Hemoglobin Variant Detection from Dried Blood Specimens by High Performance Liquid Chromatography*!

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 23, No. 6 Copyright 1993, Institute for Clinical Science, Inc. Hemoglobin Variant Detection from Dried Blood Specimens by High Performance Liquid Chromatography*! P. DANTE ROA, Ph.D., ERNEST A. TURNER, M.D.,t and MARIA D EL PILAR AGUINAGA, Ph.D. Comprehensive Sickle Cell Center, Departments o f Pediatrics,t Obstetrics, and Gynecology, Meharry Medical College, Nashville, TN ABSTRACT The convenience of dried blood filter paper specim ens for genetic screening programs has prompted us to test the stability of these specimens for hemoglobin identification by cation exchange high performance liquid chromatography. This report shows that identification of Hb AA, Hb AF, Hb AS, Hb FAS, Hb AJ, Hb FJ, Hb EF, and Hb SS can be achieved by high performance liquid chromatography even after six weeks of storage at room tem perature. Also, accurate hemoglobin quantitation can be obtained from the same samples within three weeks of storage at room temperature. The combination of dried blood samples and high performance liquid chrom atography provides an accurate system to screen for hem oglobinopathies, even after long periods of sam ple storage at am bient conditions. Introduction Blood samples collected on filter paper are utilized for genetic new born screening program s, including sickle cell disease. C onventional electrophoretic m ethodologies (cellulose acetate and citrate agar electrophoresis) are widely used to screen dried blood samples for hem oglobinopathies.2,8,10 H ow ever, if the analysis is done a few days after the collection * Send reprint requests to: Dr. M. d. P. Aguinaga, Comprehensive Sickle Cell Center, Meharry Medical College, 1005 D. B. Todd Blvd., Nashville, TN time, poor resolution of hemoglobin (Hb) bands may occur and interfere with accurate identification.9 Lim itations of the conventional techniques have prompted the use of more sensitive procedures, such as isoelectricfocusing electrophoresis (IEF) and high performance liquid chrom atography (HPLC). D etection of hem oglobin types from dried blood using IE F has been discouraged because of the appearance of fuzzy bands w hich makes difficult the identification of common hemoglobin variants.5 On the other hand, HPLC has been successfully employed for the identification of hem oglobinopathies from liquid samp le s.1,4,6,7,11,12 M oreover, it has been shown that for fetal hem oglobin quantit Support was provided by NIH Grant P60- HL38737 and GM /93/ $00.90 Institute for Clinical Science, Inc.

2 43 4 ROA, TURNER, AND AGUINAGA tatio n, H P L C is m ore accu rate and p ro v id e s b e tte r se p a ra tio n p a tte rn s than IE F.3 The present study addresses the need of using sensitive techniques, such as HPLC, for the correct identification and quantitation of hemoglobin variants from dried blood. Our results showed that all the hemoglobin variants tested (Hb AA, Hb AF, Hb AS, Hb FAS, H b AJ, Hb FJ, Hb EF, and Hb SS) could be correctly identified even after six weeks and accurately quantitated after three weeks of storage tim e at am bient conditions. Methods Newborn and adult blood specimens from individuals screened at the Comprehensive Sickle Cell Center, Meharry Medical College, Nashville, Tennessee, were collected in heparinized capillary tubes or ethylenediam inetetraacetic acid (EDTA) tubes, spotted on A & S 903 filter paper discs, and stored in the dark at room tem p eratu re (RT) for up to 10 weeks. Every seven days for 10 consecutive weeks each dried blood sample was transferred into a 1.5 ml Eppendorff tube and eluted w ith hem olysate reagent,* centrifuged at 12,000 rpm for 15 minutes at 4 C, and filtered through a 0.45 xm filter unit into another 1.5 ml tube. These hemolysate samples could be analyzed immediately or stored at 70 C for up to one year. H em olysates from anticoagulated blood samples to be used as controls were prepared in a similar manner. Hem olysates (10 jxl) from dried blood filter paper and liquid blood samples were transferred into H-style Nalgene vials for hem oglobin variant detection and quantitation using an HPLC program5 and a PolyCat A 300, stainless steel column packed with hydrophilic cationic polym er.t A gradient m ade up of m obile phase A, containing 40 mm bis-tris and 4 mm KCN at ph 6.5, and mobile phase B, containing 40 mm bis-tris, 4 mm KCN and 0.2 M NaCl at ph 6.8, was set with a 2.0 ml per m inute flow rate. The equipm ent used consisted of a 600 E multisolvent delivery system with a 700 WISP, 484 tunable UV/VIS detector interfaced w ith a B aseline 810 chrom atography work station program.$ At the end of each run, qualitative and quantitative peak integration results w ere autom atically performed by the incorporated W ater s HPLC com puter system. Results HPLC V a l id a t io n T e s t Anticoagulated blood from 157 adults with normal Hb AA patterns, was spotted on filter paper, dried, and eluted weekly for 10 consecutive weeks. The HPLC quantitative evaluations are shown in figure 1. Each point of analysis represents the results of six to 22 samples. By carefully monitoring peak chromatograms, it was possible to establish relative p e r centage (base 100) values. These findings dem onstrated a decrease in the Hb A component from 100 percent (reference liquid sample) at 0 weeks (time zero) to 78 percent at 10 weeks, i.e., dried blood filter paper samples showed an increase of degradation products and background to 22 percent after 10 weeks. Q u a l it a t iv e a n d Q u a n t it a t iv e A n a l y s is In figure 2 are shown representative chromatograms for hemoglobins AF, AS, and SS during a 10 week period. The starting Hb F component of Hb AF is five percent in the liquid control, and both can be identified after up to six weeks of * Helena Laboratories, Beaumont, TX t Polylc, Columbia, MD t Waters, Division of Millipore, Milford, MA

3 HEMOGLOBIN VARIANT DETECTION FROM DRIED BLOOD SPECIMENS BY HPLC 435 TIME IN WEEKS FIGURE 1. HPLC validation test: High performance liquid chromatography percentage trends (base 100) of normal adult Hb AA samples obtained from dried blood filter paper eluates. Slope indicates Hb AA component losses are probably due to degradation processes. storage tim e at room tem perature. The HPLC quantitative analysis is expressed in relative area percentages. T he hem o globin variant percentage trends for all blood specimens tested (Hb AA, Hb AF, Hb AS, Hb FAS, Hb AJ, Hb FJ, Hb EF, and Hb SS) showed that under the conditions reported, each hemoglobin type exhibits particular forms of degradation (figures 3 and 4). These findings dem onstrated that relative percentages losses of Hb AA after nine weeks are 22 percent or a decrease to 78 percent (figure 3, panel A), w hich is in agreem ent w ith percent RETENTION TIME IN MINUTES FIGURE 2. Representative high performance liquid chromatography chromatograms of Hb A F, Hb AS and Hb SS dried blood specimens: Dried blood filter paper eluates were prepared after 0,1, 6 and 10 weeks of specimens storage at room temperature. Hemoglobin variants were separated by cationic exchange HPLC. Quantitation of the Hemoglobin variants (relative percentage) was performed by integrating the respective peak areas.

4 436 ROA, TURNER, AND AGUINAGA Figure 3. Percentage trends of Hb AA, Hb AF, Hb AS and Hb FAS dried blood specimens by high performance liquid chromatography: Absolute percentages for each hemoglobin variant are given at each time point. Blood eluates were prepared from dried blood filter papers stored at room temperature for up to 1 0 weeks. age trend losses observed for Hb AA in the HPLC validation test (figure 1). For Hb AF, the relative percentage values for both Hb A and Hb F after nine weeks is 57 percent (figure 3, panel B), which if compared to the results obtained for Hb A alone (78 percent) accounts for an additional loss of 21 percent. In this particular case, the presence of Hb F may have accelerated the H b A degradation process. The H b AS show ed com parable relative percentage values at zero weeks (A = 91 percent, S = 89 percent), and the apparent relative stability of the components account for small losses observed (A = 14 percent, S = 24 percent), equiva F igure 4. Percentage trends of dried blood filter paper eluates for Hb AJ, Hb FJ, Hb E F and Hb SS by high performance liquid chromatography: Absolute percentages for each hemoglobin variant are given at each time point. Blood eluates were prepared from dried blood specimens stored at room temperature for up to 1 0 weeks.

5 HEMOGLOBIN VARIANT DETECTION FROM DRIED BLOOD SPECIMENS BY HPLC 437 lent to Hb A values of 86 percent and Hb S values of 76 percent after 10 weeks of storage (figure 3, panel C). W hen F is present, i.e., Hb FAS, the percentage of loss for Hb A and H b S is faster and larger, that is A = 36 percent and S = 37 percent at one week, going to a relative percentage total loss after six weeks of 47 percent for both Hb A and Hb S, while the loss for the Hb F com ponent was 44 percent. Q u a n tita tiv e d e te rm in a tio n of the abnormal Hb AJ depicts a rather stable Hb A against a more liable Hb J component at the storage conditions reported. In figure 4, p an el A, degradation is shown for Hb A = 3 percent while the Hb J com ponent reached a significant 36 percent loss after six weeks. Similar evaluations for Hb FJ, figure 4, panel B, indicated im portant percentage trend reductions for Hb F (34 percent) and greater losses for the Hb J component (83 percent) after six weeks of storage time. Although a rapid degradation pattern for the Hb J component is observed in both of these samples (Hb AJ and Hb FJ), it seems that the Hb F component in Hb FJ has a contributing effect in the rapid disappearance of Hb J. For Hb SS after a loss of 15 percent at w eek one, it dim inished to 12 percent after a 10 w eek period, equivalent to a total loss of 27 percent (figure 4, panel C). It appears that the homozygous condition of the hemoglobin has a positive effect on the stability pattern observed. A similar situation is seen with Hb EF, figure 4, panel D. The Hb E, with an initial percentage of 91 percent, went down 21 percent after nine weeks, b ut significant losses for H b F (45 percent) were observed. In this particular case, the apparent stability of Hb E is probably due to its higher starting percentage, w hereas Hb F, as previously shown, is m ore susceptible to degradation. In figures 3 and 4, the quantitation of each hemoglobin variant is given as absolute percentage and not relative percentage, in order to detect the percentage of hemoglobin variant that degrades over elapsed tim e. Discussion Va l id a t io n T e s t by HPLC T h is v a lid a tio n te s t c o rro b o ra te s reports of hem oglobin losses3 and the appearance of unknow n factors which interfere with interpretation of results w hen analyzed by conventional m eth ods.9 In this particular case, analysis of HPLC has recognized the losses and has identified the sam ple (Hb AA) tested even after a 10 week period storage time at room tem perature. Q u a l it a t iv e a n d Q u a n t it a t iv e An a l y sis by HPLC Identification of hem oglobin com ponents from dried blood filter paper eluates is possible by the use of HPLC even after several weeks of storage at am bient conditions. This may represent a good advantage in the blood screening process for hemoglobin disorders such as sickle cell, thalassem ia, and other hem oglobinopathies. The detection of small amounts of Hb AA is of particular importance to distinguish betw een a baby with FAS or FSA. Analysis by HPLC for eight different whole blood specimens has shown that d ried blood filter p aper eluates stored at room tem perature for several weeks suffered various degrees of degradation of their hemoglobin components, but the sensitivity of this m ethod has perm itted the qualitative and quantitative evaluation of these same samples. H ow ever, there are no published studies that estim ate the actual sensitivity and specificity of H P L C for h e m o g lo b in o p a thies screening.10 The aging of the dried blood or the hem olysate can cause an increase of m inor peaks in the region th at elu tes

6 438 ROA, TURNER, AND AGUINAGA from two to five m inutes and overall background; h o w ev er, reso lu tio n of major peaks is not affected by time, up to six w eeks. T he stability of the dried blood spot and sensitivity of HPLC provide an advantage to the hem oglobinopathy detection laboratory that receives dried blood samples several weeks after they have been collected (i.e., samples from foreign countries). Because of the several advantages of dried blood over cap illary and te s t tu b e sam ples for screening of hem oglobinopathies (easy m ailing and handling, transportation, decreased risk of infection from glass b reak a g e and s p illin g ), c o n tin u o u s im provem ents of detection m ethodologies are necessary, as well as procedures to m inim ize the oxidation and degradation processes of hemoglobin that occur during storage at room tem perature. Acknowledgments The authors gratefully acknowledge Dr. Bettie Nelson-Knuckles for her assistance in the statistical analysis of the data and Ms. Falicia Terrell, MT(ASMT), for the hemoglobin electrophoresis. References 1. BlSSE, E. and W leland, H.: High-performance liquid chromatographic separation of human haemoglobins. Simultaneous quantitation of foetal and glycated haemoglobins. J. Chrom. 434:95-100, G a r r i c, M. D., D e m b u r e, P., and G u t h r i e, R.: Sickle cell anemia and other hemoglobinopathies: Procedures and strategy for screening employing spots on filter paper as specimens. New Engl. J. Med. 288: , G i l b e r t - B a r n e s, E. F., K e n i s o n, K. S., S h r a g o, E., et al: Comparison of an isoelectric focusing technique and high performance liquid chromatography for determination of fetal hemoglobin levels. Amer. J. Clin. Pathol. 96: , H u is M A N, T. H. J.: U sefulness of cation exchange high performance liquid chromatography as a testing procedure. Pediatrics 83(Suppl. 5): , K u t l a r, A., O z c a n, O., B r i s c o, J. T., et al: The detection of hemoglobin variants by isoelectrofocusing using EDTA-collected and filter paper-dried cord blood specimens. Am. J. Clin. Pathol. 94: , Ou, C. N., B u f f o n e, G. J., Re i m e r, G. L., and ALPERT, A. J.: High-performance liquid chromatography of human hemoglobinous on a new cation exchanger. J. Chromatog. 266: , R o g e r s, B. B., W e s s e l s, R. A., Ou, C. N., and B u f f o n e, G. F.: High-performance liquid chromatography in the diagnosis of hemoglobinopathies and thalassemias. Amer. J. Clin. Pathol. 84: , S c h e d l b a u e r, L. M. and P a s s, K. A.: Cellulose acetate/citrate agar electrophoresis of filter paper hemolysates from heel stick. Pediatrics 83(Suppl. 5): , S c h m i d t, R. M., B r o s io u s, E. M., H o l l a n d, S., et al: Use of blood specimens collected on filter paper in screening for abnormal hemoglobins. Clin. Chem. 22: , U.S. Department of Health and Human Services. Guideline: Laboratory screening for sickle cell disease. Lab. Med. 24: , W e s s e l s, R. A., R o g e r s, B. B., O u, C. N., etal: Liquid chromatography used in diagnosis of a rare hemoglobin combination: hemoglobin S/Lepore-Boston. Clin. Chem. 32: , W i l s o n, J. B., W r i g h s t o n e, R. N., and Huism a n, T. H. J.: Rapid cation-exchange highperformance liquid chromatographic procedure for the separation and quantitation of hemoglobins S, C, and O Arab in cord blood samples. J. Lab. Clin. Med. 108: , W in t e r, W. P. and W o z e n c r a f t, L. A.: The chemistry of the decomposition of hemoglobin in filter paper blood samples used in hemoglobinopathy screening. Blood 72:76A, 214, 1988.