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Automated hplc screening of newborns for sickle cell anemia

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Automated hplc screening of newborns for sickle cell anemia

  1. 1. 704 ClinicalChemistiy 42:5 704-710 (1996) Automated HPLC screening of newborns for sickle cell anemia and other hemoglobinopathies JOHN W. EASTMAN,* RUTH WONG, CATHERINE L. Lao, and DANIEL R. MORALES Automated HPLC is used to test dried blood-spot speci- mens from newborns for hemoglobms (ITh) F, A, S, C, E, and D. We present the method and report on its perfor- mance determined during >4 years of testing 2.5 x 106 newborns. The method features automated derivation of presumptive phenotypes; quantitative quality control and proficiency testing; throughput of one specimen per minute; small sample volume; hemoglobin concentrations quantified with an mterlaboratory CV of 14-18%; retention times with interlaboratory CV of <2% and matching, within ± 0.03 miii, of laboratories and reagent lots; control of peak resolution; 0.5% detection limit for Hb S and C, and 1.0% for Hb F, A, E, and D; few interferences; and negli- gible background and carryover. Shortcomings of the method are the absence of microplate barcode identifica- tion and the need for manually pipetting the sample eluate into the microplate. INDEXING mie1s: dried blood-spot specimens #{149}Guthrie cards. chromatography, cation-exchange #{149}phenotypes In keeping with national recommendations, California legisla- tion mandates screening all newborns for sickle cell disease [1-3].This legislation was implemented for the State of Cali- fornia by the Department of Health Services’ Genetic Disease Laboratory (GDL) and Genetic Disease Branch, Berkeley, CA [4]. Because of its potential as a quantitative method of analysis amenable to automation, GDL chose cation-exchange HPLC [5-12] as the screening method. Cation-exchange HPLC has been used to screen cord blood for hemoglobinopathies [13]. Here, we report on the application of this technique to screen newborns by assaying their dried blood-spot (DBS) specimens. The method reported here is designed to resolve hemoglo- bins (Hb) F, A, 5, C, E, and D. The California program requires California Department of Health Services, Genetic Disease Laboratory, 700 Heinz St., Suite 100, Berkeley, CA 94710. *AUthOr for correspondence. Fax 510-540-2228. ‘Nonstandard abbreviations: GDL, Genetic Disease Laboratory; DBS, dried blood spot; NB, newborn; Hb, hemoglobin(s); and AU, hemoglobin concentration in area units, the area of the chromatographic peak. Received October 4, 1995; accepted January 25, 1996. that newborns with hemoglobinopathy patterns (e.g., FS, FSC, FSA, F only) be recalled for mandatory follow-up. Also, new- borns with FE patterns are recalled to differentiate EE and E//3-thalassemia. For trait patterns of FAS, FAC, and FAD, the families of the affected newborns are offered voluntary counseling. Materialsand Methods EQUIPMENT/REAGENT SYSTEM Modular instrumentation was adapted for the California pro- gram by Bio-Rad Labs., Hercules, CA. A similar integrated instrument, ASCeNT#{174},has been marketed [14]. Each of the modular instruments used in this work consists of one to three HPLC column systems run by one Model 700 chromatography workstation. Each of the column systems consists of a Model AS-I00 HRLC automated sampling system, a 20-g.tL-loop injection valve, two Model 1350 gradient elution pumps, the cation-exchange column in a Model 1250425 35 #{176}Cheater, and a dual-wavelength (415/690 nm) filter photometer (cat. no. 1961042). The 6 X 40 mm columns are packed with a nonpo- rous 7-.tm-diameter Bio-Rad MA 7 polymeric cation-exchange material. Each column can be used for as many as 500 injections. Two sodium phosphate buffers are used as eluents, run as a gradient from -4 g/L (buffer A) to 14 g/L (buffer B) at pH 6.4. Nominal conditions of analysis are a flow rate of 2 mL/min at a pressure of 25 kg/cm2 and the following gradient (time from injection in minutes/percent of eluent that is buffer B): 0.0/0%; 0.3/10%; 0.5/24%; 1.0/52%; 1.8/100%; 1.9/100%; and 2.0/0%. Including the 1-mm wash between specimens, each chromato- gram takes 3 mm. When all three columns are used, the rate of analysis is one specimen pen minute. Operation of the instrument is automated through a menu- driven software program that generates the worklist, injects the sample, controls the gradient for hemoglobin separation, mea- sures and integrates the peaks, derives the hemoglobin pattern, stores the data on electronic media, and telecommunicates the data to a remote central site. The reagent kits include whole-blood primer, wash solution, three linearity calibrators containing Hb F and A, and two lyophilized controls, one containing Hb F, A, E, and S and one Hb F, A, D, and C. To maintain the separation of hemoglobmns among all lots of the cation-exchange resin, the software that
  2. 2. Clinical Chemistry 42, No. 5, 1996 705 controls the gradient is modified when needed to accommodate any differences in the performance of the different lots. A key feature of the test system is its quantification of the concentration of the hemoglobin variants. Chromatographic peaks are reported with heights in microvolts and with areas in relative response units (AU, area units). With the integration settings used for this screening method, 1 AU is approximately three times the area in p.V-min. The three linearity calibrators are used to monitor the dose-response curve for photometer readings vs hemoglobin concentration. The reagent-instrument system must maintain the photometer readings for the linearity calibrators (-0.2, 0.4, and 0.6 g/L Hb F) within ±20% of a stated nominal value. HPLC SCREENING METHOD Specimen collection and preparation.Blood from a subject is absorbed into S&S 903 specimen collection paper (Schleicher & Schuell, Keene, N}-l). A disc 0.95 cm (3/8 in.) in diameter is punched from the DBS specimen, and the blood is eluted into 1.00 mL of water for 30 mm with periodic shaking (equivalent to a 36-fold dilution of the whole-blood specimen). The eluate is further diluted 1:6 with water and dispensed into a 96-well microplate, which is loaded onto the autosampler for injection. Worklistgenerationand reports.A worklist is generated automat- ically when the analyst sets up the run file information. At the end of each run, a Neonatal Hemoglobin Summary Report is printed out. The summary lists the identification number of each specimen and the percentages of all the hemoglobin variants found in the specimen. A Pattern Report is also printed out, listing the hemoglobin pattern (presumptive phenotype) for each specimen. This report lists, in order of decreasing concen- tration, the letter designation of each of the hemoglobins found in the specimen. Quality control. For each column system, the FAES and FADC liquid controls are run at the beginning and end of each run. At the beginning and end of each tray (microplate) of newborn specimens, eluates of blood spots containing Hb S are run as tray controls. The mean and SD values for retention time are fixed as quality-control action limits in the controller software by spec- ification. Mean and SD values for hemoglobin concentration are established by replicate analysis of the controls at GDL before they are put into use. Trays of newborn results are flagged for review by a quality-control officer when (a) the retention time of Hb S in the tray control exceeds the ±3SD limit; (b) the concentration of Hb S in the tray control exceeds the ± 3SD limit; or (c) the blank water injections give a chromatographic peak height >5000 j.tV. Because the microplates are not barcoded for identification, a positional control is also included on each tray. This is an eluate of a blank disc of blood-collection paper placed in a unique position such that it documents each tray’s number in the sequence of analysis (e.g., tray 2 must have a blank result at position 2). The positional control also serves as a water blank on each tray. DERIVATION OF PATTERNS For each newborn tested, a hemoglobin pattern presents the observed hemoglobins in order of relative concentration from the highest to lowest. GDL developed algorithms (see Appendix) for use on the chromatography workstation software so that chromatographic peaks from noise and minor hemoglobins are not included in the hemoglobin patterns. The retention time of Hb A, is similar to that of Hb E. However, Hb A2 is never expressed in a hemoglobin pattern derived for a newborn specimen because its concentration is low, and the interpretation algorithms remove such low peaks from the pattern (Appendix). Satellite hemoglobins (e.g., E1, 5,, C,) produce small chromatographic peaks that elute -0.17 mm faster than the peak of the corresponding major hemoglobin [5,7]. GDL introduced a 1:4 rule to eliminate the satellite hemoglobmns from the reported patterns. Also, based on pub- lished information on the relative percentage concentrations of Hb A in thalassemia cases [8, iS], GDL developed a 1:2 rule to differentiate patterns FAS (sickle cell trait) from FSA (S/f3- thalassemia) (see Appendix). Hb (1), an unidentified hemoglobin eluting between Hb F and Hb A, appears in the HPLC chromatograms for the linearity calibrators, the liquid controls, surrogate in-house DBS samples, and in most DBS specimens from newborns. The retention time for Hb (1) corresponds to that expected for Hb A,d [16]. The program used to derive the hemoglobin pattern adds the concentration of Hb (1) to the concentration of Hb A and reports the total concentration as Hb A (Appendix). SPECIMENS ANALYZED GDL collected data on three types of samples: the liquid Bio-Rad FAES and FADC controls, which are not processed through the punching, elution, and dilution steps of the method; surrogate DBS specimens prepared in house (GDL-DBS sam- ples); and newborns’ specimens collected on paper (NB-DBS specimens). The liquid controls contain Hb F, A, 5, C, E, D in concentrations similar to those found in actual newborn speci- mens, i.e., mostly Hb F with 10-20% Hb A and 3-10% of the other variants. The GDL-DBS samples, which contain Hb F, A, and 5, were prepared by mixing commercial bulk AS blood or cord blood with adult outdated bank blood and spotting the mixture onto specimen collection paper. These DBS sample pools contain various concentrations of the hemoglobin variants for use as controls and proficiency-test samples. For GDL-DBS pools containing Hb S, the concentration of Hb A is large (-75%), and the ratio of [Hb A]/[Hb S] does not match the ratio found in newborn specimens. Nevertheless, these samples yield satisfactory chromatograms. EVALUATION OF PERFORMANCE CHARACTERISTICS The precision and accuracy of retention times, as well as the precision of the variant quantification, were determined from data collected with 23 column systems at nine laboratories using one lot of cation-exchange resin. We also evaluated the accuracy and precision of data collected with four different lots of resin by one laboratory using three column systems. The other perfor-
  3. 3. ‘20O E a) (I) 0 100 0 400 a) Cl) 0 a- C’) a) 0 0 2 Li 0 2 706 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots mance characteristics were determined for the aggregate of 23 column systems and four lots of resin. Results PERFORMANCE ASSESSMENT Chromatograms. Fig. 1 is a chromatogram obtained from a liquid sample made by combining controls with Hb F, A, 5, C, E, and D. Fig. 2 is a chromatogram of an eluate of a DBS specimen obtained from a newborn with sickle cell disease. Precision of retention times.Table 1 compares our results with the instrument specifications. The SD specification is one-sixth the range of the retention time identification window (window ± 3SD). The observed SD for Hb S exceeds the limit some- what; nonetheless, at this precision, all cases of sickle cell disease have been correctly identified. Accuracy of retention times. The observed mean retention time (Table 1) for each hemoglobin is compared with the retention time for the center of the hemoglobin identification window in the integration software. In most cases the observed mean values are within 0.01 mm of the specified value, and in no case does the bias exceed 0.03 mm. Precision of quantification of hemoglobins. According to the speci- fications (Table 2, last column), with a one-column system (with one photometer) the interrun CV for Hb F in the middle- concentration linearity calibrator should be no more than 5%. Also, among all systems the mean should always lie between 80 000 and 120 000 AU (±20% range for matching the 23 systems). The results are in acceptable agreement with the specifications. The CV of 10.9% over all 23 column systems is equivalent to a ±2SD range of 21.8%, which is close to the ±20% range limit. On multiple column systems the observed CVs for liquid controls and DBS samples span a range from 14% at 30 000 300 Time (mm) Fig. 1. Chromatogram of a 1:1 mixture of two Bio-Rad controls containing Hb FAES and Hb FADC. Peaks (from left to right): Hb FAST, Fl, F, (1), A, E, D, S, and C. (See Appendix for hemoglobin nomenclature.) AU to 18% at 5000 AU. The 5000 AU (18% CV) is equivalent to a NIB-DBS specimen containing a hemoglobin variant at 2.5% relative concentration (in a total area of 200 000 AU). The 30 000 AU (14% CV) is equivalent to a NB-DBS specimen containing a hemoglobin variant at 15% relative concentration. Detection limits. HPLC peak criteria in the integration parame- ters are set so that Hb S and C are expressed in the hemoglobin pattern when the relative concentration of each exceeds 0.5%. For Fib F, A, E, or D the detection limit is 1.0%. To achieve these detection limits requires that the height threshold used to eliminate background noise be reduced to 500 jV. To test the practicality of this threshold, GDL submitted two GDL-DBS samples containing 1.1% and 0.8% Hb S for analysis at the satellite laboratories. In 12 weekly shipments (one sample per week) to nine laboratories (n = 106 analyses after excluding 2 results that were invalid for other reasons), there were no missed Hb S peaks. By now, >450 000 newborns have been tested with use of the 500-.tV height threshold, and there have been no known missed cases of Hb S or other clinically significant variant exceeding 0.5% in the newborns’ specimens. That a 500 .tV threshold is required is shown by results obtained during the first 3.5 years of HPLC screening, during which time we used a threshold of either 3000 or 2000 j.tV. At 3000 j.V not all satellite laboratories reported the presence of Hb 5, even though it made up 1.0% of the total hemoglobin in the GDL-DBS proficiency-test samples. However, retrospective examination of the chromatograms, which are stored on mag- netic tape, and reprocessing at a lower threshold detected all of the Fib S peaks. Also, during that period, -2.1 million newborns were screened. In rare instances Hb S, A, E, and D that were not reported in the newborn screening pattern were found in children at an older age (Table 3). In all cases, when the chromatographic raw data were electronically reprocessed at a lower threshold (e.g., 500 .tV), we found a well-resolved bell- shaped peak at the retention time of the missing variant. bOO Time (mm) FIg. 2. Chromatogram of a newborn dried blood-spot specimen with pattern ES. Peaks (from left to right):void volume and Hb FAST, Fl, F, (1), and S.
  4. 4. n Table 1. PrecisIon and accuracy of retention times. Retention time, mm x 100 Mean Mean specification F A E D S C BIas 422 422 211 211 211 211 SD 64.08 83.29 98.14 107.93 118.25 172.60 Hb Liquid samples Dried blood spots Tables 1 and 2: Datacollectedfrom nine sites using resin lot 4 on 23 columnsystems. SD specification F 61.00 83.00 98.00 107.00 118.00 172.00 CV, % A S + 3.08 + 0.29 +0.14 +0.93 +0.25 +0.60 90 0.97 1.00 1.26 1.39 1.39 1.59 97 204 63.37 84.29 117.57 2.33 1.67 1.33 1.67 1.33 2.00 61.00 83.00 118.00 1.5 1.2 1.3 1.4 1.2 0.9 -2.37 + 1.29 -0.43 1.17 1.02 1.41 2.33 1.67 1.33 1.8 1.2 1.2 The concentrations determined after reprocessing are given in Table 3. Theoretically, the detection limit of the method might be decreased by a further reduction in the threshold setting. However, all cases of sickle cell disease are detected at the current value. Specificity.In the past 4 years, GDL has been notified of 12 instances in which diagnostic follow-up results did not agree with the newborn screening hemoglobin patterns, and for which the discrepancy could be attributed to the specificity of the HPLC method (Table 4). Also, we have found that -1% of the chromatographic peaks at the retention time for Hb S have an atypical concentration ratio for [Hb A}:[Hb S] of -6:1 (usually the ratio is -1:1). The screening patterns for these newborns are FAS (also in Table 4). Hb G, an a-chain variant with four chromatographic peaks, is readily identified by visual inspection of the chromatogram. According to the rules given in the Appendix for derivation of patterns, and depending on the particular lot of cation-exchange resin in use, Hb G has been reported in newborn screening as Hb E, Hb D, or combinations of Hb E and D with Hb (2). Background and carryover. Water blanks are included on the calibration tray and on each tray of newborns’ samples for analyses. GDL staff review all chromatograms that show a peak with height >5000 .tV. These situations occur infrequently (9 times per 100 000 newborns) and do not affect newborns’ hemoglobin patterns. In some cases small spikes are observed, usually at the void volume (retention time 0.2 mm). However, the retention times are too fast, the peak widths too narrow, and the heights too low for these spikes to affect the hemoglobin patterns generated by the method algorithms (Appendix). Peak shape. Laboratory staff monitor the Hb F peak shapes for the controls and for 5% of all newborns’ specimens. By remov- Table 2. PrecIsion of quantification. No. of column Hb systems F AU No. of samples Mean SO F Liquid samples Dried blood spots A S CV, % 18 40 23b 105 23 213 23 213 103 834 101 679 29 057 7 338 F 4621 11 112 4 295 1 002 Table 3. ComparIson of the newborn screening result with the result obtaIned at an older age.a Pattern Resuft after reprocessing Case no. Newborn Older data, % of total Hb 1 FS FSaC HbA, 0.9 2 FS FSa HbA, <0.5 3 ES FSa HbA, <0.5 4 ES FSAC HbA, 0.8 5 ES FAS HbA, <1.0 6 FAd FAS HbS, 0.5 7 FA#{176} FAS FIbS, 0.88 8 F only FAS HbS, 1.08 9 FE FEa HbA, <0.5 10 FA’ FAE HbE, 0.8 11 F only HbA, 0.9, HbD, 0.8 8 Differencesattributableto prior use of higherthreshold (>500 MV). b Original newborn screening data reprocessed to reflect current 500 V threshold. r FSApatterns are reported as traitunless(Hb SJ>2 lHbAl,inwhichcase the pattern isreported as FSa forS/p.thalassernia(seeAppendix). d Follow-up initiatedfromfamilystudies. 8 HbS >0.5%. ‘Reanalysis ofNB-DBS specimen gave 1.1% HD E and a patternofFAE. g v represents an unidentifiedvariant. 4.4 10.9 14.8 13.6 A S 23 27 37838 23 97 5284 23 206 5 113 5 147 918 894 13.6 17.4 17.5 ClinicalChemistry 42, No. 5, 1996 707 8CV <5% specified. Rangeof ±20% specified.
  5. 5. 708 Eastman et al.: Screening newborns for hemoglobmnopathies by HPLC of dried blood spots Table 4. Newborn screening results dIfferIng from clinIcal foilow-up results because other Variants eluted at retention tImes for Hb S, E, and D. Screening pattern Follow-up pattern Follow.upmethodb FAV IEF FA IEF FAS 1 FAE 2 FAE 3 FAE 4 FAE 5 FAE 6 FAE 7 FAE 8 FDA 9 FDE 10 FAS 11 FAS FA FAV FAV FAC FAD/G FAD/G FAG FETak FAV IEF IEF IEF Mother CAE CAE Mother MS IEF FA IEF V represents an unidentified variant. 8 Notthesame as the cases in Table3. IEF,isoelectricfocusing;CAE,cellulose acetate electrophoresis;MS, mass spectrometry;Mother,the newborn’smotherwas tested for hemoglobinopathies. Case no. See comment Comment Hb variant elutes on HPLC as FIb S. Incidence 1/10 000 newborns (1% of FAS patterns). [FIb A1/[Hb V] - 6/1 Hb variant,not resolved by IEF, elutes on HPLC as Hb E. Same as case 1. Hb variant (not Hb C) elutes on HPLC as Hb E. Same as case 3. Same as case 3. Hb G elutes on HPLC as Hb E. (Hb D ruled out by the HPLC method.) Same as case 6. FIb G elutes on I-IPLC as FIb D. Hb Tak elutes on HPLC as Hb D. Hb variant elutes on HPLC as Kb S. [Hb Al/ [Hb V] - 1. Kb variant, not resolved by IEF, elutes on F$PLCas HbS. [Hb A]/[Hb V] - 1. ing defective columns from use in testing specimens, the staff maintain the height/area ratio for Hb F in liquid controls within a specification of 3.5 MV/AU. During a recent 12-month period when two lots of resin were in use, GDL rejected 102 (6%) of 1769 columns because of broad Hb F peaks. Resolution.Hb F, A, E, D, 5, and C are well resolved (Fig. 1). Effectsofchangingthereagentlot.In 4 years we have used four lots of resin. The CV for retention times on the different lots was comparable with that given in Table 1. The accuracy of retention times for four lots of resin was comparable with that reported in Table 1 for lot 4. In two-thirds of the measurements, the observed mean values for retention times were within 0.01 mm of the specified center of the hemoglobin identification window. The maximum observed bias was 0.03 mm. The observed mean values for all hemoglobins were the same for liquid controls and eluates from DBS samples (no matrix effect). The precision of quantification was determined by measur- ing Hb F in the linearity calibrators with three column systems at one laboratory; the CVs were <6% for each of the four lots of resin. Photometer readings for the three linearity calibrators were within acceptable limits (nominal value ±20%). CVs for measuring Hb F, A, and S in DBS samples were <16% for all lots, which is similar to the results obtained on one lot (Table 2). Linearity. For four lots of resin and the corresponding lots of linearity calibrators, the dose-response curves for the three relative concentrations gave the following multiple R2 values: lot 1, 0.977; lot 2, 0.975; lot 3, 0.976; and lot 4, 0.985. FREQUENCY DISTRIBUTION OF TOTAL HEMOGLOBINS When applied to the California newborn population, the HPLC screening method gave the following distribution for the total area for all hemoglobins: n = 151 000; mean = 208 000 AU; SD = 43 400 AU; skewness, 0.283; 1st percentile, 110 000 AU; 50th percentile, 207 000 AU; 99th percentile, 320 000 AU. Within the overall CV of 21% for the frequency distribution, the variance (SD)2 is estimated to be distributed among its compo- nents as follows: physiological 14%, DBS sample collection 14%, and HPLC methodology 72%. Discussion The HPLC screening method quantifies the relative concentra- tions of hemoglobin variants and has good reproducibility with singleton determination. Quantitative ratio rules are invoked to derive automatically the presumptive phenotype for each new- born. Setting quantitative limits allows application of routine quality-control rules. Proficiency tests are scored with the use of quantitative acceptability limits. Analyte contents measured in newborns’ DBS specimens are dependent on the adequacy of the specimen. A hemoglobinop- athies screening test result of an extremely low or high concen- tration of hemoglobin reveals specimens that are not suitable for determinations of any of the newborn screening analytes (phe- nylalanine, thyroxine, thyrotropin, uridyl transferase, etc.). In such cases a second blood specimen must be obtained from the newborn. The HPLC screening method requires only a small sample. One punch of a 0.95-cm-diameter disc from a blood-collection card is eluted in water to separate the hemoglobmns. This same
  6. 6. ClinicalChemistry 42, No. 5, 1996 709 eluate is used for the determinations of two other newborn screening analytes, phenylalanmne and uridyl transferase. According to Bio-Rad, the rapid separation of hemoglobins is possible because proteins do not penetrate the resin. Also, any degraded hemoglobins and other proteins are removed from the cation-exchange resin before the hemoglobmns of interest are eluted. The interferences from variant hemoglobmns that have reten- tion times similar to Hb S, C, E, and D (Table 4) are relatively few and do not compromise the detection of newborns with sickle cell disease. Also, degradation (if any) of hemoglobmns in a DBS sample does not interfere with the reporting of an accurate phenotype. Disadvantages of the method include the requirement for manual aliquoting and dilution of the specimen eluate into the microplate, which is subject to specimen identification error, given that a specimen may be pipetted into the wrong well of the microplate. Also, because the microplates have no barcode identification, a positional control is needed to maintain the sequence of analysis. Although the California program does not screen for Hb Barts, this variant is measurable with the HPLC screening method. The chromatographic peak for Hb Barts elutes with a retention time close to that of the void volume and can be seen, for example, in some DBS specimens from newborns with Hb E, giving a chromatogram characteristic of EJa-thalassemia. Other methods used to screen newborns for hemoglobinop- athies are cellulose acetate (basic) and citrate agar (acidic) electrophoresis and isoelectric focusing. In a large-scale screen- ing program, these methods do not compare favorably with HPLC screening, because they are not automated and quanti- tative. When electrophoresis is used, the presumptive pheno- types are derived by visual inspection, consensus decision- making, and manual data entry, all of which are subject to human error and judgment. With HPLC screening, presump- tive phenotypes are derived automatically. Quality control and proficiency testing are quantitative. Compared with other HPLC techniques such as anion-exchange chromatography, the cation-exchange chromatography used here has the advantage that hemoglobin degradation products are eluted rapidly from the column and do not interfere with quantification of the principal hemoglobmns. Results of the laboratory analyses in clinical follow-up presented in Tables 3 and 4 were determined under contract to the California Department of Health Services by the Children’s Hospital Oakland Research Institute, directed by F. Shafer, B. Lubin, and E. Vichinsky. References 1. Office of Medical Applications of Research. Newborn screening for sickle cell disease and other hemoglobinopathies. National Insti- tutes of Health consensus development conference statement, Vol. 6, No. 9. Bethesda, MD: National Institutes of Health, Public Health Service, US Department of Health and Human Services, 1987. 2. Sickle Cell Disease Guideline Panel. Sickle cell disease: screen- ing, diagnosis, management, and counseling in newborns and infants. Clinical Practice Guideline No. 6. AHCPR Pub. No. 93- 0562. Rockville, MD: Agency for Health Care Policy and Research, Public Health Service, US Department of Health and Human Services, 1993. 3. California Health and Safety Code §309.5 (West 1990 & Suppl 1995). 4. Lorey F, Cunningham GC, Shafer F, Lubin B, Vichinsky E. Universal screening for hemoglobinopathies using high performance liquid chromatography: clinical results of 2.2 million screens. Eur J Hum Genet 1994;2:262-71. 5. Wilson JB, Headlee ME, Huisman THJ. A new high-performance liquid chromatographic procedure for the separation and quanti- tation of various hemoglobin variants in adults and newborn babies. J Lab Clin Med 1983:102:174-86. 6. Ou C-N, Buffone GJ, Reimer GL. High-performance liquid chroma- tography of human hemoglobins on a new cation exchanger. J Chromatogr 1983:266:197-205. 7. Huisman THJ. Percentages of abnormal hemoglobins in adults with a heterozygosity for an a-chain and/or a chain variant. Am J Hematol 1983;14:393-404. 8. Kutlar A, Kutlar F, Wilson JB, Headlee MG, Huisman THJ. Quanti- tation of hemoglobin components by high-performance cation- exchange liquid chromatography. Am J Hematol 1984;17:39-53. 9. Rogers BB, Wessels RA, Ou C-N, Buffone GJ. High performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias; report of three cases. Am J Clin Pathol 1985;84: 67 1-4. 10. Wilson JB, Wrightstone RN, Huisman THJ. Rapid cation-exchange high-performance liquid chromatographic procedure for the sepa- ration and quantitation of hemoglobins S, C, and 0 Arab in cord blood samples. J Lab Clin Med 1986:108:138-41. 11. Huisman THJ. Separation of hemoglobins and hemoglobin chains by high-performance liquid chromatography. J Chromatogr 1987; 418:277-304. 12. Ou C-N, Rognerud CL. Rapid analysis of hemoglobin variants by cation-exchange HPLC. Clin Chem 1993;39:820-4. 13. van der Dijs FPL, van den Berg GA, Schermer JG, Muskiet FD, Landman H, Muskiet FAJ.Screening cord blood for hernogmobinop- athies and thalassernia by HPLC. Clin Chem 1992;38:1864-9. 14. Loomis SJ, Go M, Kupeli L, Bartling DJ, Binder SR. An automated system for sickle cell screening. Am Clin Lab 1990;Oct.:33-4O. 15. Weatherall DJ. The thalassaemia syndromes. Oxford, UK: Black- well Scientific Publications, 1965:268 pp. 16. Bisse E, Wieland H. High-performance liquid chromatographic separation of human haemoglobins, simultaneous quantitation of foetal and glycated haemoglobins. J Chromatogr 1988;434:95- 110. Appendix: Rules for Derivation of Hemoglobin Pattenis A list of the hemoglobmns with their retention times is given below. Numbers in parentheses are used for unidentified spe- cies. For example, Hb (1) elutes between Hb F and Hb A. Noise and minor hemoglobins. When present, Hb A is used as an internal standard. Any chromatographic peak with an area <0.1 the area of the Hb A peak is not included in the pattern. If Hb A is not present, the other adult hemoglobin (e.g., Hb S in sickle cell disease) is used as the internal standard. Hb (1).Hb (1) (possibly Hb Aid) elutes between Hb F and Hb A. The computer program adds the concentration of Hb (1) to
  7. 7. Peak name FAST Fl F A E 0 S C (1) (2) (3) (4) (5) 0.18 0.45 0.61 0.83 0.98 1.07 1.18 1.72 0.73 0.91 1.13 1.33 1.55 710 Eastman et al.: Screening newborns for hemoglobinopathies by HPLC of dried blood spots (6) 1.85 the concentration of Hb A. The total concentration is expressed in the pattern as Hb A. Hb F1 The concentration of Hb F1 (acetylated Hb F) is added to the concentration of Hb F and the sum is expressed in the pattern as Hb F. Satellite hemoglobins. Consider Hb X as the major hemoglobin and Hb Z as a potential satellite hemoglobin [5, 7]: If the concentration of Hb Z is <0.25 the concentration of Hb X, Hb Z is deleted from the pattern. In this work the combinations of major and satellite hemoglobmns are (X/Z): E/A, D/(2), D/A, SIE, S, and Cl(s). Thalassemia flag. If Hb A and Hb Y (any variant) are both present, divide the concentration of Hb Y by the concentration of Hb A. If the quotient is >2, then change the representation of Hb A in the pattern from “A” to “a”. For example, once it has been determined that Hb A and Hb S are both present, the pattern report code of Hb A is changed to Hb a if the concentration of Hb S is more than twice that of Hb A. Thus S/-thalassemia is reported as FSa. For follow-up, FAS and FSA are both reported as trait, unless [Hb S] >2[Hb A], in which case the pattern is reported as S1f3-thalassemia [8, 15]. Inadequate specimens. When the total area is <60 000 AU, the pattern report is “not determined (low area).” Similarly, when the total area exceeds 420 000 AU, the pattern report is “not determined (high area).” When a repeat analysis confirms ei- ther low area or high area, the specimen is declared made- quate for all the newborn screening analytes, and a new speci- men is requested. In the California program, the number of inadequate specimens so detected is -16 per 100 000 new- borns tested. Sample degradation. A flag is used to identify specimens with excessive concentrations of hemoglobin degradation products. In the chromatography system used, these compounds are eluted before Hb F and appear in the two identification windows defined as FAST and Fl. (In most specimens, window Fl holds Hb F1, the acetylated form of Hb F, and no degradation products.) When the total relative concentrations of Hb FAST plus Fl exceed 50%, the hemoglobin pattern is reported as “not determined (FAST exceeds 50%).” In practice, after review of the chromatogram by a quality-control officer, valid Hb pat- terns can be reported with a total [FAST + Fl] as high as 75%. In the California program the incidence of chromatograms with [FAST + Fl] >50% is <1 per 100 000 newborns tested. Many of those found are the result of improper collection of the blood sample from an umbilical line. Such specimens are designated inadequate for determining all of the newborn screening analytes. F only. If the pattern is F only, the result is printed out as “not determined (F only).” This result, which is expected only in cases of f3-thalassemia major, must be confirmed by repeat injection of the DBS eluate. Peak criteria. The peak criteria for inclusion of a species in the hemoglobin pattern are summarized in Table 5. Table 5. Peak criteria for Inclusion of a species In the hemoglobin pattern report. Retention time, mm Peak criterion for Inclusion In pattern report Always excluded Always added to F, so the total of [Fl] + [F] is reported in the pattern as F Always included Included, unless: (a) E is present and [A] <[E]/4, in which case A is deleted from the pattern, or (B) D is present and [Al <[D]/4, in which case A is deleted from the pattern Included, unless S is present and [E] <[S]/4, in which case E is deleted from the pattern Included,unless S is present and [Dl <[S]/4, in which case D is deleted from the pattern Always included Always included If F is present, the total of [(1)1 + [A] is reported in the pattern as A Included, unless D is present and [(2)] <[D]/4, in which case (2) is deleted from the pattern Always included Always included Included, unless C is present and [(5)] <[C]/4, in which case (5) is deleted from the pattern Always included

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