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Background:Availability of immunophenotypic reference values for the various leukocyte populations distributed in bone marrow may be helpful to recognize abnormal bone marrow development and, therefore, useful as first screening of individuals with suspected hematological malignancies or other hematopoietic disorders.Methods:A single tube fourcolor staining panel CD66abceCD14CD45CD34 together with a predefined gating strategy was utilized to immunologically differentiate the distribution of the major leukocyte populations in bone marrow aspirates of healthy donors. The sampleblood erythrocyte ratio was applied to assess the amount of blood contamination of marrow and account for this in the marrow value estimates.Results:The frequency of the major leukocyte populations in bone marrow of 134 normal donors were for granulocytes: mean, 69.4; SD, 10.3; monocytes: mean, 4.7; SD, 2.3; lymphocytes: mean, 18.3; SD, 8.7. The frequency of the immature cell population that included precursor cells of each of the cell lineages among other cell types were mean 5.0; SD 2.2. The mean percentage of CD34 positive cells was 1.5; SD 0.7. Our results showed further that the frequency of cell populations, of which the presence is restricted to the bone marrow e.g., CD34 progenitor cells, is influenced by the degree of peripheral blood admixture. Between the total immature cells and purity of the bone marrow, there was a significant positive correlation demonstrated, whereas a negative correlation was found between the percentages of both lymphocytes as monocytes and the purity of the bone marrow.Conclusions:With a single tubestaining panel, we obtained reference values for flow cytometric assessment of all relevant leukocyte populations present in bone marrow that can be used as a frame of reference for better recognition of individuals with abnormal hematopoiesis. In addition, we have demonstrated the influence of the degree of peripheral blood admixture in the bone marrow aspirates on those reference values. © 2008 Clinical Cytometry Society

Objectives. Familial haemophagocytic lymphohistiocytosis (FHL) is a disorder characterized by deficient cytotoxic T‐cell function and activated macrophages, owing to a defect in the perforin gene and absent perforin expression. Because symptoms of patients with systemic juvenile idiopathic arthritis (sJIA) are sometimes clinically very similar to those with FHL, we studied whether perforin expression in sJIA patients would be reduced also.Methods. We determined the perforin expression levels on two subsets of CD8+ cells (CD8+CD28−CD45RA− and CD8+CD28−CD45RA+) and natural killer (NK) cells from patients with sJIA under conventional treatment as well as before and after autologous stem‐cell transplantation (ASCT).Results. CD45RA− cytotoxic effector cells of sJIA patients (n=13) express significantly lower levels of perforin than polyarticular juvenile idiopathic arthritis (pJIA, n=9) patients [sJIA mean fluorescence intensity (MFI) 34.6; pJIA MFI 98.0] or control donors (MFI 124.6, n=5). A similar pattern was seen in the CD45RA+ subset. Also NK cells from sJIA patients expressed significantly less intracellular perforin (sJIA MFI 398.4; controls MFI 972.4). In four patients with sJIA who were treated with ASCT, a clear increase in perforin expression was found at 12 months after ASCT in both cytotoxic effector cell subsets (CD45RA− subset before ASCT MFI 13.2; 12 months after ASCT MFI 172.3).Conclusion. We conclude that perforin expression can be severely reduced in sJIA. This finding may implicate defective cytotoxicity and haemophagocytosis and could thus explain why sJIA may be complicated by macrophage activation syndrome. ASCT leads to a reconstitution of the (T cell) immune system with a normal expression of perforin.