Summary
Neutrophil granulocytes from 12 subjects with primary myeloperoxidase (MPO) deficiency (six totally deficient) and 16 patients with secondary partial MPO deficiency were tested using two different anti-MPO antibodies, in combination with either a flow-cytometric technique or an immunoalkaline phosphatase staining method. Results demonstrated three different cytofluorimetric patterns of immunoreactivity with the MPO protein: (a) a bright MPO antigenic expression, typical of patients with secondary MPO deficiency (comparable to that observed in the control group); (b) a medium MPO antigenic expression, typical of subjects with primary partial MPO deficiency; and (c) a dim MPO antigenic expression, characteristic of individuals with hereditary total MPO deficiency. No significant differences in granulocyte MPO reactivity were demonstrated for the two antibodies. Furthermore, in two individuals with complete primary enzyme deficiency, the single histogram analysis of MPO fluorescence determined by flow cytometry seemed to show that only 38% (case 1) and 44% (case 2) of neutrophils were reactive with the anti-MPO antibodies: the use of multiple histogram analysis in combination with Kolmogorov-Smirnov statistics allowed us to demonstrate that all the cells express a low density of MPO antigen. These data were more or less confirmed by the APAAP labeling method, which showed a reduced staining only in subjects with primary deficiency, while all patients with secondary deficiency had scores similar to those observed in controls (healthy subjects). Compared with the immunoenzymatic technique, the flow-cytometric procedure showed a higher sensitivity to MPO, being able to estimate even minor decreases in neutrophil MPO antigenic expression, as previously postulated by other authors. This work suggests that patients with primary MPO deficiency have different amounts of MPO antigens in the neutrophil granulocytes, and the levels of MPO fluorescence seem to decline concurrently with enzyme activity, thereby suggesting the presence of a diminished MPO production. In contrast, the normal antigenic reactivity of neutrophils from patients with acquired MPO deficiency indicates the presence of a functionally inactive form of the enzyme.
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References
Armstrong D, Dimmit S, Van Wormer DE (1974) Studies in Batten disease. I. Peroxidase deficiency in granulocytes. Arch Neurol 30: 144–152
Bakkenist ARJ, Wever R, Vulsma T, Plat H, Van Gelder BF (1978) Isolation procedure and some properties of myeloperoxidase from human leucocytes. Biochim Biophys Acta 524: 45–54
Caldwell KC, Taddeini L, Woodburn RL, Anderson GL, Labell M (1979) Induction of myeloperoxidase deficiency in granulocytes in lead-intoxicated dogs. Blood 53: 588
Campana D, Coustan-Smith E, Janossy G (1990) The immunologic detection of minimal residual disease in acute leukemia. Blood 76: 163–171
Cech P, Papathanassious A, Boreux G, Roth P, Miesher PA (1979) Hereditary myeloperoxidase deficiency. Blood 53: 403–411
Chang KS, Trujillo JM, Cook RG, Stass SA (1986) Human myeloperoxidase gene: molecular cloning and expression in leukemic cells. Blood 68: 1411–1414
Clark RA, Szot S (1981) The myeloperoxidase hydrogen peroxide halide system as effector of neutrophil-mediated tumor cell cytotoxicity. J Immunol 126: 1295–1301
Cocchi P, Mori S, Ravina A (1977) Myeloperoxidase-deficient leukocytes in streptococcal infections. Scand J Haematol 18: 113
Cordell JL, Falini B, Erber WN, Ghosh AK, Abdulaziz Z, MacDonald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal antialkaline phosphatase (APAAP complexes). J Histochem Cytochem 32: 219–229
Cramer R, Soranzo MR, Dri P, Rottini GD, Bramezza M, Cirielli S, Patriarca P (1982) Incidence of myeloperoxidase deficiency in an area of northern Italy: histochemical, biochemical and functional studies. Br J Haematol 51: 81–87
Davey F, Erber W, Gatter K, Mason D (1987) Immunophenotyping of acute myeloid leukemia by immune-alkaline phosphatase (APAAP) labeling with a panel of antibodies. Am J Hematol 26: 157–166
Davey FR, Erber WN, Gatter WN, Mason DY (1988) Abnormal neutrophils in acute myeloid leukemia and myelodysplastic syndrome. Hum Pathol 19: 454–459
Dent G, Leglise M, Pryzwansky K, Ross D (1989) Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens or oncogene expression with DNA content. Cytometry 10: 192–198
D'Onofrio G, Mancini R, Vallone R, Alfano G, Candido A, Palla M, Mango G (1983) Acquired neutrophil myeloperoxidase deficiency: an indicator of subclinical activation of blood coagulation? Blood Cells 9: 455–464
Drach J, Gattringer C, Glassl H, Schwarting R, Stein H, Huber H (1989) Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma. Cytometry 10: 743–749
Drach J, Gattringer C, Huber H (1991) Combined flow cytometric assessment of cell surface antigens and nuclear TdT for the detection of minimal residual disease in acute leukemia. Br J Haematol 77: 37–42
Dri P, Cramer R, Menegazzi R, Patriarca P (1987) Biochemical, functional and clinical aspects of hereditary human myeloperoxidase deficiency. Adv Biosci 66: 193–206
Hansen KB, Nielsen HK (1983) Myeloperoxidase-deficient polimorphonuclear leukocytes. Scand J Haematol 30: 415–419
Hayhoe FGJ, Quaglino D (1988) Haematological cytochemistry, 2nd edn. Churchill Livingstone, Edinburgh
Kastan M, Slamon D, Civin C (1989) Expression of protooncogene c-myb in normal human hematopoietic cells. Blood 73: 1444–1451
Kershaw GW, Robin H, Kronenberg H (1987) Evaluation of the Technicon H-1 hematology analyzer. Pathol 19: 305–309
Kitahara M, Eyre HG, Simonian Y, Atkin CL, Hasstedt SJ (1981) Hereditary myeloperoxidase deficiency. Blood 57: 888–893
Lanza F, Castoldi GL (1988) Basophil count in samples from chronic leukemia patients analyzed by the automated flow cytochemistry technology. Br J Haematol 68: 495–498
Lanza F, Musto P, Franze' D (1985) Hereditary myeloperoxidase deficiency syndrome. Clinical and hematological data of ten cases. Ital J Med 1: 45–52
Lanza F, Masotti M, Castoldi GL (1988) Eosinophil peroxidase deficiency detected by the Technicon H1 system. Blut 56: 143–144
Lanza F, Giuliani AL, Amelotti F, Spisani S, Traniello S, Castoldi GL (1988) Depressed neutrophil-mediated tumour cell cytotoxicity in subjects affected by hereditary myeloperoxidase deficiency and secondary neoplasia. Haematologica (Pavia) 73: 355–358
Lanza F, Fietta A, Spisani S, Castoldi GL, Traniello S (1987) Does a relationship exist between neutrophil myeloperoxidase deficiency and the occurrence of neoplasms? J Clin Lab Immunol 22: 175–180
Lanza F, Fagioli F, Gavioli R, Spisani S, Malavasi F, Castoldi GL, Traniello S (1991) Evaluation of CR1 expression in neutrophils from chronic myeloid leukemia: relationship between prognosis and cellular activity. Br J Haematol 77: 66–72
Larsson L, Schena M, Carsson M, Sallstrom J, Nilsson K (1991) Expression of the c-myc protein is down-regulated at the terminal stages during in vitro differentiation of B-type chronic lymphocytic leukemia cells. Blood 77: 1025–1032
Lehrer RJ, Cline MJ (1971) Leukocyte candidacidal activity and resistance to systemic candidiasis in patients with cancer. Cancer 27: 1211
Morishita Y, Morishima Y, Ogura M, Nagai Y, Ohno Y (1986) Biochemical characterization of human myeloperoxidase using three specific monoclonal antibodies. Br J Haematol 63: 435–444
Nauseef WM (1986) Myeloperoxidase biosynthesis by a human promyelocytic line: insight into myeloperoxidase deficiency. Blood 67: 865–872
Nauseef WM (1987) Posttranslational processing of a human myeloid lysosomal protein, myeloperoxidase. Blood 69: 1143–1150
Nauseef WM, Clark RA (1986) Separation and analysis of subcellular organelles in a human promyelocytic leukemia cell line, HL60: application to the study of myeloid lysosomial enzyme synthesis and processing. Blood 68: 442–449
Nauseef WM, Root RK, Malech HL (1983) Biochemical and immunological analysis of hereditary myeloperoxidase deficiency. J Clin Invest 71: 1297–1307
Parry MF, Root RK, Metcalf JA, Delaney KK, Kaplow LS, Richar WJ (1981) Myeloperoxidase deficiency. Prevalence and clinical significance. Ann Intern Med 95: 293–301
Pietrzyk JA, Polimaka W (1979) Immune system in patients with diabetes mellitus. Wiad Lek 32: 323–326
Presentey B (1968) A new anomaly of eosinophilic granulocytes. Am J Clin Pathol 49: 887–890
Shibata A, Bennet J, Castoldi GL, Catovsky D, Flandrin G, Jaffe E, Katajama I, Nanba K, Schmaltz F, Yam T (1985) Recommended methods for cytological procedures in hematology. Clin Lab Haematol 7: 55–74
Undritz E (1966) Die Alius Grignaschi Anomaly: der erblich konstitutionelle Peroxidase-Defekt der Neutrophilen and Monozyten. Blut 14: 129–136
Van der Shoot E, Daams M, Pinkster J, Vet R, Von dem Borne A (1990) Monoclonal antibodies against myeloperoxidase are valuable reagents for the diagnosis of acute myeloid leukemia. Br J Haematol 74: 173–178
Young IT (1977) Proof without prejudice: use of Kolmogorov-Smirnov test for the analysis of histograms from flow systems and other sources. J Histochem Cytochem 25: 935–941
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The work was supported by a grant from the National Council Research (Nr. 88.00573.44) and a grant from MPI (Nr. 88.60.06.017)
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Lanza, F., Latorraca, A., Musto, P. et al. Cytochemically unreactive neutrophils from subjects with myeloperoxidase (MPO) deficiency show a complex pattern of immunoreactivity with anti-MPO monoclonal antibodies: A flow cytometric and immunocytochemical study. Ann Hematol 63, 94–100 (1991). https://doi.org/10.1007/BF01707280
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DOI: https://doi.org/10.1007/BF01707280