Juergen Zingsem

Influence of prestorage leukoreduction and subsequent irradiation on in vitro red blood cell (RBC) storage variables of RBCs in additive solution saline-adenine-glucose-mannitol.

BACKGROUND: There exists only very few data on in vitro and in vivo effects of gamma irradiation of red blood cells (RBCs) that have been leukoreduced by filtration before a subsequent irradiation. Reported studies reflect neither the current Food and Drug Administration (FDA) nor the European recommendations on timing of irradiation and subsequent storage.

Plateletpheresis does not cause long-standing platelet-derived growth factor release into the donor blood.

BACKGROUND: Recently, long‐standing elevations of soluble growth factors released from platelets (PLTs) after contact with artificial surfaces during dialysis were described. They could be jointly responsible for the high frequency of death from cardiovascular diseases in dialysis patients. There are no comparable data on the extent and the duration of a growth factor release by plateletpheresis procedures.

Do platelets stored in additive solution really show limited osmotic balance

2008;112:4523-31. 5. Cognasse F, Payrat JM, Corash L, Osselaer JC, Garraud O. Platelet components associated with acute transfusion reactions: the role of platelet-derived soluble CD40 ligand. Blood 2008;112:4779-80. 6. Elzey BD, Schmidt NW, Crist SA, Kresowik TP, Harty JT, Nieswandt B, Ratliff TL. Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge. Blood 2008;111:3684-91. 7. Osselaer JC, Messe N, Hervig T, Bueno J, Castro E, Espinosa A, Accorsi P, Junge K, Jacquet M, Flament J, Corash L. A prospective observational cohort safety study of 5106 platelet transfusions with components prepared with photochemical pathogen inactivation treatment. Transfusion 2008;48:1061-71.

Depletion of donor-specific anti-HLA A2 alloantibodies in a hematopoietic cell transplant recipient using directed mismatched platelet transfusions

Donor-specific anti-HLA alloantibodies (DSA) are a risk factor for graft failure in HLA-mismatched allogeneic hematopoietic cell transplantation [1–4]. This seems to be particularly true when DSA can be detected at a high level in solid-phase assays [5], or are complement binding as detected in the C1q allo-antibody assay [6] and the complement-dependent cytotoxicity (CDC) crossmatch [7]. DSA may also have a negative impact on overall survival after reduced intensity conditioning [8]. With increasing use of HLA-mismatched stem cell donors and improved diagnostic methods for anti-HLA antibodies, this problem becomes clinically even more relevant. Reduction of DSA prior to transplantation is able to reduce the risk of graft failure and facilitates engraftment [4–6, 9]. However, it is still unclear which method is the most suitable for desensitization [10]. Here we report the successful desensitization prior to a HLA-A2 mismatched allogeneic hematopoietic cell transplantation (HCT) in a highly immunized patient, demonstrating the incremental effect of directed HLA-A2 mismatched platelet transfusions on the DSA level. Three months before transplantation, the patient, a then 53-year-old female, was diagnosed with acute myloid leukemia with an intermediate genetic risk profile. She received a standard 7+ 3 induction therapy with cytarabine and daunorubicin. Bone marrow control puncture on day 14 revealed induction failure and salvage therapy consisting of high-dose cytarabine and mitoxantrone was applied. In addition, the search for an allogeneic stem cell donor was initiated. The best available donor identified was a 9/10 matched unrelated donor, with the donor carrying HLA-A2 as the single antigen mismatch for HLA-A, -B, -C, -DRB1 and -DQB1. In addition, one permissive antigen mismatch for HLA DPB1 was found. During aplasia following induction therapy, the patient became refractory to platelet transfusions. Broad immunization towards HLA class I was confirmed when the patient’s serum was analyzed for HLA antibodies using a single antigen solid-phase assay (Labscreen, One Lambda, Thermo Fisher Scientific, Canoga Park, CA, USA). All sera were used undiluted and subjected to heat inactivation and filtration (AcroPrep Advance 96 Filter Plate, PALL Life Sciences, Ann Arbor, MI, USA) before being analyzed in order to reduce the so called prozone effect [11]. No antibodies against HLA-DP were present. Antibodies directed towards HLA-A2, which was also part of the paternal haplotype inherited by the patient’s two children, showed the highest mean fluorescence intensity (MFI, Fig. 1a). In contrast to ABO-isoagglutinins, which are determined by titration, the level of anti-HLA antibodies in single antigen bead assays is reported by the mean fluorescence intensity, although recent studies suggest that serial dilution of sera before subjecting them to solidphase assays may more accurately determine antibody strength in these assays [12]. The patient’s serum also reacted strongly with lymphocytes from the selected stem cell donor in a complement-dependent cytotoxicity crossmatch (CDC XM), providing the strongest evidence for the clinical relevance of the DSA. Due to the high level of DSA and the positive CDC crossmatch, desensitization was initiated prior to HSCT, using directed HLA-A2 mismatched platelet transfusions in combination with rituximab and bortezomib. Reduced intensity conditioning with fludarabine, carmustine, melphalan, and antithymocyte globulin was started * Bernd M. Spriewald bernd.spriewald@uk-erlangen.de

Comparison of Six Different Cryoprotective Agents Used for Deep Freezing and Storage of CD34+ Cells Derived from Cord Blood and Peripheral Blood Stem Cell Concentrates

BACKGROUND For cryopreservation of stem cells, a cryoprotective agent is essential. Dimethyl sulfoxide is commonly used, but has deleterious effects on cell vitality in the warmth and for the recipients of stem cells. The aim of the study was to reduce DMSO and find one cryoprotective solution suitable for stem cells of different origin. Materials: Small volumes of both stem cell apheresis products and cord blood derived stem cells were frozen using six different cryoprotective solutions. Suitability of these solutions was tested by comparing cell vitalities and recovery rates of CD45 and CD34 positive cells and colony forming unit recovery rates. RESULTS No single cryoprotective solution being significantly superior regarding all cell qualities and recovery rates could be identified. However, mixing approximately 5% DMSO with hydroxyethyl starch with a molecular weight of 450,000 Dalton showed better results for most qualities examined than DMSO alone, especially when looking at cord blood derived stem cells. CONCLUSIONS There might not be an all-in-one cryoprotective solution suitable for every purpose regarding the cryopreservation of stem cell concentrates produced from different cell sources. However, when trying to reduce the DMSO amount used, hydroxyethyl starch of a molecular weight of 450,000 Dalton is a suitable option.

Transfusion-related alloimmune neutropenia with no pulmonary complications: one donor-five cases.

BACKGROUND: Neutrophil alloantibodies are well‐known triggers of transfusion‐related acute lung injury (TRALI) and also cause immune neutropenia. Alloimmune neutropenia due to transfusion is an isolated phenomenon that is only rarely identified. Its incidence is specified in the literature as being less than one in 10,000 transfused plasma‐containing units. We expect that this phenomenon is underreported.

Comparison of Simultaneous CD34+ and CD3+ Quantification with a Modified Stem Cell Enumeration Kit on Two Different Flow Cytometers

BACKGROUND Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively. METHODS CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate). RESULTS There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine. CONCLUSIONS The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.

The effect of cell concentrations from different cell populations on the viability of umbilical blood stem cells.

BACKGROUND In addition to bone marrow or peripheral blood derived stem cells, cord blood (CB) is an alternative source for hematopoietic stem cells. This report shows the impact of higher concentrations of leukocytes, mononu- clear cells (MNCs), and CD34-positive cells on the viability of CB derived stem cells after cryopreservation. METHODS Statistical analysis of data from 5520 CB units, prepared and cryopreserved from 2003 through 2011, was performed with appropriate software. Cell concentrations for leukocytes, platelets, red blood cells (RBCs), CD34-positive leukocytes, viable leukocytes, and MNCs were determined. The proliferation and differentiation capacity was assessed in cell culture assays. RESULTS Content of leukocytes, CD34-positive leukocytes, and MNCs decreased after thawing. The recovery rate of colony forming units (CFUs) (29.05%) correlated significantly with leukocytes, platelets, RBCs, MNCs, CD34- positive leukocytes, and viable leukocytes. The recovery rate for erythroblasts (3.33%) correlated significantly with leukocytes, CD34-positive leukocytes, MNCs, and viable leukocytes. In the different cell concentration groups only RBCs showed a negative influence on viability. The concentrations of leukocytes, platelets, and CD34-positive leukocytes before cryopreservation correlated positively with the concentrations of leukocytes, CD34-positive leukocytes, MNCs as well as with the cell viability after thawing. CONCLUSIONS Increased cell concentrations in CB do not limit the recovery of CD34-positive leukocytes nor the viability of leukocytes or the number of CFUs after thawing. On the contrary, CB units with high cell concentrations show a better outcome than units with low cell concentrations. Only RBCs seem to have a negative influence on CB quality.