Effects of platelet concentrate storage time reduction in patients after blood stem cell transplantation.

OBJECTIVE: To evaluate the clinical effect of platelet concentrate (PC) transfusions after PC storage time reduction to 4 days. PATIENTS AND METHODS: This was a single-centre cohort study comparing two 3-month periods of time, before and after the reduction of PC storage time from 5 to 4 days. Seventy-seven consecutive patients with PC transfusions were enrolled after blood stem cell transplantation. Corrected platelet count increment (CCI) on the morning after transfusion, time to next platelet transfusion, need for red blood cell (RBC) transfusion and clinical bleeding symptoms were compared. RESULTS: Platelet concentrate storage time was reduced between period 1 (storage for up to 5 days, median storage time 78 h, range 11-136 h) and period 2 (storage for up to 4 days, median storage time 53 h, range 11-112 h). Patients were comparable for age, weight, body surface area, underlying disorder, type of transplantation and transfused platelet dose. The CCI increased from a median of 4 (range 0-20) to 8 (0-68) × 10(9) /l per 10(11) platelets/m(2) (P < 0·0001). Time to next PC transfusion increased from 1·1 to 2·0 days (P < 0·0001). Any bleeding symptom was noted in 20 of 36 patients (56%) vs. 9/41 patients (22%, P < 0·01). Nose bleeds, haematuria and bleeding at more than one site were significantly reduced. Frequency of RBC transfusion within 5 days after PC transfusion was reduced from 74 to 58% (P < 0·0001). CONCLUSION: Platelet concentrate storage time shortening was associated with highly significant CCI increase, reduced RC needs and lower patient numbers with bleeding events.

Semaphorin 7A inhibits platelet production from CD34+ progenitor cells.

BACKGROUND: The multifunctional protein semaphorin 7A (Sema7A) may have regulatory effects on blood cell differentiation via its receptors ß1-integrin and plexin C1. As thrombocytopenia can be treated with transfusion of ex vivo CD34(+) cell-derived megakaryocytes, we investigated the effect of Sema7A on differentiation of CD34(+) progenitor cells into megakaryocytes and platelets. METHODS: Megakaryocytes and platelets were differentiated with a specific cytokine cocktail (CC) from CD34(+) progenitor cells in the presence or absence of Sema7A. Expression of cell markers CD41, CD42a and CD61 or detection of the activation of the signal mediator focal adhesion kinase (FAK) was performed by flow cytometry, cytokine secretion by Luminex technology, and megakaryocyte cell density and morphology by microscopic studies. Sema7A levels in vivo were assessed by real-time PCR and ELISA in hematological patients undergoing chemotherapy. RESULTS: CD34(+) progenitor cells expressed the receptors for Sema7A. Expression of CD41, CD42a and CD61 was markedly reduced in the presence of Sema7A, after CC-dependent platelet production from CD34(+) progenitor cells. As revealed by microscopic analysis, megakaryocyte cell density was significantly lower in the presence of Sema7A as compared with controls. Blocking of CD29 abrogated the Sema7A-mediated inhibition. Sema7A activated FAK in CD34(+) progenitor cells and significantly increased secretion of the proinflammatory cytokines IL-6, IL-8 and GM-CSF. Finally, Sema7A levels were up-regulated in 50% of patients after chemotherapy. CONCLUSIONS: Sema7A markedly reduces the production rates of megakaryocytes and platelets from CD34(+) progenitor cells. Hence, up-regulation of Sema7A may be a major risk factor for a reduced platelet repopulation after hematopoietic stem cell transplantation.

Hepatitis E: an emerging infectious disease in Germany?

Increased frequencies of HEV infections have been reported in several industrialized countries. We suggest that this finding might be explained by a better awareness of the disease and not by an increased incidence. Although reported HEV infections increased more than 6-fold in Germany in recent years, the seroprevalence remained unchanged (2 %).

Intentional ABO-incompatible lung transplantation.

We report on a case of intentional blood group incompatible lung transplantation. A blood group O cystic fibrosis patient was mechanically ventilated and put on interventional lung assist for severe respiratory decompensation. Since timely allocation of a blood group O donor lung was impossible, an AB deceased donor lung rescue allocation was accepted and the transplant performed using a pre-, peri- and postoperative antibody depletion protocol including plasmapheresis, ivIg administration, rituximab and immunoadsorption. Nine months after the transplant the patient is at home and well.

A comparative study of adverse reactions occurring after administration of glycosylated granulocyte colony stimulating factor and/or dexamethasone for mobilization of neutrophils in healthy donors.

Both granulocyte colony-stimulating factor (G-CSF) and dexamethasone (DXM) are used for neutrophil (PMN) mobilization and collection. This prospective study was aimed to evaluate and compare the rate, severity and clinical significance of adverse reactions of these drugs alone and in combination in healthy donors. PMN mobilization was carried out using dexamethasone alone (8 mg orally; n=25) or glycosylated G-CSF alone (Lenograstim, 5 microg/kg subcutaneously, n=24) or in combination (n=23) prior to a standard granulocyte apheresis on the Spectra cell separator. The number of PMNs counted in the mobilized peripheral blood of the donors was 7.0 (3.6-20.4) x10(9)/L (DXM), 25.2 (15.5-49.7) x10(9)/L (G-CSF), and 31.6 (20.0-43.0) x10(9)/L (G-CSF+DXM), corresponding to PMN apheresis yields of 13 (8-43) x10(9)/U, 56 (34-118) x10(9)/U, and 83 (33-117) x10(9)/U, respectively. The three groups had comparable percentages of donors with at least one adverse effect (ranging from 75 to 80%), but the G-CSF-containing regimens were generally more toxic, as was reflected by higher percentages of donors with moderate to severe adverse reactions and higher overall severity scores of 2.28 (G-CSF) and 2.08 (G-CSF+DXM) compared with 1.33 in the DXM group ( p

Collection of two consecutive neutrophil concentrates for transfusion from donors mobilized with glycosylated granulocyte-CSF plus dexamethasone.

BACKGROUND: The objective of this study was to establish a mobilization and apheresis regimen for collection of two consecutive polymorphonuclear neutrophil (PMN) concentrates from the same donor. STUDY DESIGN AND METHODS: In this prospective study, 111 healthy unrelated volunteers underwent either one (Group 1, n = 57) or two consecutive granulocyte apheresis procedure (Group 2, n = 54) using the a cell separator (Spectra). Both Group 1 and 2 donors were initially mobilized with glycosylated G-CSF 6.0 micro g per kg (range, 5.2-7.0 micro g/kg) subcutaneously plus oral dexa-methasone (DXM, 8 mg) and underwent granulocyte apheresis (GA-1) 16 hours (range, 13-18 hr) after initial G-CSF+DXM. Group 2 donors were remobilized with a second DXM dose of 8 mg (n = 13), 4 mg (n = 15), 1.5 mg (n = 13), or none (n = 13), and a second apheresis (GA-2) was run 40 hours (range, 37-42 hr) after G-CSF+DXM administration and 12 hours after remobilization with DXM alone. RESULTS: Based on equivalent median preapheresis WBC and PMN counts of around 35 x 10(9) WBCs per L and 33 x 10(9) PMNs per L after initial mobilization the GA-1 yields were 85 x 10(9) PMNs per U (range, 34-150) in Group 1 and 75 x 10(9) PMNs per U (range, 35-135) in Group 2 (p = 0.14, NS). In Group 2, median preapheresis values of 19.6 x 10(9) WBCs per L (range, 9.5-37.0) and 16.6 x 10(9) PMNs per L (range, 8.8-34.8) were measured after remobilization and GA-2 yields of 49 x 10(9) WBCs per U (range, 26-113) and 42 x 10(9) PMNs per U (range, 21-95) were obtained. Borderline statistical differences in the GA-2 yields were observed from the remobilized donors: 8 mg: 36 x 10(9) PMNs per U (range, 23-60); 4 mg: 47 x 10(9) PMNs per U (range, 21-56) (p

Equivalent mobilization and collection of granulocytes for transfusion after administration of glycosylated G-CSF (3 microg/kg) plus dexamethasone versus glycosylated G-CSF (12 microg/kg) alone.

BACKGROUND: The aim of this study was to find a regimen for mobilization and collection of granulocytes that combines low-dose G-CSF administration with satisfactory PMN mobilization and apheresis at a low rate of donor adverse reactions. STUDY DESIGN AND METHODS: In a prospective study, 52 healthy unrelated volunteers received a single subcutaneous injection of glycosylated G-CSF (Lenograstim Chugai-Pharma, Frankfurt, Germany) at medians of 3.1 (range, 2.4-3.6) microg per kg plus dexamethasone (8 mg orally; n = 29) or at 11.8 (7.1-18.5) microg of lenograstim per kg (p < or = 0.0001) without dexamethasone (n = 23) and underwent standard apheresis using the PMN program of a cell separator (Spectra, COBE [now Gambro] BCT). WBC and PMN mobilization results and apheresis yields were compared and the severity and clinical significance of donor adverse reactions was evaluated. RESULTS: For the low-dose G-CSF plus dexamethasone versus the high-dose G-CSF alone group, similar mobilization results were observed for WBCs with 31.3 (19.1-44.9) x 10(9) per L versus 27.5 (19.2-44.0) x 10(9) per L (p = 0.21, NS) and PMNs with 29.0 (17.6-42.2) x 10(9) per L versus 25.2 (16.2-39.0) x 10(9) per L (p = 0.08, NS). The PMN apheresis yields were equal with 70 (39-139) x 10(9) per unit with low-dose G-CSF versus 68 (33-120) x 10(9) per unit in the high-dose G-CSF group (p = 0.83, NS). Regarding donor adverse reactions, 7 out of 29 (24%) and 8 out of 23 donors (35%) reported moderate or severe symptoms. The character of these reactions was different; symptoms of greater clinical significance and a higher need for analgesics were observed in the high-dose G-CSF group. CONCLUSIONS: A Lenograstim dose of 3 microg per kg plus DXM assures effective PMN mobilization and acceptable apheresis components. The combination of glycosylated G-CSF with DXM allows a significant dose reduction in G-CSF for PMN mobilization and collection as compared with higher G-CSF doses alone. In the high-dose G-CSF mobilization group, adverse reactions were more severe and required more analgesics.

Comparative evaluation of commonly used clones and fluorochrome conjugates of monoclonal antibodies for CD34 antigen detection.

CD34+ cell enumeration is currently the most appropriate technique for hematopoietic graft quality control. At the same time, numerous CD34 mAb have become commercially available. This study was designed to compare the commonly used clones 8G12 and QBEND-10 with the new clones 581 and BIRMA-K3. All available fluorochrome conjugates were tested: FITC, PE, and PE-Cy5 or PerCP for QBEND, BIRMA, 581, and 8G12 and FITC and PE for 581. Bone marrow from healthy donors (n = 5) and leukapheresis samples (n = 16) were stained, according to each manufacturer's protocol and analyzed using the FACScan. The following parameters were evaluated: % CD34+ cells detected and percentage of deviation from the median within each sample; mean channel fluorescence intensity of the CD34+ cells; resolution index (median channel fluorescence intensity of CD34+ cells/monocytes), % overlapping of CD34+ cell and monocyte fluorescence; proportion of CD34+ events after blocking with the same unlabeled clone; values of compensation requirements. Tables with results for each evaluated parameter separately were created, and rank points were applied. These scores represented the quality performance of the studied clones and fluorochrome conjugates and may be summarized as follows: 581 and 8G12 produced the best results, followed by BIRMA-K3 and QBEND10. The fluorochrome sequence was PE, PE-Cy5, PerCP, and FITC. However, all PE conjugates of the studied clones provided highly comparable results and conditions for CD34+ cell enumeration. When antigen coexpression must be studied and another dye than PE must be applied for CD34+ cell discrimination, the PE-Cy5 conjugates should be preferred.

Flow cytometry quantification of CD34+ cells and other leukocyte subpopulations in frozen-thawed blood cell suspensions: investigation of a new teflon container for cryopreservation of hematopoietic progenitor cells.

BACKGROUND: Cryopreservation is the only available method for the long-time maintenance of blood cells. The present study was designed to prove: (i) the reliability of multiparameter flow cytometry (MFC) for estimation of CD34+ cells in frozen-thawed cell suspensions and (ii) the acceptability of a new teflon container for the cryopreservation of hematopoietic progenitor cells. MATERIALS AND METHODS: Each of 15 ABO-compatible buffy coats (BC) were pooled, and mononuclear cells (MNC) were then separated with the Fresenius AS 104 device (n = 10). MNC harvested by apheresis were then divided into 2 portions and transferred pairwise into either the new Fresenius or into Gambro cryopreservation containers. Paired samples were frozen at controlled rates (9% DMSO final concentration) and stored at -196 degrees C in liquid nitrogen for 2 weeks. Leukocyte, MNC and differential blood counts and proportions of CD3+, CD4+, CD8+, CD14+ and CD34+ cells were assessed from the pooled BC, the apheresis products, and the frozen-thawed samples. Methyl cellulose culture assays as well as trypan blue viability staining were also carried out. RESULTS: The mean content of the divided apheresis products was 4.9 x 10(9) leukocytes with 86% MNC, 6.89 x 10(6) CD34+ cells, 2.1 x 10(5) granulocyte-macrophage colony-forming units (CFU-GM) and 7.1 x 10(5) erythroid burst-forming units (BFU-E). As expected, there were virtually no granulocytes after freezing in both types of container. The corresponding mean cell content was as follows: 6.3 x 10(6) CD34+ cells, 2.5 x 10(5) CFU-GM, and 8.1 x 10(5) BFU-E in Fresenius containers, and 6.1 x 10(6) CD34+ cells, 1.3 x 10(5) CFU-GM, and 7.7 x 10(5) BFU-E in Gambro containers. The mean MNC viability of the samples frozen in Fresenius was 81.5% and 82.7% in the Gambro containers. MFC was found to compare with stained smear differentials. CD34+ cell counts correlated with CFU-GM (0.69, p = 0.03) and BFU-E (0.63, p = 0.02) colony formation. CONCLUSIONS: The study reported here revealed no significant differences between the 2 types of storage containers. The new Fresenius teflon container could thus be recommended for cryopreservation of hematopoietic progenitor cells. MFC provided reliable data on CD34+ cell content and leukocyte subset composition of the frozen-thawed cell suspension.