1. Unexpected Hemoglobin A 1c Results Dr.M.KOTTESWARAN 1 ST YEAR BIOCHEMISTRY PG

2. CASE A 52-year-old woman with a medical history of hepatitis B, hyperlipidemia, hypertension, anemia, and depression presented to the internal medicine clinic for a routine visit. Laboratory tests 3 months previously had revealed an impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL) [reference interval, 3.9–5.6 mmol/L (70–100 mg/dL)]. Therefore, a hemoglobin (Hb) 2 A 1c analysis was performed. The initial Hb A 1c evaluation by cation-exchange HPLC (CE-HPLC) showed an Hb A 1c value of 115.8% (reference interval, 4.0%–6.0%) (Fig. 1). 2Fig. 1

3. CE-HPLC chromatogram for Hb A 1c analysis. Hb S and an aberrant Hb A 1c value of 115.8% represented the predominant Hb peaks in the chromatogram.

4. This unusual Hb A 1c result was due to potential hemoglobinopathies, we performed an Hb variant analysis with the Bio-Rad VARIANT CE- HPLC β-Thalassemia Short Program. The analysis revealed the absence of Hb A and the presence of sickle cell Hb (Hb S) (37.4%), along with normal Hb A 2 (3.2%) and Hb F (<1.0%). Also evident was another large peak (53.0%) that eluted earlier than Hb A, which we called P2. This study suggested the presence of an Hb variant with a chromatographic retention time virtually identical to that of Hb A 1c, in addition to Hb S. A subsequent Hb electrophoretic analysis at pH 6.0 (QuickGel Acid; Helena Laboratories) identified Hb S

5. Chromatogram of Hb variants analysis with the CE-HPLC β- Thalassemia Short Program. Hb S (37.4%), wild-type Hb A 2 (3.2%), and Hb F (<1.0%) were identified, but Hb A was not detected. A large peak, which we designated P2, was detected at 53.0%.

6. PATIENT FOLLOW-UP To identify the Hb variants, we investigated DNA sequences corresponding to the patient's β-globin genes. This analysis identified a substitution at codon 6 [GAG to GTG (Glu to Val)] on one allele, corresponding to Hb S, and a substitution at codon 1 [GTG to GCG (Val to Ala)] on the other allele, corresponding to Hb Raleigh The presence of these hemoglobinopathies suggested that the spurious Hb A 1c result obtained with the CE-HPLC method was due to the elution of Hb Raleigh, which has a retention time similar to that of Hb A 1c. We evaluated the Hb A 1c result with a turbidimetric inhibition immunoassay (Dimension® Clinical Chemistry System; Siemens) and obtained an Hb A 1c value of 4.1%, which was not consistent with the impaired fasting glucose concentration of 5.9 mmol/L (106 mg/dL).

7. DISCUSSION Hemoglobin A1c (HbA1c) is used routinely to monitor long-term glycemic control in people with diabetes mellitus, as HbA1c is related directly to risks for diabetic complications. The accuracy of HbA1c methods can be affected adversely by the presence of hemoglobin (Hb) variants or elevated levels of fetal hemoglobin (HbF). The most common Hb variants worldwide are HbS, HbE, HbC, and HbD. All of these Hb variants have single amino acid substitutions in the Hb β chain. HbF is the major hemoglobin during intrauterine life; by the end of the first year, HbF falls to values close to adult levels of approximately 1%. However, elevated HbF levels can occur in certain pathologic conditions or with hereditary persistence of fetal hemoglobin.

8. Homozygosity for HbS causes a serious disease (sickle cell anemia) with vascular obstruction and consequent tissue infarction. Those with HbC disease (also called HbCC disease) may have a normal hemoglobin concentration or be mildly or moderately anemic. Subjects with HbSC disease (also called sickle-hemoglobin C disease) can be affected mildly or moderately with chronic hemolytic anemia. Subjects with homozygous E disease (also called HbEE disease) are usually completely asymptomatic. HbD Punjab disease may have mild hemolysis and sometimes a mild hemolytic anemia. Subjects who are heterozygous for any of these Hb variants are usually asymptomatic and have normal red cell survival. Thus, a physician may be unaware that their patient with diabetes has one of these variants in the heterozygous form

9. Since the measurement of HbA1c is dependent on a normal erythrocyte life span, it is recommended that other tests be used to estimate glycemic control in those with sickle cell, HbC, or HbD disease. Because erythrocyte survival is normal in those with heterozygous variants, HbA1c can be used as long as the Hb variant does not interfere either with the assay method itself or with glucose binding to Hb. The presence of some variants can affect either the net charge of the hemoglobin and/or the recognition of the glycated N terminus by antibodies, resulting in erroneous HbA1c values for some methods. Therefore, the effect of each variant must be examined with each specific HbA1c method

10. HbA1c Glycation is the nonenzymatic addition of glucose to amino groups of proteins. HbA1c is a glycated hemoglobin in which glucose is bound specifically to the N-terminal valine of the hemoglobin β chain. HbA1c constitutes the major portion of the glycated hemoglobins. HbA1c can be separated based on a difference in molecular charge (CE-HPLC and electrophoresis) and based on molecular structure (immunoassays, boronate affinity chromatography, and mass spectrometry)

11. HbA1c Method Types Four basic types of methods are used most commonly to measure HbA1c: immunoassay, ion-exchange highperformance liquid chromatography (HPLC), boronate affinity HPLC, and mass spectrometry. Most immunoassays measure HbA1c specifically; antibodies recognize the structure of the N-terminal glycated amino acids (usually the first 4–10 amino acids) of the Hb β chain.(this method is affected by anti–Hb A 1c antibody or increased Hb F percentage ) Ion-exchange HPLC separates Hb species based on charge differences between HbA1c and other hemoglobins. With boronate affinity methods, m-aminophenylboronic acid reacts specifically with the cis-diol groups of glucose bound to Hb. This method measures total glycated GHB, including HbA1c and Hb glycated at other sites, and tends to demonstrate the least interference from the presence of Hb variants and derivatives.(Hb variants with decreased or increased glycation will interfere with boronate affinity chromatography ) Mass spectrometry is the IFCC reference method and generally appears to be unaffected by the presence of genetic or chemical modifications to the Hb A molecule.