Cellular therapy nowadays includes various products from haematopoietic stem cells (HSC) collected from bone marrow, peripheral blood, and umbilical cord blood to more complex adoptive immune therapy ...for the treatment of malignant diseases, and gene therapy for inherited immune deficiencies. Broader utilization of cellular therapy requires extensive quality testing of these products that should fulfil the same requirements regarding composition, purity, and potency nevertheless they are manufactured in various centres. Technical improvements of the flow cytometers accompanied by the increased number of available reagents and fluorochromes used to conjugate monoclonal antibodies, enable detailed and precise insight into the function of the immune system and other areas of cell biology, and allows cell evaluation based on size, shape, and morphology or assessment of cell surface markers, as well as cell purity and viability, which greatly contributes to the development and progress of the cell therapy. The aim of this paper is to give an overview of the current use and challenges of flow cytometry analysis in quality assessment of cellular therapy products, with regard to basic principles of determining HSC and leukocyte subpopulation, assessment of cells viability and quality of thawed cryopreserved HSC as well as the importance of validation and quality control of flow cytometry methods according to good laboratory practice.
Due to the expansion of cell therapy using not only haematopoietic stem cells (HSC) but also other leukocyte subpopulations, the loss of these cells in cryopreserved apheresis products needs to be ...evaluated. Various factors that could negatively affect post-thaw recovery, such as leukapheresis product characteristics, storage time and cryopreservation protocols have been identified.
The post-thaw recovery of HSCs, lymphocytes, NK cells and monocytes, as well as the factors that could adversely affect it were analysed in autologous and allogeneic leukapheresis products.
The lowest post-thaw recovery was observed in autologous and allogeneic CD34+ cells, with the median of 73.7% and 68.1%, respectively. In leukocyte subpopulation, the lowest post-thaw recovery was observed for CD14+ cells, both autologous and allogeneic. The highest post-thaw recovery was observed for CD3+/CD8+ cells in autologous, and for CD19+ cells in allogeneic samples. The statistically significant difference was observed between autologous and allogeneic PBSC products for CD3+ cell recovery (P = 0.031) and CD3+/CD8+ cell recovery (P = 0.009). The evaluation of factors that could adversely affect the post-thaw recovery in autologous samples showed weak negative correlations between platelet concentration and CD3+ recovery, as well as between storage time and CD3+CD8+ recovery. In allogeneic samples, a strong negative correlation was observed only between the percentage of granulocytes and CD3+, CD3+/CD8+ and CD3+/CD4+ cell recoveries.
Since various post-thaw recoveries of leukocyte subpopulations were observed, the cell therapy manufacturing centers should evaluate how their cryopreservation method and other factors affect the recovery of cell population of interest in their settings.
Background
Patients with hematological diseases are polytransfused and often immunocompromised, therefore susceptible to transfusion reactions (TR). This study aims to document the incidence of TRs ...in adult hematological patients and assess the effect of changes in the production of blood components and transfusion practice on their occurrence.
Study design and methods
Retrospective observational analysis of TRs reported from 1993 to 2019 was performed. For the analysis of the effect of changes on the incidence of TRs, the evaluated time was divided into two periods: the 1st period before the introduction of changes in production, when leukoreduced blood components were used only selectively, and the 2nd period, when semi‐automated method of production and universal leukoreduction was introduced.
Results
The decrease in the incidence of TRs was observed for both red blood cell (RBC) and platelet concentrate (PC) transfusions in the 2nd period. Since platelet additive solution has been used, a further decrease in the incidence was reported. The decrease in incidence was also observed for delayed hemolytic/serological transfusion reactions and for transfusion‐transmitted bacterial infections. Four cases of incorrect blood transfusions were uniquely related to the hematological patients, caused by antigen loss and transfusion ordering after ABO‐incompatible hematopoietic stem cell transplantation.
Discussion
Our results provided evidence that the introduction of tools offered by modern transfusion medicine: universal leukodepletion, plasma replacement with additive solutions, sensitive laboratory techniques, prophylactic antigen matching policy, informatization, and automatization, decreased the incidence of TRs and improved transfusion safety.
Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the ...quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C (p = .004) and at room temperature (p = .018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure.Chimeric antigen receptor-T (CAR-T) cell therapy is currently the best-known type of immune effector cells therapy. For CAR T-cell therapy, the determination of CD3+ T cells is necessary for the quality control of fresh leukapheresis product as starting material. The aim was to validate analytical method for quantification of percentage and absolute count of T lymphocyte subtypes (CD3+, CD4+ and CD8+ cells) in fresh apheresis products using single-platform method on flow cytometer BD FACS Canto II. Validation study included determination of precision, trueness (bias), assessment of linearity, carryover, comparison of results obtained with two different protocols on flow cytometer for CD3+ cells determination and stability study. For between-run precision coefficients of variation (CVs) were <20%, as well as bias for all T-lymphocyte subtypes. For within-run precision, CVs were <10%, except for low CD8+ cell (percentage 10.51% and viable absolute count 12.37%). Comparison of results obtained with two different protocols for CD3+ cells determination shows no statistically significant difference. Statistically significant differences between results of the analysis of CD4+ cells in fresh samples and results obtained after storage at 4 °C (p = .004) and at room temperature (p = .018) were found. In conclusion, method for enumeration of T-lymphocyte subtypes can be used in routine work on BD FACS Canto II instrument for quality assessment of fresh cell products collected by leukapheresis procedure.
•In clinical practice, a substantial proportion of platelet transfusions are routinely administered outside the guidelines.•Unnecessary platelet transfusions are an unjustified extra burden on a ...scarce healthcare resource and may also be detrimental to the patients.•One-third of all PLT transfusions were inappropriate mainly because of transfusions above the recommended threshold and unjustified double units transfusions.
Hematology patients are intensive platelet users. In clinical practice, a substantial proportion of platelet (PLT) transfusions are routinely administered outside the guidelines despite compelling evidence for recommendations. Those unnecessary PLT transfusions are an unjustified extra burden on a scarce healthcare resource and may also be detrimental to the patients. This study aims to evaluate indications and assess the appropriateness of PLT transfusion, as well as to identify common discrepancies and propose modalities for better compliance with guidelines.
The audit of all PLT orders for adult hematological inpatients was conducted over 2 months. The assessment was performed using guidelines for PLT transfusion. Patient demographic, clinical, and transfusion data were collected from hospital electronic medical records.
Based on 286 PLT orders, 344 PCs were transfused to 67 patients: 235 (82.2%) prophylactical due to low PLT count, 34 (11.9%) preprocedural and 17 (5.9%) therapeutic. Overall, 105 (36.77%) PLT transfusions were inappropriate: 78 (33.2%) of all prophylactic PLT transfusions due to low PLT count, 17 (50%) off all preprocedural and 10 (58.8%) of all therapeutical transfusion. The major reason for PLT transfusion inappropriateness was transfusion above the recommended threshold. Double units of PCs were transfused in 36.7% of all PLT transfusions and 32.4% of them were considered inappropriate.
Our audit of PLT transfusion practice found a large proportion of inappropriate PLT transfusions. Based on the most common deviations from the guidelines a variety of targeted measures for improvement are proposed.
Introduction
Peripheral blood stem cell (PBSC) harvesting requires reliable and safe vascular access. In our institution, a change of practice was implemented and the central venous catheter (CVC) ...placement for all autologous PBSC collections was abandoned in favor of a careful evaluation of peripheral venous access (PVA) for each individual patient. The aim of this prospective study was to evaluate the rate of patients with adequate peripheral veins for autologous PBSC collection and compare patient characteristics, collection efficacy, and complication rate between patients with PVA and CVC.
Method
Peripheral veins were assessed by the apheresis nurse team in all patients referred between January 2020 and July 2021 to autologous PBSC collection. Only in case of difficult venous access, CVC was inserted. Large volume leukapheresis (LVL) procedures, which processed ≥3 total blood volumes, were performed.
Results
In 65 (57%) patients PVA was used, while 49 (43%) patients required placement of short‐term CVC. Peripheral venous access was successfully used significantly more often in males (69.8%) (P = 0.010), and patients with multiple myeloma (71.0%) than in patients with non‐Hodgkin's lymphoma (35.9%) and Hodgkin's lymphoma patients (33.3%) (P < 0.001). There was a significant difference in the type of prior administered chemotherapy; in the patients who received cytostatics free chemotherapy, PVA was used more often (75.0%) (P = 0.007). In terms of the efficacy and safety of LVLs, there were no differences between procedures performed using PVA and CVCs.
Conclusion
Peripheral venous access is feasible for autologous PBSC collection in more than a half of patients, in particular in those with multiple myeloma. Changes in the treatment of multiple myeloma, using new proteasome inhibitors‐based and immunomodulatory agents that do not adversely affect peripheral veins, have enabled the use of PVA even at the high blood flow rates required by LVL. Peripheral venous access is not associated with safety issues or with a lesser collection efficiency, and it is cost‐effective as well. Each patient referred to autologous PBSC collection needs to be evaluated individually by the experienced apheresis team for the most appropriate venous access.
BackgroundPatients with hematological diseases are polytransfused and often immunocompromised, therefore susceptible to transfusion reactions (TR). This study aims to document the incidence of TRs in ...adult hematological patients and assess the effect of changes in the production of blood components and transfusion practice on their occurrence.Study design and methodsRetrospective observational analysis of TRs reported from 1993 to 2019 was performed. For the analysis of the effect of changes on the incidence of TRs, the evaluated time was divided into two periods: the 1st period before the introduction of changes in production, when leukoreduced blood components were used only selectively, and the 2nd period, when semi‐automated method of production and universal leukoreduction was introduced.ResultsThe decrease in the incidence of TRs was observed for both red blood cell (RBC) and platelet concentrate (PC) transfusions in the 2nd period. Since platelet additive solution has been used, a further decrease in the incidence was reported. The decrease in incidence was also observed for delayed hemolytic/serological transfusion reactions and for transfusion‐transmitted bacterial infections. Four cases of incorrect blood transfusions were uniquely related to the hematological patients, caused by antigen loss and transfusion ordering after ABO‐incompatible hematopoietic stem cell transplantation.DiscussionOur results provided evidence that the introduction of tools offered by modern transfusion medicine: universal leukodepletion, plasma replacement with additive solutions, sensitive laboratory techniques, prophylactic antigen matching policy, informatization, and automatization, decreased the incidence of TRs and improved transfusion safety.
Daratumumab je prvo monoklonsko protutijelo anti-CD38 koje se primjenjuje u liječenju multiplog mijeloma. Njegova primjena uzrokuje panreaktivnost u testovima prijetransfuzijskog ispitivanja. ...Panreaktivnost je posljedica vezanja monoklonskog protutijela anti-CD38 na protein CD38 na površini eritrocita, što u standardnom testiranju onemogućuje otkrivanje antieritrocitnih aloprotutijela i osiguranje podudarne krvi za transfuzijsko liječenje. Cilj rada bila je retrospektivna analiza vlastitih iskustava u rješavanju smetnja prijetransfuzijskog ispitivanja uzrokovanih monoklonskim protutijelom anti-CD38 i u transfuzijskom liječenju tih bolesnika. Prikazani su postupci za prijetransfuzijsko ispitivanje i transfuzijsko liječenje bolesnika liječenih monoklonskim protutijelom anti-CD38 koji su provedeni u Kliničkome bolničkom centru Zagreb. U istraživanju je analizirano 10-ero bolesnika liječenih daratumumabom. Prije i poslije primjene daratumumaba pretražena su antieritrocitna protutijela i određen je direktan antiglobulinski test. Pri transfuzijskom liječenju napravljeni su test pretraživanja antieritrocitnih protutijela i križne reakcije standardnim testiranjem i specifičnim postupcima imunohematoloških ispitivanja za uklanjanje smetnja monoklonskog protutijela anti-CD38. Postupci su uključivali obradu eritrocita ditiotreitolom koncentracije 0,2 M i neutralizacijski test uz primjenu reagensa DaraEx. Kod svih bolesnika testovi pretraživanja antieritrocitnih protutijela i križne reakcije bili su nakon primjene daratumumaba pozitivni, dok je direktan antiglobulinski test zbog primjene daratumumaba bio pozitivan u gotovo polovine bolesnika. Nakon obrade eritrocita ditiotreitolom 0,2 M učestalost lažno pozitivnih rezultata testova pretraživanja antieritrocitnih protutijela i križnih reakcija iznosila je oko 40%, a poslije primjene reagensa DaraEx oko 20%. Oba specifična postupka, obrada eritrocita ditiotreitolom 0,2 M i neutralizacijski test primjenom reagensa DaraEx, nisu se pokazala dovoljno pouzdanima u rješavanju smetnja uzrokovanih monoklonskim protutijelom anti-CD38. Zato je za transfuzijsko liječenje tih bolesnika nužno osigurati eritrocitne pripravke podudarne prema klinički najvažnijim antigenima u sustavima krvnih grupa Rh, Kell, Kidd, Duffy i MNS. Dobra suradnja između odjela i transfuzijske službe te postojanje protokola za prijetransfuzijsko ispitivanje i transfuzijsko liječenje ostaju preduvjet za pravodobno i sigurno transfuzijsko liječenje te skupine bolesnika.
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