Iron deficiency among Japanese whole-blood donors measured by serum ferritin.

BACKGROUND AND OBJECTIVES: A more restrictive blood donation criterion has been applied in Japan, with a maximum volume of whole blood (WB) donation of 400 mL, allowing twice a year for female donors and thrice a year for male donors. However, iron deficiency was as high as 20.5% among female donors prior to donation, increasing to 37.7% after blood donation. More than 20 years have passed since then, so we set out to investigate the present situation. MATERIALS AND METHODS: A total of 2659 (male/female: 1496/1163) donors of 400 mL WB who gave informed consent to join the study were enrolled. Serum ferritin (sFer) of first-time/reactivated (FT/RA) donors were compared with those of repeat donors, according to gender and age; those who returned for subsequent donations during the study period were also followed up. RESULTS: About one-third of FT/RA female donors had iron deficiency, possibly reflecting its high incidence among the general population. Interestingly, although sFer levels were low among pre-menopausal FT/RA female donors, these values were not much different in repeat donors, whereas significant differences were observed between FT/RA and repeat donors among post-menopausal females and in most age groups among males. As expected, donors with a normal initial sFer (≥26 ng/mL) recovered faster than those with a low initial sFer. CONCLUSION: Female donors, especially, have iron deficiency even before donation, and the rate increased compared to what was found previously. Measures to prevent iron deficiency of blood donors is required, and studies are going on in Japan.

Decreased rate of blood donors with high ABO antibody titers in Japan and the underlying factors: Comparisons between 2010 and 2021.

BACKGROUND AND OBJECTIVES: Previously (2007), it was reported that ABO antibody titers in Japanese blood donors had decreased significantly compared to 20 years before. Here we evaluated whether further decrease of antibody titers had occurred in recent years, and the potential factors associated with changes in antibody titers. MATERIALS AND METHODS: Serum/plasma from random blood donors in 2010 and 2021 (2010: 3369, 2021: 5796 donors) was classified into low, middle, and high ABO antibody titers according to the reactivity of diluted serum/plasma (2.5-fold and 20-fold) by an automated microplate system. The rates of low/high titer in the two periods were compared. Logistic regression and age-gender-BMI subgroup analyses were conducted to identify the factors that contributed to changes in antibody titers. RESULTS: Compared to 2010, the rate of donors with high ABO antibody titers was　decreased in 2021 for both anti-A and anti-B (anti-A, 2010: 23.8%, 2021: 19.3%; anti-B, 2010: 23.8%, 2021: 16.4%). In logistic regression analysis, age was found to significantly affect both anti-A and anti-B antibody titers (anti-A, adjusted odds ratio 0.36, 95% CI 0.31-0.41; anti-B, 0.42, 0.37-0.47), and BMI (0.82, 0.73-0.92) and other time-related factors (0.79, 0.71-0.88) significantly affect anti-B antibody titers. Subgroup analysis revealed decreased rate of high anti-B titers in the higher age group in 2021. CONCLUSION: The rate of high ABO antibody titers, especially high anti-B titers, was significantly decreased in 2021, and our results suggested an association with aging and obesity of blood donors as well as other time-related factors.

Trends in hepatitis E virus infection: Analyses of the long-term screening of blood donors in Hokkaido, Japan, 2005-2019.

BACKGROUND: After experiencing several cases of transfusion-transmitted hepatitis E (TT-HE) in Hokkaido, Northern Japan, hepatitis E virus (HEV) screening in blood donors, using a nucleic acid amplification test (NAT), was introduced in 2005. STUDY DESIGN AND METHODS: The frequency of HEV RNA-positive donations (2005-2019) was investigated, and the HEV RNA-positive specimens were phylogenetically analyzed. In August 2014, the 20-pooled NAT (20P-NAT) was replaced with an individual-NAT (ID-NAT) system. RESULTS: Until 2019, the frequency of HEV RNA-positive donors was 0.011% (289/2,638,685) with 20P-NAT and 0.043% (597/1,379,750) with ID-NAT, and no TT-HE cases were observed in Hokkaido. The prevalence among male, but not female donors, increased significantly between 2015 and 2019. Eighty-nine percent of HEV isolates from donors were genotype 3 and the remainder were genotype 4, and many clusters existed in each genotype. ALT levels at the time of donation were significantly higher in donors with genotype 4. Four subgenotypes, namely 3a (37%), 3b (41%), 3e (6%), and 4c (10%), comprised 94% of the total. During this period, the most identified subgenotype, 3a, transitioned to 3b. Majority of the HEV strains within the same clusters were detected in the same geographical region around the same period. Many of the human HEV isolates were shown to coexist closely with animal HEV isolates phylogenetically. CONCLUSION: In Hokkaido, multiple divergent HEV strains have been circulating, and small outbreaks of hepatitis E have occurred in the last 15 years. The results suggested that HEV NAT can contribute significantly in ensuring safety during blood transfusions.

Establishment of a novel cell-based assay using HLA-transfected cells to detect HLA antibodies.

The detection of HLA antibodies is important in clinical practice, such as platelet transfusion refractoriness and transfusion-related lung injury. However, difficulties are associated with the preparation of panel cells for conventional HLA detection systems using intact cells, such as the immunocomplex capture fluorescence analysis (ICFA). Based on an ICFA analysis, HEK293 cells stably transfected with the HLA-A locus were used instead of peripheral blood mononuclear cells (PBMC). The reactivity, sensitivity, and stability of transfectants were examined. All 20 antisera to HLA-A identified by LABScreen® Single Antigen class I (LS-SA1) were reactive to our modified-ICFA (m-ICFA) and showed the same specificities as those in LS-SA1, indicating the cell surface expression and correct antigenicity of the HLA-A locus in transfectants. The expression of HLA class I antigens was similar between transfectants frozen for 6 years and those prior to freezing. In the reaction of the anti-A24 or anti-A33 antibody vs each transfectant, the index of m-ICFA was higher than that of WAKFlow® ICFA. Our m-ICFA also showed that false negative reactions sometimes observed in capture assays may be avoided. By using HLA-A transfectants as ICFA targets, we herein developed m-ICFA. Our m-ICFA may avoid false negative reactions of capture assay like enzyme-linked immunosorbent assay and can also be carried out in almost any laboratory without cell culture facilities.

Transfusion-associated circulatory overload and high blood pressure: A multicentre retrospective study in Japan.

BACKGROUND: Transfusion-associated circulatory overload (TACO) is an adverse reaction associated with a high risk of mortality. The actual incidence of TACO and hypertension associated with transfusion in Japan is unknown. METHODS: A multicentre retrospective observational study was conducted across 23 institutions during the 1-year period of 2016. Patients were included if they developed TACO or their blood pressure (either systolic or diastolic) increased by at least 30 mmHg during the transfusion. TACO was confirmed by the primary physicians and transfusion medicine teams and recorded in the data on passive surveillance, and additional data were extracted from electronic medical records. RESULTS: In our patient cohort of 31 384 patients who underwent transfusion, the incidence of TACO and hypertension was 0·03% and 0·2%, respectively. However, 43% of the participating institutions didn't report any cases. When comparing risk factors between the TACO and hypertension groups, there were significant differences in comorbidities, such as abnormal findings on chest x-ray. Significant differences between the two groups were observed post-transfusion pulse rate, body temperature and oxygen saturation (P < 0·01). In the group of patients with hypertension, the level of BNP increased significantly after transfusion in 45% (5/11) of the patients. We identified 4 patients in the hypertension group who met the new ISBT's TACO criteria. CONCLUSION: Our study suggests that more attention should be given to TACO in Japan, particularly in terms of improving surveillance systems. For the early diagnosis of TACO, it is crucial to carefully monitor vital signs including blood pressure.

Differences among hemoglobin thresholds for red blood cell transfusions in patients with hematological diseases in teaching hospitals: a real world data in Japan.

A hemoglobin (Hb) threshold level of 7 g/dL has been proposed for red blood cell (RBC) transfusion in patients with chronic anemia in the Japanese guideline since 2005. However, Hb thresholds for hematological diseases in clinical practice and factors responsible for higher Hb thresholds remain unclear. Hb thresholds were collected for patients with hematological diseases from 32 Japanese teaching hospitals. Uni- and multivariate analyses were used to analyze relationships between Hb threshold level and various patient and hospital factors. In total, 4996 units of RBC were transfused to 1054 patients with hematological diseases in 2421 transfusions. Median age was 68 years. Myelodysplastic syndrome was the most frequent diagnosis. Overall median Hb threshold level was 6.9 g/dL. Multivariate linear regression analysis detected the following variables associated with Hb threshold level: hospital; cardiovascular disease; symptomatic anemia; and hematopoietic stem cell transplantation. Hospital was the most significant factor. Collectively, median Hb threshold level in clinical practice in Japan was similar to the guidelines. Higher Hb threshold level depended on the hospitals at which the transfusions were performed as well as patient condition. Educational approaches directed toward hospitals may be useful to promote transfusion guidelines.

Clinical significance of autologous blood transfusions in bone marrow harvest from unrelated donors.

In the Japan Marrow Donor Program (JMDP), autologous blood is collected from most unrelated bone marrow (BM) donors. We retrospectively evaluated 5772 donors who underwent BM harvest between 2010 and 2015 through the JMDP. Autologous blood was collected in 96.8% of the donors; the wastage rate was 0.6%. Allogeneic blood transfusion was not required. The mean hemoglobin (Hb) levels were 12.1 g/dL after the BM harvest (mean 891 mL) together with autologous blood transfusion (mean 596 mL). Propensity-score matching was used to adjust the backgrounds. Among donors with harvested BM of 100-400 mL, autologous blood transfusion had no impact on Hb levels or complications after BM harvest. Among donors with harvested BM of > 400 mL, more autologous blood transfusion followed by a bleeding volume of ≤ 100 mL did not confer clinical benefit to donors compared with less autologous blood transfusion followed by a bleeding volume of > 300 mL. The findings of the present study suggest that autologous blood transfusion to BM donors is excessive in terms of Hb changes and post-harvest outcomes.

Reduction in adverse transfusion reactions with increased use of washed platelet concentrates in Japan-A retrospective multicenter study.

Plasma removal by washing platelet concentrates (PCs) is effective in preventing adverse reactions to PC transfusions. The Japanese Red Cross Society (JRCS) started releasing washed PCs (WPCs) as a commercially approved blood product in September 2016. This retrospective multicenter study investigated the change in the number of transfused WPCs and the impact on the incidence of adverse reactions to PCs before and after the release. The numbers and types of transfused PCs and the adverse reactions to the PCs for a year before the start of the WPC release and for a year after the release were reported by 27 medical institutes in Japan. Transfusion information for approximately 8% of the amount of PCs supplied in Japan was analyzed during the study period. After the start of WPC release by the JRCS, the number of transfused WPCs doubled. The rate of adverse reactions to PCs decreased significantly (p = 0.0223), from 4.30% before the release to 4.05% after the release. The rates of adverse reactions to unwashed and WPCs were 4.13% and 0.84%, respectively. Allergic adverse reactions were significantly decreased after the release (3.60% before versus 3.37% after). No severe allergic reactions to WPCs were reported. The release of WPCs by the JRCS significantly reduced transfusion-related adverse reactions to PCs in Japan.

Released washed platelet concentrates are effective and safe in patients with a history of transfusion reactions.

BACKGROUND: Plasma removal by washing is an effective approach to prevent transfusion reactions by platelet concentrates (PCs). Recently, washed PCs were released by the Japanese Red Cross Society (JRCS). MATERIALS AND METHODS: This retrospective multicenter study evaluated the efficacy and safety of released washed PCs (RWPCs) between September 2016 and January 2017 in Japan. The RWPCs were prepared by washing leukoreduced apheresis PCs with the platelet additive solution, BRS-A, using automated cell processors. RESULTS: Clinical data were obtained from 91 patients and 1210 RWPC transfusions at 50 institutions. The median number of RWPC transfusions per patient was 8 (range, 1-91). RWPCs were used in 94.5% of the patients with a history of recurrent or severe transfusion reactions for preventing such reactions. Responses of RWPCs were evaluated as complete response (91.6%), partial response (8.2%), no-change (0.2%), and progression (0%) and overall response was equal across subgroups divided by patients' profiles. The median corrected count increment (CCI) at 1 and 24 h post-transfusion were 13.5 (range, 1.9-35.4) × 109/L and 3.5 (range, -13 to 53.6) × 109/L, respectively, and median CCI at 24 h was 5.5 (range, -13 to 53.6) × 109/L in patients without risk factors associated with platelet transfusion refractoriness. Transfusion reactions to RWPCs were observed in only nine transfusions (0.7%), all of which were mild allergic reactions. CONCLUSION: This study demonstrated that RWPCs were effective and safe in patients with a history of transfusion reactions. Further prospective studies on efficacy together with cost-benefit analysis in RWPCs are needed.

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.

A national survey of premedication for transfusion reactions in Japan.

BACKGROUND: Premedication before transfusion is commonly administered in clinical practice despite a lack of evidence for its efficacy. The aim of this study was to clarify the status of premedication and evaluate expert opinions regarding its use in Japanese medical institutions. METHOD: Between May and July 2016, we conducted a questionnaire survey on premedication before transfusion in 252 medical institutes that were certified by an academic society or employed transfusion experts. RESULTS: A total of 141 institutes (54.2%) responded, and hematologists (n=113) comprised the most frequent respondents. The purpose of premedication was to prevent urticaria, pruritus, and fever, and washed blood products were used for anaphylactic shock or refractory transfusion reactions before. Drugs for premedication were intravenously administered either just before or 30min before transfusion. Both inpatients and outpatients were premedicated in a similar manner, and institutional guidelines were not established. More than half of the experts recognized premedication as efficient and necessary, and premedication for previous transfusion reactions was frequently implemented, particularly for platelet transfusion or in patients with hematological diseases. Some institutions administered one or more drugs for premedication from the first transfusion. Antihistamines and hydrocortisone were the most frequently used as premedication. CONCLUSION: Our study reports the current status of premedication for transfusion in Japan. Antihistamines and hydrocortisone were most commonly used for premedication despite a lack of evidence of their use. These findings may help clarify the indications for premedication and the use of washed blood products.

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.

[Evaluation of the Fundamental Performance of 4 Latex Agglutination Reagents to Measure Anti-TP Antibody Concentration and Detailed Investigation of Decision-Mismatched Samples].

Serological diagnosis of syphilis can be made by using the serological test for syphilis (STS) method for detecting a lipid antibody and Treponema pallidum (TP) method for detecting the anti-TP-specific antibody. In STS and TP methods, the basis using latex agglutination reaction has been used in many facilities. However, in latex agglutination, false-positive results due to non-specific reaction have sometimes been obtained in reactions of a routine laboratory test reagent detecting the anti-TP antibody used in our medical laboratory. We evaluated the fundamental performance of 4 reagents to measure anti-TP antibody concentration using latex agglutination: Reagents A, B, C and D produced by SEKISUI MEDICAL, FUJI REBIO, DENKA SEIKEN and SHINO TEST, respectively. We examined the correlations between Reagent A (routine laboratory test reagent) and Reagents B, C, and D in sera from 68 patients, and we performed additional investigation by using a neutralization test, immunochromatography, Western blotting, FTA-ABS (IgG), and STS method by an automatic analyzer for 13 decision-mismatched samples. The fundamental performance of each reagent was as good as that previously reported. Eight of the 13 decision-mismatched samples were false positives due to non-specific reaction of Reagent A. In latex agglutination non-specific reaction is inevitable. However, this study strongly suggests that using a neutralization test and immunochromatography that can be performed quickly is sufficient to verify whether positive reactions are true or false.

[Team approaches to critical bleeding (massive bleeding and transfusion) - chairmen's introductory remarks. Questionnaire survey on current status of hospital clinical laboratories evaluating critical hemorrhage].

In 2007, "the Guidelines for Actions against Intraoperative Critical Hemorrhage" were established by the Japanese Society of Anaesthesiologists and the Japanese Society of Blood transfusion and Cell Therapy. The documentation of in-hospital procedures for critical hemorrhage, especially about how to select RBC units, has widely standardized hospital practice. Patients with intraoperative critical hemorrhage sometimes suffer from massive blood loss. In this situation, some patients develop coagulopathy. To treat them, we need to evaluate their coagulation status based on laboratory test results. So, we performed a nationwide questionnaire survey on the current status of hospital clinical laboratories evaluating critical hemorrhage. From the results of this survey, it was recommended that central hospital laboratories should try to reduce the turn-around time required to test for coagulation parameters as much as possible for appropriate substitution therapy. (Review).

[Contribution of central hospital laboratory to critical bleeding].

It has been reported that fibrinogen products, such as fibrinogen concentrates, cryoprecipitate (CRYO), and fresh frozen plasma, are beneficial for treating coagulopathy due to massive blood transfusion. For the appropriate use of these products, it is necessary to evaluate the status of coagulopathy and determine the trigger level of the fibrinogen concentration for the administration of fibrinogen products. In our institution, we established a treatment procedure for coagulopathy due to massive transfusion in 2011. This procedure includes determination of the trigger level for administration of CRYO (150 mg/dL), timing of sample collection for the evaluation of coagulation parameters (prothrombin time, activated partial thromboplastin time, and fibrinogen) and concentration status during the operation, and a method for rapid coagulation testing (turnaround time within 15 minutes) in critical bleeding. Since 2011, we have performed 56 rapid coagulation tests for patients suffering from critical bleeding. The average turnover time was 13 minutes. According to the rapid coagulation test results, CRYO was administered to 27 patients. These results are satisfactory for treating critical bleeding patients. We stress the need for the establishment of a rapid coagulation test system in the central hospital laboratory. (Review).

[Performance of two matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) models for identification of bacteria isolated from blood culture].

We compared the results of two bacterial identification methods: 1) a traditional method based on phenotypic identification of the causative organism using gram-staining, culture and biochemical markers and 2) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 111 isolates, including 107 strains of common bacteria species and 4 strains of 3 yeast species, were tested by the traditional method and MALDI-TOF MS method(VITEK MS and Micro flex LT). Data obtained using MALDI-TOF MS were classified as Level 1 and Level 2 according to the confidence level of identification results from the VITEK MS ver. 1.0 database (VITEK MS) and MALDI Biotyper ver. 2.0 database (Microflex LT). The proportions of measured samples identified as Level 1 were 98.2% with the VITEK MS database and 87.4% with the MALDI Biotyper database. The concordance rates of the traditional method were 93.7% with the VITEK MS database and 82.0% with the MALDI Biotyper database. Identification results of five strains were mismatched between the traditional method and MALDI-TOF MS. Their ribosomal RNA sequences were identical to the results obtained from MALDI-TOF MS. We concluded that the performance of VITEK MS is superior to that of the traditional method and Microflex LT.

[Fundamental and clinical aspects of cystatin C].

Recent progress of fundamental and clinical studies on cystatin C was reviewed. Most of key studies are indebted to prof. Grubb A and his groups. International contributions from Japanese research work are included here. The protein is a basic low molecular weight protein of 13,300 with 120 amino acid residues and pI 9.3, functioning as a cysteine protease inhibitor. With an introduction of ERM-DA471, international reference material for serum cystatin C, global standardization for immunoassay systems has been much facilitated. No serious problems are present in the pre-analytical stage. Serum reference intervals are properly set in all Asian populations including Japanese with age and gender-related differences. The protein is a powerful serum intrinsic marker for glomerular filtration rate. Estimated glomerular filtration rate (eGFRcysC) in coupled with eGFRCr will definitely be a clinical routine for early detection and prevention of altered kidney function and cardiovascular events in general population. Genetic tests clinically indicated include hereditary cystatin C amyloid angiopathy (L68Q) and adult macular degeneration (A25T) although their frequency is extremely low.

[Examination of the sample centrifugation time for emergency coagulation test].

The rapidity of coagulation testing is important for use as appropriate substitution therapy in patients with, or at risk of critical bleeding requiring massive transfusion. Whereas the ordinary method of coagulation testing is known to be slow, in a critically haemorrhaging patient, a rapid turnaround time of coagulation testing becomes indispensable. To find out if coagulation test results will be affected by a shortened centrifugation time, we measured PT (prothrombin time), APTT (activated partial thromboplastin time), FIB (fibrinogen) and PLT (platelet) in plasma, using different centrifugation times (10 min, 5 min, 3 min), and analyzed the measurements. We found that, whereas centrifugation time significantly affected the PLT count in plasma (10 min; 5.17 +/- 3.71 x 10(3)/microl, 5min; 28. +/- 26.9 x 10(3)/microl, 3min; 63.7 x 10(3)/microl), PT(10min; 14.6 +/- 5.76 sec, 5min; 14.7 +/- 5.84 sec, 3min; 14.9 +/- 6.40 sec), APTT (10min; 36.4 +/- 15.9 sec, 5min; 36.8 +/- 16.5 sec, 3min; 34.7 +/- 11.4 sec) and FIB(10min; 361 +/- 134 mg/dl, 5min; 356 +/- 132 mg/dl, 3min; 356 +/- 125 mg/dl) were not affected. These data suggest that shortening centrifugation time will have no significant effect on the value of PT, APTT and FIB, in an emergency situation.