPLC, and while the results for plasma and saliva are negative.26,27 ion mobility spectrometry.31,36,43,44 Immunoassays for amphetamine and methamphetamine gen- FORENSIC ANALYSIS OF PSYCHOSTIMULANTS erally have high cross reactivity with related drugs and have Analysis of postmortem specimens for the presence of been successfully applied to urine screens for the detection psychostimulants such as MDMA, MDEA, and MDA typi- of MDMA and MDA.30 The assays have sufficient sensitivity cally involves extraction of the drugs from tissues including for reliable detection of the drugs at the established cutoff of liver, muscle, and brain as well as from urine, central blood, 500 ng/mL and agree well with results confirmed by GC/MS. peripheral blood, and vitreous humor.45 Varying degrees The high cross reactivity of immunoassays with structures of putrefaction and postmortem redistribution of drugs similar to amphetamine and methamphetamine may result further complicate the analysis. While hair and urine are in false positives for MDMA when other substances such as suitable forensic specimens to determine the presence of the ephedrine or pseudoephedrine are present.30 drugs, peripheral blood and vitreous humor are reported to provide the best estimate of the blood concentration at the Capillary electrophoresis with electrochemical and fluores- time of death.45-48 cence detection has been successfully applied to the analysis of MDMA in urine with detection limits of 4 ng/mL with an There are various analytical techniques available for initial electrochemical detector and 50 ng/mL with a fluorescence screening, confirmation, and quantification of forensic speci- detector.32,33 Chromatographic techniques such as HPLC mens such as thin-layer chromatography (TLC), HPLC, and with fluorescence detection have been used for measurement GC/MS.45-47 TLC is a common initial screening technique of MDMA in plasma and urine with a detection limit of 25 when a method capable of detecting a broad-spectrum of ng/mL.34 Mass spectrometry methods including GC/MS and drugs in urine specimens is required. Identification is based LC/MS/MS have been reported for the analysis of plasma on Rf value and the color characteristics following exposure and saliva for MDMA and its metabolites with detection to specific staining reagents. The Toxi-Lab A® system for the limits of 6 ng/mL and 2 ng/mL, respectively.28,29 detection of basic and neutral drugs in urine specimens is able to differentiate sympathomimetic amines such as ephedrine, Hair analysis has been studied as a specimen that may be pseudoephedrine, and phenylpropanolamine from illicit dugs useful for determination of past chronic exposure to illicit such as amphetamine, methamphetamine, MDMA, and drugs.31 Hair is a complex structure that grows approxi- MDA. Sensitivity for most of the drugs in this class using mately one cm/month. During its growth, hair is exposed this procedure is approximately 500 ng/mL. to substances present in the capillary blood circulation near the follicle and substances excreted in sweat at the base of Immunoassays may also be applied to forensic drug screens the follicle. Drugs in contact with hair penetrate and embed for the presence of MDMA and related metabolites.30 in the core of the hair stock and remain in the stock for an However, as mentioned earlier, immunoassay techniques extended period of time. In the hair stock, drugs are rela- for the detection of amphetamine and methamphetamine tively protected from the environment; however, extensive have variable amounts of antibody cross reactivity to other VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 121

F O C U S : PSYCHOSTIMULANTS structurally related sympathomimetic amines including 2. Steele TD, McCann UD, Ricaurte GA. 3,4-methylenedioxymeth- pseudoephedrine and ephedrine.30 Antibody cross reactivity amphetamine (MDMA, ‘ecstasy’): pharmacology and toxicology in animals and humans. Addiction 1994;89:539-51. is variable and dependent on both the concentration of the 3. Hermle L, Spitzer M, Borchardt D, and others. Psychological effects structurally related analyte present in the specimen as well of MDE in normal subjects. Are entactogens a new class of psychoac- as the source of the antibodies used for detection. Higher tive agents? Neuropsychopharmacology 1993;8(2):171-6. levels of antibody cross reactivity occur with polyclonal an- 4. Climko RP, Roehrich H, Sweeney DR, and others. Ecstacy: a review tibody assays in comparison to monoclonal antibody assays. of MDMA and MDA. Int J Psychiatry Med 1986;16:359-72. Monoclonal antibody assays are more specific and exhibit 5. Arria AM, Yacoubian GS Jr, Fost E, and others. The pediatric forum: ecstasy use among club rave attendees. Arch Pediatr Adolesc Med less cross-reactivity to structurally related compounds and 2002;156:295-6. should be used when high selectivity is desired. Application 6. Parrott AC. Recreational ecstasy/MDMA, the serotonin syn- of immunoassay techniques for the analysis of postmortem drome, and serotonergic neurotoxicity. Pharmacol Biochem Behav specimens poses a problem due to decomposition occurring 2002;71:837-44. during the postmortem interval. This may result in the pro- 7. McCann UD, Eligulashvili V, Ricaurte GA. (+/-)3,4-methylenedioxymethamphetamine (‘ecstasy’)-induced serotonin neurotoxicity: duction of biogenic amines such as beta-phenethylamine or clinical studies. Neuropsychobiology 2000;42:11-6. tyramine that have the potential to produce a false positive 8. McCann UD, Slate SO, Ricaurte GA. Adverse reactions with 3,4- with amphetamine immunoassays due to the cross reactivity methylenedioxymethamphetamine (MDMA; ‘ecstasy’). Drug Saf with these analytes. Due to the lack of specificity associated 1996;15:107-15. with immunoassays for identification of the specific psycho- 9. Fineschi V, Masti A. Fatal poisoning by MDMA (ecstasy) and MDEA: stimulants present in the sample, confirmation of positive a case report. Int J Legal Med 1996;108:272-5. 10. Iwersen S, Schmoldt A. Two very different fatal cases associated with immunoassay results should be made using an alternate the use of methylenedioxyethylamphetamine (MDEA): Eve as deadly analytical methodology. GC/MS analysis with selective ion as Adam. Clin Toxicol 1996;34:241-4. monitoring is the analytical methodology routinely utilized 11. Randall T. Ecstasy-fueled ‘Rave’ parties become dances of death for for drug confirmation and quantification. Derivatization of English youths. JAMA 1992;268:1505-6. the drugs with heptafluorobutyric anhydride, pentafluoro- 12. Henry JA, Jeffreys KJ, Dawling S. Toxicity and deaths from 3,4-methylenedioxymethamphetamine (‘ecstasy’). Lancet 1992;340:384-7. propionic anhydride, or trifluoroacetic anhydride prior to 13. Schneider RC, Kovar KA. Analysis of ecstasy tablets: comparison of analysis improves chromatographic behavior and reduces reflectance and transmittance near infrared spectroscopy. Forensic fragmentation so that higher mass fragments can be used Sci Int 2003;134:187-95. for GC/MS selective ion monitoring which allows a more 14. Sherlock K, Wolff K, Hay AW, and others. Analysis of illicit ecstasy definitive identification and confirmation of the drugs. 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- CONCLUSION filing of seized ecstasy tablets by Raman spectroscopy. Analyst A wide range of analytical methods have been developed for 2000;125:1811-5. analysis of MDMA and related psychostimulants. Analysis by 16. Bell SE, Burns DT, Dennis AC, and others. Rapid analysis of ecstasy GC/MS is the technique that has been reported the most often and related phenethylamines in seized tablets by Raman spectroscopy. and has been applied to the widest range of specimen types. No Analyst 2000;125:541-4. 17. Bell SE, Barrett LJ, Burns DT, and others. Tracking the distribution doubt this is due to the high specificity combined with high of ‘ecstasy’ tablets by Raman composition profiling: a large scale sensitivity that is found in GC/MS applications. However, feasibility study. Analyst 2003;128:1331-5. there are a number of alternate methods using technologies 18. Ryder AG. Classification of narcotics in solid mixtures using prin- such as immunoassay, HPLC, and electrophoresis that have cipal component analysis and Raman spectroscopy. J Forensic Sci sufficient sensitivity and specificity for detection of MDMA in 2002;47:275-84. routine screening applications in the laboratory. Furthermore, 19. Cheng WC, Poon NL, Chan MF. Chemical profiling of 3,4-methylenedioxymethamphetamine (MDMA) tablets seized in Hong Kong. these alternative methods are more easily automated and more J Forensic Sci 2003;48:1249-59. suitable for high volume applications. 20. Palhol F, Boyer S, Naulet N, and others. Impurity profiling of seized MDMA tablets by capillary gas chromatography. Anal Bioanal Chem REFERENCES 2002;374:274-81. 1. Nichols DE. Differences between the mechanism of action of 21. Carter JF, Titterton EL, Murray M, and others. Isotopic characterisa- MDMA, MBDB, and the classic hallucinogens. Identification tion of 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy- of a new therapeutic class: entactogens. J Psychoactive Drugs methylamphetamine (ecstasy). 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FOCUS 22. Palhol F, Lamoureux C, Naulet N. 15N isotopic analyses: a powerful tool 36. Tagliaro F, Valentini R, Manetto G, and others. Hair analysis by using to establish links between seized 3,4-methylenedioxymethamphetamine radioimmunoassay, high-performance liquid chromatography and (MDMA) tablets. Anal Bioanal Chem 2003;376:486-90. capillary electrophoresis to investigate chronic exposure to heroin, 23. Frost M, Kohler H, Blaschke G. Analysis of ‘ecstasy’ by capillary cocaine and/or ecstasy in applicants for driving licenses. Forensic Sci electrophoresis. Int J Legal Med 1996;109:53-7. Int 2000;107:121-8. 24. Fay J, Fogerson R, Schoendorfer D, and others. Detection of 37. Pujadas M, Pichini S, Poudevida S, and others. Development and methamphetamine in sweat by EIA and GC-MS. J Anal Toxicol validation of a gas chromatography-mass spectrometry assay for 1996;20:398-403. hair analysis of amphetamine, methamphetamine, and methylene- 25. Pichini S, Navarro M, Pacifici R, and others. 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J Anal Toxicol 2001;25:144-6. 2000;107:191-9. 28. Navarro M, Pichini S, Farre M, and others. Usefulness of saliva 40. Cooper GA, Allen DL, Scott KS, and others. Hair analysis: self-re- for measurement of 3,4-methylenedioxymethamphetamine and its ported use of ‘speed’ and ‘ecstasy’ compared with laboratory findings. metabolites: correlation with plasma drug concentrations and effect J Forensic Sci 2000;45:400-6. of salivary pH. Clin Chem 2001;47:1788-95. 41. Uhl M. Determination of drugs in hair using GC/MS/MS. Forensic 29. Wood M, De Boeck G, Samyn N, and others. Development of a Sci Int 1997;84:281-94. rapid and sensitive method for the quantitation of amphetamines 42. Rothe M, Pragst F, Spiegel K, and others. Hair concentrations and in human plasma and oral fluid by LC-MS-MS. J Anal Toxicol self-reported abuse history of 20 amphetamine and ecstasy users. 2003;27:78-87. Forensic Sci Int 1997;89:111-28. 30. Stout PR, Klette KL, Wiegand R. Comparison and evaluation of 43. Tagliaro F, De Battisti Z, Groppi A, and others. High sensitivity DRI methamphetamine, DRI ecstasy, Abuscreen ONLINE amphet- simultaneous determination in hair of the major constituents of amine, and a modified Abuscreen ONLINE amphetamine screening ecstasy (3,4-methylenedioxymethamphetamine, 3,4-methylene- immunoassays for the detection of amphetamine (AMP), metham- dioxyamphetamine, and 3,4-methylene-dioxyethylamphetamine) by phetamine (MTH), 3,4-methylenedioxyamphetamine (MDA), and high-performance liquid chromatography with direct fluorescence 3,4-methylenedioxymethamphetamine (MDMA) in human urine. detection. J Chromatogr B Biomed Sci Appl 1999;723:195-202. J Anal Toxicol 2003;27:265-9. 44. Keller T. Miki A. Regenscheit P, and others. Detection of designer 31. Tagliaro F, Manetto G, Crivellente F, and others. Hair analysis for drugs in human hair by ion mobility spectrometry (IMS). 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Forensic Sci Int 3,4-methylenedioxyethylamphetamine, and 3,4-methylenedioxyam- 2002;125:142-8. phetamine with HPLC and fluorescence detection in whole blood, 34. Herraez-Hernandez R, Campins-Falco P, Verdu-Andres J. Sensitive serum, vitreous humor, and urine. Clin Chem 2000;46:1968-77. determination of methylenedioxylated amphetamines by liquid 47. Uhl M. Tandem mass spectrometry: a helpful tool in hair analysis chromatography. Analyst 2001;126:581-6. for the forensic expert. Forensic Sci Int 2000;107:169-79. 35. Kintz P, Cirimele V. Interlaboratory comparison of quantitative de- 48. De Letter EA, De Paepe P, Clauwaert KM, and others. Is vitreous termination of amphetamine and related compounds in hair samples. humour useful for the interpretation of 3,4-methylenedioxymeth- Forensic Sci Int 1997;84:151-6. amphetamine (MDMA) blood levels? Experimental approach with rabbits. Int J Legal Med 2000;114:29-35. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 123

FOCUS: PSYCHOSTIMULANTS Continuing Education Questions SPRING 2005 To receive three contact hours of basic level P.A.C.E. credit c. Hypotension for the Focus: Psychostimulants questions, insert your an- d. Tachycardia swers in the appropriate spots on the immediately following page; then complete and mail the form as directed. 2. Which of the following behavioral effects is NOT associated with common psychostimulants? NOTE: There may be more answer spaces on the answer a. Locomotor suppression sheet than needed. If so, leave them blank. Make sure the b. Sexual stimulation number of the answer space being filled matches the number c. Increased sociality of the questions being answered. d. Increased mental attention LEARNING OBJECTIVES 3. Amphetamine is used in the treatment of all of the fol- After reading the three Focus: Psychostimulants articles in this lowing disorders EXCEPT: issue, the reader will be able to: a. obesity. 1. Describe the physiologic actions for amphetamine. b. narcolepsy. 2. Describe the behavioral effects of some common c. attention-deficit hyperactivity disorder. psychostimulants. d. nasal congestion. 3. Identify several therapeutic uses for psychostimulants. 4. The mechanisms of action of amphetamine include all 4. Describe the mechanisms of action of amphetamine. of the following EXCEPT: 5. List symptoms associated with high doses of amphetamine. a. inhibition of monoamine neurotransmitter uptake. 6. List the common psychostimulants of forensic interest. b. binding to intracellular catecholamine receptors. 7. List the illicit psychostimulants that show an increasing c. inhibition of monoamine oxidase. trend of abuse in the United States. d. release of catecholamines from neurons. 8. Describe the composition of crack. 9. List the common routes of administration of cocaine. 5. An individual exhibiting symptoms of tremors, vomiting, 10. Identify the major metabolites of cocaine, MDMA, and and tonic-clonic convulsions is possibly experiencing: methamphetamine. a. withdrawal from amphetamine. 11. Describe symptoms related to toxic doses of cocaine, b. high dose amphetamine. MDMA, and methamphetamine. c. chronic tolerance to amphetamine. 12. List the drugs that are commonly referred to as ‘designer’ d. gastrointestinal complication associated with amphetamine. or ‘rave’ drugs. 13. Describe the adverse effects of MDMA and related drugs 6. All of the following drugs are likely to be included in a drug caused by chronic intoxication. screen protocol in a forensic laboratory EXCEPT: 14. Describe specimen collection for on site screening for a. cocaine. MDMA. b. methamphetamine. 15. List suitable analytical methods for analysis of MDMA c. ethylenedioxyamphetamine. and related amines in tablets, saliva, sweat, plasma, urine, d. methylenedioxymethamphetamine. hair, and vitreous humor. 7. The most frequently mentioned illicit drug reported in CONTINUING EDUCATION QUESTIONS emergency room visits over the last several years is: 1. Which of the following physiologic actions is NOT a. cocaine. attributed to amphetamine? b. methamphetamine. a. Euphoria c. methylenedioxymethamphetamine. b. Appetite suppression d. amphetamine. 124 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

F O C U S: PSYCHOSTIMULANTS 8. Crack is composed of: 15. The analytical technique most commonly used for de- a. cocaine hydrochloride. termination of MDMA levels in hair is: b. methamphetamine free base. a. GC/MS. c. methamphetamine hydrochloride. b. HPLC. d. cocaine free base. c. LC/MS. d. TLC. 9. The routes of self administration of cocaine include all of the following EXCEPT: 16. The chemical structures of most common psychostimu- a. smoked. lants share the features of: b. oral. a. phenyl ring, alkyl side chain, amine. c. intranasal. b. phenol ring, unconjugated side chain, amine. d. intravenous. c. phenyl ring, alkyl side chain, carboxylic acid. d. phenol ring, alkyl side chain, ketone. 10. Analysis of a blood sample following cocaine administration will include all of the following metabolites 17. An individual is taken to the emergency room where a EXCEPT: blood sample is collected and tested for possible cocaine a. benzoyl methyl ester. abuse. The toxicology screen shows the presence of ben- b. ecgonine methyl ester. zoylecgonine and cocaethylene. The conclusion is the: c. benzoylecgonine. a. sample is a false positive. d. ecgonine. b. chronic use of cocaine. c. use of cocaine and ethanol. 11. At toxic levels, both cocaine and MDMA cause: d. use of cocaine and amphetamine. a. fatigue. b. nausea. 18. In recent years, the percentage of emergency room visits as- c. hyperthermia. sociated with abuse of multiple drugs is approximately: d. hypothermia. a. one third. b. two thirds. 12. The group of hallucinogenic psychostimulants that are c. three quarters. often found at ‘raves’ includes all of following drugs d. one half. EXCEPT: a. MDMA. 19. The best estimate of a blood concentration of a psycho- b. MDEA. stimulant at the time of death is provided by analysis of c. MDA. a postmortem sample of: d. MDME. a. hair. b. central blood. 13. The adverse eﬀects associated with chronic use of ‘rave’ c. peripheral blood. drugs include all of the following EXCEPT: d. urine. a. renal failure. b. pancreatitis. 20. An individual is taken into custody at a rave. After 36 c. malignant hyperthermia. hours, a drug screen is ordered. Which specimen would d. rhabdomyolysis. give the best indication of abuse of MDMA at the rave? a. Plasma 14. Several techniques have been developed for onsite screening b. Saliva of ‘rave’ drugs. The onsite screens require collection of: c. Sweat a. urine. d. Urine b. hair. c. blood. d. saliva. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 125

Continuing Education Registration Form 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_____________ Check all that apply 2. Specialty: (a) biochemistry/urinalysis (b) microbiology (c) lab administration (d) hematology/hemostasis (e) education q I am an ASCLS member (f )immunology (g) immunohematology q I am not an ASCLS member q I would like to receive ASCLS membership information 3. Workplace: (a) hospital over 500 beds (b) hospital 200–499 q I have previously participated in Focus beds (c) hospital 100–199 beds (d) hospital under 100 beds q I would like information on other continuing education sources (e)private lab (f ) community blood bank (g) group practice (h) private physician (i) clinic (j) other Answers Circle correct answer (questions are on previous two pages). 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 1. a b c d e 8. a b c d e 15. a b c d e 22. a b c d e (e) over $40,000 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 5. Did these articles achieve their stated objectives? 4. a b c d e 11. a b c d e 18. a b c d e 25. a b c d e (a) yes (b) no 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 6. How much of these articles can you apply in practice? 7. a b c d e 14. a b c d e 21. a b c d e 28. a b c d e (a) all (b) some (c) very little (d) none Participant Information 7. Employment status: (a) full time (b) part time (c) student Please circle the most appropriate answers. (d) not employed (e) retired 1. Is this program used to meet your CE requirements for: 8. How long did it take you to complete both the reading (a) state license (b) NCA (c) employment (d) other and the quiz? ___________minutes 9. What subjects would you like to see addressed in future Focus articles? 126 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

TRENDS AND TECHNOLOGY Trends and Technology: Spring 2005 MARY JANE GORE ONLINE I have to say that helpfulness is a hall- studies indicate that the VITROS With this issue of Trends and Technol- mark of the Ortho-Clinical site. All of 350 Chemistry System with enhanced ogy, I decided to break with tradition its major analyzers and other product software improves throughput 10% – the tradition of not reviewing man- lines are clearly labeled as links to the to 25% and time-to-first-result up to ufacturers’ Web sites. I did not want left on the homepage. The information 12% when compared to the VITROS to create the illusion of favoritism or under “Contact Us” is extensive and 250 System. Visit www.orthoclinical. persuasion by featuring a clinical di- inviting, and not just an email address com for more information on this and agnostics Web site, but after so many that is often found online. If you want other products. years of reviewing other types of sites to contact these folks, they make it – governmental agencies, personal easy. The homepage also has links to lab-related Web sites, some associa- technical support (including holiday tion sites of interest, quality-related photos of these people, putting faces sites – I began to wonder: why not? to the voices on the phone) and techni- As always, I welcome reader input cal documentation, which brought up about sites that may be helpful – or 123 “Instructions for Use” documents, not so helpful – to review for other down to the last calibrator kit. The most readers (send your ideas to clstt@aol. impressive fact of this Web site, for me, com). This time, I visited the site of is that it is all available with a glance at Ortho-Clinical Diagnostics (www. the homepage, including headline news orthoclincal.com), a subdivision of about new products. Ortho appears to Ortho-Clinical VITROS 350 Johnson & Johnson, and found that be a company dedicated to customer when I answered the Web survey, I got service and response and its site is in- Maxell Corporation of America, a timely response to my query, which deed user friendly. leader in advanced recordable media led to me look at the site from the products, has announced new ultra- standpoint of a laboratory scientist. NEW PRODUCTS durable and ultra-reliable DVD media The VITROS 350 Chemistry System designed specifically for the medical offers easy operation, maintenance, market. Maxell’s new medical-grade and training. Whether you use it as a media incorporates the innovative primary, STAT, or back-up to the pow- MAXPRO™ Hardcoat technology to The Trends and Technology section seeks to erful VITROS 5,1 FS, the VITROS produce enhanced DVD-R media publish product and technology information 350 Chemistry System can perform that delivers the highest level of data (including black-and-white glossy photo- the work. The VITROS 350 comes protection for up to twice the archival graphs), news items (such as FDA approvals), and information about laboratory resources complete with enhanced throughput, shelf life. Maxell Medical DVD-R is of all kinds. The intent of this section is to a broad accessible menu, and new ergo- HIPAA- and DICOM-compliant, provide a cutting-edge, one-stop shop tailored nomic design. “Load-and-go” reagent and with its superior scratch, dust to the current practical needs and concerns preparation, MicroSlide™ Technology and smudge resistance and extended of clinical laboratory practitioners. Let us and up to six months’ calibration sta- archival life, is ideal for critical medical hear from you with suggestions on how to bility mean labor can be redeployed images, patient records, backup, and improve this section. Direct inquiries and for value-added tasks. Minimal in- fixed content storage. Maxell’s 700 information to Mary Jane Gore, CLS Trends strument maintenance assists with MB, 8X speed Medical DVD-R media and Technology Editor, c/o ASCLS, 6701 productivity and reduces costs with the will be available in March as a single Democracy Blvd., Suite 300, Bethesda, MD system that delivers the right results the disc in a jewel case and in 50-pack 20814, clstt@aol.com. Please send all materi- first time. Ortho-Clinical Diagnostics spindles with printable white surfaces als clearly marked NEW PRODUCTS. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 127

T R E N DS AND TECHNOLOGY for either thermal or inkjet printers. 2005 demonstrated robotic solutions in the U.S. The BioPlex 2200 system Pricing will be affordable compared that deliver speed, efficiency, and reli- is a bead-based multiplexing immu- to other backup media. Contact ability for laboratories focused on clini- noassay platform that can deliver up www.maxell.com. cal diagnostics, genomics, proteomics to 2200 results per hour. The system and drug discovery. Contact Greg will initially include a panel of assays Healthcare organizations now have Porter, Ph.D., at 919-361-5200. targeting autoimmune diagnostics. continuous access to the Joint Com- Future assays in development are in mission on Accreditation of Healthcare AxSYM Anti-HCV (hepatitis C virus) the areas of serology, infectious disease, Organizations’ Periodic Performance is a Microparticle Enzyme Immu- cardiac, and toxicology. Contact Gary Review (PPR) on the Joint Com- noassay (MEIA) for the qualitative Mantha at 510-741-4637 or email mission’s secure extranet, “JAYCO.” detection of anti-HCV IgG to HCV Gary_Mantha@bio-rad.com. The PPR is an integral component of recombinant proteins in human se- the Joint Commission’s accreditation rum or plasma containing potassium process that promotes continuous EDTA, sodium EDTA, sodium hepa- standards compliance through ongo- rin, lithium heparin, sodium citrate, ing, internal monitoring. Before mak- or potassium oxalate. Assay results, in ing the PPR continuously available, conjunction with other laboratory re- organizations had received access to sults and clinical information, may be the PPR tool 15 months after its last used to provide presumptive evidence BioPlex 2200 triennial survey and had three months of infection with HCV virus (state to complete it. Those timeframes for of infection or associated disease not Bio-Rad Laboratories recently received access have now been eliminated. The determined) in persons with signs or approval from the FDA for its new Mul- schedule for completing the PPR re- symptoms of hepatitis and in persons tispot HIV-1/HIV-2 Rapid Test. This mains unchanged until January 2006, at risk for hepatitis C infection. Visit highly sensitive test kit this year became when organizations will be expected to www.abbottdiagnostics.com for the available in the U.S. and will significantly update the PPR annually. For 2005, 2005 product releases and listings. aid in the diagnosis of HIV-1/HIV-2 the PPR process requires each accredit- (human immunodeficiency virus, types ed organization to conduct a mid-cycle FDA APPROVALS 1 and 2), the virus that causes AIDS. self-assessment against applicable Joint Bayer Diagnostics has received U.S. Contact Susan Berg at 510-741-6063 or Commission standards, develop a Plan Food and Drug Administration (FDA) email susan_berg@bio-rad.com. of Action to address identified areas of approval for its automated assay for the non-compliance and identify Measures hepatitis C virus on the ADVIA Cen- Roche announced that its first microar- of Success for validating resolution of taur Immunoassay System. Contact ray-based test, the AmpliChip CYP450 the identified problem areas. Under the Susan Hager at 781-551-7916. Test, has been cleared by the FDA for di- usual PPR process, organizations will agnostic use in the U.S. This test, which is be expected to share this information Bio-Rad Laboratories has received powered by Affymetrix microarray tech- with the Joint Commission at the mid- marketing clearance from the FDA for nology, analyses a patient’s cytochrome cycle point. Contact Charlene Hill at its new BioPlex 2200 system, a revo- P450, 2D6, and 2C19 genotypes from Charlene D. Hill 630-792-5175 or lutionary new immunoassay platform genomic DNA extracted from a blood email chill@jcaho.org. that employs multiplexing technology sample. Test results will allow physicians to analyze for multiple disease states to consider unique genetic information Tecan introduced several new labo- from single patient samples. It is the from patients in selecting medications ratory automation products at Lab first clinical diagnostics platform to and doses of medications for a wide vari- Automation 2005, the pre-eminent offer multiplexing technology on ety of common conditions such as cardiac industry meeting, held at the San Jose a fully-automated, fully-integrated diseases, pain, and cancer. This new test McEnery Convention Center. Tecan’s random access platform. The system allows physicians access to information interactive trade show booth included was unveiled last July at the American that could help to prevent harmful drug the latest unique robotic solutions. Te- Association of Clinical Chemistry interactions and to assure drugs are used can’s participation at Lab Automation meeting and will soon be available optimally. Adverse drug reactions cause 128 VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE

T R E N DS AND TECHNOLOGY a huge number of hospitalizations in documented standard operating proce- phone at 310-445-3038 or via e-mail the U.S. The new test also will, in some dures, a world-class training program at ipalmer@infotrieve.com. cases, enable patients to avoid suboptimal for all their technicians, state-of-the-art International Technidyne has struck or even harmful treatment choices. For facilities and equipment, and decades of a deal with Medical Automation Sys- patients it is extremely important to know gravimetric and metrology experience. tems for the MAS RALS to integrate whether pain killers or anesthetics might Service advantages include fast turn- ITC’s IRMA TRUpoint™ analyzer for work differently or not at all for them. around times, competitive pricing, full point-of-care blood gas monitoring More information is available at www. traceability to NIST, and fulfillment of with the widely used RALS -Plus roche-diagnostics.com. ISO and all other compliance record- data management system, marketed keeping requirements. Contact Robin by MAS. Contact Check Weber at Dade Behring (NASDAQ: DADE) has Gervasoni at 610-430-7258 or email 847-705-1802. received clearance from the FDA for robin_gervasoni@vwr.com. the use of its Advanced D-Dimer as- Abbott and Nihon Kohden Corpora- say as an aid in the diagnosis of venous Infotrieve Inc, a provider of content tion announced that they have entered thromboembolism (VTE), [deep vein software technology and information into an agreement for the commer- thrombosis (DVT) or pulmonary em- services, has announced its acquisi- cialization of automated hematology bolism (PE)]. The clearance included tion of GenSys Software, Inc, pro- diagnostic instruments for use in hos- performance data with a defined cutoff vider of the GenSys/ELN™ electronic pital laboratories and physician offices. value for the Dade Behring BCS laboratory notebook for life sciences, Under terms of the agreement, two System and Sysmex CA-1500 Sys- chemistry, and other research-intensive 5-part differential hematology instru- tem. The assay is also for use on Dade industries. The GenSys/ELN will serve ments, which are designed to offer Behring’s BCT System, and Sysmex as the anchor for Infotrieve’s life sciences red and white blood cell analysis, will CA-7000 and CA-560 Systems. electronic research platform, which has be manufactured by Nihon Kohden been designed to collectively increase and distributed by Abbott under the ARRANGEMENTS the value of organizational content CELL-DYN Pearl™ brand name. Ab- VWR International announces the and improve scientists’ existing work- bott obtains exclusive distribution recent acquisition of Alpha-Omega flow by enabling links from electronic rights for the two instruments in the Calibrations, LLC. This acquisition laboratory notebooks to discovery tools, United States and Canada and non- expands VWR’s service portfolio to now literature and scientific data, laboratory exclusive distribution rights for the include instrument calibration services product information, and integrated instruments in other countries with the and repairs for customers nationwide. retrieval capabilities for literature and exception of China and Japan. Contact Alpha-Omega Calibrations’ proven laboratory products. Contact Infotrieve Amy Woodworth at 847-935-4755. process includes a quality manual, well- Marketing Manager Ian Palmer by Instructions to Authors Detailed Instructions to Authors can be found on the ASCLS Website (www.ascls.org) by following 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. VOL 18, NO 2 SPRING 2005 CLINICAL LABORATORY SCIENCE 129

ASCLS 73rd Annual Meeting, July 26-30, 2005 AACC/ASCLS Clinical Lab Expo, July 26-28, 2005 Orlando, Florida 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! 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. Hilton in the Walt Disney World Resort Orange County Convention Center Visit www.ascls.org/conferences/2005AM/ for details and online registration.

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