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Aims: α-Naphthyl acetate esterase (ANAE) is one of the few enzymes that are histochemically detectable on formalin-fixed paraffin-embedded tissue. In bone marrow (BM) biopsies, ANAE staining highlights megakaryocytes. We investigated autopsy BM to determine whether ANAE staining intensity (SI) was associated with postmortem intervals (PMI, period between death and autopsy), and thus could allow the time of death of a patient to be deduced., Methods: ANAE-stained BM slides of 74 forensic and pathology autopsies as well as 22 biopsies were histologically evaluated and their SIs semiquantitatively graded., Results: ANAE-SIs did not differ between men and women and slightly decreased with age. Biopsies had significantly higher ANAE-SIs than pathology cases. In autopsies, ANAE-SIs were not associated with PMI, except for cases with PMI ≥7 days which were consistently ANAE-negative., Conclusions: ANAE-SIs in postmortem BM samples were independent of PMI. Thus, ANAE staining of BM megakaryocytes cannot serve as an indicator for time-since-death of a patient.

We have performed the cytochemical analysis (myeloperoxidase, ASD-chloroacetate and acid alpha-naphthylacetate esterases, Sudan black B) of blast cells from 25 acute leukemia patients, after 3, 5 and 7 days of liquid culture with conditioned medium from phytohemagglutinin stimulated leukocytes (PHA-LCM). In case of acute myeloid leukemia blast cells an increase of percentage of positive cells simultaneously with the enhancement of the cytochemical reactions was observed. This method may be useful for the precise diagnosis of poorly differentiated blasts with weak expression of cytochemical phenotype.

Monocytopoiesis and blood monocytes were investigated in nine patients with active tuberculosis and in six patients with active sarcoidosis in order to obtain information on monocyte consumption in these two types of granuloma. All patients with tuberculosis demonstrated a marked increase in proliferation activity of monocytopoiesis and premature monocyte marrow release. These changes indicate a high monocyte consumption which probably is caused by a high macrophage death rate due to the high macrophage-toxicity of tubercle bacilli. Thus, tuberculous lesions are an example of a "high turnover granuloma". In sarcoidosis monocytopoiesis showed no significant deviations from the normal. This indicates a low macrophage turnover or "low turnover granuloma". Thus, any hypothetical agent assumed to be involved in the pathogenesis of sarcoidosis would have to possess low macrophage-toxicity.

Thymocytes, bone marrow lymphocytes, as well as lymphocytes from spleen, lymphoid nodes and peripheral blood were obtained from BALB/c mice. Subpopulations of BALB/c bone marrow T-lymphocyte precursors and immature (small) and mature (large) thymocytes, as established by the percentage of terminal deoxynucleotidyl transferase (TdT) and peanut agglutinin (PNA) positive cells, were obtained by centrifugation on discontinuous density gradients. The activities of N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (BG), acid alpha-naphthyl acetate esterase (ANAE) and alpha-naphthyl acetate esterase (NAE) were determined by enzymatic assays of cell extracts of the diverse T-lymphocyte subpopulations, in order to follow their evolution with the maturation of the T-lymphocytes in the thymus. These activities were compared with that determined in lymphocytes from spleen, lymphoid nodes and peripheral blood. The glucidases BG and NAG and the esterases ANAE and NAE present a high decrease in their activities from bone marrow T-lymphocyte progenitors to immature thymocytes. BG, NAG and ANAE activities undergo an about 3-fold increase with the evolution of the thymocytes from small to large cells. Whereas the level of the NAE activity decreases (2-fold) with that evolution of the thymocytes. Lymphocytes from spleen and lymphoid nodes exhibit activities of the glucidases and, specially, the esterases marked by higher than those of thymocyte populations. Peripheral blood lymphocytes also present NAG, ANAE and NAE activities higher than in thymocytes, but their BG activity is lower.

Enzyme-cytophotometrical investigations allow evaluation of enzyme activity within single, morphologically classified cells. Special problems like development of blood cells, bone marrow cells or of macrophages may thus be studied. Knowledge about the kinetics of granulopoiesis allows a better understanding of altered functional characteristics of abnormally developed blood cells. Enzyme cytochemical microdensitometry promises to be a useful tool in haematological cytology.

Three plasma cell enzymatic reactions--ATPase, acid phosphatase (AP), alpha-naphthyl acetate esterase (alpha-NAE)--were tested in a large number of bone marrow samples from patients with multiple myeloma (MM), benign monoclonal gammapathy (BMG)--idiopathic (iBMG) and secondary (sBMG)--, polyclonal hypergammaglobulinemia (PH), Waldenström's macroglobulinemia (WM) and from normoglobulinemic subjects (NS). The purpose of the present study was to evaluate the significance of these reactions in differential diagnosis of BMG and initial or atypical MM. For each reaction results were expressed as scores and normal limits were statistically established. Our findings in MM, HP and NS are consistent with previous reports (similar enzymatic pattern in NS and PH; depressed ATPase activity, raised AP and alpha-NAE activities in most MM cases). In the BMG class, ATPase activity was normal in about 40% of iBMG, while it was depressed in other cases; most sBMG had diminished ATPase activity. AP patterns were normal for both BMG groups. alpha-NAE activity was normal in 50% of iBMG, while it was raised in the other cases. All the reactions were normal in WM. In a bayesian analysis of cytochemical patterns of NS and MM subjects ATPase showed the highest diagnostic significance, followed by alpha-NAE and AP. ATPase, therefore, seems to be the most useful criterion for the diagnosis of initial MM; low ATPase score BMG patients should therefore be carefully followed-up.

The different cells and structures of the bone-marrow stroma were studied with histochemical markers. Reticulum cells, endothelial cells and macrophages were identified by their morphologic characteristics as well as by their different positivity to alkaline-phosphatase, alpha-naphthyl-acetate-esterase and Prussian blue staining. Similarities, as well as distinguishing features of all these cells, possibly related to their common or different origin and functional properties, are described.