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Four spuriously lowered WBC counts due to in vitro leukoagglutination were reported from an automated cell counter (Coulter STKS). Leukocyte aggregates (3 to 50 cells), detected in the peripheral blood smears, included different cell types, normal (neutrophils, eosinophils, monocytes, lymphocytes) or abnormal (lymphoma cells). The phenomenon was associated with either a spurious leukoneutropenia or an underestimation of hyperleucocytosis. Leukoagglutination was extensively investigated in 3 cases : as shown in several reports, leukoagglutination may occur with various features, especially due to temperature and anticoagulant dependence. Our four cases reflected this variability. Furthermore, one case was found both temperature-dependent and anticoagulant-independent, a pattern not yet described in the literature. A common STKS graphic pattern was found in our 4 cases, suggesting that hematology analyzers such as Coulter STKS may be useful to detect leukoagglutination. In conclusion, each leukoneutropenia and/or each suggestive graphic pattern must be controlled by means of a blood smear examination in order to rule out the possibility of in vitro leukoagglutination.

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Human recombinant erythropoietin (rHu-Epo) is now extensively used in chronic renal failures; this treatment, resulting in a correction of the severe anemias seen in hemodialysed patients, may in turn lead to a resistance to rHu-Epo therapy by reason of the shortage of erythropoiesis factors, such as iron, vitamin B12 and folates. The utility of the red cell indices (MCV, MCH, RDW) for detection of early iron, folate and B12 deficiencies was studied in eighteen hemodialysed patients with end-stage renal failure treated with rHu-Epo; Microcytosis (MCV < 80 fl) was found ineffective in detecting iron deficiencies as well as macrocytosis (MCV > 100 fl) in folate and B12 deficiencies, partly due to the high incidence of associated iron and folate deficiencies. Lowered MCH (< 27 pg) was not more efficient than microcytosis in detecting early iron deficiencies. Increased RDW was the most sensitive feature for folate, iron and B12 deficiencies with respective sensitivities of 62.5%, 72% and 75%. The global specificity for detecting all deficiencies was 74%. However, high RDW values were not indicative of any type of deficiency; it may thus be concluded that RDW is a non expensive, non invasive and sensitive test, which allows a selection of hemodialysed patients treated with rHu-Epo for a complete investigation program, in order to detect early iron, B12 and folate deficiencies.

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Automated cell counters have allowed the reproducible analysis of the 5 leucocyte populations (neutrophils, eosinophils, basophils, lymphocytes and monocytes). Determination of the monocyte count by classical microscopic analysis can be inaccurate. We have compared the automated monocyte count from the coulter STKS (program 1F) with counts obtained by microscopic analysis of 1000 leucocytes and by flow cytometric analysis of cells stained simultaneously with a pan-leucocyte and a monocyte-specific antibody. The values provided by the coulter STKS are sufficiently precise to recommend the routine clinical use of this automated counter.

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The human platelet volume distribution has been shown log-normal. Coulter Counter S-Plus models, as well as JT and STKS models, give mean platelet volume and platelet distribution width parameters. A computerized mathematical method, working with an IBM-XT microcomputer, was designed to calculate median and modal volumes, as well as the percentage of platelets smaller or larger than operator definable volume thresholds, on the basis of mean platelet volume and platelet distribution width. Preliminary experiments defined that the optimal stability of results was obtained in the range 2-6 hours after the blood collection; further experiments demonstrated that the results of the mathematical analysis were reproducible and accurate, by comparison to data obtained from a channel analysis in all samples, even including macroplatelets and/or microplatelets. It was thus shown that Coulter counters working with a curve fitting process may provide a complete analysis of platelet distribution curves, which could be extensively used for studying pathological conditions involving changes in platelet parameters.

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The Coulter Counter Model S+II is an automatic analyzer designed to measure the routine hematological parameters and to carry out distribution curve analysis of platelets, erythrocytes and leukocytes. The leukocyte histogram analysis is used as a basis for calculation of both lymphocyte percentages and absolute counts. In this study, we compared the differential count obtained by light microscopy studies of 2355 blood smears with the percentage of lymphocytes in the leukocyte population as determined by the S+II. A comparison was also performed between the S+II and Hemalog D, H 6000, and Diff 3 System on a smaller sample number. There was a good correlation between lymphocyte percentages and counts given by the S+II and other methods. Furthermore, reproducibility studies, performed with the Coulter Counter Model S+II, suggested that the lymphocyte percentage given by this instrument was accurate, except for extreme values. Lack of calculation of lymphocyte parameters by the instrument was often found to correspond to spontaneous platelet aggregation and/or to abnormalities of differential counts. It is suggested that lymphocyte analysis provided by the S+II may be used as a basis for a new approach to white blood cell (WBC) analysis, which could exclude differential counts in most routine cases.

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Aqueous extracts from rabbit organs were prepared by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulphate, the 0-50 fraction was separated by ultrafiltration through Amicon XM 100 and XM 300 membranes yielding two filtrate fractions (U1 and U2) and one retentate fraction (U3). Only U1 and U3 inhibited thymidine incorporation into DNA. After a single injection of U1 from rabbit small intestine, the uptake of tritiated thymidine was decreased in mouse jejunal and colonic DNA. This effect, totally reversible after 7 hr, was found in neither the kidney nor the testis. The U1 fractions of colon and non-digestive organs (kidney, testis) were found not to exert a significant inhibition on thymidine incorporation into intestinal DNA in vivo. The U3 fraction from rabbit small intestine also decreased the uptake of tritiated thymidine in mouse jejunal and colonic DNA in vivo. However, this inhibition was irreversible and not tissue-specific. Slowing of cell migration was also noticed in the jejunum of mice injected with U1 or U3, as ascertained radioautographically by determining the position of the leading edge of the labelled cells in U1- or U3-injected mice compared with controls. A decrease of mitotic activity in U1- and U3-injected mice was recorded 8.5 hr after a single injection of small intestinal fractions. Our results suggest that U1 and U3 from rabbit small intestine contain one or more substances which may act on the G1-S transition of the cell cycle in the mouse intestine. However, only the effect of U1 is reversible and tissue specific. Our data suggest the existence of a factor, having a low molecular weight, which regulates intestinal cell proliferation.

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An aqueous extract was prepared from the mucosa of rabbit small intestine by homogenization and centrifugation at 105,000 g. After precipitation with ammonium sulfate, the 0-50 fraction (F1) and the supernatant (F2) were collected, dialysed against a phosphate buffer and tested on rats in vitro and mice in vivo. The F1 fraction was found to inhibit thymidine incorporation into rat intestinal DNA in vitro, but this effect was not found to be tissue specific (liver, kidney). Two hours after a single injection of F1 (10 mg protein content), the uptake of tritiated thymidine was decreased in jejunal and colonic DNA in mice. This effect was maximal between 2 and 4 hr and totally reversible after 7 hr; this effect was found in neither the kidney nor the testis. A slowing of cellular migration was also noticed in the jejunum and the colon. Conversely, the F2 fraction did not inhibit the synthesis of jejunal and colonic DNA either in vitro or in vivo. Our results suggest that the F1 fraction of the aqueous extract of rabbit small intestine contains one or more substances which may act either on intestinal DNA synthesis or on the GI--S transition of the cellular cycle in the mouse intestine. This reversible and specific intestinal action appears to inhibit cell proliferation and presents several of the characteristics defining a chalone.

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This paper describes a method to measure DNA synthesis in the jejunum. Everted rings of rat jejunum were incubated in the presence of (3H) thymidine. The specific activity of jejunal DNA was assayed at the end of incubation. This method was found to be valid when (1) incubation time was 30 min, (2) (3H) thymidine concentration was 90 ng/ml, and (3) paired flasks containing four, respectively, contiguous rings were compared. This method was used to investigate the existence of an intestinal chalone. An aqueous extract (S-105)of rabbit small intestine mucosa was prepared and tested for DNA synthesis inhibition in jejunum. A significant inhibition was noted, which was an hyperbolic function of the quantity of extract present in the media. The study of S-105 inhibition on other organs (kidney, liver, colon) failed to demonstrate organ specificity. However, aqueous extracts from intestinal organs were found to exert a greater inhibition than aqueous extracts form other organs. It is suggested that S-105 contains other nonspecific inhibitory fractions; our data prove the necessity of an extensive purification to demonstrate the existence of a specific chalone that regulates intestinal cell proliferation.

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