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.

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.

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.

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*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.

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.

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.

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.

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.

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.

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%.

The in vitro quality of washed, prestorage leucocyte-depleted red blood cell concentrates.

BACKGROUND AND OBJECTIVES: No data are currently available on the quality of washed prestorage leucocyte-depleted red blood cell concentrates (RCCs). MATERIALS AND METHODS: Five groups of RCCs stored in additive solution (SAG-M) were washed. The groups differed in the age of RCCs (2-5 days or 11-15 days), the temperature during the washing procedure and a 6-h storage period (4 degrees C or room temperature) and the washing solution (saline, SAG-M or 5% albumin). We measured ATP, 2,3-diphosphoglycerate (2,3-DPG), haemolysis, blood cell count, Na(+), K(+), pH, pO(2), pCO(2) and lactate, before and after the washing procedure and hourly during the 6-h postwash storage period. RESULTS: The erythrocyte ATP content increased by 2-13%, relative to the baseline value, during the washing procedure. The 2,3-DPG level decreased by 15-35% in 2-6-day-old RCCs and by 30-40% in 11-15-day-old RCCs (relative to baseline values) during the washing procedure. In RCCs that were washed and stored at room temperature, and in 2-week-old RCCs, a further decrease in 2,3-DPG of up to 40%, relative to the baseline value, was observed during the 6-h postwash time-period. CONCLUSIONS: Washing of RCCs stored in SAG-M results in a considerable, significant loss of erythrocyte 2,3-DPG, especially in older RCCs. This loss increases in during a 6-h storage period postwash, even at 4 degrees C. This loss of erythrocyte quality might well outweigh the benefits of washed SAG-M RCCs during massive transfusion in neonates.

First comparison of productivity and citrate donor load between the Trima version 4 (dual-stage filler) and the Trima Accel (single-stage filler) in the same donors.

BACKGROUND AND OBJECTIVES: Aside from new software the blood cell separator TRIMA (GambroBCT) also received a newly designed separation chamber offering a novel single stage separation technology, called Trima Accel. We evaluated this new system focusing on productivity and donor comfort by comparing it to the previous version (Trima version 4) in collecting single-donor platelet concentrates (SD-PCs) and plasma. MATERIALS AND METHODS: Each of 20 donors underwent platelet apheresis using both devices. We compared the collection efficiency (CE), the collections rate (CR), the volume of the collected plasma and the residual leukocytes. Furthermore we compared donor comfort in terms of duration of the donation, flow of citrate back to the donor and platelet and white blood cell (WBC) loss. RESULTS: While the number of collected platelets and the platelet concentration did not differ significantly between both techniques the time of the procedure was reduced by 15.6% with Trima Accel. This results in an increase of the CR and CE of 25% and 15% respectively when using Trima Accel. Log normal probability plotting of WBC counts showed that both techniques complied with the European and the US leukoreduction guidelines. The mean flow of ACDA to the donor per minute and per litre blood volume was also reduced by 20%. CONCLUSION: These data show that the Trima Accel represents a further improvement in apheresis platelet production with a better productivity and donor comfort, especially regarding the mean flow of ACDA to the donor.

Storage of single donor platelet concentrates: paired comparison of storage as single or double concentrates.

Modern cell separators allow the collection of two plateletpheresis concentrates (PCs) at one session. This study evaluates the quality of PCs stored as double concentrates in standard storage containers of two manufacturers. We collected 20 PCs that contained 4.5 x 10(11) platelets in 375 ml plasma (10 using the COBE Spectra and 10 using the Fresenius AS.TEC 204 with 500 ml bags) that were split into one unit of 3.0 x 10(11) platelets in 250 ml (3.0-PC) and one of 1.5 x 10(11) platelets in 125 ml (1.5-PC). Storage of one 3.0-PC per bag of a two-bag system corresponded to storage conditions for double PCs and storage of one 1.5-PC per bag to storage conditions of single PCs. Cell counts, blood gas analysis, glucose and lactate levels, platelet aggregation, and activation and plasma levels of beta- thromboglobulin (beta-TG) and complement factor 3a (C3a) were measured before storage and again on days 3 and 5. COBE 3.0-PCs demonstrated less pH rise, lactate production, CD 62P expression and beta-TG plasma levels, and better aggregability after storage than COBE 1.5-PCs. Fresenius 1.5-PCs had similar platelet quality to COBE 3.0-PCs. Fresenius 3.0-PCs showed a fall of pH (day 5: 6.22 +/- 0.56), the highest amount of anaerobic glycolysis compared to all other storage conditions investigated, high CD 62P- expression and beta-TG plasma levels, and impaired aggregability on days 3 and 5. The highest C3a levels were found in COBE 1.5-PCs. 3.0 x 10(11) platelets in 250 ml plasma should be stored either in one bag of the COBE system or in two 500 ml bags of the Fresenius system. The COBE two-bag system allows the storage of two PCs without loss of platelet quality. Two PCs should not be stored in the Fresenius C4L 500 ml storage containers.

Paired comparison of apheresis platelet function after storage in two containers.

Platelet quality after storage strongly depends on the pre-storage quality as well as on the storage conditions determined by the storage container. In this paired study, we evaluated two different containers (MedSep CLX and Delmed DPL-110). The Fresenius AS104 cell separator was used to prepare 17 platelet concentrates that were split and distributed into the containers to be compared. Cell counts, blood gas analysis, morphological scores, glucose and lactate levels, platelet activation, and platelet aggregation were measured before splitting at the day of preparation and after storage at day 3 and day 5. At day 3, there was no significant difference between the two bags apart from increased lactate and decreased pCO(2) concentrations in the CLX bags. At day 5 there were significantly higher lactate concentrations, pO(2) levels, and aggregation after stimulation in the CLX group, while the glucose and pCO(2) concentrations were significantly lower in these platelet concentrates as compared to the DPL-110 group. However, these parameters did not influence the functional parameters tested. While the platelet quality decreased during storage in all bags, the functional changes were nearly identical in both bags tested. We conclude that both bags are equivalent for 5-day storage of platelet concentrates.

Comparison of a new WBC-reduction system and the standard plateletpheresis protocol in the same donors.

BACKGROUND: A cell separator (Spectra, Gambro BCT) with an integrated leukoreduction system (LRS) for producing WBC-reduced single-donor platelet concentrates has been shown to result in a slightly reduced collection efficiency as compared to the former Spectra system without LRS. A novel modified system for improved collection efficiencies (LRS Turbo, Gambro BCT) was evaluated. STUDY DESIGN AND METHODS: Each of 37 donors underwent plateletpheresis using the LRS Turbo (LRS-T) and the standard LRS (LRS) of the Spectra cell separator. The collection efficiency and WBC contamination of the different techniques were compared. Platelets were counted automatically and WBCs were counted by using one or two full grids of a Nageotte chamber. RESULTS: The preseparation and postseparation numbers of RBCs, WBCs, and platelets, as well as the number of collected platelets, did not differ for the two techniques. In the LRS-T separations, the collection efficiency was 112 percent of that in the LRS procedures. Median residual WBCs in the platelet components were 0.0256 x 10(6) per LRS-T procedure and 0.0253 x 10(6) per LRS procedure. The purity of the LRS-T components was not less than that of the standard LRS components, whereas the collection efficiency of the LRS-T was significantly greater, 44.9 percent versus 40.7 percent. CONCLUSIONS: The LRS-T procedures produced platelet concentrates with WBC-reduction capacity that is comparable to that obtained with the standard LRS procedures, which have previously been described as satisfying the most stringent criteria for WBC-reduced platelets. The new technique significantly improved the collection efficiency of the plateletpheresis procedure.

The variability of compensatory erythropoiesis in repeated autologous blood donation.

BACKGROUND: Compensatory RBC production during repeated preoperative autologous blood donation (PABD) shows marked interindividual variability. This study was performed to reveal variables that might be useful to predict the amount of the erythropoietic response to PABD in an individual patient who was not iron deficient. STUDY DESIGN AND METHODS: In a retrospective study, 104 adult patients, 48 women and 56 men (mean age, 59.9 years; range, 18-82 years) who donated 3 units (450 mL) of autologous blood at weekly intervals for major surgery were investigated. Blood counts, ferritin, and net preoperative RBC production (net RBC production) were determined in all patients, and soluble transferrin receptor and endogenous levels of EPO, SCF, and IL-1beta were measured in 63 patients. Multiple linear regression analysis was used to determine whether the variance of net RBC production was attributable to baseline values of these variables. RESULTS: Net RBC production was not different in patients who received oral iron and patients who did not (384 +/- 222 mL vs. 356 +/- 158 mL). In both groups, the same two variables consistently showed a significant relationship to net RBC production: the length of the period between the third donation and the last visit was positively related (p = 0.00001 vs. p = 0.0002) and the Hct at baseline was negatively related (p = 0.0002 vs. p = 0.02) with net RBC production. The proportion of variance in net RBC production that was attributable to these two variables was 48.1 percent (r(2) = 0.481) and 34.9 percent (r(2) = 0.349), respectively. CONCLUSION: RBC production after PABD increases with increasing interval from last donation to surgery. This suggests that the interval from last donation to surgery should be maximized. This can be achieved by organizational measures in combination with the preparation of RBC concentrates in additive solution with a maximum shelf life.