Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC

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1 Clinical Chemistry 44: (1998) Molecular Pathology and Genetics Prenatal and postnatal diagnoses of thalassemias and hemoglobinopathies by HPLC Suthat Fucharoen, 1* Pranee Winichagoon, 1 Raewadee Wisedpanichkij, 3 Busara Sae-Ngow, 1 Rungrat Sriphanich, 1 Warangkana Oncoung, 1 Wanna Muangsapaya, 1 Jew Chowthaworn, 1 Sujin Kanokpongsakdi, 2 Ahnond Bunyaratvej, 3 Anong Piankijagum, 1 and Chris Dewaele 4 The conventional approach to qualitative and quantitative analyses of hemoglobin (Hb) molecules for the diagnoses of hemoglobinopathies requires a combination of tests. We used an automated HPLC (VARIANT ) system to study -thalassemia and -thalassemia syndromes in Thailand. The beta-thalassemia short program is applicable to the diagnosis of -thalassemia and -thalassemia disorders, including Hb H, EA Bart s disease, and EF Bart s disease, in adults, newborns, and fetuses. The system cannot quantify accurately certain Hb molecules, such as Hb H and Hb Bart s. The alphathalassemia short program was therefore developed and used to quantify Hb Bart s to detect -thalassemia genotypes in cord blood. This automated HPLC system is an alternative approach to the diagnosis of complicated thalassemia syndromes in Thailand and Southeast Asia. Thalassemia and hemoglobinopathies, a group of autosomal-recessive inherited human disorders, are prevalent in many parts of the world. Heterozygote screening and genetic counseling are essential for the prevention and control of severe thalassemia diseases, i.e., hemoglobin 1 Thalassemia Research Center, Institute of Science and Technology for Research and Development, Division of Hematology, Department of Medicine, Mahidol University, Nakornpathom 73170, Thailand. 2 Department of Obstetrics and Gynecology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 3 Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand. 4 Bio-Rad Laboratories, 9810 Nazarette, Belgium. *Address correspondence to this author at: Thalassemia Research Center, Institute of Science and Technology for Research and Development, Mahidol University, Salaya Campus, Puttamonthon 4 Rd., Nakornpathom 73170, Thailand. Fax ; grsfc@mahidol.ac.th. Received September 22, 1997; revision accepted January 19, (Hb) 5 Bart s hydrops fetalis ( -thalassemia 1/ -thalassemia 1), homozygous -thalassemia, and -thalassemia/hb E. Diagnoses of thalassemia and abnormal Hbs can be performed at three different stages of development: the prenatal, neonatal, and adult stages. Conventional methods, including erythrocyte indices and morphology, Hb electrophoresis, quantitation of Hb A 2,HbE, and Hb F, and detection of erythrocytes containing Hb H inclusion bodies, provide an accurate diagnosis (1 4). Although measurement of Hb A 2 and Hb E by cellulose acetate electrophoresis and elution (5) and microcolumn chromatographic (6) techniques are reproducible and accurate, they are labor-intensive and time-consuming. HPLC is a sensitive and precise method for detecting thalassemia and abnormal Hbs (7 10). It has become the preferred method for thalassemia screening because of its speed and reliability. An automatic HPLC system (VARIANT, Bio-Rad) has been developed primarily for the detection of -thalassemia disorders such as -thalassemia carriers, Hb S and Hb C. But information is quite limited about using such a system to study the complicated -thalassemia and -thalassemia syndromes in Southeast Asia (11). In this study, we used the automatic HPLC system set up with the alpha-thalassemia short (ATS) program that we developed and with the betathalassemia short (BTS) program to detect various types of thalassemias in both prenatal and postnatal specimens. Materials and Methods subjects A total of 4147 adult blood samples obtained for routine thalassemia screening were examined. Cord blood specimens, carefully collected to avoid contamination by maternal blood, were obtained from 521 newborns at the 5 Nonstandard abbreviations: Hb, hemoglobin; ATS, -thalassemia short; BTS, -thalassemia short; and MCV, mean corpuscular volume. 740

2 Clinical Chemistry 44, No. 4, Department of Obstetrics and Gynecology, Siriraj Hospital. Thirty blood samples obtained at weeks of gestation from fetuses whose parents were at risk of having severe thalassemia diseases were also studied. The project was approved by the Human Experimental Committee of Mahidol University, Thailand. procedures Erythrocyte indices and Hb concentrations were determined using an automatic cell counter, the Sysmex NE 1500 (TOA). Quantitation of Hb A 2, Hb E, Hb A, and Hb F was performed by the BTS program; quantitation of Hb Bart s was performed by the ATS program on the Bio-Rad VARIANT, a fully automated HPLC system that uses double wavelength detection (415 and 690 nm). The ATS program, developed by the manufacturer, has been designed to separate and quantitate Hb Bart s from all other Hbs ina6min run. Hb Bart s and total Hbs elute at 1.70 and 3.15 min. This program uses a3cm 0.46 cm cartridge packed with a 5- m porous silica-based weak cation exchange material. The analytes are eluted at a flow rate of 2 ml/min, using a step gradient of two phosphate buffers that differ in ph and ionic strength. An in-line prefilter is installed between the injector and the column. Quantitation is based on a specific Hb Bart s calibrator. Samples of whole blood (5 lin2mlof buffer) are hemolyzed before injection. The hemolysate (20 L) is injected onto the cartridge for analysis. All reagents were provided by the manufacturer and used according to the manufacturer s instructions. The VARIANT system was compared with an electrophoresis/elution method (5) for Hb A 2 (E) measurement and against the alkali denaturation method of Betke et al. (12) for Hb F measurement in a total of 245 healthy, -thalassemia trait, Hb E trait, or -thalassemia/hb E disease samples. In the cord-blood study Hb types, including Hb Bart s and Hb Constant Spring (Hb CS), were also confirmed by isoelectric focusing (Isolab). The quantitation of Hb Bart s by HPLC was also compared with microcolumn chromatography by analyzing 50 cord-blood samples with a commercial kit, Quik-Sep (Isolab). The presence of -thalassemia and Hb E in cord-blood samples was confirmed by dot blot hybridization between the amplified DNA and the allele-specific oligonucleotide probes (13, 14); in fetal blood samples, -thalassemia and Hb E were confirmed by reverse dot blot hybridization (15). Detection of -thalassemia 1, -thalassemia 2 (both the rightward, - 3.7, and the leftward, - 4.2, deletion types), and Hb CS was by PCR, as described previously (16 18). Results evaluation of the performance of the hplc system Chromatograms of Hbs from various phenotypes of thalassemia syndromes. The VARIANT BTS program can differentiate several Hbs during a screening study. The chromatogram of a healthy subject with Hb A 2 and Hb A is shown in Fig. 1A. Hb F was eluted at 1.38 min, Hb A at 2.64 min, and Hb A 2 at 3.84 min; the total analysis time was 6.5 min. Fig. 1B illustrates the ability of BTS program to distinguish various Hbs in the Lyphochek Hemoglobin A2/F control (Bio-Rad Laboratories). In samples with the -thalassemia trait, the chromatogram is similar to that of the nondiseased sample, but the concentration of Hb A 2 was increased. Mutations causing -thalassemia produce a deficit of -globin production that ranges from minimal ( -thalassemia) to a complete absence of -globin ( 0 - thalassemia). In homozygous -thalassemia, the chromatogram shows the prominence of Hb F; the absence of or minor amounts of Hb A, depending on the genotype of -thalassemia ( 0 -thalassemia or -thalassemia, respectively); and an unaffected or increased Hb A 2 concentration (Fig. 1C). Hb E was eluted at the same retention time as the Hb A 2 found in healthy samples or samples with the -thalassemia trait. Usually 25 30% was detected in Hb E heterozygotes, and almost 100% was detected in Hb E homozygotes (Fig. 1, D and E). The chromatogram also shows the presence of a Hb A peak in -thalassemia/hb E disease, whereas there was no Hb A in 0 -thalassemia/hb E disease (Fig. 1F). In Hb H disease, there were one or two abnormal peaks present at the retention time of 1 min, before the Hb F peak. The two peaks represent Hb Bart s and Hb H, respectively (Fig. 1G). For EA Bart s disease ( -thalassemia 1/ -thalassemia 2-EA), the chromatogram showed increased concentrations of Hb E ( 15%) and Hb F ( 2 3%; Fig. 1H). For EF Bart s disease ( -thalassemia 1/ -thalassemia 2-EE or -thalassemia 1/ -thalassemia 2-EF), the chromatogram illustrates the predominance of Hb F and Hb E, with minor amounts of Hb Bart s (Fig. 1I) In healthy newborns, the major Hb was Hb F ( 80%), and Hb A was found in small amounts (Fig. 2A). The Hb E peak was also detected in newborns with Hb E syndromes (Fig. 2B). In newborns with -thalassemia diseases, the Hb Bart s peak was also detected (Fig. 2, C and E). The pattern of the chromatogram of Hb Bart s hydrops fetalis was very specific, and the major Hb eluted at almost 0 retention time (Fig. 2C). Because the BTS program cannot quantitate Hb Bart s, the ATS program was developed to identify and quantitate Hb Bart s in -thalassemia (Fig. 2, D and F). The presence of Hb Bart s (first peak) was confirmed by an isoelectric focusing method. The second peak in the chromatogram represents other Hb derivatives. Comparison of concentrations of Hb A 2, Hb E, HB F, and Hb Bart s measured by HPLC and conventional methods. Precision studies were performed using blood samples with different concentrations of Hb A 2 and Hb F. Each sample was analyzed in 20 replicates for each HPLC assay for the within-run precision study and analyzed once a day for 10 consecutive days for the between-days precision study.

3 742 Fucharoen et al.: Thalassemia and hemoglobinopathy diagnoses by HPLC Fig. 1. Chromatograms of various genotypes of thalassemia and Hb E detected by the VARIANT BTS program. (A) Healthy genotype; (B) Lyphochek Hb A2/F control demonstrating relative positions of Hb A 2,HbF,HbS,andHbC;(C) 0 -thalassemia/ -thalassemia; (D) the Hb E trait; (E) homozygous Hb E; (F) 0 -thalassemia/hb E; (G) Hb H disease; (H) EA Bart s disease; and (I) EF Bart s disease.

4 Clinical Chemistry 44, No. 4, Fig. 2. Chromatograms of newborn Hbs detected by the BTS program (A C, E) compared with those by detected the ATS program (D and F ). (A) Healthy newborn; (B) newborn with the Hb E trait; (C and D) newborns with Hb Bart s hydrops fetalis; and (E and F ) newborns with Hb EA Bart s disease. The results showed low within-run variability in the measurement of Hb A 2 concentrations within the reference range or of increased concentrations of Hb A 2 in samples with -thalassemia and with the Hb E trait (CV %); the results of the measurement of Hb F concentrations within the reference range or of the raised Hb F concentrations in the two -thalassemia/hb E samples also showed little within-run variability (CV %). An increase in imprecision (CV %) was observed in the between-days precision study, especially in the measurement of Hb F concentrations within the reference range. Quantitative values for Hb A 2,HbE,HbF,andHb Bart s by HPLC were in agreement with values obtained using the conventional methods (Table 1). The linear regression equation of Hb A 2 and Hb E measured by

5 744 Fucharoen et al.: Thalassemia and hemoglobinopathy diagnoses by HPLC Table 1. Comparison of Hb A 2, Hb E, and Hb F levels measured by HPLC (BTS program of VARIANT) and the conventional methods. Phenotype n VARIANT Betke et al. P VARIANT Elect/elute a P Normal 192 (Hb F) (HbA 2 ) -Thalassemia trait Hb E trait Thalassemia/Hb E a Electrophoresis/elution. Hb F, % Hb A 2 /E, % HPLC (VARIANT BTS program) vs those measured by electrophoresis/elution was y x, with a correlation coefficient of Hb F measured by HPLC (BTS program) was also compared with the method of Betke et al. (12); the linear regression equation was y x, with a correlation coefficient of Although linear regression analysis of Hb F measured by HPLC showed excellent correlation with values obtained by the alkali denaturation method, the mean values of Hb F detected by HPLC were substantially higher than those determined by the alkali denaturation method (Table 1). The amounts of Hb Bart s measured by the ATS program was also comparable with those obtained by microcolumn technique; the linear regression line was y x, with a correlation coefficient of thalassemia screening by hplc Postnatal screening with adult blood. A total of 4147 adult blood specimens were screened for thalassemias and hemoglobinopathies. Subjects were from the routine hematology clinic. Apparently healthy individuals with Hb concentrations 120 g/l were excluded to avoid the effect of iron deficiency anemia. Table 2 shows that the concentration of Hb A 2 increased with the -thalassemia trait, and the Hb F concentration increased ( 1%) in 40% of those cases. Hb concentrations 10% at the position of Hb A 2 were assumed to be Hb E. The concentration of Hb E was % in the heterozygous state and % in the homozygous state. In EA Bart s disease, the Hb E concentration was %, with the appearance of an abnormal peak at the Bart s position. A presumptive diagnosis was made for the -thalassemia 1 trait when patients had a low mean corpuscular volume (MCV, L) and concentrations of Hb A 2 within the reference range without anemia. Although the VARIANT BTS program cannot measure the concentration of Hb Bart s and Hb H in Hb H disease, the abnormal peaks representing these Hbs were shown (Fig. 1G). Diagnosis of Hb H disease was also confirmed by the finding of erythrocytes containing inclusion bodies. Neonatal screening with cord blood. A total of 521 cord-blood samples were screened with the two VARIANT programs. Of these, 195 cases (37.4%) were found to have thalassemias and hemoglobinopathies. The presence of Hb E, Hb Bart s, Hb H, and an abnormal Hb was confirmed by isoelectric focusing. Dot blot hybridization with the allele-specific oligonucleotide probes detected the E -globin gene in 141 cord blood samples in which the Hb concentration at the Hb A 2 position was 2% (Table 3). Of these, 12 cases were found to be homozygous for Hb E; one case was later found to be EF Bart s disease ( -thalassemia 1/ -thalassemia 2-EE), with a Hb E concentration of 30.1% and a Hb Bart s concentration of 17.55%. The Hb E concentration in the remaining cases of homozygous Hb E ranged between 3.9% and 14.9% with a mean SD %. Two newborns carrying one allele of E were also found to have EA Bart s disease ( -thalassemia 1/ -thalassemia 2-EA) and homozygous Hbcs (CS/CS-EA), respectively. A newborn with Hb A 2 (E) (2% of total Hb) was also found to have 0 -thalassemia/hb E disease by dot blot hybridization, which dem- Table 2. Hemoglobin analysis of 4147 adult blood specimens. Phenotype n Hb A 2 /E, % Hb F, % Hb A, % Normal Thalassemia 1 trait Thalassemia trait Hb E trait Homozygous Hb E Thalassemia/Hb E EA Bart s disease ND a EF Bart s disease Hb H disease a ND, none detected.

6 Table 3. Hematologic data and hemoglobin analysis in 521 cord-blood samples. % Hb, HPLC Phenotype n Hb, g/l MCV, L MCH, a g MCHC, g/l Hb type BTS program ATS program A 2 (E) F A Bart s Genotype Normal FA / ; A / A -thal 2 trait FA Bart s / ; A / A -thal 2 homozygote FA Bart s /- ; A / A -thal 1 trait FA Bart s / ; A / A -thal 2/Hb CS CS FA Bart s / CS ; A / A Hb CS trait CS FA Bart s CS / ; A / A Hb CS homozygote CS FA Bart s CS / CS ; A / A Hb CS homozygote-hb E CS E FA Bart s CS / CS ; A / E Hb H disease FA Bart s /- ; A / A FA Bart s /- ; A / A EA Bart s disease EFA Bart s /- ; A / E EF Bart s disease EF Bart s /- ; E / E -thal/hb E disease EF / ; 654 / E Hb E homozygote EF ND / ; E / E Hb E trait EFA / ; A / E EE- -thal EF Bart s / ; E / E EE- -thal EF Bart s / ; E / E Hb E- -thal EFA Bart s / ; A / E Hb E- -thal 2/Hb CS EFA Bart s / CS ; A / E Hb E- -thal 2/ -thal EFA Bart s /- ; A / E EFA Bart s /- ; A / E EFA Bart s /- ; A / E Hb E CS CS EFA Bart s CS / ; A / E CS EFA Bart s CS / ; A / E CS EFA Bart s CS / ; A / E Abnormal Hb F Abn Hb A Abn Hb Abnormal Hb a MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; thal, thalassemia; and Abn, abnormal. Clinical Chemistry 44, No. 4,

7 746 Fucharoen et al.: Thalassemia and hemoglobinopathy diagnoses by HPLC onstrated the presence of a mutation in the IVS II nucleotide 654, a mutation of C3T in one allele and of the E -globin gene in the other. PCR detected -thalassemia determinants, including -thalassemia 1, -thalassemia 2, and Hb CS in 70 cordblood specimens, of which 15 cases also had Hb E. Of these, two were found to have Hb H disease, one had EA Bart s disease, one had EF Bart s disease, and two were homozygous for Hb CS (Hb CS/Hb CS). The concentrations of Hb Bart s determined by the ATS program of VARIANT were all 15% except for a case of homozygous Hb CS coinherited with Hb E, in which the Hb Bart s was 8.3% (Table 3). Because the Hb Bart s concentrations were similar, DNA analysis was necessary to distinguish -thalassemia syndromes among the -thalassemia 1 heterozygotes, the -thalassemia 2 homozygotes, and compound heterozygotes for -thalassemia 2/Hb CS and among Hb CS heterozygotes, -thalassemia 2 heterozygotes, and healthy individuals. Although Hb Bart s in -thalassemia 2 heterozygotes ranged between 1.2% and 2.6%, 40 cases (7.6%) of newborns with the nondiseased -globin genotype also had Hb Bart s ( %). Prenatal diagnosis with fetal blood. Cordocentesis was performed in the pregnancies at risk of having thalassemic fetuses at the gestational ages of weeks. Chromatograms of thalassemia syndromes prenatally diagnosed by the VARIANT BTS program were similar to those of the cord blood specimens. The fetuses with -thalassemia traits had similar concentrations of Hb F and Hb A to the healthy fetus (Table 4). In the Hb E heterozygote, % of the Hb E was Hb E at the Hb A 2 position, which was detected in addition to Hb A, whereas there was no Hb A in fetuses with homozygous Hb E and 0 -thalassemia/hbe disease. The latter two conditions were distinguished by DNA analysis. Hb Bart s hydrops fetalis also produced no Hb A, and only Hb Bart s was demonstrated by HPLC. Discussion The BTS program was designed to detect Hb A 2,HbF, and abnormal Hbs such as Hb S and Hb C; these were clearly separated from the normal Hb. Hb C usually migrates to the same position as Hb A 2 on electrophoresis at alkali ph and is misdiagnosed as Hb E, with a concentration of 30% for the heterozygous state (19). However, some abnormal Hbs, such as Hb E, Hb Tak, Hb D, and Hb Lepore, will also coelute with Hb A 2. Therefore, samples that have 10% Hb A 2 should be tested for the possible presence of other Hb variants. Different concentrations of Hbs may distinguish each abnormal Hb. For example, the percentage of Hb D eluted at the Hb A 2 peak in the Hb D heterozygote was 38.9%, which was higher than Hb E or Hb A 2 in the usual cases for the Hb E heterozygote (25 30%) or for the -thalassemia heterozygote (4 7%). Although the BTS program could not quantitate Hbs Bart s and Hbs that were eluted at a retention time between 0 and 1 min, BTS can qualitatively diagnose common -thalassemia diseases, including Hb H and EA Bart s or EF Bart s diseases (Fig. 1). The low variability for the measurement of Hb A 2 and Hb E was comparable with the other methods for Hb A 2 quantitation; the CV of Hb A 2 by microcolumn chromatography was 2.5%, and that by elution from cellulose acetate electrophoresis was 3.6% (5). The other automated HPLC, the DIAMAT Analyzer System, gave a CV ranging from 1.8% to 4.1% (20). The within-run precision for Hb F in this study was also appreciably better than that of other means. CVs for Hb F by radial immunodiffusion and alkali denaturation range from 3.95% to 5.74% and from 1.4% to 9.1%, respectively, whereas the CV of the HPLC method was 4.5% (21). In adults, an increased Hb A 2 in the range of 4 7% is specific for the -thalassemia trait in almost all cases (22, 23). Our study confirmed the identification of the -thalassemia trait and the Hb E trait, using the Hb A 2 (E) concentration determined by HPLC (Table 2). However, iron deficiency anemia remains a problem in differential diagnosis of -thalassemias, -thalassemias, and Hb E carriers. Iron deficiency anemia has been shown to decrease Hb A 2 and E concentrations and must be ruled out before a diagnosis is made (24). In addition, coinheritance of the -thalassemia gene in -thalassemia or Hb E also affects their phenotypic expression. The concentration of Hb E decreased proportionally with the number of -globin gene deletions, from 25 30% in the heterozygote with a nondiseased -globin genotype to 19 21% in Hb E with -thalassemia 1 or to 11 15% in EA Bart s disease (25 28). Coinheritance of -thalassemia 1 also affects MCV in the -thalassemia trait. The MCV was found to be increased Table 4. The amounts of Hb A 2 (E), Hb F, and Hb A from a normal fetus and fetuses with thalassemias and Hb E. Hb analysis, % Phenotype n Hb type A 2 /E F A Bart s Normal 1 FA Thalassemia trait 6 FA Thalassemia/Hb E 7 EF Homozygous HbE 2 EF 3.0, , Hb E trait 4 EFA Hb Bart s hydrops 10 Bart s 0 0

8 Clinical Chemistry 44, No. 4, up to L, resulting in a misdiagnosis of the -thalassemia trait in some cases. Transfusions also increase the risks of false-positive and false-negative results, especially in individuals who have received blood transfusions from Hb E donors or in Hb E individuals who have received transfusions from healthy donors. HPLC is also useful for large-scale screening of thalassemias and hemoglobinopathies in the neonatal period. Although newborns with -thalassemia cannot be distinguished from healthy newborns because -globin gene expression is not fully functional at this stage of development, the ability of the VARIANT BTS program to detect all cases of Hb E syndromes and demonstrate the absence of Hb A is a great advantage for the diagnosis of 0 - thalassemia diseases (Table 3). However, both homozygous Hb E (asymptomatic) and 0 -thalassemia/hb E diseases had similar chromatograms composed of Hb E and Hb F. Although newborns with homozygous Hb E have a tendency to have higher concentrations of Hb E (3.9 15%) than those with 0 -thalassemia/hb E disease (2%), DNA analysis is necessary to differentiate the two syndromes. Our study also showed that the qualitative demonstration of Hb Bart s and Hb H using the VARIANT BTS program aids in the diagnosis of -thalassemia syndromes during the neonatal period. All cases of Hb Bart s hydrops fetalis are detected, and all cases of Hb H disease and most cases of -thalassemia trait were also detected. The ATS program helps to quantitate the amount of Hb Bart s and provides a diagnosis for newborns with Hb H disease whose Hb Bart s was increased to 20 25% (Table 3). However, as mentioned in previous studies, diagnosis of -thalassemia cannot rely solely on the amount of Hb Bart s because the concentration of Hb Bart s in each phenotype may be similar (29, 30). It should also be noted that in the neonatal period, newborns with -thalassemia syndromes, including Hb H, EA Bart s disease, EF Bart s diseases, the -thalassemia 1 trait, and homozygous -thalassemia 2, have low MCV values, whereas those with -thalassemia syndromes still have MCV values within the reference range. This is because full synthesis of the -globin chain can be attained in the fetal stage, and the abnormalities would interfere with Hb molecules produced thereafter, resulting in abnormal erythrocyte production. In the fetal stage, the patterns of chromatograms with various genotypes of thalassemia were similar to those obtained from cord blood. Although the concentration of Hb A and Hb E were lower than those measured during the neonatal period, the disappearance of Hb A and the specific feature of chromatograms can help in prenatal diagnosis of severe thalassemia diseases. We are grateful to Don H. Keenan and the Bio-Rad Diagnostic Group for the generous loan of the VARIANT Hemoglobin Testing System and the reagents. This study was supported in part by the Prajadhipok-Rambhai Barni Foundation, National of Science and Technology Development Agency, contract number BT HIM-14-17, and a Mahidol University Research Grant. References 1. Wasi P. Population screening. In: Weatherall DJ, ed. The thalassemias. Edinburgh: Churchill Livingstone, 1983: Schmidt RM, Rucknagel DL, Necheles TF. Comparison of methodologies for thalassemia screening by Hb A 2 quantitation. J Lab Clin Med 1975;86: Efremov GD. An evaluation of the methods for quantitation of hemoglobin A 2. Results from a survey of 10,663 cases. Hemoglobin 1977;1: International Committee for Standardization in Hematology. Recommendations for selected methods for quantitative estimation of Hb A 2 and for Hb A 2 reference preparation. Br J Haematol 1978;38: Marengo-Rowe AJ. 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