Antibody studies in a patient with acute thrombocytopenia following infusion of plasma containing anti-PIA1

Immune thrombocytopenia due to passive transfer of anti-PI^A1 alloantibody has been noted as a rare but potentially dangerous complication of plasma transfusions. We report a patient with a preoperative platelet count of 241 × 10⁹/l who developed severe thrombocytopenia within 2 hr following transfusion of 2 U of fresh frozen plasma. The plasma donor was found to be a PI^A1-negative woman. The platelet count of the PI^A1-positive patient recovered within 7 days to normal values. In the frozen plasma, excessive antibody binding to GPIIb-IIIa on the recipient's platelets was detected. The antibody was shown to have anti-PI^A1-specificity. Only 40 min after transfusion of the frozen plasma, no antibody was detected in the plasma of the recipient. This case suggests that passively administered anti-PI^A1 alloantibody is immediately adsorbed onto the recipient's platelets and thus removed from circulation. Am. J. Hematol. 56:119–121, 1997. © 1997 Wiley-Liss, Inc.     

Platelet count evaluation using three automated haematology analysers compared with the immunoplatelet reference method, and estimation of possible inadequate platelet transfusion

The accuracy of three automated haematology analysers [Sysmex XE-2100 (both optical and impedance mode), Bayer Advia 120, and Beckman Coulter LH-750] was compared with the immunoplatelet reference method for platelet measurement. A total of 165 blood specimens were obtained from patients and platelet counts were determined using the four-automated haematology analyser methods and the immunoplatelet reference method. The coefficients of determination (R(2)) between the automated haematology analyser methods and the immunoplatelet reference method for the overall platelet range were >0.98. A bias study, however, showed some disagreement. The use of a coincidence correction calculation for the immunoplatelet method did not improve the correlation between the immunoplatelet method and the automated haematology analyser methods. To estimate the possibility of inadequate platelet transfusion, the number of prophylactic platelet transfusion indications determined by the automated haematology analyser platelet counts were compared with the number of transfusion indications according to the platelet counts determined by the immunoplatelet method. An additional 48 blood specimens were included in this analysis. All of the automated haematology analysers showed some disagreement in the transfusion indications when compared with the immunoplatelet method, suggesting the possibility of inadequate platelet transfusion.     

Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools

Background and Objectives Microparticles (MPs) are small phospholipid vesicles of less than 1 µm, shed in blood flow by various cell types. These MPs are involved in several biological processes and diseases. MPs have also been detected in blood products; however, their role in transfused patients is unknown. The purpose of this study was to characterize those MPs in blood bank conditions. Materials and Methods Qualitative and quantitative experiments using flow cytometry or proteomic techniques were performed on MPs derived from erythrocytes concentrates. In order to count MPs, they were either isolated by various centrifugation procedures or counted directly in erythrocyte concentrates. Results A 20-fold increase after 50 days of storage at 4°C was observed (from 3370 ± 1180 MPs/µl at day 5 to 64 850 ± 37 800 MPs/µl at day 50). Proteomic analysis revealed changes of protein expression comparing MPs to erythrocyte membranes. Finally, the expression of Rh blood group antigens was shown on MPs generated during erythrocyte storage. Conclusions Our work provides evidence that storage of red blood cell is associated with the generation of MPs characterized by particular proteomic profiles. These results contribute to fundamental knowledge of transfused blood products.     

An association of ABO non-identical platelet and cryoprecipitate transfusions with altered red cell transfusion needs in surgical patients

Background Transfusion of ABO non‐identical plasma, platelets and cryoprecipitate is routine practice even though adverse effects can occur. Methods and Materials Our hospital changed transfusion practice in 2005 and adopted a policy of providing ABO‐identical blood components to all patients when feasible. We retrospectively compared the transfusion requirements, length of stay and in‐hospital mortality in relation to ABO blood group in surgical patients who received platelet transfusions before and after this change to determine whether it resulted in any benefit. Results Prior to the change in practice, both group B and AB patients received more ABO non‐identical platelet transfusion (P = 0·0004), required significantly greater numbers of red cell transfusions (P = 0·04) and had 50% longer hospital stays (P = 0·039) than group O and A patients. Following the policy change, there was a trend for fewer red cell transfusions (P = 0·17) and length of stay in group B and AB patients than group O or A patients. Overall, the mortality rate per red cell transfusion decreased from 15·2 per 1000 to 11·0 per 1000 (P = 0·013). Conclusions These results, in the context of previous findings, suggest that providing ABO‐identical platelets and cryoprecipitate might be associated with reduction in transfusion requirements and improve outcomes in surgical patients.     

Kinetics of CXCR-4 and adhesion molecule expression during autologous stem cell mobilisation with G-CSF plus AMD3100 in patients with multiple myeloma

AMD3100, a competitive antagonist of CXCR-4, disrupts the binding of its ligand, stromal cell-derived factor-1 (SDF-1), and facilitates stem cell mobilisation in patients with haematological malignancies. This study investigated the differential kinetics of CXCR-4 and adhesion molecule expression and their impact on stem cell yield during mobilisation with granulocyte-colony stimulating factor (G-CSF) (days 1–4) followed by AMD3100 in 10 patients with multiple myeloma. A four-colour flow cytometry-based determination of CXCR-4, VLA-4, l-selectin, PECAM, LFA-1 and CD44 expression on CD34+ cells and measurement of SDF-1 concentration were performed at different time points. After G-CSF alone, CXCR-4 expression on patients’ blood and marrow CD34+ cells was significantly lower than in the healthy controls (p < 0.001), but allowed no prediction of stem cell yield. Except in the single poorly mobilising patient, AMD3100 led to a further significant decrease of CXCR4 (p = 0.001), which inversely correlated with the CD34+ counts in the blood (p = 0.005). SDF-1 level in patients’ marrow was positively correlated with CXCR-4 expression on CD34+ cells (p = 0.011). It is interesting to note that the expression of adhesion molecules remained unaffected by AMD3100 administration. Further studies will define the possible prognostic role of AMD3100 mediated changes in CXCR-4 expression for the prediction of stem cell yield attainable with this new mobilisation regimen.     

Significance of increasing adhesion of cord blood hematopoietic cells and a new method: platelet microparticles

Hematopoietic recovery after transplantation with cord blood (CB) is slower than with bone marrow (BM) and mobilized peripheral blood. Adhesion molecules (AMs) on hematopoietic cells are involved in hematopoietic cells' homing. It may be possible to enhance CB CD34+ cells engraftment by increasing their expressions of AM. Twenty-three patients with childhood acute leukemia treated with unrelated CBT were studied. It was found that the time to neutrophil recovery correlated with CXCR4 and the time to platelet recovery correlated with both CD62L and CXCR4. Platelet microparticles (PMPs) carry some AMs such as aIIb b (CD41), P-selectin (CD62P), and CXCR4, CD34+ cells express platelet-binding antigens (CD162 and CD11b). It was found that AMs were increased dramatically on CD34+ cells surface in the presence of PMPs, and CD34+ cells covered with PMPs adhered better to human umbilical vein endothelial cells and fibronectin. These findings suggested that PMPs could increase adhesion of donor's cells to host BM in CBT.     

Isotype-specific detection of ABO blood group antibodies using a novel flow cytometric method

Several methods to detect anti-A/B antibodies based on haemagglutination and haemolysis have been described. These methods measure predominantly anti-A/B immunoglobulin (Ig)M, whereas anti-A/B IgG and IgG subclasses are less well examined. We established a flow cytometry method (ABO-fluorescence-activated cell sorting; ABO-FACS) to quantify binding of anti-A/B IgM, IgG and IgG subclasses to human A or B red blood cells. Anti-A/B IgM were present in the majority of 120 blood donors, as expected from blood group typing. The sensitivity and specificity of anti-A/B IgM to predict the blood group was 93% and 96% respectively. Anti-A/B IgG was found in 34/38 blood group O samples (89%). Anti-B IgG in blood group A or anti-A IgG in blood group B was present in 4/28 (14%) and 1/28 (4%) samples, respectively, and absent in 26 AB sera. IgG2 was the predominant IgG subclass. The correlation of anti-A/B IgM and IgG in the ABO-FACS with haemagglutination titres was 0.870 and 0.783, respectively (n = 240; P < 0.001) whereas the comparison of ABO-FACS with ABO-enzyme-linked immunosorbent assay was less significant. In conclusion, ABO-FACS is a valid method to quantify anti-A/B IgM, IgG and IgG subclasses. It opens the possibility of isotype-specific monitoring of anti-A/B antibodies levels after ABO-incompatible solid organ and stem cell transplantation.     

Therapeutic efficacy of platelet components treated with amotosalen and ultraviolet A pathogen inactivation method: results of a meta‐analysis of randomized controlled trials

Background and Objectives There are conflicting data regarding the therapeutic efficacy of platelets inactivated using amotosalen and ultraviolet A light. We have performed a meta‐analysis to summarize the results of different randomized controlled trials (RCT). Materials and Methods Five RCTs reported through March 2011 met the criteria for meta‐analysis. Weighted mean difference (WMD) in corrected count increment (CCI) at 1 h, CCI‐24 h, and transfusion interval (days) and summary odds ratio (OR) of bleeding in inactivated platelet (I‐P) group vs. noninactivated platelet (C‐P) group were calculated across studies. Results Randomized controlled trials were statistically homogeneous when we analysed CCI‐24 h, and the transfusion of C‐P was associated with a higher CCI‐24 h when compared with the transfusion of I‐P (WMD, 3 × 10³; 95% CI, 2·32 × 10³–3·69 × 10³; P < 0·00001). RCTs were statistically heterogeneous when we analysed CCI‐1 h, transfusion interval and OR of bleeding. Regarding the OR of bleeding in the I‐P and C‐P groups, it varied by as much as a multiple of four among the trials, from 0·66 to 2·66. When we combined double‐blinded and high methodologic quality score RCTs, the use of I‐P was not statistically associated with an increase in the OR of bleeding when compared with the use of C‐P (OR, 0·97; 95% CI, 0·75–1·27; P = 0·84). Conclusion Although the transfusion of I‐P was associated with lower CCI‐24 h when compared with the transfusion of C‐P, this was not associated with differences in the OR of bleeding between I‐P and C‐P.     

Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

AIM: To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle. METHODS: MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC) monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry. Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion. RESULTS: Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44 splice variants CD44v4, CD44v5, and CD44v7 were all up-regulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monoclonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent. CONCLUSION: CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cycle dependent alterations of their adhesion behaviour to endothelium.     

Evaluation of perfusion changes using a 2D Parametric Parenchymal Blood Flow technique with automated vessel suppression following partial spleen embolization in patients with hypersplenism and portal hypertension

To evaluate the feasibility and potential value of 2D Parametric Parenchymal Blood Flow (2D-PPBF) for the assessment of perfusion changes following partial spleen embolization (PSE) in a retrospective observational study design.Overall, 12 PSE procedures in 12 patients were included in this study. The outcome of the study was the platelet response (PR), calculated as the percentage increase of platelet count (PLT), following PSE. To quantify perfusion changes using 2D-PPBF, the acquired digital subtraction angiography series were post-processed. A reference region-of-interest (ROI) was placed in the afferent splenic artery and a target ROI was positioned on the embolization territory of the spleen on digital subtraction angiography series pre- and post-embolization. The ratios of the target ROIs to the reference ROIs were calculated for the Wash-In-Rate (WIR), the Time-To-Peak (TTP) and the Area-Under-the-Curve (AUC). Comparisons between pre- and post-embolization data were made using Wilcoxon signed-rank test and Spearman''s rank correlation coefficient (r). Afterwards, the study population was divided by the median of the TTP before PSE to analyze its value for the prediction of PR following PSE.Following PSE, PLT increased significantly from 43,000 ± 21,405 platelets/μL to 128,500 ± 66,083 platelets/μL with a PR of 255 ± 243% (P = .003). In the embolized splenic territory, the pre-/post-embolization 2D-PPBF parameter changed significantly: WIR_pre-PSE 1.23 ± 2.42/WIR_post-PSE 0.09 ± 0.07; -64 ± 46% (p = 0.04), TTP_pre-PSE 4.41 ± 0.99/TTP_post-PSE 5.67 ± 1.52 (P = .041); +34 ± 47% and AUC_post-PSE 0.81 ± 0.85/AUC_post-PSE 0.14 ± 0.08; -71 ± 18% (P = .002). A significant correlation of a 2D-PPBF parameter with the PLT was found for TTP_pre-PSE/PLT_pre-PSE r = -0.66 (P = .01). Subgroup analysis showed a significantly increased PR for the group with TTP_pre-PSE >4.44 compared to the group with TTP_pre-PSE ≤4.44 (404 ± 267% versus 107 ± 76%; P = .04).2D-PPBF is an objective approach to analyze the perfusion reduction of embolized splenic tissue. TTP derived from 2D-PPBF has the potential to predict the extent of PR during PSE.     

Infection with TT virus, a novel transfusion-transmissible DNA virus, in haemophiliacs and in blood products

We evaluated the characteristics and rate of infection with TT virus (TTV), a novel DNA virus, in Japanese haemophiliacs. TTV DNA was measured in 60 haemophiliacs by semi-nested polymerase chain reaction. Co-infection with hepatitis C virus (HCV), hepatitis G virus (HGV) and human immunodeficiency virus (HIV) was also evaluated. In addition, the rate of detection of TTV DNA in blood products was evaluated. TTV DNA was detected in 35/60 haemophiliacs (58.3%). There were no differences in the backgrounds or characteristics between haemophiliacs with and without TTV infection, except for higher levels of IgG and IgM in patients with TTV infection. In patients infected with TTV of types other than type 1, which are rarely detected in Japan, the rate of co-infection with HCV of imported types was high; TTV of types other than type 1 in Japanese haemophiliacs were probably transmitted by imported blood products. TTV DNA was detected in over half of the blood products tested, but TTV DNA concentrations in these products were lower than in the serum of haemophiliacs.     

HLA-B27 determination using serological methods. A comparison of enzyme immunoassay and a microlymphocytotoxic test with flow cytometry and a molecular biological assay

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results: one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.