A comparative study on culture conditions and routine expansion of amniotic fluid-derived mesenchymal progenitor cells.

BACKGROUND: Amniotic fluid (AF) cell populations will be applied in perinatology. We aimed to test the feasibility of large-scale cell expansion. STUDY METHODS: We determined the best out of three published expansion protocols for mesenchymal progenitors (AF samples, n = 4) in terms of self-renewal ability. Characterization was performed based on morphology, surface marker analysis, cytogenetic stability, and differentiation potential. The conditions for the best self-renewal ability were further determined in a consecutive series (n = 159). RESULTS: The medium containing fetal bovine serum (FBS), epidermal growth factor, insulin, transferrin, and tri-iodothyronine, combined with seeding on gelatin-coated wells, best stimulated the growth of cells with mesenchymal features, as demonstrated by flow cytometry; however, only osteogenic differentiation was possible. Large-scale testing (n = 44) failed to confirm a robust self-renewal ability. Better results were obtained (n = 88) using optimized FBS or an increased initial cell density. Eventually over 81% of cultures continued growing after the initial medium change and had mesenchymal features but failed differentiation assays. DISCUSSION: Routine in vitro expansion of AF-derived mesenchymal cells remains problematic. Despite an increase in successful cell cultures from 40 up to 80% using optimized serum and an increased cell density, eventually cells failed to demonstrate differentiation abilities. Routine isolation and expansion from unselected AF samples remains a challenge.

CD34+ marrow progenitors from MDS patients with high levels of intramedullary apoptosis have reduced expression of alpha4beta1 and alpha5beta1 integrins.

Excessive intramedullary apoptosis is central in the pathogenesis of myelodysplastic syndromes (MDS). Growth-inhibiting cytokines, the Fas/FasLigand pathway, and autoreactive cytotoxic T-lymphocytes have been identified to be important proapoptotic factors in MDS. In normal hematopoiesis, alpha4beta1 and alpha5beta1 integrin-mediated interactions between progenitors and fibronectin are critical for progenitor cell survival. In this study, we have used flow cytometry to quantify the expression levels of members of the beta1 integrin family on CD34(+) marrow progenitors in 27 untreated patients with MDS, three with s-AML, and 25 control subjects. In MDS, we observed that nonapoptotic progenitors significantly downregulate cell surface expression levels of alpha4 and beta1 integrin chains compared with healthy controls. Downregulation of alpha4, beta1, and also alpha5 was present in MDS patients with > or =25% apoptotic progenitors, irrespective of their French, American, British subcategory. Reduced cell surface expression levels of alpha4, alpha5, and beta1 did also correlate with decreased in vitro adhesiveness to fibronectin fragments. Therefore, our observations suggest that downregulation of alpha4beta1 and alpha5beta1 integrins on CD34(+) progenitors could be a newly identified proapoptotic mechanism in MDS.

Circulating myeloid and lymphoid precursor dendritic cells are clonally involved in myelodysplastic syndromes.

Circulating myeloid and lymphoid precursor dendritic cell (pDC) counts were determined in peripheral blood from 22 patients with myelodysplastic syndromes (MDS) by a single-platform flow cytometric protocol. The absolute count of myeloid and lymphoid pDC, as well as their relative number (as proportion of mononuclear cells or total leukocytes) was significantly lower in MDS (n=22) than in healthy controls (n=41). In 11 patients with chromosomal aberrations, purified pDC were examined by interphase fluorescence in situ hybridization. This revealed clonal involvement of myeloid as well as lymphoid pDC in all of them. These data therefore strongly suggest that myeloid and lymphoid pDC share a common precursor. Whether reduced peripheral blood counts of pDC contribute to the immunological abnormalities observed in MDS remains to be investigated.

Patients with high-risk myelodysplastic syndrome can have polyclonal or clonal haemopoiesis in complete haematological remission.

The clonality of mature peripheral blood-derived myeloid and lymphoid cells and bone marrow haemopoietic progenitors from 18 females with myelodysplasia (MDS) (five refractory anaemia, RA; one RA with ringed sideroblasts, RARS; three chronic myelomonocytic leukaemia, CMML; four RA with excess of blasts, RAEB; five RAEB in transformation, RAEB-t) was studied by X-chromosome inactivation analysis. Using the human androgen-receptor (HUMARA) assay, we analysed the clonal patterns of highly purified immature CD34+ 38- and committed CD34+ 38+ marrow-derived progenitors, and CD16+ 14- granulocytes, CD14+ monocytes, CD3+ T and CD19+ B lymphocytes from peripheral blood. In high-risk patients (RAEB, RAEB-t), clonality analysis was performed before and after intensive remission-induction treatment. All patients, except one with RA, had predominance of a single clone in their granulocytes and monocytes. The same clonal pattern was found in CD34+ progenitor cells. In contrast, CD3+ T lymphocytes were polyclonal or oligoclonal in 14/18 patients. X-chromosome inactivation patterns of CD19+ B cells were highly concordant with CD3+ T cells except for two patients (one RA, one CMML) with monoclonal B and polyclonal T lymphocytes, therefore suggesting a clonal mutation in a progenitor common to the myeloid and B-lymphoid lineages or the coexistence of MDS and a B-cell disorder in these particular patients. After high-dose non-myeloablative chemotherapy, polyclonal haemopoiesis was reinstalled in the mature myeloid cells and immature and committed marrow progenitors in three of four patients achieving complete haematological remission. Therefore we conclude that most haematological remissions in MDS are associated with restoration of polyclonal haemopoiesis.

Polyclonal primitive hematopoietic progenitors can be detected in mobilized peripheral blood from patients with high-risk myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) form a heterogeneous group of clonal hematopoietic disorders with unfavourable prognosis. Allogeneic bone marrow transplantation is the only potentially curative treatment, but remains limited to a small subgroup of younger patients with HLA-compatible donors. As autologous stem cell transplantation is currently being explored as an alternative treatment strategy for MDS, more information needs to be acquired regarding the clonal nature of the progenitor cells in these autografts. Therefore, we have analyzed the clonal patterns of highly purified hematopoietic progenitors and their mature daughter cells in mobilized peripheral blood collections produced from five female patients with high-risk MDS in complete hematologic remission. X-chromosome activation patterns of flow-sorted immature (CD34 + 38low, CD34 + 33low) and committed (CD34 + 38high, CD34 + 33high) progenitors were studied with the polymerase chain reaction-based HUMARA assay. In four patients, a polyclonal remission was shown in all stem cell subpopulations and their mature daughter cells whereas one patient was found to remain skewed in all fractions, except T lymphocytes. This study provides strong evidence that polyclonal immature hematopoietic progenitors can be mobilized and harvested in patients high-risk MDS after treatment with high-dose chemotherapy.

Osmotic effects of dilution on erythrocytes after freezing and thawing in glycerol-containing buffer.

Red blood cells frozen in 1.7 M and particularly in 2.2 M of glycerol retain a high degree of integrity upon thawing as long as the dilution procedure of the cryoprotectant is slow and preferentially compensated by the addition of sorbitol. As the nonpenetrating cryoprotectant sorbitol induces initial cell shrinkage, cell swelling upon dilution of the cryoprotectants may not lead to hemolysis. However, rapid dilution of glycerol even with buffer containing up to 0.50 M of sorbitol cannot be achieved without provoking considerable hemolysis. Due to the relative slow rate at which glycerol leaves the cells, membrane damage to the younger cell populations remains considerable and is even more pronounced in the older cell groups. The dramatic osmotic changes occurring during the dilution process lead to the formation of aberrant cell populations as demonstrated by the red cell size frequency distribution curves.

Comparative evaluation of three flow cytometers for reticulocyte enumeration.

Flow cytometric determination of reticulocytosis is progressively replacing the manual microscopic method. We evaluated three flow cytometers (Becton Dickinson FACScan, Coulter EPICS Profile II, Ortho Cytoron Absolute) for reticulocyte enumeration, using thiazole orange. For each sample, 30,000 cells were analysed. In order to comparatively evaluate the three instruments, reticulocytes were counted by manually gating the erythroid population and evaluating the gated population for fluorescence characteristics. The different instruments showed good linearity and precision. No carry-over was observed. Orthogonal regression analysis of reticulocyte counts of 100 healthy blood donor samples and 108 haematological patient samples showed a good mutual comparability between all three instruments tested, although the paired t-test showed a significant difference between the Cytoron and both the FACScan and the Profile. Despite minor statistical differences, the three instruments can be considered equivalent for daily routine reticulocyte enumeration.

The prevention of erythrocyte swelling upon dilution after freezing and thawing.

Cellular swelling of erythrocytes exposed to Me2SO during freezing and thawing may lead to hemolysis upon dilution of the cryoprotectant with pure electrolyte buffer. Excessive cell swelling is effectively avoided by exposing the RBC to the nonpenetrating sorbitol after thawing and before dilution. Due to the initial reduction in volume by sorbitol, cell swelling upon dilution may not cause hemolysis particularly with concentrations of 0.05 to 0.15 M of sorbitol in the diluting electrolyte buffer. Membrane damage incurred during freezing and thawing is particularly pronounced with the older red cell population, while the younger population membrane integrity can be preserved to an optimal degree.

K2- or K3-EDTA: the anticoagulant of choice in routine haematology?

The choice of K2- or K3-EDTA as the preferred anticoagulant for blood count remains controversial. We compared the effect of different concentrations of both anticoagulants on normal blood. In optimal conditions (appropriate anticoagulant concentration and measurements done between 1 and 4 h after phlebotomy), no marked differences are seen between either EDTA salt. Important discrepancies appear, however, in less optimal conditions, as often happens in day to day practice. The packed cell volume, when measured on centrifuged blood, decreases with increasing anticoagulant concentrations and this is most pronounced with the K3 salt. This phenomenon has been reported by different authors and is ascribed to shrinking of erythrocytes in an hypertonic medium. Automated instruments react in a different way, their MCV is not influenced by K3-EDTA concentrations up to ten times normal, while K2-EDTA, at high concentrations, results in a slight increase in MCV, as measured with three of the instruments. With most instruments, the accuracy of the white cell count is not markedly influenced. However, when measured with the Unipath CD 3000 all tested blood samples, taken in high K3 (but not in K2) concentrations, showed an appreciable decrease in the leucocyte count (to less than 50% of the original value, at a concentration of 15 g/l, 24 h after blood collection). Measurement of RDW and automated differentials is also influenced by the choice of anticoagulant when determinations are done in less than optimal conditions. We conclude that the choice of anticoagulant, its use at an appropriate concentration and the age of the blood sample are important matters and should be given due consideration.(ABSTRACT TRUNCATED AT 250 WORDS)

Reticulocyte count using thiazole orange. A flow cytometry method.

Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.

Problems related to CPD preserved blood used for NEQAS trials in haematology.

A study of the results of the Belgian National External Quality Assessment Scheme (NEQAS) in haematology, revealed marked differences in MCV values obtained with instruments of different types. For this reason the citrate-phosphate-dextrose (CPD) preserved blood used as NEQAS material was analysed daily with a Coulter S-Plus IV and with a cross-calibrated Ortho ELT-800. The observed differences in MCV are apparently due to differences in measuring apparatus and also to the changing characteristics of the erythrocytes. Ageing of CPD blood during 1 to 4 days (postal delivery) influenced markedly the MCV as measured with the Ortho ELT systems. These time- and instrument-related factors influence the measurements, they interfere with the statistical evaluation of NEQAS results and can lead to errors in the evaluation of individual performers.