Further evaluation of the world health organization international reference reagent for Haemoglobin A2 measurement.

INTRODUCTION: The accurate measurement of HbA2 is essential for the detection of ß-thalassaemia carriers and as no single calibrant is used by the various manufacturers of analysers, differences are seen in results obtained. The World Health Organization International Reference Reagent for HbA2 (WHO IRR 89/666) was made available to diagnostic laboratories in the 1980s and remains the only international reference material available. A previous study (2015) demonstrated that the WHO IRR remained suitable for use as an HbA2 standard as tested by 52 participants in the UK NEQAS Haematology Abnormal Haemoglobins Programme. This study was undertaken to include simultaneous analysis of three whole blood specimens over a range of HbA2 values with the WHO IRR and to include participants from laboratories outside of the UK. METHOD: Three whole blood specimens with HbA2 levels ranging from 2.4% to 5.7% and the WHO IRR were distributed to 56 laboratories located in 14 different countries. Participants were requested to test the specimens at defined intervals and return results accompanied by chromatograms or electropherograms produced. RESULTS: Differences found in results from different analyser groups reflect the bias found in the 2015 study in that bias is seen according to the methodology used and also varies in relation to the level of analyte being measured. CONCLUSION: Results of measurements from whole blood specimens and the lyophilized WHO IRR standard did not show any deterioration of the IRR, and it remains suitable for use. Linearity and calibration of analysers remain a problem.

Identification and quantification of hemoglobins in whole blood: the analytical and organizational aspects of Capillarys 2 Flex Piercing compared with agarose electrophoresis and HPLC methods.

BACKGROUND: The present study was conducted to evaluate the analytical performance and the organizational aspects of Capillarys 2 Flex Piercing system (CFP) respect to agarose electrophoresis and HPLC methods in hemoglobinopathies screening. METHODS: The measurement of imprecision in HbA 2 and HbF quantification was verified on HbA 2 CFP control and on three samples; 74 whole blood samples were used to evaluate migration time imprecision of hemoglobin variants S, C and E (HbS, HbC, and HbE); to compare methods, 451 samples were tested on CFP and HPLC; reference values were verified as value distribution in 160 blood donors and at ROC curve analysis on 449 samples from routine analysis. RESULTS: Imprecision: the analytical CV % s ranged from 1.25 to 3.9 at HbA 2 quantification, the CV % was 3.78 at HbF quantification; the running time imprecision for HbS and HbC and HbE ranged from 0.20 to 0.69 % . Method comparison: at regression analysis findings were HbA 2: CFP=1.21×HPLC­0.64, HbF: CFP=1.31×HPLC−0.75, HbS: CFP=1.10×HPLC−3.24. Reference values: the HbA 2 95th percentile range was 2.5­2.8; HbF was undetectable in 154 out 160 samples tested; at ROC curve analysis the best combination of sensitivity and diagnostic efficiency was obtained using 2.2 and 3.0, as reference values, for HbA 2 and 1.1 as the upper reference limit for HbF. Organizational aspects: with respect to the procedures currently implemented in our laboratory CFP requires 2 h less time and obviates the need for some manual steps. CONCLUSIONS: The quantification, reproducibility and diagnostic efficiency provided by CFP in identification and quantification of hemoglobins appear accurate. In addition, the use of primary tubes allows improved safety, and the avoidance of some manual steps, that prolong working time and are a source of possible errors.

Inter-method differences and commutability of control materials for HbA2 measurement.

The intermethod variability of control materials and patient blood samples for the measurement of hemoglobin A2 (HbA2) were compared. A set of 54 blood samples and 10 control materials were analyzed in duplicate by HPLC and microcolumn methods. For each set of methods the distances of the materials from the regression line of patient blood results (expressed as normalized residuals) were calculated. Four out of ten controls had normalized residuals exceeding three standard deviations from the regression line. Moreover, total Hb and Hb derivatives analysis proved that only a minority of the controls could be considered similar to patients' blood samples. Intermethod calibration performed "a posteriori" by the two best performing control materials improved intermethod variability among all the five tested methods. We conclude that the use of high resolution HPLC methods together with appropriate commutable control materials allows for better harmonization of results in the field of diagnosis of hemoglobin disorders in research and clinical practice.