Mirasol PRT system inactivation efficacy evaluated in platelet concentrates by bacteria-contamination model

Background/Aim. Bacterial contamination of blood components, primarily platelet concentrates (PCs), has been identified as one of the most frequent infectious complications in transfusion practice. PC units have a high risk for bacterial growth/multiplication due to their storage at ambient temperature (20 ± 2°C). Consequences of blood contamination could be effectively prevented or reduced by pathogen inactivation systems. The aim of this study was to determine the Mirasol pathogen reduction technology (PRT) system efficacy in PCs using an artificial bacteria-contamination model. Methods. According to the ABO blood groups, PC units (n = 216) were pooled into 54 pools (PC-Ps). PC-Ps were divided into three equal groups, with 18 units in each, designed for an artificial bacteria-contamination. Briefly, PC-Ps were contaminated by Staphylococcus epidermidis, Staphylococcus aureus or Escherichia coli in concentrations 102 to 107 colony forming units (CFU) per unit. Afterward, PC-Ps were underwent to inactivation by Mirasol PRT system, using UV (l = 265-370 nm) activated riboflavin (RB). All PC-Ps were assayed by BacT/Alert Microbial Detection System for CFU quantification before and after the Mirasol treatment. Samples from non-inactivated PC-P units were tested after preparation and immediately following bacterial contamination. Samples from Mirasol treated units were quantified for CFUs one hour, 3 days and 5 days after inactivation. Results. A complete inactivation of all bacteria species was obtained at CFU concentrations of 102 and 103 per PC-P unit through storage/ investigation period. The most effective inactivation (105 CFU per PC-P unit) was obtained in Escherichia coli setting. Contrary, inactivation of all the three tested bacteria species was unworkable in concentrations of ≥ 106 CFU per PC-P unit. Conclusion. Efficient inactivation of investigated bacteria types with a significant CFU depletion in PC-P units was obtained - 3 Log for all three tested species, and 5 Log for Escherichia coli. The safety of blood component therapy, primarily the clinical use of PCs can be improved using the Mirasol PRT system

Influence of applied CD34+ cell dose on the survival of Hodgkin's lymphoma and multiple myeloma patients following autologous stem cell transplants

Background/Aim. Autologous stem cell transplants (ASCTs) improve the rate of overall survival (OS) in patients with hematological malignancies such as multiple myeloma (MM) after induction chemotherapy, aggressive non-Hodgkin's lymphomas (NHL), and relapsed, chemotherapy-sensitive Hodgkin's lymphoma (HL). The study aim was to evaluate influence of applied CD34+ cell quantity on clinical outcome, as well as early post-transplant and overall survival (OS) of HL and MM patients following ASCT. Methods. This study included a total of 210 patients (90 HL/120 MM) who underwent ASCT. Stem cell (SC) mobilization was accomplished by granulocyte-colony stimulating factor (G-CSF) 10–16 μg/kg body mass (bm) following chemotherapy. For proven poor mobilizers, mobilization with G-CSF (16 μg/kgbm) and Plerixafor (24 or 48 mg) was performed. To our best knowledge, it was the first usage of the Plerixafor in our country in the ASCT-setting. Harvesting was initiated merely at "cut-off-value" of CD34+ cells ≥ 20 × 106/L in peripheral blood with "target-dose" of CD34+ cells ≥ 5 × 106/kgbm in harvest. The CD34+ cell count and viability was determined using flow cytometry. Results. The majority of HL patients (76.7%) were infused with > 5.0 × 106/kgbm CD34+ cells, while 68.3% of MM patients were treated by approximately 4.0–5.4 × 106/kgbm CD34+ dose, respectively. Beneficial response (complete/partial remission) was achieved in 83.3% (HL) and 94.2% (MM) patients. Among parameters that influenced survival of HL patients with positive response to the therapy, multivariate analysis (pre-ASCT performance status, CD34+ cell quantity applied, rapid hematopoietic, i.e. lymphocyte and platelet recovery) indicated that higher CD34+ cell dose used, along with pre-ASCT performance status correlated with superior event-free survival (EFS) and OS following ASCT. In MM patients, multivariate analysis (renal impairment, infused CD34+ cell quantity, early platelet recovery) indicated that the number of CD34+ cells infused was the most important parameter that influenced both EFS and OS after ASCT. Conclusion. Data obtained in this study undoubtedly confirmed that CD34+ cell dose applied is an independent factor that may contribute to superior clinical outcome and OS of HL and MM patients following ASCT

Impact of stem cell source on allogeneic stem cell transplantation outcome in hematological malignancies

Background/Aim. Peripheral blood (PB) is used more frequently as a source of stem cells (SCs) for allogeneic transplantation. However, the influence of cell source on the clinical outcome of SC transplantation is not yet well established. The aim of this study was to compare the results of PBSC transplantation (PBSCT) with bone marrow transplantation (BMT) on the basis of engraftment, frequency and severity of immediate (mucositis, acute Graft versus Host Disease - aGvHD) and delayed (chronic GvHD - cGvHD) complications, as well as transplant-related mortality (TRM), transfusion needs, relapses and overall survival (OS). Methods. We analyzed 158 patients, women/men ratio 64/94 median age 29 (range 9-57), who underwent allogeneic SC transplantation between 1989 and 2009. All included patients had diseases as follows: acute myeloid leukemia (AML) - 39, acute lymphoblastic leukemia (ALL) - 47, chronic myeloid leukemia (CML) - 32, myelodysplastic syndrome (MDS) - 10, Hodgkin’s lymphoma (HL) - 2, multiple myeloma (MM) - 3, granulocytic sarcoma (GrSa) - 3, severe aplastic anemia (sAA) - 22. The patients underwent transplantations were divided into two groups: BMT group (74 patients) and PBSCT group (84 patients). Each recipient had HLA identical sibling donor. SCs from bone marrow were collected by multiple aspirations of iliac bone and from PB by one “Large Volume Leukapheresis” (after recombinant human granulocyte colony stimulating factor, rHuG-CSF) application (5-12 μg/kgbm, 5 days). Conditioning regimens were applied according to primary disease, GvHD prophylaxis consisted of combination of a cyclosporine A and methotrexate. Results. Engraftment, according to the count of polymorphonuclear and platelets, were significantly (p < 0.001) faster in the PBSCT vs BMT group. The needs for transfusion support were significantly (p < 0.01) higher in the BMT group. Those patients had more frequently oropharingeal mucositis grade 3/4 (33.3% vs 10.0%, p < 0.05). There were no significant differences in the incidence of aGvHD and cGvHD between the two groups. The patients who underwent PBSCT had more frequently extensive cGvHD in comparison with the BMT group (29.1% vs 11.29%, p < 0.05). SC source (SCS) had no significant influence on the TRM (21.62% vs 23.8%, p = 0.64) and the incidence of relapses (21.6% vs 29.7%, p = 0.32). Finally, the patients treated by BMT had a significantly better OS (logrank 2.33, p < 0.05). Conclusion. SCs harvesting from PB resulted in improved cell yield, faster engraftment, as well as in a decrease of immediate transplantation related complications with a reduced treatment cost. Allogeneic PBSCT were associated with more frequent extensive cGvHD, while the influence of SCS in TRM and relapses was not observed. Finally, the longterm OS was better in the patients treated by BMT. To verify impact of SC source on transplantation (PBSCT vs BMT) overall efficacy, more larger randomized clinical studies are needed

Overexpression of MRP1/ABCC1, Survivin and BCRP/ABCC2 Predicts the Resistance of Diffuse Large B-Cell Lymphoma to R-CHOP Treatment

Background: Approximately 40% of patients with diffuse large B-cell lymphoma (DLBCL) experience treatment resistance to the first-line R-CHOP regimen. ATP binding cassette (ABC) transporters and survivin might play a role in multidrug resistance (MDR) in various tumors. The aim was to investigate if the coexpression of ABC transporters and survivin was associated with R-CHOP treatment response. Methods: The expression of Bcl-2, survivin, P-glycoprotein/ABCB1, MRP1/ABCC1, and BCRP/ABCC2 was analyzed using immunohistochemistry in tumor specimens obtained from patients with DLBCL, and classified according to the treatment response as Remission, Relapsed, and (primary) Refractory groups. All patients received R-CHOP or equivalent treatment. Results: Bcl-2 was in strong positive correlation with clinical parameters and all biomarkers except P-gp/ABCB1. The overexpression of MRP1/ABCC1, survivin, and BCRP/ABCC2 presented as high immunoreactive scores (IRSs) was detected in the Refractory and Relapsed groups (p < 0.05 vs. Remission), respectively, whereas the IRS of P-gp/ABCB1 was low. Significant correlations were found among either MRP1/ABCC1 and survivin or BCRP/ABCC2 in the Refractory and Relapsed groups, respectively. In multiple linear regression analysis, ECOG status along with MRP1/ABCC1 or survivin and BRCP/ABCG2 was significantly associated with the prediction of the R-CHOP treatment response. Conclusions: DLBCL might harbor certain molecular signatures such as MRP1/ABCC1, survivin, and BCRP/ABCC2 overexpression that can predict resistance to R-CHOP

An evidence-based and risk-adapted GSF versus GSF plus plerixafor mobilization strategy to obtain a sufficient CD34+ cell yield in the harvest for autologous stem cell transplants

Background: Plerixafor is a bicyclam molecule with the ability to reversibly bind to receptor CXCR-4 thus leading to an increased release of stem cells (SC) into the circulation. This study aims to evaluate the efficacy of G-CSF plus plerixafor versus G-CSF alone mobilizing regimens on the basis of CD34+ cell yield and engraftment kinetics following hematopoietic SC transplants. Methods: The study incorporated 173 patients with plasma cell neoplasms (PCN), Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), undergoing mobilization and following autologous SC-transplant. For patients with mobilization failure and those predicted to be at risk of harvesting inadequate CD34+ yields (poor-responders), plerixafor was administered. Data was collected and compared in relation to the harvesting protocols used, cell quantification, cell-engraftment potential and overall clinical outcome. Results: A total of 101 patients received plerixafor (58.4 %) and the median CD34+increase was 312 %. Chemotherapy-mobilized PCN-patients required less plerixafor administration (p = 0.01), no difference was observed in lymphoma groups (p = 0.46). The median CD34+cell yield was 7.8 × 106/kg bm. Patients requiring plerixafor achieved lower, but still comparable cell yields. Total cell dose infused was in correlation with engraftment kinetics. Patients requiring plerixafor had delayed platelet engraftment (p = 0.029). Conclusions: Adequately selected plerixafor administration reduces ''mobilization-related-failure'' rate and assure a high-level cell dose for SC transplants, with superior ''therapeutic-potential'' and safety profile. The mobilization strategy that incorporates ''just-in-time'' plerixafor administration, also leads to a reduction of hospitalization days and healthcare resource utilization. For definitive conclusions, further controlled/larger clinical trials concerning correlation of CD34+ cell count/yield, with hematopoietic reconstitution are required

Relative frequency of immature CD34+/CD90+ subset in peripheral blood following mobilization correlates closely and inversely with the absolute count of harvested stem cells in multiple myeloma patients

Background/Aim. Stem cells (SCs) guarantee complete/longterm bone marrow (BM) repopulation after SC-transplants. The aim of the study was to evaluate absolute count of total SCs (determined by ISHAGE-sequential-gating protocol – SCish) and relative frequency of immature CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB) as predictive factors of mobilization and apheresis product (AP) quality. Methods. Mobilization included chemotherapy and granulocytegrowth- factor (G-CSF). Harvesting was performed by Spectra- Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the “stem-cell-region” using FC- 500 flow-cytometer. In this study, the original ISHAGEsequential- gating protocol was modified by introduction of anti-CD90-PE monoclonal-antibody into the analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleated-cell count, absolute SCish count in μL of the PB or the AP, percentage of the CD90+SCish expressed to SCish and absolute CD90+SCish count in μL of the PB or the AP. Results. The absolute count of total SCish and CD90+SCish was significantly higher (p = 0.0007 and p = 0.0266, respectively) in the AP than in the PB samples. The CD90+SCish/total SCish indexes from PB were higher than indexes from the AP (p = 0.039). The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the PB also showed a significant (p = 0.0002) inverse relationship with total SCish count in the AP. Patients with less than 10% CD90+SCish in the PB had evidently higher (p = 0.0025) total SCish count in the AP. Conclusion. We speculate that lower CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still not fully resolved immature SC cytomorphological/ biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear – whether we should follow the absolute count of total SCish, or, whether we should test for relative frequency of CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger investigations concerning the correlation of circulating and harvested SCs with patients' hematopoietic recovery. [Project of the Ministry of Defence of the RS, Project MF/VMA 9/17-19 and Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41031

Flow cytometry analysis of platelet populations: usefulness for monitoring the storage lesion in pooled buffy-coat platelet concentrates

Background. Early detection of the platelet storage lesion is still a challenge in transfusion practice. Using flow cytometry, we evaluated the appearance of the storage lesion, based on the expression of platelet activation markers, in total platelets and platelet populations. Materials and methods. Buffy-coat-derived platelet concentrates were stored under standard conditions for 5 days. The expression of activation antigens CD42b, CD36, CD62p and phosphatidylserine on total platelets and populations of small, medium-sized and large platelets was analysed by flow cytometry on storage days 1, 3 and 5. Results. The activation/lesion on total platelets and each platelet population was detected on storage day 3, by the increased expression of CD36. On the same day, increased expression of CD42b and CD62p was detected, but only on large platelets. Small and medium-sized platelets had increased CD62p expression only on day 5. Externalisation of phosphatidylserine was not detected. Discussion. Evaluation of the level of expression of various activation markers on different platelet populations could be an additional valid analysis in cell quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation

Autologous transfusions for elective surgery - from existing approaches to upcoming challenges

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Relative frequency of immature CD34+/CD90+ subset in peripheral blood following mobilization correlates closely and inversely with the absolute count of harvested stem cells in multiple myeloma patients

Background/Aim. Stem cells (SCs) guarantee complete/longterm bone marrow (BM) repopulation after SC-transplants. The aim of the study was to evaluate absolute count of total SCs (determined by ISHAGE-sequential-gating protocol – SCish) and relative frequency of immature CD34+/CD90+ (CD90+SCish) subset in peripheral blood (PB) as predictive factors of mobilization and apheresis product (AP) quality. Methods. Mobilization included chemotherapy and granulocytegrowth- factor (G-CSF). Harvesting was performed by Spectra- Optia-IDL-system. The SCsish were determined as a constitutional part of CD34+ cells in the “stem-cell-region” using FC- 500 flow-cytometer. In this study, the original ISHAGEsequential- gating protocol was modified by introduction of anti-CD90-PE monoclonal-antibody into the analysis of CD90 expression on SCish (CD90+SCish). The results were presented as a percentage of SCish per nucleated-cell count, absolute SCish count in μL of the PB or the AP, percentage of the CD90+SCish expressed to SCish and absolute CD90+SCish count in μL of the PB or the AP. Results. The absolute count of total SCish and CD90+SCish was significantly higher (p = 0.0007 and p = 0.0266, respectively) in the AP than in the PB samples. The CD90+SCish/total SCish indexes from PB were higher than indexes from the AP (p = 0.039). The relative frequency of CD90+SCish showed a highly significant inverse correlation with the absolute count of total SCish in both, the PB and AP (p = 0.0003 and p = 0.0013 respectively). The relative frequency of CD90+SCish from the PB also showed a significant (p = 0.0002) inverse relationship with total SCish count in the AP. Patients with less than 10% CD90+SCish in the PB had evidently higher (p = 0.0025) total SCish count in the AP. Conclusion. We speculate that lower CD90+SCish yield in the AP is not a consequence of an inferior collection efficacy, but most likely a result of several still not fully resolved immature SC cytomorphological/ biophysical features. Therefore, following the mobilization by chemotherapy G-CSF, some logical questions appear – whether we should follow the absolute count of total SCish, or, whether we should test for relative frequency of CD90+SCish prior to harvesting. To reach the final conclusions, it is essential to conduct further controlled and larger investigations concerning the correlation of circulating and harvested SCs with patients' hematopoietic recovery. [Project of the Ministry of Defence of the RS, Project MF/VMA 9/17-19 and Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 41031

Follicular lymphoma patients with a high FLIPI score and a high tumor burden: A risk stratification model

Background/Aim. The widely accepted Follicular Lymphoma International Prognostic Index (FLIPI) divides patients into three risk groups based on the score of adverse prognostic factors. The estimated 5-year survival in patients with a high FLIPI score is around 50%. The aim of this study was to analyse the prognostic value of clinical and laboratory parameters that are not included in the FLIPI and the New Prognostic Index for Follicular Lymphoma developed by the International Follicular Lymphoma Prognostic Factor Project (FLIPI2) indices, in follicular lymphoma (FL) patients with a high FLIPI score and high tumor burden. Methods. The retrospective analysis included 57 newly diagnosed patients with FL, a high FLIPI score and a high tumor burden. All the patients were diagnosed and treated between April 2000 and June 2007 at the Clinic for Hematology, Clinical Center of Serbia, Belgrade. Results. The patients with a histological grade > 1, erythrocyte sedimentation rate (ESR) ± 45 mm/h and hypoalbuminemia had a significantly worse overall survival (p = 0.015; p = 0.001; p = 0.008, respectively), while there was a tendency toward worse overall survival in the patients with an Eastern Cooperative Oncology Group (ECOG) > 1 (p = 0.075). Multivariate Cox regression analysis identified a histological grade > 1, ESR ± 45 mm/h and hypoalbuminemia as independent risk factors for a poor outcome. Based on a cumulative score of unfavourable prognostic factors, patients who had 0 or 1 unfavourable factors had a significantly better 5-year overall survival compared to patients with 2 or 3 risk factors (75% vs 24.1%, p = 0.000). Conclusion. The obtained results suggest that from the examined prognostic parameters histological grade > 1, ESR ± 45 mm/h and hypoalbuminemia can contribute in defining patients who need more aggressive initial treatment approach, if two or three of these parameters are present on presentation