Uwe Cassens

The role of caspases in cryoinjury : caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells

Cryopreserved cells and tissues are increasingly used for stem cell transplantation and tissue engineering. However, their freezing, storage, and thawing is associated with severe damage, suggesting the need for better cryopreservation methods. Here, we show that activation of caspase‐3 is induced during the freeze‐thaw process. Moreover, we demonstrate that prevention of caspase activation by the caspase inhibitor zVAD‐fmk strongly improves the recovery and survival of several cryopreserved cell types and hematopoietic progenitor cells. A short preincubation with the caspase inhibitor after thawing also enhances the colony‐forming activity of hematopoietic progenitor cells up to threefold. Furthermore, overexpression of Bcl‐2, but not the blockade of the death receptor signaling, confers protection, indicating that cryoinjury‐ associated cell death is mediated by a Bcl‐2‐controlled mitochondrial pathway. Thus, our data suggest the use of zVAD‐fmk as an efficient cryoprotective agent. The addition of caspase inhibitors may be an important tool for the cryopreservation of living cells and advantageous in cell transplantation, tissue engineering, and other genetic technologies.

Inflammatory mediators in bronchoalveolar lavage fluid and plasma in leukocytopenic patients with septic shock-induced acute respiratory distress syndrome

OBJECTIVES The acute respiratory distress syndrome (ARDS) is a frequent complication of severe sepsis and a major cause of death in patients with hematologic malignancy during chemotherapy-induced leukocytopenia. Inflammatory mediators are important modulators of host response to injury and have been found to be increased in the bronchoalveolar lavage (BAL) fluid of nonleukocytopenic patients with ARDS. Since inflammatory cytokines in plasma of nonleukocytopenic patients seem to be efficient predictors of the course of ARDS, we examined this hypothesis in leukocytopenic patients with septic shock-induced ARDS. DESIGN Prospective, observational study. SETTING Intensive care unit (ICU) of a university hospital. PATIENTS Nineteen patients with leukocytopenia (white blood cell count of <1/nL) following cytoreductive chemotherapy for malignant disorders and severe sepsis with shock-induced ARDS (Murray score of >2.5). INTERVENTIONS BAL and plasma sampling and ICU management. MEASUREMENTS AND MAIN RESULTS The proinflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 were measured in the BAL aspirates and in plasma samples, both obtained within 18 hrs after onset of ARDS. Hemodynamic and oxygen metabolism data were measured immediately before plasma samples were taken and BAL was performed. Of the 19 patients studied, nine patients responded to ICU treatment (e.g., mechanical ventilation as indicated by PaO2/FIO2, FIO2, shunt volume, and course of pulmonary infiltrates), whereas ten patients did not respond. BAL cytokine concentrations were significantly increased in nonresponders in comparison with responding patients (TNF-alpha, p = .021; IL-6, p = .008; IL-8, p = .019). In contrast, we did not observe any differences between the groups in terms of plasma cytokine concentrations. CONCLUSION Determination of cytokine concentrations in BAL samples may be useful for evaluation of severity and course of ARDS in leukocytopenic patients, whereas measurement of plasma cytokines is not helpful.

The influence of blood group differences in allogeneic hematopoietic peripheral blood progenitor cell transplantation.

BACKGROUND: Severe immunohematologic complications after ABO‐mismatched allogeneic blood peripheral blood progenitor cell (PBPC) transplantation (PBPCT), including pure red cell aplasia and immune hemolysis, have been described. Although several studies have addressed this issue, the clinical influence of blood group differences on transfusion requirements and survival is still discussed controversially, especially in the case of PBPCT.

A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

Processing of peripheral blood progenitor cell components in improved clean areas does not reduce the rate of microbial contamination

BACKGROUND: Microbial contamination of peripheral blood progenitor cell components (PBPCs) may cause severe complications in immunosuppressed recipients. Therefore, principles of Good Manufacturing Practice (GMP) are applicable for processing of PBPC components to reduce potential risks of contamination.

A novel true volumetric method for the determination of residual leucocytes in blood components

Background and Objectives Accurate determination of residual leucocytes [white blood cells (WBC)] in blood components is of high clinical importance. To date, several labour‐intensive, time‐consuming or expensive techniques have been used for this purpose.

Factors affecting the efficacy of peripheral blood progenitor cells collections by large-volume leukaphereses with standardized processing volumes.

BACKGROUND:  Peripheral blood progenitor cell (PBPC) collections should be safe and efficient. Therefore, the influence and risk factors in large‐volume leukaphereses (LVL) with standardized blood volumes was investigated.

Autologous red cells derived from cord blood: collection, preparation, storage and quality controls with optimal additive storage medium (Sag-mannitol)

Summary.  To investigate whether packed red cells (PRCs) prepared from autologous cord blood‐packed red cells (AC‐PRCs) could be used as an alternative for homologous‐packed red cells (H‐PRCs), we developed a system to collect and prepare AC‐PRCs and determined standard storage parameters during 35 days of storage in extended storage medium (Sag‐mannitol). We collected and fractionated cord blood from 390 newborns. The amount and quality of the AC‐PRCs were analysed. The bacterial contamination rate was 1·84%. Twelve AC‐PRCs were stored for 35 days, and standard laboratory parameters were measured at day 1 and day 35. The initial laboratory parameters of the AC‐PRCs were similar to the parameters of the H‐PRCs. After 35 days, the AC‐PRCs displayed an increased haemolysis rate compared to H‐PRCs (1·1 versus 0·2%) and also a significant decreased adenosine triphosphate value (1·2 versus 2·3 µmol L−1). Haemoglobin, haematocrit and pH were comparable in both groups. AC‐PRCs meet the quality criteria for H‐PRCs after 35 days. Utilizing a closed collection system for cord blood and an extended storage medium will increase safety and quality and facilitate the routine transfusion of autologous red cells derived from cord blood.

Individual Patient-Dependent Influence of Erythrocyte Lysing Procedures on Flow-Cytometric Analysis of Leukocyte Subpopulations

Background: It is known from the literature that the use of erythrocyte lysing reagents in flow-cytometric analysis results in a significant loss of immunolabelled cells, e.g. T helper and T supressor lymphocytes (CD4+/CD8+ T cells) or stem cells (CD34+ cells). Although WBC subset-specific variations in the sensitivity to lysing reagents are already described, the same variations are possible for each individual. In the present study we investigate the individual and patient-dependent loss of CD4+ T cells in blood samples by the use of a true volumetric flow cytometer (TVC) and a new no-lyse no-wash procedure. Material and Methods: Blood of 50 individuals was used for flow-cytometric counting of CD4+ T cells, and cells were counted by the use of a lyse no-wash and a no-lyse no-wash protocol (600 single measurements). The reproducibility of the loss of CD4+ T cells was measured by 10 individually prepared samples out of a single blood sample; each sample was measured 3 times (n = 30). Results: The mean lysing-dependent CD4+ T-cell loss out of the sample from a single individual was 15% (range 12–18%). The reproducibility of the determination from the same individual was high (coefficient of variation (CV) = 12%). Measuring the concentration of CD4+ T cells in blood samples of different individuals, the mean loss of CD4+ T cells was 9.8% (range 0–23%), interestingly with a high variability and a CV of 65% (n = 600). Therefore, most current flow-cytometric protocols with erythrocyte lysing procedure do not allow reproducible and accurate determinations of CD4+ T cells because of individual leukocyte losses. Conclusion: Lysing-dependent cell loss does not represent a constant or calculable parameter in leukocyte subclass enumeration. Beside the leukocyte subset- specific differences in sensitivity to erythrocyte lysing reagents, patient disease and drug treatment may influence the stability of leukocytes under investigation. Accordingly, the present study shows large lysing-dependent interindividual differences in counting results of CD4+ T cells.

Flow Cytometry in Transfusion Medicine: Development, Strategies and Applications

Nowadays, flow cytometry represents an essential tool for the daily work in laboratories for transfusion medicine. After cytophotometry of immobilized cells, flow cytometry was developed since the 1960s using moving fluorescence-stained cells. This technique enabled the determination of several thousand cells per second, e.g. from blood, bone marrow, or organ-associated cell suspensions. The introduction of monoclonal fluorescence-labeled antibodies in diagnostics has further accelerated the development of flow cytometry. In the meantime, numerous cellular and nuclear properties can simultaneously be measured on single cell level in automated devices at high speed and precision. Internal standards and quality control systems further increased the accuracy of flow cytometric analyses. Flow cytometry is therefore likewise suitable for routine diagnostics in patients, quality control of blood components and scientific purposes in transfusion medicine. Nevertheless, many flow cytometric applications are related to counting of rare events or detection of cells with low antigen expression. Therefore, several pre-analytic and analytic factors (e.g. erythrocyte lysing procedures, cell gating strategies, etc.) may essentially affect the accurate determination of cell numbers and functions. In this issue, TRANSFUSION MEDICINE AND HEMOTHERAPY starts a recurrent sequence of reviews focused on ‘Flow Cytometry in Transfusion Medicine’. In order to introduce the flow cytometry series, this review summarizes the development, principles, and strategies of flow cytometry as an increasing diagnostic tool in medical laboratories. Also, current and possibly future flow cytometric applications in transfusion medicine are epitomized, e.g. quality control of blood products, immunohematologic diagnostics, hematopoietic progenitor cell transplantation, adoptive immunotherapy, and further therapeutic applications. In following issues of this journal, reviews of notable authors will then focus on special applications and give more detailed insights into single diagnostic fields.