CD16 antigen density on neutrophils in chronic myeloproliferative disorders.

Using flow cytometry, we quantitatively examined the density of the CD16 (IgG Fc receptor III) antigen on neutrophils in healthy control subjects, in patients with neutrophilia due to bacterial infection, and in patients with chronic myeloproliferative disorders (chronic myeloid leukemia [CML], polycythemia vera, or essential thrombocythemia). The density was expressed as the mean fluorescence intensity of neutrophils stained with fluorescein isothiocyanate-labeled anti-CD16 monoclonal antibody. We also determined leukocyte alkaline phosphatase activity semiquantitatively in the same population. The mean (+/- SD) density of the CD16 antigen on neutrophils in patients with CML (n = 13; 240.4 +/- 134.8) was lower (P<.001 ) than in healthy control subjects (n = 25; 656.6 +/- 238.0), and the density was also lower than in patients with bacterial infection (n = 15; 671.5 +/- 288.1), polycythemia vera (n = 7; 552.6 +/- 99.9), or essential thrombocythemia (n = 11; 671.5 +/- 411.5). The density of the CD16 antigen was 300 or more in all healthy control subjects and in all patients examined, except for those with CML. The CD16 antigen density was less than 300 in 10 of the 13 patients with CML. Leukocyte alkaline phosphatase activity was also low in 10 of the 13 patients with CML. These findings indicate that flow cytometric analysis of the density of neutrophil CD16 antigen is useful for the differential diagnosis of CML from other chronic myeloproliferative disorders.

[A case of red blood cell(RBC) agglutination on peripheral blood smear and effect of cefpirom sulfate].

We observed red blood cell (RBC) agglutination on peripheral blood smear and an abnormal RBC distribution histogram that suggested the influence of cefpirom sulfate. The patient was receiving cefpirom sulfate when RBC agglutination and the abnormal pattern were recognized, but these abnormalities disappeared as soon as the drug was withdrawn. When the peripheral blood sample was re-examined after warming for an hour at 37 degrees C, the abnormal pattern almost disappeared and RBC agglutination became minimal. We also observed cold agglutination reaction by using O type washed RBCs from a healthy individual that were reacted with cefpirom sulfate, but found only minimal agglutination at RT. As the drug concentration and plasma concentration were increased, we observed more agglutination at 4 degrees C, but this agglutination disappeared after incubation for an hour at 37 degrees C. The cold agglutinin titer was normal. We thus believe that the cause of RBC agglutination in this case was caused by cefpirom sulfate.

Unusual eosinophilia not detected by an automated haematological analyser in a patient with liver cirrhosis.

The use of automated haematological analysers to differentiate leucocytes has become more widespread. Unusual eosinophilia in a 57 year old man with liver cirrhosis, caused by hepatitis C infection, and abnormal blood counts detected using a manual method (eosinophils, 50%) was not detected by an automated analyser using the electrical impedance method (0.3%) or the optical method (14.1%). It is important to check blood films when cell counts are apparently abnormal, even for automated haematological examination.

[Discrepancy of platelet numbers between automated blood cell analysis and manual counting in the patients with thrombocytopenia].

Automated blood cell analyzer can measure the numbers of blood cell and a histogram patterns of WBC, RBC and Platelet (PLT), and calculate the blood cell indices (RDW, MPV, PDW, etc). It is important clinically in the patients with thrombocytopenia (below 10 x 10(4)/microliter) to measure correct platelet number. However platelet number with automated blood cell analyzer was influenced by the changes in erythrocyte morphology or various PLT conditions. We subclassified the patients with thrombocytopenia into 5 groups according to the histogram pattern of PLT, and compared platelet numbers among NE-8000 analysis (Sysmex), H*2 analysis (Bayer's) and manual method in each group. In the group of abnormal histogram pattern of PLT, platelet numbers obtained with NE-8000 or H*2 were significantly low comparing to manual numbers. About 70% of samples of which Platelet Distribution Width (PDW) was abnormal showed discrepant values between automated analysis and manual eye count. In conclusion, discrepant low platelet count would be obtained by automated analysis and therefore should be carefully interpreted in the patients with thrombocytopenia and abnormal platelet histogram pattern.

[A quantitative measurement of polymorphonuclear leukocyte oxidative metabolism using plasma depleted blood by flow cytometry].

We investigated a method measuring the superoxide production in peripheral polymorphonuclear leukocytes (PMN) in whole blood by flow cytometer using DCFH-DA as marker dye. The DCFH-DA which is colorless in its form and is freely permeable into blood cells is converted into DCF, a fluorescent substance, by oxidative reaction of superoxide in PMN. Thus, the superoxide production in PMN is measurable by counting number of fluorescence positive cells and their intensity by flow cytometer. However, when the DCFH-DA is added to the whole blood, a considerable amount of it is trapped not only in PMN but also in red blood cell, thus giving rise an inconsistent incorporation of the DCFH-DA in PMN due to differences in number of red blood cell among samples. Furthermore, there are several anti-oxidative factors in blood plasma. In order to avoid possible effects of plasma oxidative factors and red blood cell-numbers in whole blood on the measurement of PMN-superoxide production, we prepared plasma depleted blood by washing and the number of red blood cells was adjusted to 5 x 10(6) microliters before addition of DCFH-DA. This improved reproducibility and gave within assay coefficient of variance (CV) of 3.62% for a sample with normal value and 7.09% for a low value sample. The value obtained by this method correlated well with those of NBT reduction test (r = -0.887). It is concluded that this method is simple, reliable and useful for the clinical determination of superoxide production instead of NBT test.

[Decrease of CD16 antigen density on granulocytes in chronic myeloid leukemia].

We examined the fluorescence intensity of CD16 antigen, which represents the density of CD16 antigen, on granulocytes by flow cytometry in 15 healthy subjects and 15 patients with neutrophilia due to inflammatory diseases and 10 patients with chronic myeloid leukemia (CML). The fluorescence intensity of CD16 antigen was significantly lower in patients with CML than in healthy subjects and also than in patients with neutrophilia. These data indicate that 1) density of CD16 antigen on granulocytes decrease in CML, and 2) analysis on the density of granulocyte CD16 antigen is useful for differential diagnosis of CML from inflammatory diseases with neutrophilia.

[Mixed type cryoglobulinemia with discrepancy of platelet counts measured with an automated cell counter and by manual counting method].

We experienced an abnormal blood sample which showed larger numbers of white blood cells and platelets counted by the electric resistance system and of platelets in mechanical counting by the flow system, than those in manual counting. Many precipitates were observed in this blood sample at room temperature or 4 degrees C, but were not observed at 37 degrees C. The precipitates were clarified to be caused by the mixed type (IgA, IgG and IgM) cryoglobulin by using an immunofluorescence method and an ouchterlony method. These data indicate that precipitation of cryoglobulin at room temperature induce falsely high value of platelet count in mechanical counting systems.

[Intracellular alkaline phosphatase activities in myelodysplastic syndrome].

We examined the intracellular alkaline phosphatase (NIAP) activities in peripheral neutrophils in 15 normal controls, 4 patients with myelodysplastic syndrome, and 4 with chronic myeloid leukemia. NIAP activities were decreased in myelodysplastic syndrome in comparing to normal controls (p less than 0.01). These data suggest that measurement of NIAP activity is useful for supporting a diagnosis of myelodysplastic syndrome.

[Correlation between intracellular alkaline phosphatase activities and incidence of toxic granules in neutrophilia].

We examined the intracellular alkaline phosphatase (NIAP) activities in peripheral neutrophils in 15 healthy subjects and 15 patients with inflammatory diseases, and compared them with the percentages of neutrophils with cytoplasmic toxic granules (NTG), which is thought to be induced by infection. NIAP activities were markedly increased in patients and significantly correlated with the percentages of NTG. These data indicate that measurement of NIAP activity is useful for the diagnosis of infections and/or inflammatory diseases.