Relevance of flow cytometric enumeration of post-thaw leucocytes: influence of temperature during cell staining on viable cell recovery.

BACKGROUND AND OBJECTIVES: Our post-thaw cell recovery rates differed substantially in interlaboratory comparisons of identical samples, potentially due to different temperatures during cell staining. MATERIALS AND METHODS: Viable CD34(+) cells and leucocyte (WBC) subtypes were quantified by multiparameter single-platform flow cytometry in leucapheresis products collected from 30 adult lymphoma and myeloma patients, and from 10 paediatric patients. After thawing, cells were prepared for analysis within 30 min between thawing and acquisition, at either 4°C or at room temperature. RESULTS: For cell products cryopreserved in conventional freezing medium (10% final DMSO), viable cell recovery was clearly lower after staining at 4°C than at RT. Of all WBC subtypes analysed, CD4(+) T cells showed the lowest median recovery of 4% (4°C) vs. 25% (RT), followed by CD3, CD34 and CD8 cells. The recovery was highest for CD3γδ cells with 44% (4°C) vs. 71% (RT). In the 10 samples cryopreserved in synthetic freezing medium (5% final DMSO), median recovery rates were 89% for viable CD34 (both at 4°C and RT) and 79% (4°C) vs 68% (RT) for WBC. CONCLUSIONS: The post-thaw environment and, potentially, the cryoprotectant impact the outcome of cell enumeration, and results from the analysis tube may not be representative of the cells infused into a patient.

Interleukin-3 promotes the expression of E-NPP3/CD203C on human blood basophils in healthy subjects and in patients with birch pollen allergy.

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is upregulated in response to FcepsilonRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC(50): 0.1-1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FcepsilonRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7.

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD. METHODS: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+) bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.

Characterization of distinct consecutive phases in non-genotoxic p53-induced apoptosis of Ewing tumor cells and the rate-limiting role of caspase 8.

To dissect the p53-dependent apoptotic pathway, events following induction of temperature sensitive (ts) p53val138 were studied in a Ewing tumor cell line. Transcriptional deregulation of p53 targets first observable after 1 h at 32 degrees C preceded activation of caspases and the break-down of mitochondrial respiratory activity. Activation of caspases was first observed 4 h after p53 induction. Using peptide inhibitors we identified activation of caspase 8 upstream of caspases-9 and -3. Although the caspase 8 specific inhibitor z-IETD.fmk did not affect translocation of BAX to the mitochondrial membrane and cytochrome C release it almost completely blocked cleavage of the prototype caspase substrate PARP and DNA fragmentation while enforcing mitochondrial depolarization and production of reactive oxygene species (ROS). Activation of caspase 8 did not involve death-domain receptor signaling. Expression of BCL2 only partially suppressed caspase activation but blocked apoptosis. Replacement of the N-terminus of p53val138 by the related VP16 transactivation domain created a ts p53 with a tanscriptional activity indistinguishable from p53val138 until the time of caspase activation. However, the VP16 - p53 fusion failed to trigger caspases and subsequent induction of the ROS producing gene pig3 paralleled by complete loss of apoptotic activity. These results indicate that p53-dependent transcriptional deregulation, triggering of the caspase cascade and the mitochondrial break-down occur in a timely ordered sequence coordinated by the genuine p53 amino terminus and suggest caspase 8 and PIG3 as key regulatory elements in this process. Oncogene (2000) 19, 4096 - 4107

CD99 expression in T-lineage ALL: implications for flow cytometric detection of minimal residual disease.

Expression of CD99 is higher on immature than on mature T cells. We postulated that this marker could be used to assess minimal residual disease (MRD) in T-lineage acute lymphoblastic leukemia (T-ALL). In diagnostic bone marrow (BM) samples from 27 children with T-ALL, expression of CD99 on leukemic lymphoblasts by flow cytometry was in median 7.7 times higher than on normal T lymphocytes from within the same sample. In 85% of cases, leukemic MFI values were higher than the mean MFI+2 s.d. of normal populations. We applied CD99 to study MRD in 39 follow-up samples from 15 consecutive T-ALL patients, and compared the results with those obtained with the well-established MRD-marker terminal deoxynucleotidyl transferase (TdT). Either antibody was combined in four-color flow cytometry with CD7, surfaceCD3, and cytoplasmicCD3. We found that CD99 was a valid complement to TdT in quantifying T-ALL MRD. Given a considerable interpatient variability, CD99 could be favorably used in nine patients, and TdT in other five patients. Both approaches showed a similar very low nonspecific background throughout 12 weeks from diagnosis (in median 0.002% of nucleated BM cells in patients with non-T ALL). We conclude that CD99 is a highly informative tool for MRD detection in T-ALL, bearing the advantage of surface expression in contrast to TdT.

Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow.

We studied the differentiation profiles of B cell precursors (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molecules, the stage-specific patterns were found to differ in certain details from previously published concepts. Among the earliest BCP, a subset of CD34+ CD10(lo) precursors was repeatedly observed in addition to the well characterized CD34+ CD10(hi) CD19+ majority of cells. Only two-thirds of these CD34+ CD10(lo) cells expressed CD19. However, uniformity of phenotypic features, absence of T lineage markers, and the regeneration kinetics after chemotherapy suggest the B lineage affiliation of the CD34+ CD10(lo) precursors in general. In the more mature BCP, expression of CD10, CD20, cytoplasmic and surface mu chains (c mu and s mu) was observed to overlap more than previously recognized. We found that CD20 and c mu appear early during B cell ontogeny (already on CD34+ BCP), and that CD10 is lost late, following the onset of s mu expression. Differences between normal and post-chemotherapy BM specimens regarding the phenotypic appearance of BCP were exclusively due to differences in the subset composition, as post-chemotherapy samples showed a preponderance of immature stages. Our observations may build a framework for comparing leukemic cells with their normal counterparts to define possible leukemia-associated aberrations useful for residual disease studies.

Kinetics of chimerism during the early post-transplant period in pediatric patients with malignant and non-malignant hematologic disorders: implications for timely detection of engraftment, graft failure and rejection.

The monitoring of chimerism by PCR has become a routine diagnostic approach in patients after allogeneic bone marrow or peripheral blood stem cell transplantation. Nevertheless, a temporal correlation between molecular and hematologic assessment of engraftment has not been clearly established. To address this issue, and to determine the potential clinical implications of early kinetics of mixed chimerism, we have investigated 66 allogeneic stem cell transplantations (SCTs) in 58 pediatric patients suffering from different types of leukemia (n = 44) or non-malignant hematologic disorders (n = 14) by close molecular monitoring during the first days and weeks after transplantation. Patient- and donor-derived hematopoiesis were assessed at 1- to 3-day intervals in peripheral blood samples by PCR analysis of highly polymorphic microsatellite loci (STR-PCR). Detection of an increasing, and ultimately dominant donor-specific allelic pattern, which we defined as molecular engraftment, preceded hematologic engraftment by a median of 7 days (range 1-17 days) in all patients investigated. PCR analyses during the first days after transplantation facilitated detection of molecular engraftment according to the above definition by day +14 (range day +2 to day +14), thus permitting prediction of successful engraftment (upper limit of the two-sided confidence interval po = 6%) while the peripheral leukocyte counts were mostly below 200/microl. In three cases, however, the criteria for molecular engraftment were not fulfilled by day +14. These patients also failed to show hematologic engraftment, and required a second transplantation. Close monitoring by STR-PCR showed that graft rejection and autologous recovery can occur early and with very rapid dynamics. Molecular analysis of specific leukocyte subsets isolated by flow-sorting enabled sensitive assessment of changes in the pattern of chimerism which had escaped detection in assays using whole white blood cell (WBC) samples. This approach facilitated the identification of expanding or decreasing recipient cells, and permitted early detection of impending rejection or relapse. Moreover, monitoring of the dynamics of chimerism allowed rapid assessment of the response to therapy. Our observations provide support for the concept of initiating genotype analyses early after SCT and monitoring at rather short intervals to permit timely evaluation of clinically relevant processes, and to provide a basis for early implementation of treatment.

Lineage-specific chimaerism after stem cell transplantation in children following reduced intensity conditioning: potential predictive value of NK cell chimaerism for late graft rejection.

Chimaerism of FACS-sorted leucocyte subsets (CD14+, CD15+, CD3-/56+, CD3+/4+, CD3+/8+, CD19+) was monitored prospectively between days +14 and +100 in 39 children undergoing allogeneic stem cell transplantation with reduced intensity-conditioning regimens. Cell subsets exceeding 1% of nucleated cells were subject to cell sorting. Chimaerism was analysed by dual-colour FISH and/or by short tandem repeat-polymerase chain reaction. The chimaerism pattern on day +28 was evaluated with regard to its correlation with graft rejection. Of 39 patients, nine patients had donor chimaerism (DC) in all subsets. Mixed/recipient chimaerism (MC/RC) was detectable within T cells in 62%, within NK cells in 39% and within monocytes and granulocytes in 38% of the patients. The correlation of secondary graft rejection with the chimaerism pattern on day +28 revealed the strongest association between RC in NK-cells (P<0.0001), followed by T cells (P=0.001), and granulocytes and monocytes (P=0.034). Notably, patients with RC in T cells rejected their graft only if MC or RC was also present in the NK-cell subset. By contrast, none of the children with DC in NK cells experienced a graft rejection. These observations suggest that, in the presence of recipient T-cell chimaerism, the chimaerism status in NK-cells on day +28 might be able to identify patients at high risk for late graft rejection.

CD34-positive cell proportions in peripheral blood correlate with colony-forming capacity.

Blood samples were examined from 25 children with malignancies during hematopoietic recovery following chemotherapy-induced aplasia and from 9 children undergoing tonsillectomy. The proportion of CD34-positive peripheral blood mononuclear cells (PBMNC) evaluated by flow cytometry was compared with the number of colonies (granulocyte-macrophage colony-forming units, CFU-GM; mixed-lineage colony-forming units, CFU-GEMM; and erythroid burst-forming units, BFU-E) grown in methylcellulose medium within 2 weeks. A mean of 1387 myeloid colonies (495-4480) per 10(5) PBMNC seeded developed from 13 samples with detectable CD34 populations (between 0.9% and 5.6%), whereas only 152 (9-386, p = 0.002) and 65 (12-137, p = 0.005) colonies were formed from 12 patient and from 9 control samples in which the percentage of CD34-positive cells was too low for analysis. Linear regression analysis revealed that CD34 positivity correlates with colony-forming capacity (p = 0.0008, r = 0.782). Flow cytometric evaluation of the CD34 proportions can thus predict the in vitro colony-forming capacity of peripheral blood prior to leukapheresis.

Detection of residual disease in pediatric B-cell precursor acute lymphoblastic leukemia by comparative phenotype mapping: a study of five cases controlled by genetic methods.

We recently investigated samples of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and normal bone marrow (BM). We found that leukemic blasts, compared to their physiologic counterpart cells, frequently display aberrant phenotypes with respect to levels of expression of certain antigens. Using multiparameter flow cytometry, these differences enabled us to trace leukemic cells admixed to normal BM, which suggested that this approach might be a useful strategy for minimal residual disease detection. In the present study, we used the same multiparameter approach ("comparative phenotype mapping") to prove that such quantitative phenotypic differences really exist between malignant and normal BCP when simultaneously present in the BM. We demonstrate this by five exemplary follow-up BM samples from patients with BCP-ALL, all of which showed phenotypically aberrant cells according to levels of expression of CD10, CD11a, CD19, CD34, CD44, or CD45RA, as well as according to altered orthogonal light scattering properties. We confirmed the leukemic nature of these cells by polymerase chain reaction-based detection of bcr1/abl transcripts, and of leukemia clone-specific immunoglobulin heavy chain rearrangements in only the suspicious cells when sorted by flow cytometry, but not in normal BCP or non-B cells. Comparative phenotype mapping thus allows one to distinguish between normal and leukemic cells, and we show that it may enable rapid, specific, and quantitative detection of residual/resurgent leukemia in BCP-ALL.

Comparative phenotype mapping of normal vs. malignant pediatric B-lymphopoiesis unveils leukemia-associated aberrations.

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.

SPECT study of visual fixation in schizophrenia and comparison subjects.

BACKGROUND: The consistent association of impaired eye movements and schizophrenia suggests a relationship between the neurobiology of the illness and visual pursuit systems. Visual fixation (VF), an eye "movement" task at zero velocity, is the simplest such abnormality in schizophrenia patients and their relatives. METHODS: We used a VF task for a functional imaging study. Six neuroleptic-free schizophrenia patients and eight gender and mean age matched comparison subjects had SPECT scans with 20 mCi of Tc99-HMPAO, during VF on a simple blue line intersection. MEDX data saved in ANALYZE format for SPM 95 was used to generate paired t-test statistical data for display in Talairach space, with rCBF changes given as Z-scores. RESULTS: Patients, compared to controls, had increased rCBF in both the parahippocampal gyrus (bilaterally) and in the right fusiform gyrus. They had decreased rCBF in the left frontal cortex, including medial and superior frontal gyri and anterior cingulate. Overall, compared to controls, patients had medial temporal lobe hyperperfusion along with left prefrontal hypoperfusion. CONCLUSIONS: These findings are consistent with the hypothesized imbalance between the medial temporal and frontal lobes that is postulated for schizophrenia. It was of interest that the relative rCBF differences between schizophrenia patients and controls in this small sample were observable with this cognitively non-demanding visual fixation task.

Elevation of CD5+ B lymphocytes in schizophrenia.

BACKGROUND: A variety of immunologic alterations have been observed in patients with schizophrenia. These findings have lent support to theories that autoimmune mechanisms may be important in some patients with the illness. The CD5+ B lymphocyte, a B-cell subset associated with autoimmune disease, has been the subject of two previously published studies yielding disparate results. METHODS: In this study, we used immunofluorescent flow cytometry to measure CD5+ B cells, total B and T cells, and CD4 and CD8 subsets in patients with schizophrenia and in normal control subjects. RESULTS: A significantly higher percentage of patients with schizophrenia, relative to normal control subjects, exhibited an elevated level of CD5+ B cells (27.6% vs 6.7%). Antipsychotic withdrawal had no effect on CD5+ B-cell levels, suggesting that medication effects were not the cause of this difference. No other studied lymphocyte subsets differed between the two groups. CONCLUSIONS: A subset of patients with schizophrenia have elevated levels of CD5+ B cells. This finding replicates an earlier study by another group and provides further evidence suggestive of autoimmune manifestations in schizophrenia.

Relation of familial schizophrenia to negative symptoms but not to the deficit syndrome.

OBJECTIVE: Although a family history of schizophrenia has been associated with negative symptoms, family history is inconsistently related to the presence of the deficit syndrome. METHOD: The authors assessed family history and the deficit syndrome in 99 patients with DSM-III-R-diagnosed schizophrenia who were assessed during clinical treatment. Of these 99 patients, 45 were assessed both while antipsychotic free and during antipsychotic treatment to index their treatment response. RESULTS: Patients with (N=39) and without (N=60) a family history of schizophrenia had similar proportions of the deficit syndrome. Yet family history and deficit syndrome categorizations identified a group with greater negative symptoms on the Positive and Negative Syndrome Scale. Those with a family history had greater emotional withdrawal, poor rapport, and lack of spontaneity. Groups with and without the deficit syndrome similarly differed in these symptoms but also in affective blunting, motor retardation, and passive or apathetic social withdrawal. The study involving antipsychotic-free and antipsychotic treatment phases showed main medication effects explaining positive, psychopathology, depression, and activation symptoms but not negative symptoms. Only patients without a family history had improved negative symptoms with antipsychotic treatment. CONCLUSIONS: Patients with a family history of schizophrenia had greater and more treatment-resistant negative symptoms than those without a family history. They were not more likely to have the deficit syndrome. The group with a family history had more pathology only in negative symptoms related to psychosocial function. The stable negative symptoms specifically related to the genetic vulnerability to inherit schizophrenia might be those associated with psychosocial functioning.

Functional maturation of dendritic cells by exposure to CD40L transgenic tumor cells, fibroblasts or keratinocytes.

Tumor antigen pulsed dendritic cells (DCs) can induce anti-tumor immunity. We studied strategies for the reliable generation of such a tumor vaccine by functional maturation of DCs via interaction of CD40 with its ligand (CD40L, CD154). Exposure of immature DCs to CD40L transgenic cells, soluble recombinant human CD40L molecules or lipopolysaccharide induced expression of the co-stimulatory molecules, CD80 and CD86, and supported an allogeneic mixed leukocyte reaction. In contrast, the release of IL-12, an important mediator of anti-tumor immunity, and antigen-specific expansion and IFNgamma secretion of lymphocytes, was strongly triggered only by DCs exposed to CD40L transgenic cells.

Isolation stress increases tyrosine hydroxylase mRNA in the locus coeruleus and midbrain and decreases proenkephalin mRNA in the striatum and nucleus accumbens.

Isolation of adult animals represents a form of psychological stress from which the animals cannot escape. In order to assess the effect of this stressor on neurochemical substrates in the brain, we assessed behavior and measured tyrosine hydroxylase and proenkephalin mRNA levels in selected brain areas by in situ hybridization histochemistry. Tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (LC) were significantly and progressively increased by 18, 42 and 68% after 7, 14 or 28 days of isolation, respectively. TH mRNA in the midbrain was transiently increased by isolation. Levels were significantly elevated by 34 and 48% above group-housed controls in the ventral tegmentum and the substantia nigra, respectively, after 14 days of isolation. In the forebrain, proenkephalin (PE) mRNA levels were found to be transiently decreased by 29% in the anterior and medial aspects of the caudate-putamen and the nucleus accumbens after 7 or 14 days of isolation stress, but the levels returned toward control levels after 28 days of isolation. Behavioral tests indicate that isolated animals progressively became more aggressive with duration of stress and showed a small but significant decrease in locomotor activity. The results demonstrate that a physically noninvasive stressor such as isolation of adult male rats can produce significant alterations in brain neurochemistry. The neurochemical responses observed may represent a brain mechanism designed to help the organism adapt to or protect from the deleterious effects of chronic psychological stress.

The composition of CD34 subpopulations differs between bone marrow, blood and cord blood.

Our previous data obtained by flow cytometry and by clonogenic assay had consistently shown a lower cloning efficiency of hematopoietic progenitor cells in bone marrow (BM) compared to those in blood (PB) or in cord blood (CB). Also, recent clinical reports have described more rapid reconstitution after PB than after BM transplantation. We have applied two- or three-color flow cytometric analysis using monoclonal antibodies directed against the stem- and progenitor cell antigen CD34, in combination with other cell surface markers. We report significant differences in the composition of progenitor cells contained in BM (238 specimens from 53 healthy donors and from patients in remission), PB (301 samples from 92 patients with or without cytokine support) and CB (n = 37). Leukapheresis products (Pher, n = 69) were included in the study as well as positively selected CD34+ cells obtained from BM (BMsel, n = 2), PB (PBsel, n = 28) and CB (CBsel, n = 5). We used monoclonal antibodies directed against CD7, CD19, CD34, CD38, CD45RA and glycophorin A. The highest proportion of CD34+ cells (in % of the MNC) was found in BM (mean 5.37% +/- 4.5). In the other sources, the mean values were 1.79% +/- 2.46 (PB), 1.48% +/- 1.81 (Pher) and 1.1% +/- 1.69 (CB). However, BM was the only source in which a considerable proportion of the CD34+ cells coexpressed the B cell antigen CD19 (mean 30.1%, median 28, range 0 to 84%). The amount of earlier myeloid progenitors as determined by their non-expression of the CD45RA antigen was lowest among BM CD34+ cells (26.7% +/- 16.6). In the other sources, the respective values were 57.5% +/- 17.9 (PB), 63.6% +/- 13.9 (Pher) and 70.4% +/- 16.1 (CB). These results were confirmed by subtype analyses of the CD34+ cells positively selected from the three sources. Enrichment showed minor CD34+ subpopulations to be identified. The mean proportions of B cell progenitors were 0.11% +/- 0.24 (PBsel) and 1.3% +/- 1.42 (CBsel) of the CD34+ cells. The CD34+ cells from all cell sources coexpressed GPA (median 0.15%, range 0 to 1.8%) and CD7 (median 0.25%, range 0 to 1.2%). The proportion of CD38- cells ranged from 0.7 to 4% of the CD34+ MNC. Thus, despite higher CD34 counts in BM, the relative proportions of myeloid progenitors are higher in PB and in CB. This suggests that, if timely reconstitution depends on the number of CD34+ cells transplanted, the mean number of "stem cells' (SC) required is 1.4-fold (for myeloid cells) or 2.2-fold (for earlier myeloid cells) higher for BM than for PB.

Characterization of hematopoietic stem cells.

On the basis of density-separated mononuclear cells isolated from bone marrow, peripheral blood, and cord blood, we have repeatedly shown good correlation between two-color flow cytometric (FACS) CD34 analysis and colony formation in the clonogenic assay. We have analyzed the distributions of CD34 subpopulations in these three stem cell sources using patients' and donors' bone marrow biopsies (n = 196), cord blood samples from full-term deliveries (n = 14), and peripheral blood from patients mobilized by chemotherapy and/or cytokine treatment (n = 258). Irrespective of absolute cell counts, the mean (+/- SD) proportions of CD34+ cells were clearly higher in bone marrow (5.6 +/- 4.6% of mononuclear cells) than in peripheral blood (1.9 +/- 2.6) and cord blood (1.7 +/- 2.6). However, two-color FACS analyses revealed significant differences among these cell sources with regard to their distribution of CD34 subpopulations: B-cell progenitors coexpressing CD34 and CD19, at considerable concentrations of > 0.5%, were only found in bone marrow (mean 30 +/- 24.3% of CD34+ mononuclear cells, median 28.7%, minimum 0%, maximum 83.3%). In addition, CD34+ cells in S/G2M phase were never detected in peripheral blood or cord blood, but only in bone marrow at a concentration of 10-15% of CD34+ mononuclear cells. On the other hand, the proportions of relatively immature myeloid progenitors, as characterized by not expressing CD45RA and by higher clonogenic capacity, were significantly higher in cord blood (76.7 +/- 17.2) and peripheral blood (58.2 +/- 17.5) than in bone marrow (26.4 +/- 16.7). These data were confirmed by analysis of apheresis products and of progenitors positively selected from different cell sources, and they may explain why, in autologous transplantations of analogous amounts of CD34+ cells, peripheral blood is superior to bone marrow. We conclude from our results that if successful transplantation and timely recovery depend on the number of CD34+ cells transplanted, the mean amount of stem cells required is 1.4- (for myeloid cells) or 2.2-fold (for early myeloid cells) higher for bone marrow than for peripheral blood.

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)

Spiking fevers with clozapine treatment.

Clozapine often causes low-grade fever and less frequently spiking fever. We describe three cases of spiking fever that occurred in the first 3 weeks of clozapine therapy. A new set of side effects of clozapine is identified, which includes spiking fever, respiratory and gastrointestinal symptoms, and neutrophilia. Possible mechanisms are discussed.