Liquid Storage of Hematopoietic Stem Cells Versus Proliferative Potential Colony-Forming Unit Granulocyte-Monocytes: Validation of Cell Processing.

BACKGROUND: The material for transplantation must be of the highest quality. As far as we know, short-term storage is one of the crucial points of stem cell banking. According to the quality assurance system in a stem cell bank, each step of cell processing must be validated. The aim of this study was to assess the influence of short-term storage conditions into a clonogenic assay. METHODS: Material was collected from mobilized peripheral blood by means of leukapheresis from 15 patients. Samples were stored at 4°C and 20°C; samples were evaluated on the day of leukapheresis and after 24 hours and after 48 hours of storage. The number of colony-forming unit granulocyte-monocyte (CFU-GM) precursors was analyzed with the use of in vitro culture. The material was evaluated before freezing and after thawing. RESULTS: The average number of CFU-GM precursors in the material stored at 4°C before freezing on the day of collection was 84/10(5) nuclear cells (nc) and after 24 hours and 48 hours of storage was, respectively, 62/10(5) nc (P = .011719) and 36/10(5) nc (P = .02088). The average of the CFU-GM precursors in material stored at 20°C after 24 hours and 48 hours of storage amounted to 33/10(5) nc (P = .004439) and 2/10(5) nc (P = .00346), respectively. CONCLUSIONS: In our study, the number of colonies of CFU-GM after 24 hours and 48 hours of storage, both at 4°C and 20°C, was significantly reduced compared with the number of colonies on the day of collection. Significantly greater numbers of CFU-GM precursors were observed in the material stored before freezing at 4°C in comparison with the material stored at 20°C.

Banking of Hematopoietic Stem Cells: Influence of Storage Time on Their Quality Parameters.

BACKGROUND: Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. METHODS: Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen. The evaluation was performed after thawing the probes. Viability of nuclear cells was assessed after incubation in Trypan blue, CD34+ phenotype cells were determined by means of cytometry with the use of 7-aminoactinomycin D (7-AAD), and an analysis of the proliferative potential of granulocyte-monocyte precursors was performed. For comparative statistical analysis, the material was divided into 3 groups according to storage time: A: <1 month (n = 45); B: 1-12 months (n = 50); C: >12 months (n = 31). RESULTS: In the examined groups, similar median values were observed of nuclear cell viability (A, 86%; B, 87%; and C, 83%) and CD34+ cells (95%, 94.5%, and 95.8%, respectively). A gradual, nonsignificant, reduction in the median of granulocyte-monocyte precursors was found: 68 × 10(4)/kg of body weight (kg bw), 48.5 × 10(4)/kg bw, and 47 × 10(4)/kg bw, respectively. Statistical analysis with the use of the Kruskal-Wallis test showed a P value of >.05 for all variables. CONCLUSIONS: There were no significant differences in the viability of nuclear cells, CD34+ cells, and proliferative potential granulocyte-monocyte precursors between groups. Storage for up to 76 months does not change the essential quality parameters, and HSCs could be qualified for distribution.

Analysis of the effect of cryoprotectant medium composition to viability of autologous hematopoietic cells collected by leukapheresis.

OBJECTIVES: Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. MATERIAL AND METHODS: Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). RESULTS AND CONCLUSIONS: The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.

Microbial contamination of peripheral blood and bone marrow hematopoietic cell products and environmental contamination in a stem cell bank: a single-center report.

Hematopoietic stem cells (HSC) derived from peripheral blood (PB) and bone marrow (BM) are frequently used for autologous and allogenic transplantations. Establishing quality control at appropriate steps of the stem cell preparation process is crucial for a successful transplantation. Microbial contamination of haematopoietic stem cells is rare but could cause a potentially mortal complication of a stem cells transplantation. We investigated the microbiological contamination of PB (291 donations) and BM (39 donations) products. Microbial cultures of 330 donations between January 2012 and June 2013 were retrospectively analyzed after the collection and preparation steps. The microbiological analysis was performed with an automated system. Hematopoietic stem cells were processed in a closed system. Additionally, in this report the environment of the working areas of stem cell preparation was monitored. We analyzed microbial contamination of the air in a class I laminar air flow clean bench at the time of preparation and in the laboratory once per month. We reported 9 (2.73%) contaminated HSC products. The most frequent bacteria isolated from PB and BM products were Bacillus species. Coagulase-negative staphylococci and Micrococcus species were the most frequent micro-organisms detected in the air microbial control. Microbial control results are necessary for the safety of hematopoietic stem cell products transplantation. Microbial control of hematopoietic stem cell products enables an early contamination detection and allows for knowledgeable decision making concerning either discarding the contaminated product or introducing an efficient antibiotic therapy. Each step of cell processing may cause a bacterial contamination. A minimum of manipulation steps is crucial for increasing the microbial purity of the transplant material. Also, the air contamination control is essential to ensure the highest quality standards of HSC products preparation.

Factors affecting neutrophil recovery after autologous bone marrow-derived stem cell transplantation in patients with acute myeloid leukemia.

Bone marrow is currently regarded as the most appropriate source of stem cells for patients with acute myeloid leukemia (AML) undergoing autologous transplants. A total of 55 adult patients with AML in first complete remission receiving autologous bone marrow-derived stem cell (BMSC) transplantation (BMSCT) were analyzed to determine factors affecting the rate of neutrophil recovery. All patients were treated with standard induction and three to four courses of consolidation chemotherapy and, after collection of BMSC, conditioned with BuCy2. The median time to neutrophil reconstitution was 30 (10-62) days and was delayed in 24 patients. Neutrophil recovery was faster in patients who had received granulocyte-macrophage progenitors (CFU-GM) dose >23.5 x 10(4)/kg, CD34(+) cells >3.2 x 10(6)/kg, and mononuclear cells (MNCs) >3 x 10(8)/kg. The speed of neutrophil recovery correlated with the number of transplanted CFU-GM progenitors (P = .0077) and MNCs (P = .0015). CFU-GM progenitors dose was the only factor close to significance in univariate analysis of neutrophil engraftment. Probability for neutrophil recovery was higher in patients transplanted with a higher dose of MNCs. These data suggested that the content of CFU-GM progenitors and MNCs within the bone marrow graft was the most important factor for the quality of neutrophil recovery after autologous BMSCT in AML patients.

Increased risk for invasive aspergillosis in patients with lymphoproliferative diseases after autologous hematopoietic SCT.

The risk of invasive aspergillosis (IA) is considered to be low among autologous HSCT recipients, but an increase in the incidence has been observed recently in this setting. The aim of the study was to assess the influence of immunosuppressive drugs (steroids, rituximab, fludarabine, thalidomide), used in treatment of lymphoid malignancies during 6 months of pretransplant period, on IA incidence after autologous HSCT. A total of 109 patients with non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD) and multiple myeloma (MM), conditioned with carmustine, etoposide, cytarabine, melphalan or melphalan and transplanted with PBSC, were analyzed prospectively. Patients were monitored with twice-weekly galactomannan test. High-resolution computed tomograhy of the chest and bronchoscopy were performed in case of positive galactomanan test, persistent fever or pulmonary infiltrates. Documented IA was diagnosed in nine (8%) patients (three proven, six probable). The incidence of IA was comparable in NHL, HD and MM patients and not influenced by age, advanced disease or conditioning regimen. Factors significant for development of documented IA by univariate analysis were treatment with fludarabine (P=0.008) or rituximab (P=0.039). The only factor predicting documented IA by multivariate analysis was treatment with fludarabine (P=0.008). Patients treated with fludarabine or rituximab in pretransplant period are at risk of IA and require close monitoring and/or anti-mould prophylaxis.

[Effect of 2-chlorodeoxyadenosine, arabinoside cytosine and doxorubicin on acute myeloid leukemia clonogenic cell proliferation]. / Wplyw 2-chlorodeoksyadenozyny, arabinozydu cytozyny i doksorubicyny na wzrost komórek klonogennych ostrych bialaczek szpikowych.

We examined the influence of 2-chlorodeoxyadenosine (2-CDA) in concentration 300 nM/l alone and in combination with cytosine arabinoside (ara-C; 10(-7) M/l and doxorubicine (drb; 1 microgram/ml) on the proliferation of acute myeloid leukemia (AML) clonogenic cells (L-CFU) in agar/liquid culture system. After preincubation of blast cells with 2-CDA or ara-C as a single agent the number of colonies was reduced, reaching 70% of control (p < 0.05). Exposure of AML blasts to drb resulted in a greater reduction of L-CFU proliferation than in experiments with 2-CDA or ara-C alone (p < 0.05). Coadministration of 2-CDA in conjunction with ara-C or drb, and additionally with ara-C and drb caused the inhibition of L-CFU growth in comparison with control experiments by 52%, 78% and 61%, respectively (p < 0.05). In further experiments we studied the effect of ara-C and drb together with 2-CDA in different concentrations (20, 100 or 300 nM/l). After preincubation with 2-CDA in concentration 20 nM/l no change in blast colony formation was observed in relation to controls which comprised ara-C and drb only (p < 0.05). The increase of 2-CDA concentration to 100 or 300 nM/l in combination with ara-C and drb significantly reduced the growth of AML clonogenic cells (p < 0.05). The greatest, 90%, inhibition of L-CFU proliferation was observed after the exposure of blast cells to ara-C, drb and 2-CDA in concentration 300 nM/l (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytokines modify 2-chlorodeoxyadenosine (2-CdA) toxicity in acute myeloid leukemia clonogenic cells (L-CFU)]. / Cytokiny modyfikuja toksycznosc 2-chlorodeoksyadenozyny (2-CdA) wobec komórek klonogennych ostrych bialaczek szpikowych (L-CFU).

We investigated the effect of 2-chlorodeoxyadenosine (2-CdA) in concentrations: 10, 20, 50, 100, 150 and 300 nM/l on the clonal growth of acute myeloid leukemia clonogenic cells (L-CFU), after 24 h preincubation with recombinant granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha) or interleukin 3 (IL-3) and in conditioned medium obtained from phytohemagglutin stimulated lymphocytes (PHA-LCM). 2-CdA in concentration 300 nM/l significantly reduced (30%) the number of blast colonies in comparison with control experiments (p < 0.05). After preincubation of blast cells with cytokines (20% PHA-LCM, or GM-CSF--100 ng/ml, or IL-3--5 U/ml) an increase of 2-CdA cytotoxic effect on L-CFU clonal growth was observed. In experiments with 2-CdA (300 nM/l) and GM-CSF or PHA-LCM or IL-3 the number of blast colonies was reduced in 70%, 50% and 50%, respectively, (p < 0.05). Preincubation of blasts with IL-1, did not effect the 2-CdA--induced growth inhibition of CFU.

Activated lymphocytes in the marrow cell suspension decrease the mafosfamide-induced CFU-GM cytotoxicity.

The aim of the study was to assess whether other cells, besides erythrocytes, may influence the cytotoxic effect of mafosfamide (maf) during ex vivo bone marrow purging from residual tumor cells before autologous transplantation. It was shown that the presence of normal granulocytes, blast cells from acute myeloid leukemia-patients (AML) and lymphoma cells from patients with chronic lymphocytic leukemia (CLL) during maf incubation did not change the maf-induced growth inhibition of CFU-GM. Similar observation was made in experiments with resting lymphocytes. However, when phytohaemagglutinin- and pokeweed mitogen-preincubated lymphocytes were present in the marrow cell suspension, significant decline of the maf-related CFU-GM cytotoxicity was observed. These results suggest that besides erythrocytes also the activated lymphocytes in the marrow mononuclear suspension may change the final effect of maf purging.

[T lymphocytes from patients with Hodgkin's disease suppress erythroid progenitor growth from autologous marrow]. / Limfocyty T chorych na ziarnice zlosliwa hamuja wzrost klonalny prekursorów erytroidalanych szpiku autologicznego.

The effect of peripheral blood T lymphocytes from 42 patients with advanced Hodgkin's disease (grade III and IV) on the autologous marrow erythroid colony formation was studied in diffusion chamber culture. It was found, that unfractionated T lymphocytes suppress the BFU-E--(burst forming unit erythroid) and CFU-E--(colony forming unit erythroid)--derived colony formation by releasing an inhibitory activity. The suppression of colony formation was noted already at 0.25 x 10(5) cell concentration. In experiments with 0.5 x 10(5) and 1.0 x 10(5) T cells the inhibitory effect was increased. Subsequently it was shown, that this inhibition was generated by radioresistant, CD8+ and HLA-DR- subset of T cells. In control experiments, T lymphocytes from healthy subjects had no influence on the erythroid colony formation.

[Production of activity stimulating an increase of clonal granulocytic precursors by normal B lymphocytes under the influence of various mitogens]. / Wytwarzanie aktywnosci pobudzajacej wzrost klonalny prekursorów granulocytarnych przez limfocyty B, poddane dzialaniu róznych mitogenów.

The effect of mitogen-stimulated (concanavalin A, Con A; phytohemagglutinin, PHA; pokeweed mitogen, PWM; Staphylococcus aureus Cowan I, SAC I) normal B lymphocytes on the clonal proliferation of granulocytic progenitors from marrow of healthy subjects (CFU-dG) was studied in diffusion chamber culture. PWM-, SAC- and Con A-stimulated B lymphocytes produced an humoral activity that increased the CFU-dG-derived colony formation. The highest growth-stimulating effect was induced by SAC I-preincubated B lymphocytes and to a lesser degree by PWM- or Con A-stimulated B cells. In contrast, PHA-preincubated and unstimulated B lymphocytes revealed no effect on the CFU-dG proliferation.

Interferon-gamma-mediated suppression of erythroid progenitor growth by a HLA-DR- and CD4-positive subset of T lymphocytes in acute myeloid leukemia.

In this study, we examined the effects of peripheral blood T lymphocytes from patients with acute myeloid leukemia (AML) on marrow-derived erythroid progenitors (BFU-DE and CFU-DE) growth in an in vivo culture by using the plasma clot diffusion chamber (DC) technique. The application of double-compartment chambers (each compartment separated by a membrane filter) makes the investigations of humoral effects of T lymphocytes upon marrow erythroid progenitors proliferation possible. T lymphocytes of AML-patients in the absence of a statistically significant number of monocytes suppressed the growth of BFU-DE and CFU-DE from T lymphocyte- and adherent cell-depleted marrows. The inhibition ability was restricted to the CD4-positive enriched fraction obtained from T cells by using the negative selection technique. In contrast, the CD8-positive enriched fraction had no effect on erythroid colony formation. Autologous and allogeneic BFU-DE and CFU-DE were similarly affected by the CD4-positive T cells. Treatment of T cells with monoclonal antibodies against HLA-DR before cocultures, completely abrogated the suppression of BFU-DE and CFU-DE-derived colony formation. Suppressive activity detected in the CD4-positive T cells was also totally abolished by treatment with anti-interferon-gamma antibodies; whereas the inhibition was retained after 30 Gy radiation. Under these experimental conditions, resting T lymphocytes from healthy subjects did not affect the erythroid colony formation. Our data show that in AML-patients, a circulating HLA-DR-positive, less radiosensitive subset within the CD4-positive T cells is capable of inducing an interferon-gamma-mediated suppression of erythropoiesis, at least in DC culture.

[Clonal proliferation of less mature granulocytic precursors (CFU-dG) under the influence of neoplastic B lymphocytes in low grade non-hodgkin's lymphomas]. / Proliferacja klonalna mniej dojrzalych prekursorów ukladu granulocytarnego (k.u.-dG) pod wplywem nowotworowych limfocytów B chloniaków nie-ziarniczych o niskim stopniu zlosliwosci.

The effect of peripheral blood lymphoma B cells from low grade malignancy non-Hodgkin's lymphoma patients (Kiel classification: lymphocytic, centrocytic, centroblastic-centrocytic lymphomas) on clonal growth of normal marrow granulocytic precursors (CFU-dG) was studied. Using in vivo diffusion chamber culture method it was shown that all the tested lymphoma cells enhanced the CFU-dG growth by releasing humoral factor(s). The effect of stimulation was cell-concentration and cell-source dependent. The colony stimulating activity produced by centrocytic lymphoma cells had higher potency than that released by centroblastic-centrocytic lymphoma cells, whereas lymphocytic lymphoma cells showed the lowest ability to generate the CFU-dG growth-promoting activity (p < 0.01). It was shown that the CFU-dG growth enhancing effect was independent of marrow-derived macrophages and T lymphocytes (p > 0.05). Normal peripheral blood B lymphocytes used as a control did not show any substantial effect on the granulocytic precursors proliferation.

[Effect of bloodletting on the proliferative activity of erythroid progenitor cells from the bone marrow of patients with polycythemia and polycythemia vera]. / Wplyw upustów krwi na aktywnosc proliferacyjna prekursorów erytroidalnych szpiku chorych na czerwienice prawdziwa i objawowa.

We assessed the effect of phlebotomy on the proliferative activity of less (BFU-E) and more (CFU-E) mature bone marrow-derived erythroid progenitors from patients with polycythemia vera (PV) and polycythemia symptomatic (PS) in vivo in diffusion chamber culture. The cloning efficiency of erythroid progenitors under the effect of normal and increased erythropoietin (Epo) concentrations in PS was comparable with controls, whereas in PV the BFU-E and CFU-E-derived colony formation was significantly higher (p less than 0.01). In PV, the erythroid progenitors formed markedly more colonies in cultures stimulated with higher Epo concentration (p less than 0.01). The results of this study indicate that phlebotomy both in PV and PS does not affect the reactivity of erythroid progenitors to various Epo concentrations.

[Capacity of erythroid progenitor cells of the human bone marrow for clonal growth in diffuse chamber culture]. / Zdolnosc wzrostu klonalnego komórek ukierunkowanych szeregu erytroidalnego szpiku ludzkiego w hodowli w komorach dyfuzyjnych.

The ability of human marrow erythroid progenitors growth in diffusion chamber culture has been assessed. Chambers containing cells (2 X 10(5)) suspended in clot were implanted into peritoneal cavity of anemic rats. The anaemia was induced by phenylohydrazine (50 mg/kg body weigh). Erythroid colonies were counted on day 1, 2, 3, 5, 7, 9, 11, 13 and 15. Two types of colonies formed by erythroid progenitors (BFU-D-E, CFU-D-E) were determined using criteria of colony size and culture duration. The count of colonies formed by CFU-D-E was highest on day 2 (29 +/- 4) and underwent reduction in subsequent days. Colonies formed by BFU-D-E were seen beginning of day 5 and their number peaked on day 7 and 9. These results indicate that the best time for determining the growth of human marrow CFU-D-E and BFU-D-E in diffusion chamber culture is on day 2 and day 7-9, respectively.

Human normal B lymphocytes produce a growth-promoting activity for granulocyte progenitors in diffusion chamber culture after stimulation with pokeweed mitogen.

We have measured the effect of normal B lymphocytes on more primitive granulocyte progenitors (CFU-dG) clonal growth in double-diffusion chamber culture in vivo. It was found that pokeweed mitogen (PWM) stimulated B cells produce a growth-promoting activity which augments the CFU-dG--derived myeloid colony formation in a dose-dependent fashion. All colonies formed under the experimental conditions were composed exclusively of granulocytes at different stages of maturation. Unstimulated B lymphocytes did not effect the CFU-dG clonal proliferation.

[Proliferative activity of granulocyte progenitors from the bone marrow of patients with chronic myeloid leukemia in 14-day culture in vivo]. / Aktywnosc proliferacyjna komórek ukierunkowanych szeregu granulocytarnego szpiku chorych na przewlekla bialaczke szpikowa w 14-dniowej hodowli in vivo.

The proliferative activity of marrow granulocyte progenitors (CFU-dG) from patients with chronic myeloid leukemia (CML) and healthy subjects was investigated in 14th-day culture using diffusion chamber technique in vivo. On each day the number of clusters (20-50 cells) and colonies (over 50 cells) was estimated. In addition, on the day 4, 7 and 9 the count of CFU-dG in the S-phase of cell cycle was determined. During the first 9 days the number of clusters and colonies successively increased both in CML and control, but this result was more pronounced in experiments with CML-CFU-dG. In the period between days 9 th and 14th a decrease in the proliferative activity of CFU-dG could be observed. During the whole culture-time the number of colonies was markedly higher than that of clusters. The percentage of CML-CFU-dG in S-phase was comparable to that noted in controls.

Usefulness of the assessment of erythroid progenitors growth for differential diagnosis of primary and secondary polycythemias.

Erythropoietic progenitors from bone marrow of patients with polycythemia vera (PV), secondary polycythemia (SP) and healthy subjects (HS) were cultured in plasma clot diffusion chambers in vivo. The chambers were inserted into the peritoneal cavities of rats, which 24 and 2 h before implantation received an injection of phenylohydrazine. Control experiments were done without erythropoietin (Epo) stimulation. Colonies after 2 and 7 days of culture were considered to be formed by mature erythropoietic progenitors (CFU-D-E) and burst forming cells (BFU-D-E), respectively. PV-erythroid progenitors, both BFU-D-E and CFU-D-E produced markedly more colonies than those from SP and HS, especially in experiments without Epo stimulation (p less than 0.01). The plating efficiency in SP was comparable to that noted in HS (p greater than 0.05). These results have led us to postulate that the study of erythroid progenitor clonal proliferation in plasma clot diffusion chamber can be helpful in the differential diagnosis of PV and SP, when other clinical and laboratory findings are not sufficiently convincing.