Rapid Quantitation of Hemoglobin S by Isoelectric Focusing*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 24, No. 5 Copyright 1994, Institute for Clinical Science, Inc. Rapid Quantitation of Hemoglobin S by Isoelectric Focusing* M. NARAHARI REDDY, Ph.D. and RALPH A. FRANCIOSI, M.D. Department of Pathology, Medical College of Wisconsin, and Children s Hospital of Wisconsin, Milwaukee, WI ABSTRACT The feasibility of isoelectric focusing (IEF) to determine hemoglobin S (HbS) at a faster turn-around-time and to resolve the HbS and hemoglobin A (HbA) in presence of high concentrations of hemoglobin F (HbF) is evaluated. The IEF procedure is faster, and the results can be obtained in less than 45 minutes. The resulting data are comparable to gel electrophoresis. It is a superior procedure in resolving both HbS and HbA in the presence of high HbF and, therefore, a desirable technique to use for infants and children. Further, IEF is simpler than the gel electrophoresis, relatively inexpensive, easily adaptable for routine use, and suitable for stat conditions. Introduction Hemoglobinopathies related to sickle cell trait and disease (SS, SC, Sp-thalassemia) are some of the common genetic disorders with an incidence of approximately 1 in 400 of all newborns in USA.1 In children up to 5 years of age, sickle cell anemia can develop into at least three potentially lethal complications: (1) bacterial infections, which sometimes can be fulm inant; (2 ) acute splenic sequestration; and (3) aplastic crises.2 Any one or combination of these conditions can be catastrophic to the patient with a 30 to 50 percent increased mor tality rate. Since sickle cell crisis occurs mainly owing to polymerization of HbS under low oxygen conditions, patients undergoing general anesthesia are at risk for an increased morbidity and mortality. Therefore, a need exists for rapid identification and quantitation of HbS to manage patients with sickle cell trait and disease.3,4,5 One of the common laboratory procedures that is used for the separation and quantitation of HbS is electrophoresis at alkaline and acidic conditions followed by densitometric scanning. It requires approximately 3 hours for completion and, therefore, is not amicable for stat situations. This inability to obtain results at a faster turn around time is one of the major draw-backs of electrophoresis, especially in critical situations such as sickle cell crisis. In addition, with sam /94/ $00.90 Institute for Clinical Science, Inc. * Address reprint requests to: M. Narahari Reddy, Ph.D., Children s Hospital of Wisconsin, Pathology Department, 9000 West Wisconsin Avenue, Milwaukee, WI

2 402 REDDY AND FRANCIOSI pies containing high levels of HbF, gel electrophoresis is not a sensitive technique to resolve HbS, as well as HbA. Under these conditions, recognition of the presence of HbA also is critical in order to distinguish patients with sickle cell disease (homozygous HbS) from sickle cell trait (heterozygous HbS). In view of these difficulties, the electrofocusing technique has been evaluated to provide faster turnaround time for results and to obtain an accurate identification and quantitation of HbS, as well as HbA, in the presence of high HbF levels. Materials and Methods Whole blood samples from 19 children (age 1 to 16 years), containing different levels of HbS were collected in tubes containing ethylenediam inetetraacetic acid (EDTA). Total hemoglobin in these samples varied from 8.7 to 15.2 g/dl (mean, 11.4 g/dl), and the percent of HbS levels varied from 5.5 to 77.4 percent (mean, 34.7 percent). All samples were stored at 4 C until analyzed. Reagents and supplies for gel electrophoresis* and for isoelectric focusingt were purchased. E le c tro p h o re sis w as carrie d out according to the protocols supplied by the Beckman Instruments.6 Briefly, 3 to 5 xl of hemolyzed samples were applied to agarose membrane and subjected to electrophoresis using 50 mm barbital buffer, ph 8.6, for 25 minutes at 150 V. The gel was stained with paragon blue stain and the separated hemoglobins were quantitated (as percent) densitometrically by scanning at 600 nm. Isoelectric focusing was performed on one mm-thick precast agarose films containing ampholytes in the ph range of 6.0 to 8.O.7 Hemolysates were made by add * Beckman Instruments, Arlington Heights, IL t Isolab, Inc., Akron, OH ing 10 jjli of well-mixed EDTA-whole blood to 50 fjd of 0.05 percent KCN in water. From this, 3 xl were applied to the gel, air-dried for two to three minutes, and then subjected to electrofocusing for 15 minutes using 10 W and about 1500 V. A constant tem perature of 13 C was maintained throughout the procedure. The separated hemoglobins were fixed for 5 minutes by soaking in 10 percent trichloroacetic acid, followed by washing in water for 5 minutes, and then air-dried for 5 minutes. They were quantitated (as percent) by densitometrically scanning at 415 nm without subjecting the gel to any staining methods. High pressure liquid chromatography was performed exactly as per the protocol described by Ou et al.8 Results and Discussion The isoelectric focusing patterns and their corresponding densitometric scans for HbS in presence Hbs A, F, and C are presented in figures 1 and 2, respectively. The HbS resolved well from the other hemoglobins, including from HbF and HbA, without any ambiguity (figure 1, lanes A, C, and D). This discreet separation was observed even in presence of high concentrations of hemoglobin F (lanes A and C of figure 1); IEF technique provided better resolution of HbS without any trailing or smudging. This better separation in turn resulted in sharper peaks in the densitograms with good baseline separation (A and C, figure 2). Accuracy of the IE F technique to quantitate HbS was evaluated by comparing the results from IEF with that from electrophoresis using split-samples from 19 different patients with varying concentrations of HbS. The regression curve shown in figure 3 yielded the following data: Y (IEF) = x (electrophoresis) ; and a correlation coefficient (r) = These two different

3 RAPID QUANTITATION OF HEMOGLOBIN S BY ISOELECTRIC FOCUSING 403 B F ig u r e 1. Resolution of samples with different hemoglobin phenotypes by isoelectric focusing. Phenotypes of these sam ples are, (A) AS; (B) AC; (C) SC; and (D) AS. The resolved bands represent: 1 = Acetylated HbF, 2 = HbA, 3 = HbF, 4 = HbS, 5 = HbA2, and 6 = HbC. 5 6 (-) techniques resulted in almost identical mean values (sample size, n = 19) of 34.7 percent and 34.2 percent for electropho resis and for IEF, respectively. Electrophoresis cannot resolve HbS in presence of high levels of HbF.6 Moni toring the HbS in infants and children is always a problem. Under persistent high levels of HbF, this can be a problem even in children of older ages. Therefore, the IEF technique was evaluated to resolve and quantitate HbS in presence of differ ent levels of HbF. For this, three differ ent hemolysates from patients of known AS or AA phenotypes were mixed with a hemolysate obtained from cord blood. The final composition of hemoglobins in these three samples, determined by high pressure liquid chromatography,8 was Sample 1: HbF = 90.6 percent and HbA T" 2 X1 6 1! F ig u r e 2. D ensitom etric patterns of the resolved hemoglobins for I 3 11i the samples given in fig /I s li L ure 1. The resolved hemo - it s = icrr-1 globins were scanned at 415 nm without any stain ing. Different hemoglo 0 bins in these samples are: 1 = Acetylated HbF, 2 = 4 2 HbA, 3 = HbF, 4 = HbS, 5 = HbA2, and 6 = HbC. j I 1 1 A -w J -

4 404 REDDY AND FRANCIOSI Electrophoresis (% Hb) F ig u r e 3. Correlation of quantitative HbS results (given as percent of total Hb) from gel elec trophoresis (ph 8.2) and isoelectric focusing, repre senting regression line (----- ) with 95 percent con fidence limits (----- ). The obtained linear regression equation: Y (IEF) = x (electropho resis); r = Nineteen samples containing 8.7 to 15.2 g/dl of total hemoglobin were used, and the HbS percent was calculated from the HbS peak, as percent area, after densitometric scanning at 415 nm. F ig u r e 4. Resolution of HbA (lane B), and dif ferent concentrations of HbA and HbS (lanes C and D) in presence of high concentration of HbF by isoelectric focusing (figure 4A), and their corresponding d en sito m etrically scanned patterns (figure 4B, C, and D). Concentra tions of different hemoglo bins in these sam ples, determined by high per formance liquid chroma tography (HPLC), were: HbA = 9.4 percent and HbF = 90.6 percent (lane B); HbA = 0.6 percent, HbS = 10.1 percent and HbF = 89.3 percent (lane C); HbA = 7.3 percent, HbS = 1.8 percent and HbF = 90.9 percent (lane D). The resolved hemo globins represent, 1 = A cetylated H bf, 2 = HbA, 3 = HbF, 4 = HbS. = 9.4 percent; Sample 2: HbF = 89.3 percent, HbS = 10.1 percent, and HbA = 0.6 percent; Sample 3: HbF = 90.9 percent HbS = 1.8 percent, and HbA = 7.3 percent. When these samples were subjected to IEF, HbS as well as HbA were clearly resolved from the HbF without any trail ing or smudging (figure 4, sample 1 = lane B, sample 2 = lane C, sample 3 = lane D). However, when split samples from these were used for the routine electrophoresis at ph 8.2, no clear sepa ration of either HbS (lane C) or HbA (lanes B and D) was observed (figure 5), indicating the superiority of the IEF pro cedure to resolve HbS or HbA in the presence of high concentration of HbF. This superiority of the IEF technique was evident since even HbF at a concen tration as high as 90 percent of the totals, Hbs S and A were resolved clearly and allowed to obtain accurate results for the concentrations of HbS at as low as 1.8 percent (lane D, figure 4) and HbA at as low as 0.6 percent (lane C) of the totals.

5 RAPID QUANTITATION OF HEMOGLOBIN S BY ISOELECTRIC FOCUSING ^ B L J The time requirements for completion of electrophoresis and IEF are about 130 minutes for the electrophoresis (30 min utes for washing and centrifugation of red cells to remove plasma proteins, 2 min utes for hemolysate preparation, 10 min utes for sample application and drying, 35 minutes for electrophoresis, 45 min utes for staining, washing and drying, and 5 minutes for densitometric scan ning); and about 45 minutes for the iso electric focusing (2 minutes for hemoly sate preparations, 5 minutes for sample application and drying, 15 minutes for focusing, 15 minutes for fixing, washing and drying, and 5 minutes for densito metric scanning). Further, if electropho resis has to be performed also at acidic ph, often a requirement for an accurate identification of HbS under high levels of HbF, an additional 60 to 90 minutes will be necessary to obtain the results. Therefore, final results from the IEF procedure can be obtained in relatively less time than by the electrophoresis. The reasons for this are that the IEF, unlike the electrophoresis, can be per formed from the hemolysate using whole blood. It requires only 15 minutes for actual focusing and can be scanned I 4 05 F ig u r e 5. Resolution of HbA (lane B), and two different levels of HbA and HbS (lanes C and D) in presence of high con centration of HbF by gel electrophoresis at ph 8.2 (figure 5A), and their cor responding densitometrically scanned patterns (figure 5B, C and D). Same samples that were used for the IE F were also used for the electro phoresis. Type of hemo globin present and its concentration in these samples are given in the legend for figure 4. immediately after focusing without going through elaborate staining, destaining and drying steps. If results are needed at a faster time, the IEF plate can be scanned immediately after the focusing step without going through the wash cycle, thereby saving about an additional 10 minutes. Because IEF resolves hemo globins into extrem ely narrow and focused bands, it allows scanning of the resolved hemoglobins at a higher molar absorptivity wavelength of 415 nm, (Soret band) to quantitate, thus eliminat ing the need of use of extensive washing and staining steps. In summary, IEF simplifies the whole protocol for identification and quantita tion of HbS. Further, under emergency conditions, IEF provides a general idea of HbS immediately after focusing by observing bands visually. By simplifying sample preparation and elim inating staining and destaining steps, results can be obtained in about 20 minutes. This procedure is, in our opinion, extremely advantageous for infants who have to undergo emergency surgical intervention and those with sickle cell anemia who are in crisis for their immediate manage ment. Although, accurate identification

6 40 6 REDDY AND FRANCIOSI and quantitation of HbS can be accomplished by other methodologies including high performance liquid chromatography.8 IEF is a preferred one because of its ease-of-operation and rapid turnaround-time. References 1. Brittenham GM, Schechter AN, Noguchi CT. Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes. Blood 1985;65: Pearson HA. A neonatal program for sickle cell anemia. Adv Pediatr 1986;33:381^ Harkness DR. Sickle cell trait revisited. Am J Med. 1989;87: Gaston MH, Verter JI, Woods G, et al. Prophylaxis with oral penicillin in children with sickle cell anemia. New Engl J Med 1986;314: Black J. An isoelectric focusing m ethod to detect hemoglobin variants in newborn blood samples including (J-thalassemias. Hemoglobin 1988;12: Instructions for use of Paragon Hemoglobin Electrophoresis (Beckman Instructions A). Beckman Instruments, Inc., Brea, CA Instructions for use of Resolve-Hb : A complete kit for analysis of adult and neonatal blood by isoelectric focusing electrophoresis. IsoLab Inc., Akron, OH 44321, Ou CN, Buffone GJ, Reimer GL, Alpert AJ. High performance liquid chromatography of human hemoglobins on a new cation exchanger. J. Chromatogr 1983;266: