Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.     

Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.     

Reticulocyte changes after experimental anemia and erythropoietin treatment of horses.

Availability of recombinant human erythropoietin (EPO) has facilitated use to enhance red blood cell production, and therefore aerobic performance, in human and equine athletes. Recombinant human EPO promotes growth and differentiation of equine erythroid precursor cells, but in some horses repeat administration induces immune interference with endogenous EPO resulting in fatal anemia. Although blood reticulocyte parameters acquire unique changes in humans treated with EPO, with manual enumeration methods, horses were not considered to release reticulocytes from the bone marrow into circulation, even under severe erythropoietic stress. The goals of this study were to determine whether reticulocytes could be detected and characterized in horses that are anemic or have been treated with EPO using a modern hematology analyzer. Anemia was induced in six horses by removal of 30 ml of blood/kg of body wt over 24 h. After 28 days, the horses were treated twice with 55 U/kg of EPO (Eprex), and after 65 days they were treated thrice with 73 U/kg of EPO. Blood samples were analyzed with the ADVIA120 instrument every 3-5 days and bone marrow samples 7 days after anemia and EPO treatments. Analysis of blood reticulocyte parameters by ANOVA in a randomized complete block design determined that anemia and EPO induced significant (P < or = 0.05) increases in red cell distribution width and reticulocyte mean cell volume. Parameters changed only after EPO treatment were cellular hemoglobin concentration mean, mean cell volume, reticulocyte concentration, proportion of macrocytic reticulocytes, and reticulocyte cellular hemoglobin. These findings indicate that horses under erythropoietic stress and after EPO treatment release reticulocytes with unique characteristics into circulation.     

Flow cytometric analysis of blood cells stained with the cyanine dye DiOC1[3]: reticulocyte quantification

The fluorescent dye 3,3'-dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes less than immature erythrocytes congruent to platelets less than leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, new methylene blue, brilliant cresyl blue (BCB), and acridine orange (AO). However, the population of imRBC as quantified by DiOC1[3] fluorescence is a subset of reticulocytes (reticulocytes as determined by BCB assay). The advantages and disadvantages of using DiOC1[3], AO, or pyronine Y as reticulocyte stains are discussed.     

Evaluation of leukocyte number by using an automated blood cell counter and a traditional hematological method in animals irradiated with gamma rays

Female mice were irradiated with a single whole body dose of 7 Gy of gamma-rays. Leucocyte numbers were monitored in the peripheral blood using automated blood cell counter Coulter counter and a traditional hematological method with a light microscope in the Bürker chamber. Reticulocyte numbers, RNA blood concentration, spleen weight and morphological changes in spleen and bone marrow were also studied. In the period between 15th-19th days after irradiation the numbers of leucocytes obtained by CC counting were manifold higher than those obtained by microscope counting. Since this period is characterised by a steep increase in the reticulocyte number and RNA concentration in blood as well as by increased weight of spleen as the result of marked regeneration of extramedullar erythropoiesis, leukocytes as well as reticulocytes are assumed to be additionally registered by the automated counter CC in this period, probably due to a higher resistance of reticulocytes to the lysing agent Zapoglobine.     

In-depth validation of acridine orange staining for flow cytometric parasite and reticulocyte enumeration in an experimental model using Plasmodium berghei

Flow cytometry is potentially an effective method for counting malaria parasites, but inconsistent results have hampered its routine use in rodent models. A published two-channel method using acridine orange offers clear discrimination between the infected and uninfected erythrocytes. However, preliminary studies showed concerns when dealing with Plasmodium berghei-infected blood samples with high numbers of reticulocytes.In hyperparasitemic or chronic P. berghei infection, enhanced erythropoietic activity results in high numbers of circulating immature reticulocytes. We show that even though the protocol offered good discrimination in newly infected animals, discrimination between infected erythrocytes and uninfected reticulocytes became difficult in animals with hyperparasitemia or chronic infections maintained with subcurative treatment. Discrimination was especially hampered by increased nucleic acid content in immature uninfected reticulocytes. Our data confirms that though flow cytometry is a promising analytical tool in malaria research, care should still be taken when analysing samples from anemic or chronically infected animals.     

Preferential invasion of reticulocytes during late-stage Plasmodium berghei infection accounts for reduced circulating reticulocyte levels

Insufficient circulating reticulocytes have been observed during severe malarial anaemia in both human and murine infection, and are often attributed to reduced production of red cell precursors. However, a number of Plasmodium species display a preference for invading reticulocytes rather than erythrocytes. Thus, the reduction in circulating reticulocyte numbers may arise as a result both of increased parasitization and lysis of reticulocytes, as well as decreased production. We have analysed both circulating reticulocyte numbers and the percentage of infected reticulocytes during murine Plasmodium berghei infection. We found a large reduction in circulating numbers when compared with an equivalent chemically induced anaemia. However, mathematical analysis of parasite and red cell numbers revealed the preference of P. berghei for reticulocytes to be approximately 150-fold over that for erythrocytes, leading to increased destruction of reticulocytes. Although erythropoietic suppression is evident during the first week of P. berghei infection, this preferential infection and destruction of reticulocytes is sufficient to mediate ongoing reduced levels of circulating reticulocytes during the latter stages of infection, following compensatory erythropoiesis in response to haemolytic anaemia.     

Significant Biochemical,Biophysical and Metabolic Diversity in Circulating Human Cord Blood Reticulocytes

Background

The transition from enucleated reticulocytes to mature normocytes is marked by substantial remodeling of the erythrocytic cytoplasm and membrane. Despite conspicuous changes, most studies describe the maturing reticulocyte as a homogenous erythropoietic cell type. While reticulocyte staging based on fluorescent RNA stains such as thiazole orange have been useful in a clinical setting; these ‘sub-vital’ stains may confound delicate studies on reticulocyte biology and may preclude their use in heamoparasite invasion studies.

Design and Methods

Here we use highly purified populations of reticulocytes isolated from cord blood, sorted by flow cytometry into four sequential subpopulations based on transferrin receptor (CD71) expression: CD71high, CD71medium, CD71low and CD71negative. Each of these subgroups was phenotyped in terms of their, morphology, membrane antigens, biomechanical properties and metabolomic profile.

Results

Superficially CD71high and CD71medium reticulocytes share a similar gross morphology (large and multilobular) when compared to the smaller, smooth and increasingly concave reticulocytes as seen in the in the CD71low and CD71negativesamples. However, between each of the four sample sets we observe significant decreases in shear modulus, cytoadhesive capacity, erythroid receptor expression (CD44, CD55, CD147, CD235R, and CD242) and metabolite concentrations. Interestingly increasing amounts of boric acid was found in the mature reticulocytes.

Conclusions

Reticulocyte maturation is a dynamic and continuous process, confounding efforts to rigidly classify them. Certainly this study does not offer an alternative classification strategy; instead we used a nondestructive sampling method to examine key phenotypic changes of in reticulocytes. Our study emphasizes a need to focus greater attention on reticulocyte biology.     

A dose-dependent effect of recombinant erythropoietin on the reticulocyte population of rats

The effect of human recombinant erythropoietin (rEPO) on the reticulocyte count and the reticulocyte maturation distribution in rats was measured with an automatic reticulocyte analysis system (AURAS). The strongest reaction was found on the third day after a single application of rEPO with respect to reticulocyte count, maturation distribution, and the count of immature reticulocytes. At this time these parameters show a distinct dose dependency. The results indicate a possible basis for a simple rEPO bioassay using normocytemic rats.     

Evaluation of the reticulocyte micronucleus assay in patients treated with radioiodine for thyroid cancer

In the case of accidental radiation exposure, biological dosimetry has an important role. Previous studies have indicated that the flow cytometric micronucleus (MN) assay in human transferrin receptor positive reticulocytes (Tf-Ret) in blood could be a sensitive biomarker for chromosome damage. In the present investigation, the utility and sensitivity of this method was studied in 44 young patients from Belarus, who were treated with 131I for thyroid cancer. Red marrow (RM) is the critical organ in radioiodine therapy (RIT). In our patients, it was exposed to 100-700 mSv low-dose rate irradiation within 2-4 days. About 3 days after 131I administration, the frequency of micronucleated-Tf-Ret (f(MN-Tf-Ret)) increases within 1 day to a maximum and declines in the following 2-5 days to its value before treatment. A total dose of 100 mSv was easily detectable. The sensitivity of the assay after acute irradiation may be 50 mSv. The method should be useful for monitoring individuals after a radiation accident, provided blood samples can be obtained within a few days after exposure. The time-course of f(MN-Tf-Ret) is interpreted using a model, which considers the exponential exposure of red marrow in RIT as well as the kinetics of erythroblast maturation and reticulocyte migration into the peripheral circulation. Similar modelling was done on published data of MN in immature mouse erythrocytes. Striking similarities in the kinetic and in the yield of MN-induction were found between these two species. This lends support for the use of the mouse as a model for the MN-induction in humans.     

Effect of acute anemia or polycythemia on blood flow and iron transport in bone marrow

Iron has been shown to be the limiting factor for erythropoiesis. The anemia and polycythemia effect on iron supplied to the bone marrow has been studied in a group of rabbits, by modifying the hematocrit without altering of the blood volume. The cardiac output and the percentage of blood flow to the skeleton was measured using 57Co and 113Sn radiolabelled microspheres, before and after the exchange of blood by plasma or red blood cells concentrates. In addition, ferrokinetic measurements were performed with 55Fe and 59Fe. The production of an acute anemia induced an increase in the cardiac output from 156 +/- 35 to 239 +/- 89 ml/min/kg and a decrease in the percentage of the total blood flow to the skeleton from 7.58 +/- 2.51 to 4.63 +/- 1.8. The production of an acute polycythemia induced a decrease in the cardiac output (97 +/- 28 ml/min/kg) and an increase in the percentage of the total blood flow to the bone marrow (11.69 +/- 4.03). However, in both cases, the absolute amount of blood flow and iron flow to the bone marrow were similar to the controls. These studies demonstrate that anemia or polycythemia per se do not determine the iron supply to the bone marrow.     

Quantitative Analysis of Mechanisms That Govern Red Blood Cell Age Structure and Dynamics during Anaemia

Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.     

CD45-assisted PanLeucogating for accurate,cost-effective dual-platform CD4+ T-cell enumeration

BACKGROUND: North American and European guidelines for dual-platform (DP) flow cytometry recommend absolute CD4 T-cell counts to be calculated from two parameters: the absolute lymphocyte counts obtained on a hematology analyzer and the percentages of CD4+ cells among lymphocytes (CD4%/lympho) obtained by flow cytometry. Nevertheless, the identification of lymphocytes is error-prone: a poor match between these common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocyte counts (white cell counts [WCC]) and CD4% among the gated CD45+ leucocytes (CD4%/leuco) can be determined with greater accuracy. METHODS: We introduced "PanLeucogating," i.e., we used total leucocytes as the common denominator for improving the precision of DP absolute CD4 counting. Correlations and Bland-Altman tests were used for statistical analysis. RESULTS: First, 22 stabilized blood product samples were provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy and precision of CD4 counts were documented using PanLeucogating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single-platform (SP) methodology, including both volumetric and bead-based techniques. A particularly high correlation and comparable precision of absolute CD4 counts were observed between the SP volumetric method and DP PanLeucogating (R(2) = 0.990; bias 6 +/- SD 17%). The SP volumetric method showed lower levels of agreement with the DP lymphocyte gating (R(2) = 0.758; bias 14 +/- SD 51%) and with the SP bead-based method (R(2) = 0.923; bias 4 +/-SD 31%). CONCLUSIONS: These observations show that DP leucocyte counts (WCC) should replace lymphocyte counts as the "common denominator" although CD4%/lympho values can, as an extra step, be also provided readily if requested. When coupled with quality control for WCC on hematology analyzers, the DP method with CD45 PanLeucogating represents a robust CD4 T-cell assay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4, antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer.     

The red blood cells in growing guinea pigs. (Cavia cobaya). II. Communication: erythropoiesis and red blood cells in the postnatal development period

Bone-marrow smears of 175 guinea pigs aged 1-27 days and venous blood samples of 351 animals aged 1-25 days were prepared for cell counting. A significant increase of erythroblasts were found between life day 1 and 2; normoblasts increased in number synchronously with a decrease of erythroblasts after the 5th day. The percentage of the erythroid bone marrow increased from 10 to 14 during the developmental period. Beyond the perinatal period the red blood picture is characterized by the following changes: a decrease of erythrocyte count, hematocrit, hemoglobin, mean corpuscular volume, and mean corpuscular hemoglobin; a constant mean corpuscular hemoglobin concentration; an increase of the reticulocyte count. The decrease of the red cell count is compensated by a decreasing oxygen affinity attained by an important increase of 2,3-DPG. Nevertheless, the stimulus for a raising erythropoiesis remains constant which can be shown by the growing percentage of erythroid cells and reticulocytes. The difference between the human postnatal development and that of the guinea pig becomes obvious. Cell counts in dependence of body masses in postnatally growing guinea pigs, veil the perinatal finding of the increase in erythrocytes up to the 5th day and the decrease of the mean corpuscular volume after the 3rd day.     

Flow cytometric analysis of micronucleated reticulocytes in mouse bone marrow

This laboratory has previously reported a flow cytometric procedure for quantitatively analyzing mouse peripheral blood reticulocytes for micronucleus content. The current study extends this line of investigation by evaluating whether these same flow cytometric scoring procedures can be applied to the analysis of mouse bone marrow samples. To validate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collected 20, 40 or 60 h after administration. A set of 5 untreated animals was included to provide an indication of spontaneous micronucleus frequencies. The cells were fixed with ultracold methanol, treated with ribonuclease, and labeled with anti-CD71 antibody (FITC conjugate) and propidium iodide. This fixing and labeling procedure resulted in the resolution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry. The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10?000 total reticulocytes per bone marrow sample. In addition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocytes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronucleated reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly indicated.     

A peculiar pattern of expression of the transferrin receptor (CD71) by reticulocytes in patients given recombinant human erythropoietin (rHuEPO): a novel marker for abuse in sport?

To overcome the limitation of the currently adopted direct method to detect recombinant Human Erythropoietin (rHuEpo) abuse in sport, indirect analysis of blood parameters are increasingly used as part of the anti-doping strategies. The aim of the present work is to identify whether immunophenotype modifications on erythroid cells may be indicative of previous rHuEPO administration. The study was conducted on dialyzed patients under treatment with rHuEPO (DPT). Dialyzed patients without rHuEPO therapy (DP) and volunteer donors (H) were used as controls. The analysis of erythroid cells immunophenotype, performed using a multiparametric flow cytometry technique, showed a peculiar pattern of CD71 expression following rHuEPO treatment. In particular CD71 showed an increased expression in mature and intermediate reticulocytes and a surprisingly decreased expression in immature reticulocytes. In conclusion, the analysis of reticulocyte maturation stages with TO/CD71 double staining may be considered as a valid alternative indirect method for the detection of rHuEPO abuse.     

Absolute TCD4+ counting by a minimalist dual-platform flow cytometric method

The aim of this work was to compare the performance of an absolute TCD4+ counting method based on total WBC gating versus the standard lymphocyte (Ly) gating method, in order to develop a flow cytometric (FCM) minimalist strategy for TCD4+ enumeration. METHOD: 132 routine peripheral blood samples, mainly from HIV infected patients, were labelled with CD3-FITC/CD4-PE/CD45-PECy5 and analyzed by two gating methods: a) standard method based on Ly immunological gating (CD45++SSClow), followed by the determination of CD3+CD4+ percentage and absolute number (# calculation using Ly # from hematological analyser (HA); b) total WBC immunological gate on biparametric scatter CD45/CD4, followed by CD4++SSClow percentage determination and absolute number calculation using WBC absolute number from hematological counter without using the WBC differential. Moreover on 63 samples Ly # based on Ly % from FCM and WBC counting from HA was compared with Ly # from HA. RESULTS: The TCD4+/microL ranged from 3 to 3277 and the statistical analysis results showed: a) linear regression: r2 = 0.9847; b) Bland & Altman analysis: difference mean = -56.22; agreement range = +95.68 / -208.12; c) the mean of result difference/mean value*100 between two methods was -9.06%; d) comparison between regression line and the boundaries for acceptable residual values based on regressed confidence limits found by A. Kunkl et al showed regression line within boundaries near the upper limits. The Ly/microL count ranged from 635 to 8752. The statistical analysis results showed: a) linear regression: r2 = 0.9764; b) Bland & Altman analysis: difference mean = -362.93; agreement range = +134.51 / -860.37; c) the mean of result difference/mean value*100 between two methods was -16.12%. CONCLUSIONS: Our results suggest a fair agreement between the two gating methods, but the one based on total WBC gate gives TCD4+/microL counts systematically higher than the standard method. This finding can be attributed to a systematic lower estimation of Ly% by HA.     

网织红细胞在消化道肿瘤化疗前后的变化及临床意义

目的:探讨采用网织红细胞监测消化道肿瘤患者化疗前、后骨髓造血功能恢复的临床价值。方法:采用SysmexXE2100全自动细胞分析仪对60例消化道恶性肿瘤患者化疗过程中白细胞数(WBC)、血小板(PLT)、网织红细胞百分率(RET%)、未成熟网织红细胞指数(IRF)进行动态监测。结果:化疗后骨髓造血功能受到抑制,RET%和IRF从化疗后第3天开始出现明显下降,差异有统计学意义(P<0.01),在化疗后第14天开始出现回升,其中IRF值较第7天显著升高,两者间差异具有统计学意义(P<0.01);WBC和PLT则于化疗后第7天出现明显下降,差异具有统计学意义(P<0.01),化疗后第21天开始出现回升。结论:未成熟网织红细胞指数是预测化疗后骨髓造血恢复的较敏感指标。     

Optimization of flow-cytometric discrimination between reticulocytes and erythrocytes

An automatized technique to count reticulocytes by means of flow cytometry is described. Blood samples were stained by the fluorescent dye acridine orange without the use of fixative. Scatter and red fluorescence of the blood cells were measured in a flow cytometer. A discrimination between reticulocytes and erythrocytes was only achieved by using logarithmic amplification. The discrimination was better in peak mode than in area mode. The optimum dye concentration was 0.5 mg/liter acridine orange. At lower dye concentrations, not all reticulocytes were measured, whereas at higher dye concentrations the degree of discrimination between reticulocytes and erythrocytes decreased. There was a suitable discrimination between reticulocytes and erythrocytes. The reticulocyte numbers were scored by flow cytometry as well as by microscope for blood samples with 0.1-14% reticulocytes. The correlation between both methods was close.