Use of 2 years of patient data to estimate intra-laboratory total imprecision of HbA(1c) measured by multiple HPLC analyzers.

BACKGROUND: Analytic imprecision is used to assess the acceptability of HbA(1c) methods performed on a single analyzer. When multiple analyzers are used interchangeably in a laboratory, the analytic imprecision is usually increased and can obscure the detection of a genuine HbA(1c) trend or result in an artefactual patient trend. We have estimated the imprecision of HbA(1c) testing of patient specimens by three HbA(1c) analyzers independent of reference sample analysis. METHODS: Over 2 years, approximately 150,000 HbA(1c) measurements were obtained from any one of three different Bio-Rad VARIANT II HPLC analyzers operated in a large reference laboratory. We tabulated the HbA(1c) measurements of paired intra-patient blood samples drawn within 30 days of each other. We calculated the standard deviations of duplicates (SDD) of the intra-patient HbA(1c) pairs grouped by the following time intervals: 0-3 days, 4-6 days, 7-9 days, 28-30 days. The SDDs were then regressed against time with extrapolation to zero time representing the random analytic error. RESULTS: At a mean HbA(1c) of 7.16%, the total analytic imprecision (coefficient of variation [CV]) is 3.6%. CONCLUSIONS: This variation is remarkably low, given that the HbA(1c) measurements were obtained over a 2-year period on any one of three analyzers and the long-term within-analyzer CV was usually 2.3-3.1% as assessed by reference control analysis. This approach could be extended to all HbA(1c) analyzers since unlike reference control statistics, the patient-derived random error should allow easy comparison of analytic imprecision among different analytical systems.

Novel method for screening for the presence of haemoglobin S in blood.

A high-performance liquid chromatography (HPLC) method designed for the measurement of haemoglobin (Hb)A(1c) in blood was investigated for use as a screening method for the presence of HbS in blood. In the Bio-Rad VARIANT II HbA(1c) method, HbS was found to have a specific retention time and percentage Hb value that enabled the detection of HbS in blood. Other Hb variants did not have the same combination of retention time and percentage Hb as HbS. The HPLC method was superior to the HbS solubility test in ease of performance and readability. Also, the proposed method showed less interference than the solubility test and could be used with samples from all age groups. The proposed method takes 3 min per sample to perform and is thus suitable for large-scale screening.

Analytical evaluation of the Bio-Rad variant II automated HbA(1C) analyzer.

OBJECTIVES: To evaluate the analytical performance of the Bio-Rad Variant II HbA(1C) analyzer (a completely automated system for the quantification of glycohemoglobin [HbA(1C)] in blood). DESIGN AND METHODS: The analytical parameters of precision, linearity and analytical range were assessed and HbA(1C) results from the Variant II were compared to HbA(1C) results from the Bio-Rad Variant (a method certified by the National Glycohemoglobin Standardization Program). The effect of a variety of hemoglobin variants on HbA(1C) obtained on the system was investigated. RESULTS: Total imprecision was less than 5% and the results compared well with those from an established method. The method has a wide analytical range with no carryover between specimens. CONCLUSION: The HbA(1C) method on the Variant II gives acceptable analytical performance.

Laboratory investigation of hemoglobinopathies and thalassemias: review and update.

Structural hemoglobin (Hb) variants typically are based on a point mutation in a globin gene that produce a single amino acid substitution in a globin chain. Although most are of limited clinical significance, a few important subtypes have been identified with some frequency. Homozygous Hb C and Hb S (sickle cell disease) produce significant clinical manifestations, whereas Hb E and Hb D homozygotes may be mildly symptomatic. Although heterozygotes for these variants are typically asymptomatic, diagnosis may be important for genetic counseling. Thalassemia, in contrast, results from quantitative reductions in globin chain synthesis. Those with diminished beta-globin chains are termed beta-thalassemias, whereas those with decreased alpha-chain production are called alpha-thalassemias. Severity of clinical manifestations in these disorders relates to the amount of globin chain produced and the stability of residual chains present in excess. The thalassemia minor syndromes are characterized clinically by mild anemia with persistent microcytosis. Thalassemia intermedia (i.e., Hb H disease) is typified by a moderate, variably compensated hemolytic anemia that may present with clinical symptoms during a period of physiologic stress such as infection, pregnancy, or surgery. The thalassemia major syndromes produce severe, life-threatening anemia. alpha-Thalassemia major usually is incompatible with extrauterine life; beta-thalassemia major presents in infancy and requires life-long transfusion therapy and/or bone marrow transplantation for successful control of the disease. Double heterozygosity for certain structural variants and/or thalassemia syndromes may also lead to severe clinical disease. Several guidelines have been published that outline the required steps for hemoglobinopathy and thalassemia investigation. The availability of HPLC has streamlined many of these requirements, allowing an efficient stepwise diagnostic strategy for these complex disorders.

Comparison of hemoglobin A1C results by two different methods on patients with structural hemoglobin variants.

OBJECTIVES: The objective was to compare hemoglobin A1C (HbA1C) results obtained by two methods based on different analytical principles for individuals with a structural hemoglobin variant. DESIGN AND METHODS: Hemoglobin A1C results were obtained using the Bio-Rad Variant (based on cation exchange chromatography) and the Bayer DCA 2000 (based on an immunological reaction) on individuals with a structural hemoglobin variant. The identity of the hemoglobin variant was confirmed by high pressure liquid chromatography (HPLC) and electrophoresis. RESULTS: Hemoglobin A1C results obtained by the two methods on individuals with S, C, D, and E trait were in close agreement. CONCLUSION: The Bio-Rad Variant and Bayer DCA 2000 produce equivalent hemoglobin A1C results on patients with S, C, and E trait. With appropriate correction, correlation of hemoglobin A1C results from the Bio-Rad Variant for individuals with D trait was good (r = 0.927). Glycohemoglobin results obtained by the two methods for some unusual structural hemoglobin variants were in close agreement.

Enzyme immunoassay of valproic acid using the Abbott ABA-200 analyzer.

The enzyme immunoassay of valproic acid using EMIT reagents was developed for an Abbott ABA-200 analyzer. The number of tests per package that could be obtained using this method was at least three times that suggested by the reagent manufacturer. The reproducibility of the method was assessed by within-run and day-to-day reproducibility studies and the accuracy of the method was assessed by comparison of results obtained to those obtained using a gas-chromatographic method and by performance on an inter-laboratory therapeutic drug monitoring quality control programme.