Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials

We have used five independent variables on a flow cytometer to discriminate and to quantify the cellular components within both blood and bone marrow aspirates. The signals were stored in list mode by which a five-dimensional space was created. The cells--differentiated into: 1) erythrocytes, 2) reticulocytes, 3) nucleated erythroid cells, 4) platelets, 5) lymphocytes, 6) monocytes, 7) neutrophils, 8) eosinophils, and 9) immature leukocytes--had to meet unique criteria with regard to their characteristics in the created five-dimensional space in order to be classified in a specific cell category. Forward and orthogonal light-scattering signals were matched with three fluorescence variables to obtain discrimination without necessitating erythrocyte lysis. Thiazole orange (binding predominantly to RNA) and LDS-751 (principally detecting DNA) were used to differentiate erythrocytes, platelets, reticulocytes, and nucleated cells. A monoclonal antibody, CD45, conjugated with phycoerythrin, was used to aid in discriminating between lineages of nucleated cells.     

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.     

Discriminating between damaged and intact cells in fixed flow cytometric samples

A vital, nucleic acid stain (LDS-751) was used to discriminate intact from damaged cells in a flow cytometer even after the samples had been fixed with paraformaldehyde. Three major cell populations with different fluorescence properties with LDS-751 were found in the fixed samples. Cells not staining or only dimly staining with LDS-751 were identified as erythrocytes and platelets, respectively. Cells staining with intermediate amounts of LDS-751 were found to be intact cells, while cells intensively stained were identified as damaged cells. Confirmation of the identity of the populations was obtained by light microscopic examination of the sorted populations and by correlating the fluorescent signals of FDA and LDS-751 in nonfixed cell preparations. Agglutinated cells could also be identified by the increased fluorescent signal in the LDS-751 channel as compared with single cells. The spectral properties of this dye permit excitation at 488 nm with emission in the far red portion of the spectrum. This allowed two-color immunofluorescence to be combined with the intact/damaged cell discrimination on fixed samples. Therefore, intact single cells could be distinguished during flow cytometric analysis, increasing the accuracy of the immunofluorescence measurements. The visualization of the multidimensional data was facilitated using color to discriminate cell populations depicted in multiple perspectives.     

In vivo radioprotection by 5-androstenediol: stimulation of the innate immune system

We showed previously that 5-androstenediol stimulates myelopoiesis, increases the numbers of circulating neutrophils and platelets, and enhances resistance to infection in gamma-irradiated mice. We have extended those studies to include monocytes, natural killer (NK) cells, eosinophils and basophils, and we have measured the activation marker CD11b using flow cytometry. Androstenediol (160 mg/kg) was administered subcutaneously to female B6D2F1 mice 24 h before whole-body gamma irradiation. Androstenediol treatments increased the blood levels of neutrophils, monocytes and NK cells in unirradiated animals; decreased the numbers of circulating eosinophils; and ameliorated radiation-induced decreases in neutrophils, monocytes, NK cells, erythrocytes and platelets. The androstenediol treatments had no significant effect on the numbers of circulating B cells or T cells. CD11b labeling intensity on monocytes was decreased slightly after androstenediol treatment. In contrast, radiation or androstenediol alone caused increases in CD11b labeling intensity on NK cells. Androstenediol and radiation combined caused a marked increase in NK cell CD11b. The results indicate that androstenediol increases the numbers of the three major cell types of the innate immune system (neutrophils, monocytes and NK cells), that androstenediol-induced changes in blood elements in irradiated animals persist for at least several weeks, and that there is a significant positive interaction between radiation and administration of androstenediol in the activation of NK cells.     

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.     

Detection of granulocyte reactive oxygen species formation in whole blood using flow cytometry.

We have developed a technique for analysis of granulocyte reactive oxygen species formation in whole blood using flow cytometry and two color immunofluorescence. This technique relies upon the use of specific fluorescent dye (LDS-751) to stain nucleated cells, eliminating erythrocytes from analysis. Using LDS-751, forward angle light scatter, and 90 degrees side scatter, a granulocyte gate, monocyte gate, and lymphocyte gate were identified. Analysis with multiple FITC conjugated monoclonal antibodies demonstrated greater than 95% purity of a flow cytometrically identified granulocyte population in whole blood without physical manipulation of the blood. Utilizing 2'7' dichlorofluorescein diacetate (DCFH-DA), we were able to measure granulocyte intracellular reactive oxygen species production. Dose response curves were obtained for the effect of granulocyte agonists phorbol myristate acetate, FMLP, and heat fixed Staphylococcus aureus on reactive oxygen species production. The techniques described in this paper should be useful for measuring granulocyte activation in vivo with flow cytometry.     

Combined blood cell counting and classification with fluorochrome stains and flow instrumentation.

A multiparameter flow cytophotometer was used to count and classify fixed human blood cells fluorochromed with a mixture of ethidium bromide (EB), brilliant sulfaflavine and a blue fluorescent stilbene disulfonic acid derivative (LN). The system measures light scattered by the cells and absorption at 420 nm for all cells. In addition, nuclear EB fluorescence (540 leads to 610 nm) and cytoplasmic fluorescence from LN (366 leads to 470 nm), brilliant sulfaflavine (420 leads to 520 nm) and EB exicted by energy transfer from LN (366 leads to 610 nm) are measured for all nucleated cells. This information is sufficient to perform red and white blood cell counts and to classify leukocytes as lymphocytes, monocytes, basophils, eosinophils or neutrophils. Light scattering and/or nuclear and cytoplasmic fluorescence values may be further analyzed to obtain the ratio of immature to mature neutrophils. Counts produced by the system are in reasonable agreement with those obtained by electronic cells counting and examination of Wright's-stained blood smears; some discrepancies appear to be due to systematic errors in the manual counting method.     

Identification of normal and leukemic granulocytic cells with merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytes and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in erythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single agent stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

Identification of Normal and Leukemic Granulocytic Cells with Merocyanine 540

After fixation in a modified Bouin's solution, the acid dye merocyanine 540 stained granules in granulocytic cells intensely. In immature granulocytes, such as promyelocytes and myelocytes, granules stained pink to violet. In some leukemic myeloblasts, promyelocytos and monocytes, granules also stained deep pink to violet. In more mature granulocytes, such as metamyelocytes, bands, and neutrophils, granules stained bright red to orange. In eosinophils and basophils, granules stained deep red. Granules of the type described were not visualized in normal plasma cells, lymphocytes, monocytes, or megakaryocytes. In normoblasts, cytoplasm stained diffusely red. Cytoplasmic staining in crythroblasts became darker as the cell matured, probably reflecting hemoglobin content. Used as a single a p t stain, merocyanine 540 may be useful in distinguishing normal and leukemic granulocytic cells from other types of blood cells.     

版纳鱼螈外周血细胞观察

以濒危两栖动物版纳鱼螈(Ichthyophis bannanica)为材料,应用瑞氏-姬姆萨混合染色法与血细胞计数法观察并统计了版纳鱼螈各种外周血细胞的形态特征和数量比例.结果表明,版纳鱼螈的外周血液中红细胞数量较多,呈卵圆形、椭圆形、梭形和梨形,平均含量为2.57 ×105个/mm3.白细胞数量较少,多呈近圆形,平均含量为0.72×103个/mm3.白细胞中,淋巴细胞最多,其次为单核细胞、嗜中性粒细胞、嗜碱性粒细胞和嗜酸性粒细胞.血栓细胞数量较少,常数个集合在一起.同时,将此研究结果与鱼类、爬行类和其他两栖类的血细胞比较,进而探讨了版纳鱼螈的进化地位.     

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.     

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides.

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.     

Increased light scattering resolution facilitates multidimensional flow cytometric analysis

Multidimensional flow cytometry identifies cell populations as clusters in a space created by the analysis of multiple parameters simultaneously. Optimal use of this multidimensional space requires each of the individual parameters to provide additional information for cell population discrimination as well as maximum utilization of the dynamic range available for each parameter. In this study we improve the visualization of the information present in light scattering signals from leukocytes to facilitate multidimensional flow cytometric analysis. Optimization of cell preparation techniques are essential to obtain high resolution light scattering signals that give complete separation of the granulocytes, monocytes, and granular and nongranular lymphocytes. The angle at which the forward scattered light was collected was modified to enhance the separation between leukocyte populations. Although orthogonal light scattering signals separate granular and nongranular lymphocytes, the resolution and dynamic range could not be displayed using linear or logarithmic functions. By applying a polynomial function to the orthogonal light scattering signals, all leukocyte populations could be displayed while maintaining high resolution. The combination of high resolution light scattering with a nonlinear display resulted in an equally spaced distribution of the cell populations distinguished by correlating forward and orthogonal light scattering signals. Using this approach, peripheral blood neutrophils, eosinophils, basophils, monocytes, and granular and nongranular lymphocytes were shown to occupy distinct locations in the correlation of orthogonal and forward light scattering. Surprisingly, the basophilic granulocytes were located close to granular lymphocytes and monocytes rather than near neutrophils and eosinophils.     

Haematological characteristics of albacore, Thunnus alalunga (Bonnaterre), and skipjack, Katsuwonus pelamis (Linnaeus)

Observations on haematological characteristics of albacore, Thunnus alalunga , and skipjack Katsuwonus pelamis , tunas, were made on blood samples collected from specimens immediately after they were caught and from an immobilized, restrained albacore sampled serially over a 24 h period. Results indicated eight types of blood cells in the peripheral circulation of both species. Lymphocytes were the most common leucocyte followed, in decreasing order, by neutrophils, eosinophils and monocytes in fish sampled immediately after being landed. Variations in differential and total white blood cell counts were observed in the immobilized, restrained albacore. High packed cell volumes and haemoglobin concentrations, typical for fast swimming fishes, were found in both species. Red blood cell counts were similar to those of other teleosts, and reticulocytes expressed as a percentage of mature erythrocytes were 6.1% and 5.2% for albacore and skipjack, respectively.     

团头鲂外周血细胞的显微结构及细胞化学特征

采用常规瑞氏染色和细胞化学染色方法对团头鲂(Megalobrama amblycephala)外周血细胞的显微结构及细胞化学特征进行了观察。在团头鲂外周血细胞中可区分出六类细胞: 红细胞、淋巴细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞和血栓细胞。其中淋巴细胞是除红细胞外含量最多的细胞, 其次分别为血栓细胞、单核细胞、嗜中性粒细胞、嗜酸性粒细胞。成熟红细胞多为卵圆形, 表面光滑, 胞核呈椭圆形或圆形, 染色质较为致密; 淋巴细胞多呈圆形, 胞质较少, 胞核常偏位; 单核细胞多为圆形, 胞核呈圆形或椭圆形, 胞质内可见空泡状结构; 嗜中性粒细胞近似圆形, 胞核常偏于细胞一侧, 呈分叶状、肾形或椭圆形, 核质界限清晰; 嗜酸性粒细胞一般为圆形, 胞核为肾形或椭圆形, 胞质中充满紫红色颗粒; 血栓细胞形态多样, 主要有椭圆形、纺锤形、长杆状和泪滴形, 核质比较大。淋巴细胞呈α-醋酸萘酚酯酶(ANAE)阳性, 呈过碘酸-雪夫(PAS)、氯乙酸AS-D萘酚酯酶(AS-DCE)弱阳性, 呈苏丹黑B(SBB)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)及过氧化物酶(POX)阴性; 单核细胞呈POX、ACP强阳性, PAS、SBB、AS-DCE和ANAE为阳性, 呈AKP阴性; 嗜中性粒细胞除PAS和ANAE为弱阳性外, 其他染色结果和单核细胞相同; 嗜酸性粒细胞呈POX、ANAE强阳性, SBB、ACP阳性, PAS及AS-DCE则为弱阳性, 呈AKP阴性; 血栓细胞呈PAS、AS-DCE及ANAE弱阳性, 呈SBB、ACP、AKP及POX阴性。团头鲂外周血细胞的显微结构及细胞化学特征与其他鱼类具有相似之处, 但亦有其明显的物种特异性。该研究结果可作为监测团头鲂健康状态的依据, 为其养殖及病理诊断提供基础资料。     

A novel cell surface antigen, 4C8, is expressed on human eosinophils

BACKGROUND: A novel monoclonal antibody, anti-4C8, reacted with human peripheral lymphocytes and monocytes but not with neutrophils. In this study, we investigated whether the 4C8 antigen is expressed on human peripheral eosinophils. METHODS: Expression of the 4C8 antigen on eosinophils was analyzed by flow cytometry and molecular analysis of the antigen was performed with eosinophils by Western blotting. RESULTS: Among human peripheral granulocytes, the 4C8 antigen was expressed on CD16-negative cells but not on CD16-positive cells. The 4C8 antigen also appeared to be expressed on eosinophils. To confirm the latter finding, eosinophils were purified from peripheral blood. On flow cytometric analysis, anti-4C8 antibody reacted with purified eosinophils. On Western blotting analysis, anti-4C8 reacted with a single band of 80 kDa in lysates from purified eosinophils. The correlation between the percentage of eosinophils determined by May-Giemsa staining and the percentage of 4C8-positive/CD16-negative cells among granulocytes was good (r = 0.91, P < 0.0001). CONCLUSIONS: Only a few cell surface antigens are available to distinguish human peripheral eosinophils from neutrophils. The novel cell surface antigen, 4C8, is a useful new marker of human eosinophils.     

Effects of 4-nonylphenol on blood cells of the African catfish Clarias gariepinus (Burchell, 1822)

In the present work, the destructive effects of the 4-nonylphenol on one of the most economically important Nile fishes, namely African catfish (Clarias gariepinus) were studied. Apoptosis, erythrocytes alterations, micronucleus test and blood parameters count were used as biological indicators to detect those effects. After exposure to sublethal concentrations of 4-nonylphenol (0, 0.05, 0.08 and 0.1 mg/l), apoptotic red blood cells with many malformations and micronucleated erythrocytes were recorded. Decrease in the blood parameters such as red blood cells (RBCs), hemoglobin (Hb), package cell volume (PCV), mean corpuscular hemoglobin concentration (MCHC), platelets, white blood cells (WBCs), lymphocytes, basophils, monocytes and increase in mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), neutrophils, eosinophils indicated the negative effects of 4-nonylphenol. It was concluded that, the 4-nonylphenol caused genotoxicity in erythrocytes with many malformations in shape and number indicated with other blood parameters.     

Comparative analysis of different flow cytometry-based immunophenotypic methods for the analysis of CD59 and CD55 expression on major peripheral blood cell subsets

BACKGROUND: Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH). METHODS: In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube. RESULTS: Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP. CONCLUSIONS: In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.     

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.     

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.