Screening for hemoglobins S and C in newborn and adult blood with a monoclonal antibody in an ELISA procedure

Summary To facilitate the screening of blood for the presence of hemoglobins S or C, we devised an enzymelinked immunoassay (ELISA). The ELISA procedure incorporated a murine monoclonal antibody (mAb), ^S-1, which recognized both Hb variants but did not react with Hb A, Hb A₂ or Hb F. Hemoglobins in cord or adult hemolysates were coated on the surface of wells of polystyrene microtiter plates and treated with ^S-1 mAb, followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After addition of tetramethylbenzidine substrate solution, a deep blue color developed, signifying the presence of Hb S or Hb C. The ^S-1 mAb ascites fluid could detect purified Hb S and Hb C when diluted to over 1/512,000 and cord blood hemolysates containing Hb S or Hb C when diluted to 1/128,000. Although maximal reactivity was achieved using undiluted hemolysates, the ELISA system could easily detect Hb S and Hb C in cord blood hemolysates when diluted 10^–4. The sensitivity of the ELISA was 1%, which exceeds the lowest quantities of these variants normally found in cord blood. In addition, we found that the ELISA procedure was suitable for detecting Hb S/Hb C in whole blood as well. The entire assay could be conducted on multiple samples in less than 1 h, thus providing a specific, sensitive, rapid and simple screening technique for Hb S and Hb C in cord or adult blood.Abbreviations ELISA enzyme linked immunoassay - mAb monoclonal antibody - Hb hemoglobin(s) - TBS tris-buffered saline - PBS phosphate-buffered saline - BSA bovine serum albumin     

Application of a monoclonal antibody specific for the delta chain of hemoglobin A2 in the diagnosis of beta thalassemia

We have developed a murine monoclonal antibody (mAb) specific for the delta chain of hemoglobin (Hb) A2 that does not cross-react with alpha, beta, or gamma chains. The mAb reacted with Hb P-Nilotic (beta delta hybrid), but not with Hb Lepore-Boston (delta beta hybrid), indicating an epitope consisting of positions 116 (Arg) and 117 (Asn) or 125 (Gln) and 126 (Met) of the delta chain. By using this antibody, we have established a simple and rapid enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of Hb A2 in adult, cord, and fetal hemolysates. We analyzed 70 adult, 8 newborn, and 19 fetal hemolysates from normal subjects and those with various hemoglobinopathies. The mean percentage of Hb A2 was 2.5 for normal adults, 5.4 for beta thalassemic (beta thal) heterozygotes, and less than 0.1% in beta thal fetal samples. We were able to distinguish and characterize certain phenotypes of beta thal patients such as beta thal heterozygotes, beta 0 thal homozygotes, and C beta 0 thal, and C beta + thal double heterozygotes with the aid of this and other mAbs we have generated. This technique is a valuable addition to current methods for the diagnosis of beta thal based on quantification of Hb A2.     

Generation of a monoclonal antibody specific for Hb G-Philadelphia [alpha 2(68)(E17)Asn----Lys beta 2] and development of an immunoassay

A murine hybridoma was generated which secreted a monoclonal antibody (Mab) that specifically recognized the alpha 2(68)(E17)Asn----Lys beta 2 substitution of Hb G-Philadelphia. Hybridomas were produced by fusion of RBF/DnJ immune splenic lymphocytes with FOX-NY murine myeloma cells and selected in adenine-aminopterin-thymidine (AAT) medium. Culture fluids were screened by ELISA for antibody reacting with Hb G-Philadelphia but not Hb A. One such culture was cloned by limiting dilution, expanded and injected into pristane-primed, cyclophosphamide-suppressed BALB/c mice for ascites production. An enzyme-linked immunoassay was developed by conjugating hemoglobin in hemolysates or purified hemoglobins to the plastic surface of wells of a microtiter plate. The ascites fluid containing the Hb G-Philadelphia Mab was added to the wells followed by goat anti-mouse IgG conjugated with horseradish peroxidase. After the addition of substrate (tetramethylbenzidine), a deep blue color developed, signifying a positive reaction. We analyzed 58 hemolysates (17 adult, 41 cord) containing a G-variant along with 28 control hemolysates (12 cords comprising FA, FAC, FAS, FSS, FCC phenotypes; 16 adults consisting of AA, AS, SS, SC, S-beta thal, AD-Los Angeles phenotypes). Of the 58 hemolysates containing a G-variant, 53 were positive by ELISA and confirmed by radioimmunoassay (RIA). Four of the five hemolysates negative for Hb G-Philadelphia were shown to be Hb G-Montgomery by RIA. None of the control hemolysates were positive. The assay could be completed in 1 hr and represents a technological advance in hemoglobin identification.     

Characteristics of a rapid,point‐of‐care lateral flow immunoassay for the diagnosis of sickle cell disease

Sickle cell disease (SCD) is a common and life‐threatening hematological disorder, affecting approximately 400,000 newborns annually worldwide. Most SCD births occur in low‐resource countries, particularly in sub‐Saharan Africa, where limited access to accurate diagnostics results in early mortality. We evaluated a prototype immunoassay as a novel, rapid, and low‐cost point‐of‐care (POC) diagnostic device (Sickle SCAN?) designed to identify HbA, HbS, and HbC. A total of 139 blood samples were scored by three masked observers and compared to results using capillary zone electrophoresis. The sensitivity (98.3–100%) and specificity (92.5–100%) to detect the presence of HbA, HbS, and HbS were excellent. The test demonstrated 98.4% sensitivity and 98.6% specificity for the diagnosis of HbSS disease and 100% sensitivity and specificity for the diagnosis of HbSC disease. Most variant hemoglobins, including samples with high concentrations of HbF, did not interfere with the ability to detect HbS or HbC. Additionally, HbS and HbC were accurately detected at concentrations as low as 1–2%. Dried blood spot samples yielded clear positive bands, without loss of sensitivity or specificity, and devices stored at 37°C gave reliable results. These analyses indicate that the Sickle SCAN POC device is simple, rapid, and robust with high sensitivity and specificity for the detection of HbA, HbS, and HbC. The ability to obtain rapid and accurate results with both liquid blood and dried blood spots, including those with newborn high‐HbF phenotypes, suggests that this POC device is suitable for large‐scale screening and potentially for accurate diagnosis of SCD in limited resource settings. Am. J. Hematol. 91:205–210, 2016. © 2015 Wiley Periodicals, Inc.     

Compound heterozygosity for hemoglobin C and Korle-Bu: moderate microcytic hemolytic anemia and acceleration of crystal formation [corrected] [published erratum appears in Blood 1994 May 15;83(10):3105]

We report here that compound heterozygosity for hemoglobin Korle-Bu (HbKB) and HbC (beta 6 Glu-->Lys) is associated with moderate chronic hemolytic anemia with microcytosis. To understand the pathogenesis of this syndrome, we have studied the effect of Hb Korle-Bu (KB = beta 73 Asp-->Asn) on the crystallization of HbC. We have previously established that fetal Hb (HbF) inhibits the crystallization of HbC. In contrast, HbS accelerates crystallization affecting the pathogenesis of SC disease. We now report on in vitro crystallization of mixtures of HbKB, HbC, and various amounts of HbF and the native hemolysate of a child who is a compound heterozygote for HbKB and HbC. At 6 months of age, the propositus' hemolysate contained 55% HbKB, 39% HbC, and 6% HbF. Crystal formed within 2 minutes compared with 30 minutes for the mixture of 40% HbC:60% HbS and with 180 minutes for 40% HbC:60% HbA. The morphology of the crystals formed was cubic, in contrast with the tetragonal crystals observed in CC and SC disease. Early crystals did not exhibit "sharp edges" until 45 minutes. Purified HbKB formed aggregates but not crystals after 24 hours. Isopycnic gradients showed that the KB/C compound heterozygotes have red blood cell (RBC) densities intermediate between the AC and CC phenotype and similar to SC disease. The surface residue beta 73, known to participate in areas of interaction of the deoxy HbS polymer, can now be assigned to areas of contact in HbC containing crystals. The hemolysis observed in the HbKB/C compound heterozygote is likely to be secondary to the acceleration of Hb crystallization. The microcytosis and increased RBC density is clearly the consequence of the presence of HbC, but the basis of the increased RBC pathology compared with AC trait, despite the low proportion of HbC (35% to 40%), remains to be elucidated.     

Innovative PCR without DNA extraction for African sickle cell disease diagnosis

Objectives: Hemoglobin (Hb) disorders consist of thalassemia and Hb structural variants, of which the major forms are associated with severe anemia and/or vascular occlusion. Current diagnostic techniques are highly accurate and mostly based on isoelectric focusing, high-performance liquid chromatography or mass spectrometry, which often require advanced laboratory equipment. In sub-Saharan Africa, the Hb disorders are mainly associated to the pathological variants hemoglobin S (HbS) and HbC. Unfortunately, until now, it is not easy to get a diagnosis of these disorders in this area. In this study, we tested the performance of a new molecular diagnostic tests on qualified samples.

Methods: The Human Hb S/C Lamp assay is a new polymerase chain reaction test able to detect HbS, HbC and HbA alleles without DNA extraction, directly on fresh or frozen blood samples, or on dried blood spots (DBS). In this study, we compared the genotyping of 248 blood samples (56 whole blood and 192 DBS) with this LAMP assay to the routine diagnostic methods performed in the genetics lab at the university hospital of Liège.

Results: Our results show that the LAMP method can detect HbS and HbC with an accuracy of 100%. Moreover, this test can be used for the neonatal screening because we did not observe any interference with fetal Hb.

Discussion: To our knowledge, this method is the only molecular assay that can be performed directly on dried blood cards without DNA extraction, lowering handling, turnaround time and costs.     

A novel monoclonal antibody based diagnostic test for alpha-thalassemia- 1 carriers due to the (-SEA/) deletion

The presence of minute amounts of embryonic zeta-globin chains in adult hemolysates is a marker for carriers of alpha-thalassemia-1 resulting from (--SEA/) deletion. Recently, we developed a murine monoclonal antihuman embryonic zeta-globin chain antibody, 8E8. By using this antibody, we have now established a slot-blot immunobinding assay for the rapid detection of zeta-globin chains in adult hemolysates. zeta- globin chains were found to be present in 30 blood samples obtained from individuals who were carriers of alpha-thalassemia-1. In another 30 blood samples from individuals who were not carriers of the (--SEA/) deletion, zeta-globin chains were not detected. This simple diagnostic test can be used in appropriate populations to identify those couples at risk of conceiving fetuses afflicted with the Hb Bart's hydrops fetalis syndrome due to homozygous alpha-thalassemia.     

Generation of transgenic mice expressing human hemoglobin E

Hemoglobin E (HbE, beta26 Glu-->Lys) is the most common abnormal Hb variant in the world, and found in greatest frequency in Southeast (SE) Asia. In the United States, HbE is the third most prevalent variant (after HbS and HbC); and its now increasing frequency is due to immigration from SE Asia. HbE homozygotes present a benign clinical picture, but when HbE is coupled with beta0-thalassemia or HbS, variably severe hemoglobinopathies arise. To date, there are no transgenic animal models of HbE-related diseases. We report here the creation of transgenic mice expressing human HbE as a step toward creating animal models for HbE-related diseases. The betaE mice exhibit red blood cell hypochromia and target cells consistent with those observed in human patients exhibiting HbE trait. Furthermore, the transgenic HbE hemolysates contain increased amounts of Hb oxidation products.     

A test tube method for quantitation of hemoglobin A2 using DE 52 cellulose.

A test tube method for quantitation of Hb A2 which is an adaption of the microcolumn chromatographic method is described. The method uses microangular DEAE cellulose (DE 52, microgranular and preswollen, Whatman Inc.) equilibrated with a 0.2 M glycine-0.1% KCN solution, pH 7.25. Hemoglobin A and F become bound to the resin while Hb A2 is in supernatant fraction allowing its quantitation. The method is simple, rapid, inexpensive and accurate, and allows one technician to determine the level of Hb A2 in at least 50 samples a day. The method can be used with whole blood, hemolysates and blood collected on filter paper.     

Identification and quantification of Hb C with an enzyme-linked immunosorbent assay

A highly specific enzyme-linked immunosorbent assay (ELISA) was developed for the rapid identification and quantification of hemoglobin C in hemolysates. The procedure involves coating the surface of microtiter wells with Hb C and then addition of monospecific rabbit antibodies that recognize the unique beta 6 GLU----LYS substitution in Hb C. Next, an antibody to rabbit gamma-globulin conjugated with alkaline phosphatase is added, followed by substrate; a yellow color is formed due to the enzymatic hydrolysis of the substrate, which can be measured spectrophotometrically. For quantification purposes, a hemolysate containing Hb C is introduced just prior to the addition of the Hb C antibody. This results in blocking the attachment of the anti-Hb C to the Hb C coated to the plastic surface. Upon addition of anti-rabbit gamma-globulin conjugate and substrate, there is a consequent reduction or elimination of color formation. Since the degree of diminution of color formation is dose-dependent, standard curves can be developed for quantification of Hb C in unknowns. Of the total hemoglobin, the amounts of Hb C in heterozygotes averaged 27.3 +/- 5.7% by ELISA and 25.1 +/- 3.9% by radioimmunoassay (RIA). In SC individuals the corresponding values were 30.2 +/- 10.1% by ELISA and 24.7 +/- 10.9% by RIA. In homozygotes, Hb C values averaged 83.2 +/- 4.2% by ELISA and 85.0 +/- 6.6% by RIA. Subjects with Hb C beta(+)-thalassemia had 66.5 +/- 3.7% Hb C as measured by ELISA and 63.5 +/- 9.1% as determined by RIA. The ELISA procedure offers distinct advantages for Hb C identification and quantification over other techniques in parameters such as specificity, sensitivity, and rapidity.     

A monoclonal antibody-linked immunoassay for hemoglobin H disease

Summary A murine monoclonal antibody (mAb) was generated that recognizes hemoglobin (Hb) H, the tetrameric form (₄) of human -globin chains. The antibody ₄-1 (1, ) does not react with Hbs A, F, Bart's, or isolated chains, indicating that the antibody recognizes an epitope comprised of multiple chains. A simple, rapid, and sensitive enzyme immunoassay was established to detect and quantitate Hb H in hemolysates from subjects with Hb H disease. The globin level in these patients was also measured using the monoclonal antibody -1, which is specific for chains of Hb A₂. With these assays, 20 hemolysates from subjects with Hb H disease' ten from normal adults and ten from newborn babies were analyzed. The percent of Hb H ranged from 1.5% to 25% in Hb H patients. There was a significant average reduction (32%) in chains in these samples as compared with the normal average adult value. The decreased expresion of chains thus results in a reduction of the levels of normal Hbs A and A₂ and accumulation of ₄, causing Hb H disease.Work supported by NIH grant HLB-41544     

Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins

Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Koln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.     

Development of a fluorescence immunochromatographic assay for the detection of zeta globin in the blood of (--(SEA)) α-thalassemia carriers

Southeast Asian deletion (--(SEA)) α-thalassemia is an inherited monogenic disorder of human hemoglobin, and embryonic globin ζ (hemoglobin ζ, zeta globin chain or Hb zeta chain) has been shown to be a marker that can be used for the identification of carriers of the (--(SEA)) α-thalassemia deletion. In this work, a fluorescence immunochromatographic assay (FL-ICA) was established to detect the zeta globin chain in the hemolysates of carriers of the (--(SEA)) α-thalassemia deletion. This assay can be completed within 10min using a simple UV detector and does not suffer from interference from the red background color of the hemolysate. A total of 314 blood samples were tested by FL-ICA and ELISA. The results of these assays were confirmed by PCR, the standard technique for genetic disease testing. The sensitivity and specificity of this novel FL-ICA were 100% and 98.0%, respectively; the corresponding values for the ELISA performed simultaneously were 100% and 99.2%, respectively. In conclusion, a new FL-ICA-a simple, fast, convenient, low-cost method-was developed that may be useful in both high-throughput screening and individual detection of the (--(SEA)) α-thalassemia deletion in carriers. Additionally, this qualitative FL-ICA may enlighten the development of a new systems for analysis of other target molecules using whole-blood samples.     

Hemoglobin variant HbG-coushatta (beta-22 Glu --> Ala) found by dissociation of blood glucose from values of HbA1C measured by HPLC

OBJECTIVE: Unexpectedly low values of HbA1C, measured by HPLC compared with their blood glucose levels were found in three related persons. We investigated whether this discrepancy was due to abnormal hemoglobin. PATIENTS AND METHODS: HbA1C was measured by latex agglutination and ordinary HPLC. For further examination, a hemoglobin specimen from the 82-year-old female case was prepared and analyzed by PolyCAT A chromatography, ESI/MS and MS/MS. The HbA1C levels of the three cases measured by HPLC were lower than those measured by latex agglutination. The elution profiles on HPLC of the three gave an unusual peak between HbA1C and HbA0. RESULTS: PolyCAT A chromatography revealed two additional peaks which were not present in normal hemolysates. These peaks were revealed to correspond to abnormalities of HbA0 and HbA1C. The amounts of HbA0 and HbX were almost the same (1:0.85) and their glycation ratios were almost equal (5.2% and 5.9%). ESI/MS showed that the woman's intact globin contained an abnormal beta(X)-chain in addition to the normal alpha(A)- and beta(A)-chains. The molecular weight of this abnormal beta(X)-chain was 58 Da lower than that of the normal beta(A)-chain. Glutamate at the 22nd amino acid residue of the beta(A)-chain was replaced by alanine in the abnormal beta(X)-chain.This variant of Hb was revealed to be the same as HbG-Coushatta (beta-22 Glu --> Ala) from the library of variant Hb. Family studies showed that the variant was inherited as a dominant trait. CONCLUSION: The dissociation was due to underestimation of HbA1C in the measurement by HPLC which excluded glycated variant Hb.     

A Test Tube Method for Quantitation of Hemoglobin A2 Using DE 52 Cellulose

A test tube method for quantitation of Hb A₂ which is an adaption of the microcolumn chromatographic method is described. The method uses microgranular DEAE cellulose (DE 52, microgranular and preswollen, Whatman Inc.) equilibrated with a 0.2 M glycine-0.1 KCN solution, pH 7.25. Hemoglobin A and F become bound to the resin while Htr A₂ is in the supernatant fraction allowing its quantitation. The method is simple, rapid, inexpensive and accurate, and allows one technician to determine the level of Hb A₂ in at least 50 samples a day. The method can be used with whole blood, hemolysates and blood collected on filter paper.     

Hemoglobin switching in sheep: a comparison of the erythropoietin- induced switch to HbC and the fetal to adult hemoglobin switch

Stimulation of sheep erythropoietic progenitor cells by erythropoietin (epo) has been studied with regard to its effect on the pattern of hemoglobin production. An analysis of hemoglobin (Hb) synthesis in BFU- E- and CFU-E-derived colonies from fetuses either homozygous for HbA (AA) (homozygous also for the beta c gene responsible for HbC production) or HbB (BB) (lacking the beta c gene) indicated the following. Colonies derived from precursor cells from 51- and 89-day fetuses exhibited small but detectable increments of HbB synthesis with prolonged incubation in vitro. This response was not dependent on the epo concentration. Erythropoietic precursor cells from a 124-day BB fetus were already committed to HbB synthesis, since HbF production was replaced by HbB on successive days in vitro as erythroid colonies matured; this switch was not affected by varying the epo concentration. In contrast, progenitor cells from a 124-day AA fetus responded to higher doses of epo by forming colonies in which more HbC was made at the expense of both HbF and HbA. Erythropoietic stress did not result in induction of HbF in vivo or in erythroid colonies derived from CFU-E in young adult BB sheep, whereas our prior studies had shown induction of HbC synthesis under analogous conditions in colonies derived from young adult AA sheep. We conclude that the epo-induced HbF (or HbA) to HbC switch and the fetal to adult hemoglobin switch are regulated by different mechanisms.     

Hb Cardarelli [beta86(F2)Ala-->Pro]: a new unstable and hyperaffine variant in association with beta(+)-thalassemia

Hb Cardarelli [beta86(F2)Ala-->Pro] is a new unstable and high oxygen affinity variant found in several members of a family from Naples, Southern Italy. A detailed structural and functional characterization of the variant was performed on two subjects, at both the protein and DNA level. The first patient exhibited 43% of the variant hemoglobin (Hb) without major hematological problems. The proband showed 82% of the abnormal Hb in association with beta(+)-thalassemia (thal) that caused relevant erythrocytosis requiring frequent phlebotomies. Structural investigation of the Hb variant by mass spectrometric methodologies identified the amino acid replacement as Ala-->Pro at beta86. The corresponding DNA mutation GCC-->CCC at codon 86 of the beta-globin gene was assessed by both DNA sequencing and amplification refractory mutation system (ARMS) techniques. Functional studies carried out on whole blood and diluted hemolysates from both patients demonstrated increased oxygen affinity, decreased Bohr effect, reduced heme-heme interaction and nearly halved 2,3-diphosphoglycerate (2,3-DPG) and chloride effects.     

Monoclonal antibody-based methods for quantitation of hemoglobins: application to evaluating patients with sickle cell anemia treated with hydroxyurea

Abstract: High-titer monoclonal antibodies (mAb) were raised against chromatographically purified human hemoglobin (Hb) species. These mAb were specific for either Hb A, Hb F, Hb S or Hb C. Based on these antibodies, which were directly conjugated with either fluorochromes or an enzyme (horseradish peroxidase), we developed immunoassays for determining the Hb profile in the peripheral blood; an enzyme-linked immunosorbent assay (ELISA) for determining the absolute and relative quantities of various Hb species and one-step immunolabeling for fluorescence microscopic and flow cytometric analyses of the distribution of RBC with respect to their Hb types. We utilized these methods for monitoring the Hb F level and the percentage of Hb F-containing cells in patients with sickle cell anemia undergoing treatment with hydroxyurea.     

Enzyme immunoassay for the identification of hemoglobin variants

We have prepared monospecific antibodies to Hbs D-Los Angeles, J-Baltimore, O-Arab and J-Paris-I and developed an enzyme immunoassay (ELISA) for their identification in hemolysates. Hbs in adult or cord blood hemolysates were coated to the wells of microtiter plates and reacted with the appropriate antisera followed by the detection system which contains anti-rabbit IgG/peroxidase conjugate and the substrate tetramethylbenzidine. Sixty-nine samples were tentatively considered to contain the above hemoglobin variants by isoelectrofocusing and the identity of 83% of them was confirmed by ELISA. Some of the non-reacting hemolysates were shown by amino acid sequence analysis to contain Hbs Korle-Bu, D-Ibadan, G-Copenhagen and the new variant Chandigarh. This ELISA offers specificity and simplicity for the confirmatory identification of hemoglobin variants.     

Effects of Hemoglobin Variants on Hemoglobin A1c Values Measured Using a High-Performance Liquid Chromatography Method

Hemoglobin A1c (HbA1c) is routinely used to monitor long-term glycemic control and for diagnosing diabetes mellitus. However, hemoglobin (Hb) gene variants/modifications can affect the accuracy of some methods. The potential effect of Hb variants on HbA1c measurements was investigated using a high-performance liquid chromatography (HPLC) method compared with an immunoturbimetric assay. Fasting plasma glucose (FPG) and HbA1c levels were measured in 42 371 blood samples. Samples producing abnormal chromatograms were further analyzed to characterize any Hb variants. Fructosamine levels were determined in place of HbA1c levels when unstable Hb variants were identified. Abnormal HPLC chromatograms were obtained for 160 of 42 371 samples. In 26 samples HbS was identified and HbA1c results correlated with FPG. In the remaining 134 samples HbD, Hb Louisville, Hb Las Palmas, Hb N-Baltimore, or Hb Porto Alegre were identified and HbA1c did not correlate with FPG. These samples were retested using an immunoturbidimetric assay and the majority of results were accurate; only 3 (with the unstable Hb Louisville trait) gave aberrant HbA1c results. Hb variants can affect determination of HbA1c levels with some methods. Laboratories should be aware of Hb variants occurring locally and choose an appropriate HbA1c testing method.