Using red cell phenotyping (RCP) and/or cytogenetics (CYT) we identified 19 patients with persisting mixed chimerism (MC) among 231 patients transplanted with partially T cell-depleted stem cell grafts from HLA-identical siblings. Persisting MC is defined as MC for more than 2 years in patients without any evidence of relapse. Median leukemia-free survival in these patients was 150 (range, 50-218) months. Diagnoses were ALL (n ‫؍‬ 10); AML (n ‫؍‬ 2); CML (n ‫؍‬ 2); NHL (n ‫؍‬ 2); MDS (n ‫؍‬ 1); MM (n ‫؍‬ 1) and SAA (n ‫؍‬ 1). Purpose of this study was the longterm follow-up of MC and definition of patterns of chimerism in the various subsets of PBMCs and granulocytes. Using a PCR-STR technique CD3 + /CD4 + (T4 lymphocytes), CD3 + /CD8 + (T8 lymphocytes), CD45 + /CD19 + (B lymphocytes), CD45 + /CD14 + (monocytes), CD45 + /CD15 + (granulocytes) and CD3 − /CD56 + (NKcells) were analyzed. The majority of patients with persisting MC were conditioned with a less intensive conditioning regimen and had little GVHD. Sequential monitoring of the chimerism resulted in a group of patients (n ‫؍‬ 7) with very slow transient mixed chimerism that resulted in complete DC after median 7 years. Another nine patients had a relatively high percentage of persisting autologous cells for a median of 12 years and in three patients we observed a stable low percentage of autologous cells. Only two out of 19 patients (AML-CR1, CML-CP1) relapsed during follow-up. Both patients had a relatively high percentage of autologous cells. Chimerism in granulocytes and PBMC subsets was analyzed at a median of 8 years after SCT in nine patients. In five patients mixed chimerism simultaneously detected by RCP and CYT was associated with MC in all subsets. Within each individual patient the percentages of donor and recipient cells were very different between the different subsets. Two CML-CP1 patients were mixed chimera in only two subsets and in one patient these subsets represented pending relapse. In another two patients mixed chimerism with a very low number of autologous red cells was not found in the PBMCs because of the different sensitivity level of the RCP and the PCR-STR technique. We conclude that in patients with persisting mixed chimerism after partially T cell-depleted SCT a remarkable number of patients had lymphoid malignancies, the majority of the patients were conditioned with less intensive conditioning regimens and the mixed chimerism was not correlated with relapse. Chimerism in granulocytes and PBMC subsets did show great intra-individual differences in the subsets and these data correlated well with RCP and CYT data with the exception of the NK cells. Leukemia (2002) 16, 13-21.

BackgroundTo study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin‐V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death.MethodsApoptosis was induced in Jurkat cells by γ‐irradiation, incubation with camptothecin (CPT), or cytosine β‐D‐arabinofuranoside (Ara‐C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze‐thaw procedure. Various AnV/PI− CD34+ fractions were cultured in a single‐cell single‐well (SCSW) assay.ResultsJurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI− cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80–90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI− fractions (overall range 23–62%). Within total AnV+ and AnV+/PI− populations, only a minority of CD34+ cells showed ISEL positivity (range 4–8% and 0.8–6%, respectively). Different fractions of AnV+/PI− CD34+ cells did have clonogenic capacity.ConclusionsPCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo‐nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV‐fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI− CD34+ cells in the SCSW assay. Cytometry 47:24–31, 2002. © 2001 Wiley‐Liss, Inc.

This paper describes the measurement of S phase DNA content in human bone marrow subpopulations using a single laser method for bivariate analysis of DNA content and cell‐surface immunofluorescence (s‐IF). Low density (<1.077 g/ml) bone marrow cells were labeled with a panel of unconjugated monoclonal antibodies (MoAb) for the lymphoid (CD2 + CD19), T‐lymphoid (CD2), B‐lymphoid (CD19), erythroid (anti‐glycophorin‐A), myelomonocytic (CD13, CD33; single and as cocktail) and monocytic (CD14) lineages. A fluorescein isothiocyanate (FITC)‐conjugated goat anti‐mouse label was used as second step. Unfixed, MoAb‐labeled cells were incubated for 24 h with a hypotonic propidium iodide solution for DNA staining. Cells were analysed on a single‐laser flow cytometer, operating at 488 nm. The effect of the combined staining protocol upon both s‐IF and DNA stainability was evaluated. Only a slight decrease (mean: 29.0%) in s‐IF intensity was observed after DNA staining. The percentages of immunofluorescent cells in the bone marrow samples of 10 normal individuals before and after DNA staining were essentially unchanged for all the MoAbs used. The DNA histograms of the immunophenotypically defined subpopulations were of excellent quality with a mean coefficient or variation of 1.8%. This procedure allows the assessment of very low levels of S‐phase DNA content, as measured in normal low density blood cells of 8 healthy volunteers (mean 0.07%). Considerable differences in percentages of cells with S‐phase content were seen between the four main bone marrow subpopulations (P<0.0001). The erythroid hematopoietic lineage showed the highest proportion of S‐phase DNA cells (26.4% ± 4.7%), the lymphoid lineage the lowest (2.5% ± 0.8%) while the monocytic lineage (6.1% ± 0.9%) and the myelomonocytic lineage (8.5% ± 1.4%) were in between. Within the lymphoid lineage the percentage of S‐phase cells was significantly lower in T‐cells (2.0% ± 0.7%) compared to B‐cells (5.1% ± 1.5%) (P<0.0001). We conclude that bivariate measurement of S‐phase DNA content and s‐IF is feasible with use of a single laser flow cytometer and that this approach provides important additional biological information. The value of this method to study hematologic malignancies is demonstrated in a bone marrow sample from a patient with erythroleukemia.

Summary.Pgp is expressed on normal haemopoietic progenitor cells. The significance of the efflux pump in protecting normal progenitors for anthracycline toxicity is not defined and is the subject of this study. Pgp was measured in CD34 þ progenitors with a rhodamine efflux assay. A high efflux, modulated by verapamil, was only found in a distinct subpopulation (20-30%). Pgp measured by the monoclonal antibody antibody (MoAb) MRK-16 was low in the rhodamine dull, but significantly (P < 0 . 04) higher than in the rhodamine bright cells. Reverse transcriptase polymerase chain reaction (RT-PCR) of MDR1 mRNA showed a very weak signal in both populations. In a single-cell clonogenic assay, rhodamine dull cells appeared less sensitive to anthracyclines (IC 50 daunorubicin 0 . 005 mg/ml; adriamycin 0 . 03 mg/ml) compared to rhodamine bright cells (IC 50 daunorubicin 0 . 0025 mg/ml; adriamycin 0 . 01 mg/ml). Furthermore, verapamil significantly (P < 0 . 05) potentiated anthracycline toxicity only in the rhodamine dull cells, proving its Pgp-specific modulating effect. Rhodamine dull cells gave larger and more mixed colonies compatible with a more primitive origin. Although detection with MoAbs and RT-PCR revealed a low Pgp level, functionally this Pgp appeared to be very important in protecting primitive progenitors against anthracycline toxicity. This protection can be jeopardized by administration of Pgp modulators.

Flow cytometric analysis of bone marrow aspirates and blood samples in 42 patients with chronic myelogenous leukemia (CML) at various disease stages was performed to determine the size of the S-phase compartment of bone marrow and blood. 25 healthy controls were studied for comparative information with both DNA-flow cytometry (DNA-FCM) and ³H-thymidine autoradiography. A correction procedure was applied for peripheral nucleated cell admixture in bone marrow aspirates. The fraction of peripheral nucleated cells in bone marrow aspirates (F_pb) in individual patients was considerable, especially in those with a very high white blood cell count ( > 100 × 10⁹/1). The size of the S-phase compartments of bone marrow (% S_bm) in patients with CML at diagnosis and in patients at apparent hematological remission was of the same order of magnitude as in normal bone marrow. However, in 3 out of 4 patients at malignant metamorphosis in which the % S_bm could be reliably determined, this percentage was significantly higher than normal (p = 0.013). In 4 out of 11 patients at malignant metamorphosis aneuploidy was noticed. From these findings it is concluded that bone marrow cell proliferation in CML patients at diagnosis and during apparent remission is not essentially different from normal. However, at malignant metamorphosis changes occur in ploidy level and proliferative activity, which can be detected by DNA-FCM already in an early phase.