The impact of erythrocyte lysing procedures on the recovery of hematopoietic progenitor cells in flow cytometric analysis.

Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34+ cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell-enriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform [1-PF]). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34+ cells (50.8% for CD45+ cells) in unselected HCTs and a mean decrease of 52% for CD34+ cells (49.8% for CD45+ cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)-counted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34+ cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34+ cells in nonvolumetric flow cytometric (2-PF) protocols.

A noninvasive strategy for screening prospective blood donors for anemia.

BACKGROUND: The reliability of capillary hemoglobin (Hb) as an indicator for eligibility to donate blood is discussed controversially. Therefore, a noninvasive alternative with acceptable predictive values was established and evaluated. STUDY DESIGN AND METHODS: Donor candidates were selected according to their Hb level. The first donation was performed 6 weeks after this selection step. A venous blood sample was collected from all donors at the end of their donation and a postdonation Hb determination was performed. Donors with acceptable postdonation Hb values were permitted to donate next time without any predonation Hb measurement. Donors with low postdonation Hb values were permitted to donate only after a venous Hb measurement had shown an acceptable value. Sensitivity and specificity were determined by comparing the gold standard (i.e., venous Hb measurement) with the presented method of Hb estimation for 19,534 donors. RESULTS: Taking the postdonation Hb as an indicator for eligibility saved 97 percent of donors from being tested unnecessarily by capillary Hb measurement. This procedure resulted in a specificity of 92.6 percent and a sensitivity of 37.9 percent for Hb cutoff levels of 135 and 125 g per L for men and women, respectively. The sensitivity increased rapidly to 100 percent for Hb levels below 105 g per L. The average deviation from true Hb level was 6 g per L. CONCLUSION: The presented noninvasive method distinctly saves time and expenditure without endangering blood donors.

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. STUDY DESIGN AND METHODS: This single-center study is based on 143 patients receiving PBPCT after standard or reduced-intensity conditioning. The influence of blood group differences in the ABO, Rh, and Kell systems on red blood cell, platelet, and plasma transfusion requirements; length of hospitalization in transplantation unit; survival; and occurrence of graft-versus-host disease was investigated. Additionally, the influence of the conditioning regimen and irregular antibodies on the measures mentioned above was analyzed. RESULTS: Multivariate analysis demonstrated that minor and bidirectional ABO mismatch (p = 0.028) and Rh difference (p = 0.020) independently led to poorer survival. The Kell difference did not show significant influences on the measures mentioned above. A clinically relevant influence of blood group differences on transfusion requirements could not be demonstrated. Irregular antibodies also did not show significant influences. CONCLUSION: These findings indicate an influence of blood group differences in PBPCT on survival and must be studied in further detail.

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. STUDY DESIGN AND METHODS: In a total of 724 autologous LVL performed at our center, either 4x or 6x the patient's blood volume (PBV) was processed. The group with processing 4x the PBV showed a median of 31 circulating CD34+ cells per microL, and the group with processing 6x the PBV had a median of 13 CD34+ cells per microL before LVL. Individual clinical factors, laboratory factors, and apheresis run variables influencing the yields of PBPCs were retrospectively analyzed. Furthermore, the changes of laboratory variables and adverse effects during LVL were investigated. RESULTS: Multivariate analysis identified "age,""circulating CD34+ cells," and "percentage of mononuclear cells" as only factors influencing the yields of PBPCs. Altogether, processing 6x versus 4x the PBV did not result in significantly higher yields of CD34+ cells for the total group, but requested PBPC yields were achieved more often after processing 6x the PBV in patients below 20 CD34+ cells per microL blood. Processing 6x versus 4x the PBV showed a significant difference for the decrease of platelets, but not for any other laboratory variable. Adverse effects were recorded in 4.97 percent of LVL without accumulation in one group. CONCLUSION: In particular, patients with low amounts of circulating CD34+ cells profited from enlarged LVL demonstrating higher PBPC yields but comparable rates of adverse effects.

Cryopreservation reduces the concentration of detectable bacteria in contaminated peripheral blood progenitor cell products.

BACKGROUND: Microbial contamination of PBPC products PBPCPs may cause severe clinical complications. There-fore, we investigated the influence of cryopreservation on the sensitivity to detect bacterial contaminations in PBPCPs. STUDY DESIGN AND METHODS: Expired PBPCPs (n = 29) were thawed, and defined concentrations of Staphylococcus epidermidis or Escherichia coli were inoculated into each bag. After 60 minutes of intermixing, a representative aliquot was drawn and cultured on Mueller-Hinton agar for 24 hours. Then, the products were cryopreserved for 24 hours, and the procedure was repeated as mentioned above. The total numbers of CFUs were counted before and after cryopreservation. RESULTS: A mean concentration of 2529 CFUs per mL of S. epidermidis was determined before cryopreservation versus 2182 CFUs per mL after cryopreservation, demonstrating a decrease of detectable colonies (p < 0.05). For E. coli, the mean numbers were 424 CFUs per mL before cryopreservation and 343 CFUs per mL after cryopreservation, also showing a decrease (p < 0.05). CONCLUSION: The cryopreservation reduces the concentration of detectable bacteria in contaminated PBPCPs. Especially in sterility testing of PBPCPs with low bacterial contamination, this phenomenon could lead to false-negative results with severe clinical consequences.

High-grade loss of leukocytes and hematopoietic progenitor cells caused by erythrocyte-lysing procedures for flow cytometric analyses.

All current-flow cytometric techniques use erythrocyte-lysing procedures before leukocyte analysis. We investigated the impact of four lysing procedures with different flow cytometric techniques on the loss of leukocytes and hematopoietic progenitor cells in blood samples. A total of 280 determinations out of 10 samples were measured by two flow cytometers (FCMs), using a FACS-Calibur (Becton Dickinson) and a particle-analyzing system (PAS) with a "true volumetric unit" (Partec). All samples were prepared with four different commercially available erythrocyte-lysing reagents (n = 10, respectively). CD34(+) cells were determined in relation to counted leukocytes with both FCMs (dual platform determinations, 2-PF). In addition, further immunologic and nuclear staining determinations of cells with and without erythrocyte-lysing procedures were performed in the "true volumetric unit" (single platform mode 1-PF) using the PAS system (n = 10, respectively). In the 2-PF mode, both systems showed identical results for CD34(+) cells (r = 0.997). The comparison of 1-PF and 2-PF modes with immunologic stainings revealed a mean decrease of 34.5% for absolute amounts of CD45(+) cells [in detail: Becton-Dickinson (BD) lysis 40%; Ortho Diagnostics (OD) lysis 31%; Uti lyse (UL) 38%; Cylyse (CL) 29%] and of 41.3% for absolute concentration of CD34(+) cells [in detail: BD lysis 45%; OD lysis 40%; UL lysis 45%; CL lysis 34%] by the lysing procedures. In contrast, the nuclear stainings revealed a mean leukocyte loss of only 5% for the nonlysed samples and of 12% for lysed samples. All investigated lysing procedures induced a large loss of leukocytes and progenitor cells, obviously due to cell membrane destruction as demonstrated for identical samples in the 1-PF and 2-PF modes by immunologic and nuclear staining methods.

Circulatory arrest during PBPC apheresis in an unrelated donor.

BACKGROUND: Nowadays, the collection of PBPCs by apheresis from healthy donors is a routine method. The mobilization with rHu G-CSF and the apheresis procedures are usually well tolerated without severe side effects. STUDY DESIGN AND METHODS: We report a severe complication in a 41-year-old unrelated female donor who was allowed to donate PBPCs and was mobilized with 10 microg of G-CSF per kg per day. During PBPC apheresis, she experienced a circulatory arrest after 132 minutes and processing of 7078 mL of blood (twice the donor's blood volume). RESULTS: Immediate cardiopulmonary resuscitation restored sinus rhythm and regulatory respiration without sequelae. Subsequent cardiologic examinations (heart catheterization, electrophysiologic testing, tilting table test) resulted in the diagnosis of a neurocardiogenic syncope. Other cardiac or circulatory disorders could be excluded. The implantation of a cardiac pacemaker was recommended to the donor. The 4-year-old recipient was successfully transplanted with the partial product collected until the arrest occurred. The patient received a total of 2.54 x 106 CD34+ cells per kg of body weight. CONCLUSION: After exclusion of other cardiac diseases, the diagnosed neurocardiogenic syncope probably induced the circulatory arrest during apheresis rather than the administration of G-CSF.

Viral modulation of cell death by inhibition of caspases.

Caspases are key effectors of the apoptotic process. Some of them play important roles in the immune system, being involved in the proteolytic maturation of the key cytokines, including interleukin 1beta (IL-1beta) and IL-18. The latter directs the production of interferon gamma (IFN-gamma). Among pathogens, particularly viruses express various modulators of caspases that inhibit their activity by direct binding. By evading the apoptotic process, viruses can better control their production in the infected cell and avoid the attack of the immune system. Targeting the maturation of the key cytokines involved in the initiation of (antiviral) immune response helps to avoid recognition and eradication by the immune system. The three main classes of caspase inhibitors frequently found among viruses include serine proteinase inhibitors (serpins: CrmA/SPI-2), viral IAPs (vIAPs) and p35. Their molecular mechanisms of action, structures and overall influence on cellular physiology are discussed in the review below.

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.