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Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source
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Towards the development of a certified reference material for hemoglobin A2.

Renata Paleari1, Amalia Muñoz, Andrea Mosca

  • 1Centre for Metrological Traceability in Laboratory Medicine (CIRME), Department of Science and Biomedical Technology, Laboratorio Interdisciplinare di Tecnologie Avanzate (LITA), University of Milan, Milan, Italy.

Clinical Chemistry and Laboratory Medicine
|November 2, 2010
PubMed
Summary

A new lyophilized reference material for hemoglobin A2 (HbA2) analysis was developed and found to be homogeneous, stable, and commutable. This material will help improve standardization of HbA2 measurements across different laboratory methods.

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Area of Science:

  • Clinical Chemistry
  • Biochemistry
  • Analytical Chemistry

Background:

  • The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) established a working group to standardize hemoglobin A2 (HbA2) analysis.
  • A key objective was to create a certified reference material for HbA2, collaborating with the Institute for Reference Materials and Measurements (IRMM).
  • This study details the characterization of an initial batch of this candidate reference material.

Purpose of the Study:

  • To develop and characterize a stable, homogeneous, and commutable reference material for HbA2 quantification.
  • To assess the suitability of lyophilized blood as a secondary reference material for HbA2 analysis.
  • To lay the groundwork for improved inter-method variability in HbA2 testing.

Main Methods:

  • Preparation of lyophilized material from healthy donor whole blood, removing non-red blood cells and lysing red cells.
  • Analysis of HbA2 content using seven HPLC, three electrophoretic, and two capillary electrophoresis (CE) methods.
  • Evaluation of homogeneity, long-term stability (1 year at +4°C and -20°C), and short-term stability after reconstitution.
  • Assessment of commutability by analyzing the material alongside 54 fresh blood samples using specific HPLC and CE methods.

Main Results:

  • All tested chromatographic and electrophoretic methods showed consistent results between the study material and fresh blood.
  • The prepared lot demonstrated homogeneity in lyophilized powder content per vial.
  • The HbA2 concentration remained stable for 1 year at +4°C and -20°C, and for over 2 weeks after reconstitution at +4°C.
  • The study material exhibited good commutability (normalized residual of 0.9) with routine methods.

Conclusions:

  • Lyophilization of blood for HbA2 analysis does not introduce matrix effects or inhomogeneity.
  • The developed material is commutable with key routine methods (Bio-Rad Variant II, Beckman PA800).
  • This candidate reference material, when assigned a value by a reference measurement procedure, will be suitable for calibrating routine HbA2 methods, enhancing standardization.