Screening for Leukocyte Peroxidase Deficiencies by Means of Flow Cytometry: Application to the Study of Prevalence, Pathology and Genetics

Abstract

To gain an insight into the prevalence, pathology and genetics of peroxidase deficiencies it is necessary to study a large number of cases. This means screening large population groups. In classical haematology the detection of peroxidase deficiencies is only possible by special peroxidase staining techniques that are not commonly used in daily routines. Thus, the first case of myeloperoxidase (MPO) deficiency was discovered by chance by means of peroxidase staining. Descriptions of similar cases remained rare until the introduction of the first Technicon Hemalog Analyser and the more modern versions now produced by Bayer (H-1, H-3, ADVIA). The unique detector system of these instruments automatically combines two-angle laser diffraction of red cells and leukocyte differentiation based on their peroxidase activity. Fig.19.1 shows the work flow of a complete blood count. The “erythrogram” obtained by measuring both the volume and haemoglobin concentration of approximately 50,000 red blood cells allows an insight into red cell morphology. Differentiation of white blood cells is displayed by plotting their size against their peroxidase activity, as measured after exposure to a peroxide/chromogen reagent. In the normal PEROX diagram of these instruments, the white blood cells are clearly separated into five clusters corresponding to neutrophils (N), eosinophils (E), monocytes (M), lymphocytes (L) and LUC (large unstained cells) (Fig.19.2A). Basophils are differentiated in a separate operation: a sample of blood is treated with an acid buffer, which deprives all the white cells of their protoplasm (“protoplasmic stripping”), leaving the bare nuclei and differentiating round nuclei from more complex ones. Only basophils withstand this treatment and are counted in a separate BASO channel. This allows routine detection not only of complete peroxidase deficiency of certain types of white blood cells but also of all degrees of partial deficiency, with the possibility of quantifying the defect.