A flow cytometric assay for D8/17 B cell marker in patients with Tourette's syndrome and obsessive compulsive disorder

D8/17 is proving to be an important diagnostic and treatment tool in patients with Tourette's syndrome (TS) and obsessive compulsive disorder (OCD). The present study represents a direct comparison of immunofluorescence microscopy and flow cytometry in terms of their relative efficiency in the detection of the DB/17 B cell marker. Analysis of 41 patients with OCD or TS and 31 control subjects by immunofluorescence microscopy and flow cytometer demonstrated an average of 6.5% of D8/17-positive B cells in controls with an average of 19.6% in OCD patients. Using the flow cytometer, values of 6.2% for controls and 22.4% for OCD patients were obtained. The flow cytometer test was able to count many more cells, induce less operator error and offer faster analysis while correlating strongly with the microscopic technique. Overall in our sample, the FACS assay test has a sensitivity of 77% and a specificity of 87%. The ease of use and close correlation to disease activity makes this an ideal test for clinical laboratories.     

Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay

BACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression. PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression). METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases. RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion. CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening. IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.     

A flow cytometric method for rapid selection of novel industrial yeast hybrids

We rapidly produced and isolated novel yeast hybrids by using two-color flow cytometric cell sorting. We labeled one parent strain with a fluorescent green stain and the other parent with a fluorescent orange stain, and hybrids were selected based on their dual orange and green fluorescence. When this technique was applied to the production of hybrids by traditional mating procedures, more than 96% of the isolates were hybrids. When it was applied to rare mating, three hybrids were identified among 50 isolates enriched from a population containing 2 x 10(6) cells. This technology is not dependent on genetic markers and has applications in the development of improved industrial yeast strains.     

The applicability of the flow cytometric sperm chromatin structure assay in epidemiological studies. Asclepios

The impact of demographic, lifestyle, and seminal factors on the sperm chromatin structure assay (SCSA) parameters was evaluated in a population of 277 healthy Danish men. This cohort was established within the framework of a European Concerted Action on occupational hazards to male reproductive capability in order to examine the possible reproductive effects of exposure to styrene or pesticides. The SCSA measures the susceptibility of sperm DNA to in-situ acid-induced denaturation, by multiparameter flow cytometric analysis after staining with the DNA-specific fluorescent dye acridine orange. The green versus red bivariate cytogram patterns were quite variable among donors, showing a wide heterogeneity of sperm DNA denaturability. Nevertheless, in those cases where we had the possibility to measure two semen samples from the same donor, the cytogram pattern remained stable over time (0.64 < r < 0.78). Analysis of variance demonstrated that the SCSA results can be influenced by the age of the donor (P < 0.0001), smoking habits (P < 0.05), the presence of leukocytes and immature germ forms in the ejaculate (P < 0.0001), and the duration of sexual abstinence (P < 0.0001). Furthermore, the relationship between the SCSA data and sperm concentration, morphology, and vitality was weak (-0.22 < r < -0.46). Therefore, the SCSA provides independent and complementary measurements of semen quality and is thus a useful tool for epidemiological studies, but the effects of some confounders should be accounted for in the survey design and analysis.     

Localized malignant mesothelioma. A clinicopathologic and flow cytometric study

Six examples of malignant mesothelioma appearing as a localized pleural mass are described. There were four women and two men, ranging in age from 42 to 76 years. A history of asbestos exposure was obtained from three patients. The tumors ranged in size from 2.8 to 10 cm. Two were pedunculated and four were sessile with broad-based pleural attachments. Histologically, three tumors were purely epithelioid and three were biphasic. Immunohistochemical stains in all six cases were positive for cytokeratin and negative for carcinoembryonic antigen. Five were also positive for epithelial membrane antigen. Five were negative for Leu-M1, while one showed focal staining in a peripheral membrane pattern. Electron microscopy in two purely epithelioid tumors showed long, thin microvilli, well-developed desmosomes, and numerous tonofilaments. Flow cytometry showed an aneuploid DNA content in four tumors and a diploid content in one. Flow cytometry in five cases identified a DNA aneuploid cell population in four tumors and a diploid population in one. Three patients showed signs of local recurrences 4, 7, and 18 months after excision and died of their disease 12, 10, and 24 months after diagnosis, respectively. Three patients are well with no evidence of disease 8, 24, and 96 months after diagnosis. These findings indicate that malignant mesotheliomas of the pleura may rarely appear as a localized mass. The biologic behavior of such tumors is difficult to predict, but some patients survive disease-free for a long time after surgical excision.     

Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A

The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI). From the results obtained, FM3A cells proved to be susceptible to CPE. A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively. These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells). Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates.     

Preparation of fine needle aspiration biopsy samples for flow cytometric analysis

In order to improve the quality of flow cytometric (FCM) DNA histograms, a new preparatory method was tested on samples obtained by fine needle aspiration biopsy (FNAB) of breast tumors. Twenty-four samples were obtained in vivo (group 1), and 20 were obtained from surgically resected specimens (group 2). Tumors from both groups were aspirated twice each. The first sample was injected directly into 70% ethanol, whereas the second sample was pretreated with a mixture of Tween-20 and citric acid solution (Tween-20 CA) before ethanol fixation. The coefficient of variation (CV) of G0-G1 peaks of Tween 20 CA-pretreated samples varied from 1.85 to 5.10 (mean, 3.3) in group 1 and from 1.87 to 3.72 (mean, 2.77) in group 2. The CV of G0-G1 peaks of ethanol-preserved samples ranged from 2.28 to 7.22 (mean, 5.23) in group 1 and from 1.78 to 4.04 (mean, 3.48) in group 2. The CV values of histograms obtained by the new protocol were significantly lower (group 1, P < .05; group 2, P < .01).     

A strong prognostic variable in endometrial carcinoma: flow cytometric S-phase fraction

BACKGROUND: The purpose of this study was to determine the role of flow cytometric S-phase fraction as a prognostic factor in patients with endometrial adenocarcinoma. METHODS: The study included 80 patients with endometrial adenocarcinoma of endometrioid type who were followed regularly between 1984-1995 in the Department of Obstetrics and Gynecology at Cerrahpasa Faculty of Medicine in Istanbul, Turkey. The method employed for the flow cytometric analysis was modified from Hedley et al. RESULTS: The S-phase fraction was identified as the most significant variable associated with death from endometrial carcinoma of endometrioid type by the Cox proportional hazards model. The risk of death was significantly higher in patients with S-phase values greater than 20%. Aneuploidy and DNA indexes were also significant prognostic variables. CONCLUSIONS: The S-phase fraction is considered to be a significant prognostic variable in identifying those patients with endometrial carcinoma who have a poor prognosis. The authors believe that S-phase fraction distinguishes those patients who may benefit from additional treatment approaches.     

Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms

Genetic damages are frequently found in both tumor and normal cells at carcinogen exposed areas in the patients with upper aerodigestive tract cancer. These phenomena are explained by the multistage process and/or field cancerization theories. The c-erbB-2 proto-oncogene has been amplified in many human tumors including breast, stomach, kidney and lung cancers. To study the possible evidence of multistage process and/or field cancerization in the development of gastric adenocarcinoma, the amplification statuses of c-erbB-2 proto-oncogene using the Southern hybridization technique were evaluated at the 45 gastric adenocarcinoma specimen sets consisting of tumor tissue, adjacent normal tissue (within 2 cm of the primary tumor), metastatic tissue and normal stomach tissue (at least 5 cm away from primary tumor). As a result, c-erbB-2 proto-oncogene at 2 specimen sets (4.4%) was amplified 2- to 4-fold to normal control status. In these 2 cases, c-erbB-2 proto-oncogene at histologically normal tissue adjacent to tumor tissue was amplified. And, the metastatic tissue of 1 case also exhibited c-erbB-2 proto-oncogene amplification of which the degree was less than that of tumor tissue. From these results, we were able to suspect that c-erbB-2 proto-oncogene amplification in the normal tissue adjacent to tumor tissue could be a biomarker of premalignant changes in a small proportion of gastric adenocarcinoma patients. And, this result might suggest the possible role of multistage process and/or field cancerization in the development of gastric adenocarcinoma.     

A flow cytometric method for counting very low levels of white cells in blood and blood components

Reduction of white cells (WBCs) in blood components may reduce the risk of virus transmission and HLA alloimmunization. Filtration provides a means by which to achieve high-efficiency WBC reduction. A method has been developed using flow cytometry to quantitate the number of WBCs in WBC-reduced packed red cells or platelet concentrates. This method uses a detergent and propidium iodide (PI) solution to label the WBC nuclei and incorporates a known amount of fluorescein isothiocyanate (FITC)-labeled chicken red cells (cRBCs) into the mixture as an indicator of the volume examined. The number of observed WBCs per mL is calculated as follows: Number of PI WBC nuclei events/Number of FITC cRBC events x Number of FITC cRBCs added to mixture/Volume of blood in mixture. The method may allow the detection of WBCs at a concentration as low as 0.01 per microliters (10/mL) in a blood sample. It is an efficient method of collecting data, as it requires less than 10 minutes per sample. This flow cytometric technique is suitable for research purposes and for quality control of WBC-reduced blood components, because it is precise and can be used to quantitate WBCs in large or small numbers in a sample.     

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophil surface antigens, and monoclonal antibody production and characterization

Enzyme-linked immunosorbent assay and flow cytometric methods to screen hybridoma culture supernatants for antibodies to bovine neutrophils (surface antigen-specific) were optimized. Sensitivity of the 2 methods was compared. A panel of 14 murine monoclonal antibodies (MAB) to surface antigens of bovine polymorphonuclear neutrophilic leukocytes (neutrophils) was produced by hybridoma technology, and their isotypes were determined by whole-cell ELISA. Monoclonal antibody reactivity with neutrophils, eosinophils, and lymphocytes isolated on phosphate-buffered saline solution and on Ficoll-sodium diatrizoate were compared. Biochemical characterization of antigens recognized by MAB was performed by immunoblot analysis. Neutrophil plasma membranes were isolated on sucrose gradients (20, 32, and 50%) and purified for polypeptide characterization. Neutrophil surface proteins were characterized by external labeling with 125I. The flow cytometric method was proven to be more sensitive and rapid than ELISA to screen hybridoma supernatants. This method allowed light-scatter gating of live neutrophil populations for analysis, which eliminated nonspecific binding of antibodies to contaminating cells and dead neutrophils. The optimal conditions for flow cytometric analyses were 5 x 10(5) neutrophils and 1 micrograms of fluorescein-labeled F(ab')2/assay as the second antibody. The optimal conditions for hybridoma screening by ELISA were neutrophil concentration of 2.5 x 10(5)/well, using a 96-well polystyrene microtitration plate as solid support, and 2,2'-azino-di[3-ethyl-benzthiazoline sulfonate (6)] with H2O2 as the chromatogenic substrate. Tissue culture plates as solid support and 3,3', 5,5'-tetramethyl benzidine, with H2O2 as the chromogenic substrate, were equally as sensitive. Panel MAB reacted differently with neutrophils, eosinophils, and lymphocytes. Isolation of these cells from blood on Ficoll-sodium diatrizoate generally did not alter MAB reactivity. Coomassie blue-stained gels of neutrophil plasma membrane proteins contained about 25 polypeptide bands, 13 of which were major bands. Autoradiography revealed about 11 surface proteins, 5 of which were heavily labeled with 125I. Monoclonal antibody S7G8 identified a 65-kd protein and MAB S8G10 identified 65- and 70-kd proteins. On the basis of molecular weight, MAB S7G8 and S8G10 are comparable to human CD15, CD16, and CD64 molecules. The MAB generated in this study are potential candidates to discern bovine neutrophil function and heterogeneity.     

Combining morphology and flow cytometric immunophenotyping to evaluate bone marrow specimens for B-cell malignant neoplasms

We studied the evolutionary history of two homologous proteins of the human complement system, factor H (FH) and the alpha chain of the C4b binding protein (C4bpalpha), and included in this study the related proteins from the barred sand bass (P. nebulifer) and the nematode C. elegans. Phylogenetic trees inferred from individual short consensus repeats (SCRs) and divergence among repeats from different genes suggest that human FH has a much closer evolutionary relationship to putative complement components from P. nebulifer and C. elegans than does the C4bpalpha. This indicates that a member of the alternative pathway of the complement system (FH) has an ancient origin, while a homologous member of the classical pathway (C4bpalpha) appeared later in evolutionary history as a result of gene duplication. The ancient evolutionary position of FH is in agreement with the suggestion that the alternative pathway of the complement system is older than the classical pathway. Phylogenetic analysis also shows that the sand bass cofactor protein SBP1 and cofactor related protein SBCRP-1 have diverged very recently.     

Heterogeneity in early and advanced gastric carcinomas by flow cytometric DNA analysis

We investigated 230 systematically sampled fresh specimens from 12 early and 26 advanced gastric cancer patients by DNA flow cytometry for heterogeneity in DNA content. Fifty-eight percent of the 12 early gastric cancers were uniformly diploid and 42% were uniformly aneuploid. Fifty-four percent of advanced cancers were uniformly diploid in superficial layers and 42% were uniformly diploid in deep layers, whereas 46% were uniformly aneuploid in superficial layers, and 50% were uniformly aneuploid and 8% were heterogeneously aneuploid and diploid in deep layers. Both diploid and aneuploid samples were obtained from 15% for advanced cancers, but ploidy heterogeneity did not occur in early cancers. Heterogeneity for DNA index (more than one aneuploid DNA index) occurred in 46% of whole thickness of advanced cancers, in 19% of superficial layers of advanced cancers, and in 8% of early cancers. We concluded that DNA ploidy determination using superficial layer specimens may be reliable in early gastric cancer but must be interpreted with care in advanced cancer.     

DNA flow cytometric analysis in patients with operable non-small cell lung carcinoma

Fresh surgical specimens of tumors from 60 patients with previously untreated non-small cell lung carcinoma (NSCLC) who underwent radical surgery between January 1991 and October 1992 were investigated by means of flow-cytometry. The nuclear DNA measurement was carried out using a Facscan (Becton, Dickinson, USA). Analysis of the DNA content was performed in all 60 patients whilst cell cycle analysis was possible in 41 cases (68.3%). Forty-two of the 60 cases (70%) were aneuploid and 18 (30%) were diploid. The overall mean value of DNA index was 1.5. Diploid NSCLC were compared with aneuploid tumors: no significant differences in age distribution, sex ratio, histology and staging were found between the two groups (P > 0.05). An S-phase proportion of more than 10% was found in 30 out of 41 patients (73.2%). Early cancer deaths were reported in four patients (6.6%): the aneuploidy rate was very close in these patients (75%) and in the remaining surviving patients (69.6%). An S-phase proportion of more than 10% was found in 100% of early cancer deaths and in 70.2% of the remaining cases; such a difference seems of some importance although it was not statistically significant (P = 0.071). In conclusion, flow-cytometry studies seem to be a useful tool in the understanding of the biological behavior of patients with NSCLC. In the present prospective report there were no significant correlations between DNA measurements and clinical outcome, however, these results suggest that a high S-phase proportion should be seen as a possible prognostic indicator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular rejection post heart transplantation is associated with positive flow cytometric cross-matching

28 isolates of canine parvovirus type-2 (CPV-2) were obtained from dogs with hemorrhagic gastroenteritis in Italy. The antigenic structure of CPV-2 isolates was characterized, using four discriminating monoclonal antibodies. In addition, four vaccinal strains were examined. Similar to reports from Australia and the United Kingdom, a much higher prevalence of CPV-2a (25/28 isolates) was observed than the other variant type, CPV-2b (3/28 isolates). DNA fragments (2.2 kbp) of representative strains of CPV-2, CPV-2a and CPV-2b were amplified by the polymerase chain reaction (PCR) and the products were digested by the restriction enzymes (RE) RsaI, HpaII, HindIII and PvuII. The RvaI enzyme allows the differentiation of CPV-2 from CPV-2a and CPV-2b.     

Prognostic significance of DNA flow cytometric analysis in patients with nasopharyngeal carcinoma

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a prevalent malignant tumor among Southern Chinese. Previously, the authors described the prognostic significance of a serum antibody assay to a recombinant Epstein-Barr virus Bam HI-Z replication activator protein (ZEBRA) in NPC patients with long term follow-up. In this study, the authors further reported the use of DNA flow cytometry (DNA-FCM) as an additional technique for determining the prognosis of NPC patients in the same series. METHODS: One hundred and forty-three archival biopsies from 110 NPC patients were deparaffinized and subjected to DNA-FCM analysis. DNA ploidy state and various proliferative indices (PI) of the tumors were correlated with patient survival and frequency of recurrence. RESULTS: Among the biopsies analyzed, 119 were histologically positive NPC and 24 were negative. Fifty-one tumor biopsies that fulfilled the guideline criteria of the DNA Cytometry Consensus Conference were correlated with the clinical manifestations of the patients. Among them, 43 tumors (84%) were DNA diploid and 8 (16%) were aneuploid. Two PI, S-phase fraction (SPF) and proliferation fraction (PF), appear to be potentially useful prognostic indicators. For example, PF in patients who developed locoregional recurrence (15.1%) and distant recurrence (16.4%) after radiation therapy both were significantly higher than PF in patients who were in complete remission (8.2%) (P = 0.0005 and P = 0.004, respectively). Significant differences in SPF between patients with distant recurrence (10.6%) and those in remission (5.7%) also was found (P = 0.005). Using Kaplan-Meier analysis, patients with high PF, high SPF, and aneuploid tumors had significantly poorer 12-year survival rates (35%, 26%, and 28%, respectively) than those patients with low PF, low SPF, and diploid tumors (77%, 67%, and 59%, respectively) (P < 0.0009, P < 0.004, and P < 0.01, respectively). CONCLUSIONS: Determination of tumor PI and DNA ploidy state by DNA-FCM at diagnosis of NPC can be potentially useful in selecting a poor prognostic subgroup of NPC patients. These parameters may enable oncologists to plan for more stringent treatment strategies such as hyperfractionated and accelerated radiation therapy or concomitant chemoradiotherapy for these patients.     

Increased apoptosis of adult rat lymphocytes after single neonatal vitamin A treatment (hormonal imprinting). A flow cytometric analysis

Newborn rats were treated with a single dose of vitamin A (retinol) and apoptosis of peripheral lymphocytes was studied by flow cytometry in adult age. Vitamin A treatment (hormonal imprinting) caused a moderate, however significant elevation in the number of apoptotic lymphocytes after three months. Dexamethasone or Concanavalin-A alone did not influence apoptosis significantly. However, in the neonatally retinol treated rats dexamathasone significantly elevated the quantity of apoptotic lymphocytes related to the control or Concanavalin-A treated control cells. The results call attention to the prolonged effect of hormonal imprinting in a new index and to the possible dangerous effects in human, neonatally treated with vitamin A.     

Use of acridine orange in: flow cytometric evaluation of erythropoietic cytotoxicity

Cytotoxic insult to bone marrow frequently impairs the proliferating and maturational abilities of erythroid cells. Typically, a ratio of enucleated, immature polychromatic erythrocytes (PCE) to mature normochromatic erythrocytes (NCE) is used to assess cytotoxicity in the micronucleus (MN) assay. The effects of cyclophosphamide (CP) on PCE/NCE ratio in rat bone marrow and spleen were assessed by a newly developed flow cytometric procedure using glutaraldehyde-fixed, acridine orange (AO)-stained cells, and compared to manual scoring of PCE/NCE in Wright stained slides. Comparison of methods showed that manual and flow cytometric determination of PCE were not statistically different. Several other parameters of cytotoxicity could be simultaneously assessed because the method allowed use of unfractionated whole bone marrow/spleen cell samples. Absolute numbers of total nucleated cells (TNC), a ratio of TNC to total erythrocytes (TE), and determination of RNA content within the PCE population demonstrated dose- and time-dependent effects with CP treatment. Shifts in RNA content were particularly sensitive, correctly identifying all CP-treated from control specimens, even in those samples where PCE/NCE ratio was similar. The AO methodology provided a more rapid, statistically-superior, and thorough approach in the assessment of bone marrow and spleen cytotoxicity than the conventional manual method of scoring PCE/NCE ratio alone.     

Use of flow cytometric CD34 analysis to quantify hematopoietic progenitor cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mononuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only 1%-9% of the CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA.(ABSTRACT TRUNCATED AT 250 WORDS)