T-cell subsets in autologous and allogeneic peripheral blood stem cell concentrates.

BACKGROUND AND OBJECTIVES: Regulatory T cells (Tregs) and other T-cell subsets are of importance in the setting of autologous and allogeneic stem cell transplantations. We conducted a study to assess the content of peripheral blood stem cell concentrates and related apheresis parameters in the autologous and allogeneic setting. MATERIAL AND METHODS: We characterized 53 donors, patients and peripheral blood stem cell concentrates (PBSC) regarding the content of CD45(+) cells, lymphocytes, CD3(+) cells, CD3(+) CD4(+) T cells, CD3(+) CD4(+) CD25(+) T cells, CD3(+) CD4(+) CD25(+) CD127(low/negative) Tregs and CD34(+) cells and calculated cell yields, recruitment factors and collection efficiency for all cell types. We compared allogeneic data with autologous data. RESULTS: Autologous PBSC show significantly lower concentrations of T-cell subsets compared to allogeneic PBSC (17,112/µl CD4(+), 14,858/µl CD4(+) CD25(+) and 1579/µl CD3(+) CD4(+) CD25(+) CD127(low/negative) Tregs in autologous compared to 65,539/µl CD4(+), 44,208(+) /µl CD4(+) CD25(+) and 5040/µl CD3(+) CD4(+) CD25(+) CD127(low/negative) Tregs in allogeneic PBSC, respectively), in contrast to CD34(+) concentrations (5342/µl CD34(+) in autologous compared to 2367/µl CD34(+) in allogeneic PBSC, respectively). Accordantly, all T-cell yields are lower in the autologous setting compared to allogeneic PBSC. However, recruitment factor and collection efficiency of all cell types are higher in autologous compared to allogeneic PBSC, but not all parameters differ significantly when groups are compared. CONCLUSION: T-cell subsets and especially Tregs are a substantial part of PBSC transplantation, as considerable recruitment during apheresis occurs. In large volume apheresis, the collection efficiency of Treg is comparable to that of CD34(+) cells, while recruitment factors are even higher.

The novel allele, HLA-B*08:113, identified in a German cord blood donor and his mother.

The novel allele HLA-B*08:113 identified in two related individuals of Caucasian origin is described.

Comparison of a new microscopic system for the measurement of residual leucocytes in apheresis platelets with flow cytometry and manual counting.

BACKGROUND AND OBJECTIVES: Since 2001, all blood components in Germany must be leucocyte depleted. Recently, a new method for quality control of depletion was introduced. Our study aimed at the validation of the method for routine use in apheresis platelet concentrates. MATERIALS AND METHODS: We compared the new ADAM-rWBC device with manual counting in the Nageotte chamber and flow cytometry, two standard methods, by measuring residual leucocytes in 40 units of apheresis platelet concentrates and in six geometrical dilution series. RESULTS: Cell counts of residual leucocytes in the 40 units were below 10(6) cells per component with all methods, although mean cell counts were approximately 5 and 6 times higher in flow cytometry and ADAM-rWBC, respectively, compared to the Nageotte chamber. No unit with <10(6) leucocytes was regarded as contaminated. The dilution series showed acceptable accuracy, especially in the range around the cut-off (approximately 4·5 cells/µl in components with a volume of 220 ml) for regarding a concentrate as contaminated with leucocytes. No sample spiked with more than 4·5 cells/µl was counted as having less. CONCLUSION: In comparison with manual counting and flow cytometry, the ADAM-rWBC device performed equally. The method is suitable for routine screening of leucocyte contamination of apheresis platelets.

The novel allele, HLA-B*07:68:02, identified in a German cord blood donor and her father.

The novel allele HLA-B*07:68:02 identified in two related individuals of Caucasian origin is described.

Human neutrophil alloantigen genotype frequencies among blood donors with Turkish and German descent.

Antibodies against the human neutrophil antigens (HNA) are able to stimulate transfusion reactions, autoimmune and neonatal neutropenia. The aim of this study was to determine the HNA allele frequencies in the largest ethnic minority group in Germany in comparison with the German population for predicting the risk of alloimmunization and associated transfusion reactions, as well as the risk of developing neonatal neutropenia for the newborn of racial mixed couples. However, there exists no data about HNA genotype distribution in Turkish population. DNA was isolated from blood samples of 119 German and 118 Turkish blood donors and typed them for HNA-1, -3, -4, and -5 by using a commercial polymerase chain reaction kit with sequence-specific primers (SSP-PCR) and compared the HNA genotype distribution of both groups. In German blood donors, the gene frequencies for HNA-1a and HNA-1b were 0.391 and 0.601, for HNA-3a and -3b, 0.744 and 0.256, for HNA-4a and -4b, 0.908 and 0.092, and for HNA-5a and -5bw, 0.731 and 0.269. In Turkish blood donors, we observed 0.420/0.564, 0.737/0.263, 0.881/0.119, and 0.754/0.246 for HNA-1a/1b, -3a/3b, -4a/4b, and -5a/5bw. No statistic significant difference between genotypes in these populations was observed. This study is the first to report HNA gene frequencies in a Turkish population. It showed that there is no difference of HNA genotype in blood donors with Turkish descent in comparison with German blood donors. The alternating transfusion of blood and blood components is no increased risk for developing alloantibodies against HNA antigens. In pregnancy of mixed couples no special screening programs for HNA are necessary.

Association of blood group A with early-onset ovarian hyperstimulation syndrome.

PURPOSE OF THE STUDY: Ovarian hyperstimulation syndrome is a potentially life-threatening complication during controlled ovarian stimulation for fertility treatment. Since no association of this condition with ABO blood groups was known, we compared ABO antigens with severity and onset of symptoms in a case-control study. PATIENTS AND METHODS: One hundred and twenty-one patients, mainly Caucasians, were hospitalized because of ovarian hyperstimulation syndrome after receiving in vitro fertilisation, in the period from January 2000 to February 2007. Severity of symptoms, pregnancy rate and ABO blood group were collated. The ABO blood group distribution was compared to four independent control groups. RESULTS: Blood group A was markedly more frequent and blood group O less frequent in patients with ovarian hyperstimulation syndrome compared to the blood group distribution in all control cohorts. The odds ratio for patients undergoing controlled ovarian stimulation with blood group A versus O to develop the early-onset form of this condition was 2.171 (p-value 0.002). No association for late-onset form could be found. The overall pregnancy rate was 50.4% and three times higher in the group of late-onset ovarian hyperstimulation syndrome compared to the early-onset form. Four patients developed thromboses in the jugular or subclavian vein, none of whom had blood group O. CONCLUSION: Blood group A may be associated with early-onset ovarian hyperstimulation syndrome in Caucasians. Depending on further studies, this possible association may be considered for an individualized hormone dosing in controlled ovarian stimulation.

Identification of the novel allele, HLA-A*01:234, in the mother of a German cord blood donor.

HLA-A*01:234 was identified by next-generation sequencing and confirmed by Sanger sequencing.

Identification of the novel allele, HLA-C*01:136 in a German cord blood donor originating from Azerbaijan.

HLA-C*01:136 identified by next generation sequencing and confirmed by Sanger sequencing.

High-dose treatment with carboplatin, etoposide, and ifosfamide followed by autologous stem-cell transplantation in relapsed or refractory germ cell cancer: a phase I/II study. The German Testicular Cancer Cooperative Study Group.

PURPOSE: This trial evaluated the toxicity and efficacy of high-dose carboplatin, etoposide, and ifosfamide followed by autologous stem-cell transplantation in patients with refractory or relapsed germ cell cancer. PATIENTS AND METHODS: Between August 1989 and September 1992, 74 patients with refractory or recurrent germ-cell tumors received one cycle of escalating doses of carboplatin (1,500 to 2,000 mg/m2), etoposide (1,200 to 2,400 mg/m2), and ifosfamide (0 to 10 g/m2). Before high-dose therapy, two cycles of conventional-dose cisplatin, etoposide, and ifosfamide were administered to assess tumor responsiveness. Seventy-four patients were assessable for toxicity and 68 for response. RESULTS: The doses of carboplatin 1,500 mg/m2, etoposide 2,400 mg/m2, and ifosfamide 10 g/m2 appeared to be safe. At this dosage, we treated 20 patients and observed World Health Organization (WHO) grade 3 and 4 hematotoxicity (100%), nausea (100%), diarrhea (30%), and hepatotoxicity (10%). All patients developed granulocytopenic fever. At carboplatin doses of 1,500 mg/m2, kidney toxicity was mild, with a median maximum creatinine level of 1.4 mg/dL (range, 1.1 to 3.0 mg/dL). However, at carboplatin doses of 1,750 and 2,000 mg/m2, we observed nonacceptable nephrotoxicity and neurotoxicity. Two (3%) patients died of treatment-related complications. Six patients required hemodialysis, which was temporary in five patients and permanent in one. Objective responses were obtained in 43 of 68 (63%) patients, including 21 (31%) complete remissions (CRs) and 14 (20%) inoperable partial remissions (PRs) with marker normalization. The median observation time of surviving patients was 12 months (range, 2 to 32). The probabilities of overall survival, event-free survival, and the relapse-free survival at 2 years were 44% (SD 8%), 35% (SD 6%), and 67% (SD 9%), respectively. Patients with disease refractory to conventional-dose pretreatment had a poor prognosis, with only one of 23 patients surviving event-free at 7 months after high-dose chemotherapy (HDT). In contrast, 24 of 45 (53.3%) patients with sensitive disease survive event-free with a probability of event-free survival at 2 years of 50% (SD 8%). CONCLUSION: High-dose carboplatin, etoposide, and ifosfamide plus autologous stem-cell transplantation can be used in refractory and relapsed germ cell cancer with acceptable toxicity, and represents an effective, potentially curative salvage treatment.

Hematopoietic rescue after high-dose chemotherapy using autologous peripheral-blood progenitor cells or bone marrow: a randomized comparison.

PURPOSE: To compare autologous bone marrow (BM) with peripheral-blood progenitor cells (PBPC) as hematopoietic rescue after high-dose chemotherapy (HDCT). PATIENTS AND METHODS: From January 1991 until April 1993, 47 consecutive patients with relapsed or refractory germ cell tumors were randomized to either BM harvest or collection of PBPC mobilized by chemotherapy plus granulocyte colony-stimulating factor (G-CSF). After additional conventional-dose salvage treatment, all patients received HDCT with carboplatin 1,500 mg/m2, etoposide 2,400 mg/m2, and ifosfamide 10 g/m2 with either BM or PBPC rescue. RESULTS: Forty-six patients were assessable for hematologic reconstitution, and one patient died on day +4 before engraftment. Rescue using PBPC resulted in a significantly shorter recovery time to neutrophil counts more than 500/microL (10.0 v 11.0 days, P < .01), neutrophil counts more than 1,000/microL (10.0 v 12.0 days, P = .001), and platelet counts more than 20,000/microL (10.0 v 17.0 days, P < .01), as well as in fewer days to transfusion independence from RBCs (8.0 v 12.0, P < .05) and platelets (9.0 v 12.0, P < .01) and fewer days of intravenous (IV) antibiotics (9.0 v 11.0, P < .05). However, no statistical differences in transfusion requirements or in other clinical outcome variables were observed. Overall survival and event-free survival also were not different in the two study arms. CONCLUSION: We conclude that the use of PBPC mobilized by chemotherapy plus G-CSF results in sustained trilineage reconstitution after HDCT, which occurs more rapidly as compared with BM. The earlier hematologic reconstitution in patients with PBPC rescue significantly reduces the time to transfusion independence.

Fractionated total body irradiation and high-dose VP-16 with purged autologous bone marrow rescue for children with high risk relapsed acute lymphoblastic leukemia.

Twenty-two children with ALL in high risk second (n = 13), third or subsequent complete remission (n = 9) were treated with high-dose VP-16 60 mg/kg and fractionated total body irradiation (fTBI) 12 Gy, 2 x 2 Gy daily followed by autologous BM rescue. Prior to transplantation all patients had been treated according to intensive German BFM front-line or BFM relapse protocols. In all cases the marrow was purged using monoclonal antibodies attached to magnetic microspheres. All patients engrafted. There was no severe toxicity related to the pre-transplant high-dose chemoradiotherapy. Two patients died in the early course of transplantation from infections (Legionella and Aspergillus). Sixteen patients relapsed within 259 days (median 109 days); 13 died from leukemia. Four patients are alive in CR at a median of 1328 days with a Karnofsky score of 100%. The Kaplan-Meier estimation shows a probability of event-free survival (EFS) of 18% and a probability of relapse of 80%. Considering the otherwise poor prognosis of these children the results are acceptable although the high relapse rate is still disappointing. We conclude that high-dose VP-16 and fTBI combined with ABMT is a curative treatment for some children and should therefore be considered for those who lack an HLA-identical sibling donor. In future better therapy concepts are needed either in pre-transplant conditioning regimens or in post-transplant treatment schedules.

Immunogenetic and serological investigations in nonpregnant and in pregnant women with a history of recurrent spontaneous abortions. German RSA/IVIG Study Group.

In the context of a controlled multicenter study on intravenous immunoglobulin (IVIG) treatment of patients with a history of unexplained recurrent spontaneous abortions (RSA), a number of controversial immunological parameters were evaluated prior to and during pregnancy with respect to their diagnostic and/or prognostic significance. A total of 390 serum samples from 52 patients were investigated. Sharing of 2 or more HLA (A, B, DR, DQ) antigens was significantly more frequent in RSA couples than in controls. The rate of cytotoxic or Fc-receptor (FcR)-blocking antibodies was not significantly lower in RSA patients than in individuals with normal pregnancies. Both tumor necrosis factor-alpha (TNF-alpha) levels and IgG anticardiolipin antibodies (IgG-ACA) were significantly increased in the patient group. While the occurrence of HLA sharing, cytotoxic/FcR-blocking antibodies and IgG-ACA did not correlate with the outcome of pregnancy, TNF-alpha levels were found to be significantly higher in patients with subsequent miscarriage than in those with successful pregnancy. IgG-ACA, if present, significantly decreased during the course of successful pregnancy but remained high in patients with subsequent abortion. It is concluded that the diagnostic and/or prognostic value of HLA sharing and cytotoxic/FcR-blocking antibodies has been overestimated while TNF-alpha and ACA levels are potential diagnostic markers and/or exhibit prognostic significance in subgroups of RSA patients.

Separation of peripheral blood derived stem cells in children.

We performed 28 separations in 10 children from 14 to 44 kg (median: 24 kg) using either the Cobe Spectra or the Fresenius AS 104 cell separator. For children below 20 kg, human albumin solution was used for the last 200 ml of the priming procedure instead of NaCl. Blood flow was reduced to 30 - 50 ml/min depending on the childrens' size and weight to prevent citrate reactions. Within 149 - 337 minutes we processed 3 x the patients' total blood volume and collected 326.9 x 10(6) (76.5-1, 140.0) MNC and 2.03 x 10(6) (0.14-10.12) CD34+ cells per kg body weight. These results were comparable to the results we previously obtained in adults. We conclude from these initial results that PBSC separations with both devices can be adapted successfully to the needs for paediatric patients.

Collection of hyperconcentrated platelets with Trima Accel.

BACKGROUND AND OBJECTIVES: Lowering the plasma content in single-donor platelet (PLT) concentrates well below 30% implies the need to collect platelets at very high concentrations. Trima Accel (TA) is validated for collection below 4000 x 10(3) PLTs/microl. We evaluated its performance at 5000 x 10(3) PLTs/microl. MATERIALS AND METHODS: Twenty blood donors underwent apheresis with TA twice collecting either a hyperconcentrated or a standard single-donor platelet concentrate with a target platelet concentration of 5000 or 1200 x 10(3) PLTs/microl, respectively. We analysed the collection efficiency, the collection rate and the quality of the collected by-plasma. RESULTS: We collected 20 hyperconcentrated and 20 standard units containing 2.56 +/- 0.5 and 3.39 +/- 0.4 x 10(11) PLTs at a concentration of 4518 +/- 978 and 1374 +/- 166 x 10(3) PLTs/microl in 45 +/- 8 and 39 +/- 6 min resulting in a collection efficiency of 47.5 +/- 10.0 and 70.7 +/- 7.9% and a collection rate of 5.9 +/- 1.4 and 8.8 +/- 1.5 x 10(9) PLTs/min, respectively (all results expressed as mean +/- standard deviation). The collected by-plasma showed a very high grade of cell purity and a satisfactory recovery of the clotting factors. CONCLUSION: Although TA is a suitable device for PLT collection at very high concentrations, improvements are desirable to further increase the productivity above its currently validated upper collect concentration limit.

Comparison of the performance of microtube column systems and solid-phase systems and the tube low-ionic-strength solution additive indirect antiglobulin test in the detection of red cell alloantibodies.

To compare the performance of seven currently available test systems in the detection of erythrocyte alloantibodies (ab), we tested in parallel 446 sera samples containing red cell ab [368 sera samples with ab that are assumed to be clinically significant (cs-ab) and 78 sera samples with ab that are assumed to be of minor clinical significance (ms-ab)] using the tube spin low-ionic-strength solution (addition method) indirect antiglobulin test (tube LISS-IAT), three microtube column agglutination techniques (DiaMed-ID, Ortho BioVue and Bio-Rad Scangel), one affinity adherence test system (CLB/Mast CellBind Screen) and two solid-phase tests [Biotest Solidscreen II and Immucor Capture-R Ready-Screen (4)]. To address the specificity of the three test systems under routine conditions, results of 4566 patient samples obtained using the tube LISS-IAT, results of 5205 patient samples obtained using the Scangel and results of 3560 samples obtained using the Capture-R were evaluated. The DiaMed-ID detected 344 cs-ab and 43 ms-ab, BioVue 333 cs-ab and 48 ms-ab, Scangel 348 cs-ab and 62 ms-ab, CellBind Screen 346 cs-ab and 47 ms-ab, Solidscreen 330 cs-ab and 38 ms-ab, Capture-R 358 cs-ab and 45 ms-ab and LISS-IAT 159 cs-ab and 12 ms-ab. In routine practice, erythrocyte cs-ab could be identified in 61 (67.8%) of 90 reactive sera (specificity: 98.6%) in the tube LISS-IAT, in 169 (58.7%) of 288 (94.4%) in Bio-Rad Scangel and in 101 (51.0%) of 198 reactive sera (94.3%) in Capture-R. We conclude that the sensitivity of the microcolumn, affinity adherence and solid-phase test systems in the detection of cs-ab was similar and was markedly superior to that of the conventional tube LISS-IAT. All high-sensitive test systems produced higher rates of false positives and ms-ab compared to the tube test. An individual cost-benefit analysis, considering the recent knowledge about the clinical significance of weak-reactive cs-ab, should be performed in every institution to decide whether and if so which high-sensitive screening system should be applied.

Hyperconcentrated platelets stored in additive solution: aspects on productivity and in vitro quality.

BACKGROUND AND OBJECTIVES: New platelet (PLT) additive solutions (PASs) allow a plasma carryover of < 30% in PLT concentrates. This implicates the need to collect apheresis PLT concentrates at very high PLT concentrations: so-called dry PLTs (DPs). We used the TRIMA, with software version 4 (TRIMA V4), to collect such DPs and investigated the in vitro quality of these PLTs when stored in the new modified PAS-III (PAS-IIIM). MATERIALS AND METHODS: TRIMA V4 was programmed to collect 6.0 x 10(11) PLTs at a concentration of 5000 x 10(3) PLTs/microl. Two DPs were pooled, split into four equal parts and diluted to obtain secondary pools (SPs) consisting of 70% PAS-III/30% plasma, 70% PAS-IIIM/30% plasma, 80% PAS-IIIM/20% plasma or 100% plasma. In vitro testing was performed on days 0, 1, 5 and 7. Collection efficiency (CE), collection rate (CR) and PLT yield were calculated for each donation. RESULTS: Thirty-two runs with TRIMA V4 were performed, collecting 6.58 +/- 0.74 x 10(11) PLTs at a concentration of 4255 +/- 914 x 10(3)/microl in 99 +/- 19.9 min, resulting in a CE of 65.3 +/- 8.2% and a CR of 6.92 +/- 1.6 x 10(9) PLTs/min. On day 0, 34-37% of the PLTs in the units prepared for storage were already activated. PLTs stored in 70% or 80% PAS-IIIM showed superior in vitro quality compared to PLTs stored in PAS-III. CONCLUSIONS: TRIMA V4 is a suitable device for the collection of DPs. Nevertheless, improvements are desirable to further increase the ability to concentrate PLTs at very high levels. The storage of apheresis-derived PLTs in PAS III-M is a very promising approach, even at a plasma carryover of < 30%.

Effective derosetting of mononuclear cells isolated by immunomagnetic beads.

A rapid and effective method for the derosetting of peripheral mononuclear cells (PMNC) isolated by CD8 monoclonal antibody (MoAb) coated immunomagnetic beads (IMB) is described. PMNC are positively selected and enriched by CD8-IMB and then derosetted from the IMB by a polyclonal antibody (PoAb) preparation against mouse Fab. Our experiments confirm the feasibility of this method with a high derosetting efficiency.

Sporadic occurrence of Diego A antigens and antibodies in Berlin.

BACKGROUND: The antigen Dia is common among Mongols (5-12%), but rare in Caucasians (< 1%). OBJECTIVE: The accidental discovery of Diego A (Dia) antibodies in a South American tourist and in an elderly German patient prompted us to investigate the frequency of Dia antigens in the German population. DESIGN: 1,352 German random blood donors, all living in Berlin or in the areas around, were tested for the presence of the Dia antigen on their red blood cells. All donors were Caucasians. RESULTS: Dia was found in 12 individuals (0.89%). CONCLUSIONS: We think that Dia is a low-frequency antigen in the European population. The admixture of Mongol genes following wars or migration is only of low or no importance.