Rapid quantitation of hemoglobin S by isoelectric RAPID QUANTITATION OF HEMOGLOBIN S BY ISOELECTRIC

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Text of Rapid quantitation of hemoglobin S by isoelectric RAPID QUANTITATION OF HEMOGLOBIN S BY ISOELECTRIC

  • 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 o f Wisconsin,

    Milwaukee, WI 53226

    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 electropho­ resis. 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-thalas- semia) are some of the common genetic disorders with an incidence of approxi­ mately 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 fu lm inant; (2 ) acute splenic sequestration; and (3) aplastic crises.2 Any one or combination of these condi­ tions can be catastrophic to the patient with a 30 to 50 percent increased mor­

    * Address reprint requests to: M. Narahari Reddy, Ph.D., Children’s Hospital of Wisconsin, Pathology Department, 9000 West Wisconsin Avenue, Mil­ waukee, WI 53226.

    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 mor­ tality. 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 proce­ dures 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-

    401 0091-7370/94/0900-0401 $00.90 © Institute for Clinical Science, Inc.

  • 4 0 2 REDDY AND FRANCIOSI

    pies containing high levels of HbF, gel electrophoresis is not a sensitive tech­ nique 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 electrofo- cusing 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 electro­ phoresis* and for isoelectric focusingt were purchased.

    E le c tro p h o re s is w as ca rrie 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 elec­ trophoresis 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 quanti­ tated (as percent) densitometrically by scanning at 600 nm.

    Isoelectric focusing was performed on one mm-thick precast agarose films con­ taining ampholytes in the pH range of 6.0 to 8 .O.7 Hemolysates were made by add­

    * Beckman Instruments, Arlington Heights, IL 60004.

    t Isolab, Inc., Akron, OH 44313.

    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 , respec­ tively. 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 pres­ ence of high concentrations of hemoglo­ bin 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 com­ paring 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 fol­ lowing data: Y (IEF) = 0.873 x (electro­ phoresis) + 3.905; and a correlation coef­ ficient (r) = 0.9929. These two different

  • RAPID QUANTITATION OF HEMOGLOBIN S BY ISOELECTRIC FOCUSING 4 0 3

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    F ig u r e 1. Resolution of samples with different hem oglobin 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.

    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 H bF .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

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    F ig u r e 2. D ensito - m e tric p a tte rn s of the resolved hemoglobins for the samples given in fig­ ure 1. The resolved hemo­ globins were scanned at 415 nm without any stain­ ing. D ifferent hem oglo­ bins in these samples are: 1 = Acetylated HbF, 2 = HbA, 3 = HbF, 4 = HbS, 5 = HbA2, and 6 = HbC.

  • = 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.

    4 0 4 REDDY AND FRANCIOSI

    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 correspond- ing d e n s ito m e tr ic a lly 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