Corrigendum to "Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE" [Journal of Immunological Methods 447 (2017) 57-64].

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness.

Flow cytometric quantitation of platelet phagocytosis by monocytes using a pH-sensitive dye, pHrodo-SE.

Antibody-mediated phagocytosis of platelets using a flow cytometric monocyte-based phagocytosis assay (FMPA) has been shown to predict the outcome of platelet transfusion. The easy adherence between platelets and monocytes even in the absence of an antibody is regarded as one of limitations of the FMPA. To improve the FMPA for prediction of transfusion outcome, we used the pH-sensitive dye pHrodo succinimidyl ester (pHrodo-SE), which has weak fluorescence at neutral pH and has increased fluorescence intensity in low pH conditions such as in lysomes. Platelets stained with pHrodo-SE were sensitized with an HLA class I monoclonal antibody (w6/32 clone) or anti-HLA class I containing antisera. The platelets were incubated with monocyte-enriched mononuclear cells. Phagocytic activity was assessed by the percentage of monocytes that phagocytosed platelets. Sensitization of platelets with w6/32 significantly increased platelet phagocytosis by monocytes in dose- and time-dependent manners. Anti-HLA class I antibody-containing sera caused platelet phagocytosis in a cognate antigen-antibody-dependent manner. There was a significant correlation (r=0.69, p<0.01) between phagocytic index and titer of HLA class I antibody measured by lymphocyte immunofluorescence test-flow cytometry. In addition, the phagocytic index obtained by FMPA with pHrodo-SE was significantly higher than that obtained by FMPA with the previously used dye, carboxyfluorescein diacetate succinimidyl ester, when platelets were sensitized by w6/32 and anti-HLA class I antibody-containing sera. Because of the higher resolution and higher sensitivity than those of the previous method, the pHrodo-SE-based FMPA may be suitable for more precise quantitation of phagocytosis activity, which would enable qualitative evaluation of transfusion effectiveness.

Comparison between in vitro properties of washed platelet concentrates suspended in M-sol and those in BRS-A, both of which were prepared with an automated cell processor.

BACKGROUND: Washed platelet concentrate (WPC) is prepared manually in general, but automated preparation is desirable to minimize variation in the WPC quality and enhance WPC production. Recently, the software was improved for an automated cell processor (ACP) to control all processes of WPC preparation. M-sol and BRS-A, which are mixtures of medical solutions, are widely used for WPC preparation with a manual method in Japan. In this study, we prepared WPC suspended in M-sol (WPC-M) or BRS-A (WPC-B) with the ACP, and compared their in vitro properties during 7-day storage. STUDY DESIGN AND METHODS: PC was divided into two equal aliquots for WPC-M and WPC-B. A divided PC, medical solutions and disposable materials were set in the ACP, and it was started to prepare WPC-M or WPC-B on Day 0. Prepared WPC was stored on a flatbed shaker until Day 7. RESULTS: The pH of WPC-M and WPC-B was maintained above 6.8 during the 7-day storage. The differences in aggregation (%), HSR (%), P-selectin expression, GPIbα expression, and phosphatidylserine expression between WPC-M and WPC-B were minimal until Day 3. CONCLUSION: The in vitro properties of WPC-B are not markedly different from those of WPC-M until Day 3.

Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke.

Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs) as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs). Platelet concentrates (PC) were harvested from healthy volunteers and made into single donor-derived PL (sPL). The PL mixtures (mPL) were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA). The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO) agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS). No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

Repeated exposure rather than the total volume of transfused components may influence the incidence of allergic transfusion reactions.

BACKGROUND: The plasma fraction of blood components has an essential role in the etiology of allergic transfusion reactions (ATRs). The difference of incidences of ATRs between fresh-frozen plasma (FFP) and platelet concentrates (PCs), in which plasma is the main component, is not clearly understood. This study compares the frequency of ATRs to FFP versus PCs on both first and subsequent (nonfirst) transfusions and considers the factors influencing the risk of ATRs. STUDY DESIGN AND METHODS: Five hospitals agreed to systematically collect and share 2 years of data (January 2010 through December 2011). This was a retrospective observational analysis of data including the number of transfusion episodes and ATRs for FFP and PCs on first-transfusion patients (without transfusion history) and previously transfused patients. RESULTS: The incidence of ATRs to PCs (2.51%) was significantly higher than to FFP (1.68%) on subsequent transfusions (p < 0.001). On the other hand, there were no significant differences in the incidences of ATRs to FFP (2.67%) and PCs (2.82%) on first transfusions. This discrepancy was most pronounced among males: FFP versus PCs on first transfusions, 2.02% versus 2.60% (p = 0.30); and on subsequent transfusions, 1.58% versus 2.46% (p = 0.0007). Among females, FFP versus PCs on first transfusions was 3.59% versus 3.13% (p = 0.61) and on subsequent transfusions was 1.87% versus 2.61% (p = 0.029). CONCLUSION: Repeated exposure rather than the total volume of transfused components may influence the incidence of ATRs.

Anti-M Antibody Induced Prolonged Anemia Following Hemolytic Disease of the Newborn Due to Erythropoietic Suppression in 2 Siblings.

Hemolytic disease of the newborn (HDN) arising from MNSs incompatibility is rare, with few reports of prolonged anemia and reticulocytopenia following HDN. We report the younger of 2 male siblings, both of whom had anti-M-induced HDN and anemia persisting for over a month. Peripheral reticulocytes remained inappropriately low for the degree of anemia, and they needed multiple red cell transfusions. Viral infections were ruled out. Corticosteroids were given for suspected pure red cell aplasia. Anemia and reticulocytopenia subsequently improved. Colony-forming unit erythroid assay revealed erythropoietic suppression of M antigen-positive erythroid precursor cells cultured with maternal or infant sera containing anti-M. In conclusion, maternal anti-M caused HDN and prolonged anemia by erythropoietic suppression in 2 siblings.

Storage of volume-reduced washed platelets in M-sol additive solution for 7 days.

BACKGROUND: Volume-reduced washed platelets (VR-wPLTs), which are prepared by concentrating platelets (PLTs) into a smaller volume of additive solution (AS), may prevent not only circulatory overload, but also adverse reactions caused by plasma components. Although VR-wPLTs may be quickly degraded due to high PLT concentrations, few studies have examined the effects of storage on VR-wPLTs. We examined here the in vitro properties of VR-wPLTs prepared with M-sol AS during their storage for 7 days. STUDY DESIGN AND METHODS: Platelet concentrates (PCs) were divided into two equal aliquots (control group and test group). After the centrifugation of both aliquots and removal of as much supernatant as possible, the pellet of the control group was resuspended in 160 mL of M-sol while that of the test group was resuspended in 80 or 40 mL of M-sol. The wPLTs of both groups were stored in polyolefin bags with agitation at 20 to 24°C for 7 days. RESULTS: The pH values of both groups were maintained at higher than 7.0 during the 7-day storage. Differences in %disk, CD62P, annexin V, percent hypotonic shock response, and aggregation values between the test group and control group were small for at least 2 days after washing. CONCLUSIONS: The in vitro properties of VR-wPLTs were not markedly degraded for at least 2 days. Therefore, the storage properties of PLTs may be maintained in VR-wPLTs prepared at blood centers until they are administered to patients in hospitals.

Incidence of transfusion-related adverse reactions per patient reflects the potential risk of transfusion therapy in Japan.

OBJECTIVES: To describe the frequency of adverse reactions (ARs) after transfusion on both per transfused patient and per transfused unit bases. METHODS: We performed a retrospective analysis of data available from records of 6 hospitals on the total number of transfusions and documented ARs between January 2008 and December 2009 for RBCs, fresh-frozen plasma (FFP), and platelet concentrates (PCs). RESULTS: The incidence of ARs to RBCs, FFP, and PCs per transfused unit was 0.6%, 1.3%, and 3.8%, respectively. The incidence of ARs to RBCs, FFP, and PCs per patient was 2.6%, 4.3%, and 13.2%, respectively-almost 3-fold higher. Most RBC-ARs were febrile nonhemolytic transfusion reactions and allergic reactions, whereas most FFP-ARs and PC-ARs were allergic reactions. CONCLUSIONS: The incidence of ARs per transfused patient may reflect better the potential risk of transfusion with blood components, taking into account the characteristics of the transfused patient.

Online reporting system for transfusion-related adverse events to enhance recipient haemovigilance in Japan: a pilot study.

BACKGROUND: A surveillance system for transfusion-related adverse reactions and infectious diseases in Japan was started at a national level in 1993, but current reporting of events in recipients is performed on a voluntary basis. A reporting system which can collect information on all transfusion-related events in recipients is required in Japan. METHODS: We have developed an online reporting system for transfusion-related events and performed a pilot study in 12 hospitals from 2007 to 2010. RESULTS: The overall incidence of adverse events per transfusion bag was 1.47%. Platelet concentrates gave rise to statistically more adverse events (4.16%) than red blood cells (0.66%) and fresh-frozen plasma (0.93%). In addition, we found that the incidence of adverse events varied between hospitals according to their size and patient characteristics. CONCLUSION: This online reporting system is useful for collection and analysis of actual adverse events in recipients of blood transfusions and may contribute to enhancement of the existing surveillance system for recipients in Japan.

Impact of chemiluminescent enzyme immunoassay screening for human parvovirus B19 antigen in Japanese blood donors.

BACKGROUND: To reduce the risk of human parvovirus B19 (B19V) transmission through contaminated blood for transfusion and plasma-derived products, the Japanese Red Cross (JRC) Blood Centers introduced B19V antigen screening by chemiluminescent enzyme immunoassay (CLEIA-B19V) in 2008. STUDY DESIGN AND METHODS: Donor samples that were positive by CLEIA-B19V screening were tested for B19V DNA. The sensitivity of CLEIA-B19V was tested using samples of all three genotypes and B19V DNA-positive donations. B19V DNA-positive donations and pooled plasma were quantitatively assayed for B19V DNA. B19V DNA-positive donations were phylogenetically analyzed by polymerase chain reaction direct sequencing. RESULTS: The sensitivity of CLEIA-B19V was inferred to be approximately 6.3 log IU/mL with the genotype samples and 6.4 log IU/mL with B19V DNA-positive donor samples. Of 417 CLEIA-B19V-positive samples from 1,035,560 donations in Hokkaido, Japan, 101 were positive for B19V DNA. The 198 strains of B19V DNA-positive donations in Hokkaido over the past 15 years clustered exclusively with Genotype 1. After introduction of CLEIA-B19V, the viral load for B19V DNA in all 772 pooled plasma for fractionation from donors in nationwide Japan did not exceed 4 log IU/mL. CONCLUSION: CLEIA-B19V can detect all three genotypes of B19V (viral load >6.3 log IU/mL) and limit the viral load (<4 log IU/mL) in pooled plasma, and thus such screening has further reduced the risk of transfusion-transmitted B19V infection. These results show that CLEIA-B19V screening at the JRC Blood Centers can be an alternative approach to comply with recommendations regarding B19V in the United States and Europe.

Impact of fresh-frozen plasma from male-only donors versus mixed-sex donors on postoperative respiratory function in surgical patients: a prospective case-controlled study.

BACKGROUND: To reduce the risk of transfusion-related acute lung injury (TRALI), plasma products are mainly made from male donors in some countries because of the lower possibility of alloimmunization; other countries are considering this policy. The advantage of male-only fresh-frozen plasma (FFP) should be examined in a prospective case-control study. STUDY DESIGN AND METHODS: This study compared pulmonary function after the transfusion of FFP derived from either male donors only (FFP-male) or mixed donors (FFP-mixed) in informed surgical patients treated at a tertiary university hospital in Japan. The factors contributing to pulmonary distress (PD) after transfusion were then statistically examined. RESULTS: Eighty-two patients participated in this study (FFP-male, n = 55; FFP-mixed, n = 27). Nineteen patients developed PD (PaO(2)/FiO(2) ratio [P/F] < 300) within 6 hours after transfusion: seven had congestive pulmonary edema (transfusion-associated circulatory overload), five had permeability pulmonary edema (possible TRALI), and seven had no apparent pulmonary edema. A multivariate logistic regression analysis revealed that the use of cardiopulmonary bypass and preoperative liver dysfunction were significantly associated with a P/F of less than 300 (odds ratios [ORs], 8.95 [p = 0.004] and 6.54 [p = 0.005], respectively), while the use of FFP-male was significantly associated with the absence of PD (OR, 0.219; p = 0.022). All the patients with possible TRALI had received either white blood cell or granulocyte antibody-positive FFP. The lysophosphatidylcholine level was not correlated with PD. CONCLUSIONS: Our data suggests that the use of FFP derived from male donors may be advantageous for posttransfusion pulmonary function, although PD is also determined by background characteristics.

Identification of two distinct populations of endothelial progenitor cells differing in size and antigen expression from human umbilical cord blood.

Endothelial progenitor cells (EPCs) have been isolated from peripheral blood, bone marrow, and umbilical cord blood (CB) and determined to be in heterogeneous populations; however, specific variations in their characteristics remain to be clarified. In this study, we observed that mononuclear cells (MNCs) of CB change in morphology to differentiate into mature endothelial cells (EC) after 6 weeks of culture. In early days of culture along with the differentiation, two distinct populations of EPCs were detected, defined by two-dimensional dot plots (forward scatter vs side scatter) with flow cytometry, namely, relatively small cells (S-EPCs) and relatively large cells (L-EPCs). S-EPCs were found to express CD34 but not CD14, while the converse was the case for L-EPCs. When CD34(+)/CD14(-) cells and CD34(-)/CD14(+) cells were isolated from original MNCs of CB and cultured independently, S-EPCs and L-EPCs were derived from CD34(+)/CD14(-) and from CD34(-)/CD14(+) cells, respectively. Furthermore, when the two EPCs at day 7 were separated by cell sorter and recultured, there was no crossover in terms of CD34 and CD14 expression. While expression of VE-cadherin and vascular endothelial growth factor receptor-2 (VEGFR-2) on L-EPCs was significantly greater than on S-EPCs, levels of CD31 were lower. In addition, L-EPCs exhibited greater proliferative ability on stimulation with VEGF. Although these two EPCs expressed different phenotypes, including growth factor receptors, and had different proliferative ability, they both eventually differentiated into mature ECs after more than 3 weeks of culture.

[Current status and problem of platelet transfusion for hematopoietic diseases: results from a questionnaire survey].

We carried out a survey on platelet transfusions performed in hospitals certified by the Japanese Society of Hematology. The average values of the pretransfusion platelet count (trigger value) for the day on which the transfusion was ordered, and the values on each day in the interval between the order and actual transfusion, were compared with the Guidelines. The average trigger value in aplastic anemia and myelodysplastic syndrome patients (A group) (1.41 x 10(4)/microl) for the same day on which the transfusion was ordered was higher than the Guideline, whereas those patients with hematological disorders undergoing chemotherapy (B group) and hematopoietic stem cell transplantation (C group), namely 2.08 x 10(4)/microl and 2.1 x 10(4)/microl, respectively, were acceptable values when compared with the Guidelines. On the other hand, in all groups, the transfusion trigger values at one or two days after ordering were higher than the Guidelines, being 2.56 x 10(4)/microl in the A group, 3.15 x 10(4)/microl in the B group, and 2.59 x 10(4)/microl in the C group. We have tried to formulate platelet count criteria for ordering a transfusion based on one day before the actual transfusion, because these platelet counts on ordering were relatively high. The criteria are 1.0-1.5 x 10(4)/microl in group A, 3.0 x 10(4)/microI in group B, and 3.0 x 10(4)/microl in group C. In order to perform platelet transfusion according to the Guidelines, the platelet count on ordering should be decreased as we proposed.

[Survey on the threshold for platelet transfusions].

We carried out a survey on platelet transfusions performed in nine general hospitals. We evaluated 303 adults who received a total of 24455 units over 1864 platelet transfusions. The underlying diseases were hematologic disorders with chemotherapy (59.7%), hematologic disorders without chemotherapy (15.5%), hematopoietic stem cell transplantation (18.5%), and others (2.0%). The patient platelet count before transfusion (platelet trigger value) was measured in only 77.1%. The platelet trigger value differed greatly between the hospitals, with an average of 2.2 x 10(4)/microl, a minimum of 1.3 x 10(4)/microl, and maximum of 3.2 x 10(4)/microl. Only 55.3% of the platelet transfusions carried out complied with the Platelet Transfusion Guideline published by the Ministry of Health, Labour and Welfare. The hospitals surveyed could be divided into those who gave mainly around 10 units and those who gave over 10 units. The total dose of platelets transfused was more in the hospitals that used mainly 15 or more unit-PCs than in the hospitals that used mainly 10 unit-PCs. These results indicate that platelet transfusion may be greatly reduced by complying with the 2 x 10(4)/microl of platelet transfusion threshold and by selecting less than 10 units of PC per transfusion.