Chihiro Homma

Reduction in adverse reactions to platelets by the removal of plasma supernatant and resuspension in a new additive solution (M-sol).

BACKGROUND: Leukodepletion reduces but does not eliminate adverse reactions to platelet concentrate (PC). As an alternative strategy, plasma reduction or washing of platelets should be considered. However, the efficacy of this strategy is still unclear.

Storage of platelets in a novel additive solution (M-sol), which is prepared by mixing solutions approved for clinical use that are not especially for platelet storage

BACKGROUND: To reduce adverse reactions due to platelet (PLT) transfusion, medical solutions on the market, such as saline and ACD‐A, are used to replace the plasma of PLT concentrates in Japan; however, they are not strongly preservative. Here, an attempt was made to develop a novel additive solution (M‐sol) having the ability to preserve PLTs stably, with only approved solutions for clinical use.

Comparison between in vitro qualities of platelets washed with commercially available additive solutions and those washed with M-sol.

Background and Objectives  We previously developed a novel additive solution (M‐sol) with a high ability to preserve the in vitro qualities of platelets (PLTs) in washed PLTs Here, we compared the ability of M‐sol with that of commercially available additive solutions (ASs) to preserve the in vitro qualities (pH, mean PLT volume, %disc, P‐selectin, %hypotonic shock response and aggregation) of PLTs at a low plasma concentration.

Comparison between bacterial growth in platelets (PLTs) washed with M‐sol and that in PLT‐rich plasma

Platelets (PLTs) washed with additive solution (AS) are useful for the reduction of adverse reactions such as anaphylaxis or febrile nonhemolytic reactions induced by PLT concentrate (PC) transfusion. We previously developed a novel AS (M-sol), which is able to maintain the in vitro qualities of PLTs at a low plasma concentration for a longer period than other commercially available AS. Improvement of AS may lead to extending the shelf life of washed PLTs. Since M-sol can be aseptically prepared by simply mixing commercially available solutions approved for clinical use, this solution is already being used to prepare washed PLTs at some blood centers and hospitals in Japan. Bacterial contamination is currently the most frequent infectious risk related to transfusion. Due to storage at room temperature, PCs are more prone to grow bacteria than red blood cells. Since plasma contains certain factors such as complement components that inhibit bacterial growth, decreasing the plasma concentration by washing of PC may promote bacterial growth in washed PLTs. Here we compared bacterial growth in PLTs washed with M-sol with that in PLT-rich plasma (PRP) during a 7-day storage period. Apheresis PC, which was processed 4 or 5 days after collection, was divided into two equal aliquots (control group and test group). After centrifugation (2560 ¥ g, 10 min) of both aliquots and removal of as much supernatant as possible, the pellet of the control group was resuspended in 170 mL of ABO-identical fresh-frozen plasma that had been prepared from CPD whole blood within 6 hours after donation and stored at -20°C until use. In the test group, the pellet was resuspended in 170 mL of M-sol. The control group (176 8 mL, 109 ¥ 10 18 ¥ 10/mL, 5 units under Japanese standard, plasma concentration 100%) and test group (175 7 mL, 104 ¥ 10 18 ¥ 10/mL, 5 units, plasma concentration 4.54%) were stored at 20 to 24°C on a flatbed shaker (50-60 cycles/min) in a polyolefin bag (KBP600FPN, Kawasumi Co., Ltd, Tokyo, Japan). The bacterial strains used in this study were: Bacillus cereus (ATCC#10876), Propionibacterium acnes (ATCC#6919), Staphylococcus epidermidis (ATCC#49134), Staphylococcus aureus (NBRC#13276), Escherichia coli (ATCC#11775), and Serratia marcescens (ATCC#14756). Fresh bacterial culture was prepared by incubating a single colony in liquid medium (BacT/ALERT BPA or BPN, bioMérieux, Inc., Durham, NC). Bacterial culture diluted with saline was inoculated into the control group and test group so that the final concentration of bacteria in each group was less than 10 colony-forming units (CFUs)/mL (Day 0). The PLT suspensions including bacteria were stored with agitation at 20 to 24°C until Day 7. To count bacterial colonies, aliquots sampled from the test group and the control group were plated as undiluted samples (200 mL/ plate ¥ five plates) and as serial 1-in-10 dilutions (100 mL/ plate) on agar plates. The aerobic bacteria were incubated for 24 hours or more at 37°C under aerobic conditions on agar plates (Trypto-Soya agar plate, Nissui Pharmaceutical Co. Ltd, Tokyo, Japan). P. acnes was incubated on blood agar plates (Brucella HK agar plate, Kyokuto Pharmaceutical Industrial Co. Ltd, Tokyo, Japan) for 7 days at 30°C under anaerobic conditions. Two-tailed paired t test was used for analysis of differences between the control group and test group. Significance was accepted at a p value less than 0.05. Bacterial concentrations of S. epidermidis in the test group were significantly lower than those in the control group on Days 3 and 4 (Table 1). Similarly, bacterial concentrations of S. aureus in the test group were significantly lower than those in the control group on Days 1 and 2 (Table 1). Previously, the decreased iron concentration in PC due to iron chelation was reported to suppress the growth of S. epidermidis and S. aureus, indicating that iron is a critical factor for the proliferation of these two bacteria. Therefore, growth suppression of those bacteria by washing is thought to be due to a reduction of the essential nutritional content. The exhaustion of essential nutrients by plasma removal may have larger effects on the growth of S. epidermidis and S. aureus than the reduction of suppressive effects of plasma components on bacterial growth. Furthermore, it was reported that pH increase in PLT suspension inhibits the proliferation of S. epidermidis. The pH of PLTs washed with M-sol (7.47-7.64) is higher than that of PRP (6.91-7.09) 24 hours after washing. Therefore, the higher pH may be another reason for the growth suppression of S. epidermidis in the test group. Bacterial concentrations of B. cereus in both groups were comparable on Days 2, 3, and 7, while those in the test group were significantly lower than those in the control group on Day 1, which may be due to a delay in entering the vegetative state in M-sol compared to that in plasma. The growth of P. acnes (anaerobic bacteria) was negligible in both groups during 7 days of storage under aerobic conditions (Table 1). In case of E. coli and S. marcescens, bacterial concentrations in the test group were increased in a time-dependent manner, while that in the control group was negligible or under the detectable limit during 7 days of storage (Table 1). Since Gramnegative bacteria are susceptible to growth inhibition

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

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.

Effects of helicopter transport on red blood cell components

BACKGROUND There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. MATERIALS AND METHODS Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. RESULTS During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. DISCUSSION These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance.

Influence of a 24-hour interruption of agitation on in vitro properties of platelets washed with M-sol during 7-day storage

We would like to make a contribution toward the recent publication in TRANSFUSION by Mestra and coworkers. Firstly, the authors said that this is the first report of transfusion-transmitted leishmaniasis in an immunocompromised patient in Colombia. However, identification of the Leishmania infection was demonstrated neither in transfused blood components nor in blood donors. Antibodies against Leishmania spp. were detected in the patient’s serum samples as part of a study to rule out renal transplant-transmitted visceral leishmaniasis. However, it is not clear if the serologic Leishmaniapositive result of the patient’s serum corresponded to a sample taken before or after receiving transfusions. To our understanding, if no data related to the patient’s infection status before the transfusions have been clearly provided and, additionally, as Leishmania infection in transfused blood components and/or blood donors has not been demonstrated by the authors, it is very difficult to agree that this certainly represents a case of transfusiontransmitted visceral leishmaniasis. From the transfusion point of view it would have been interesting to know whether or not the transfused blood components were leukoreduced and, if so, when the leukoreduction was performed, that is, before storage or at the bedside, since various studies have been published on the efficacy of whole blood, fresh plasma, and red blood cell filters used for leukoreduction in reducing the number of Leishmania spp. parasites in blood components and thereby minimizing the potential risk of Leishmania transmission through blood transfusions. This information could have thrown light on the probability of the parasite being transmitted to the patient through the transfusion of a contaminated blood component taken from an asymptomatic donor. This case was also published in 2006 as allograft kidney dysfunction associated with infection with amastigotes of Trypanosoma cruzi. However, neither polymerase chain reaction (PCR) nor any other antigen detection technique was performed to demonstrate that the amastigotes in the renal parenchymal corresponded to T. cruzi. To distinguish Chagas disease from Leishmania infection, it would have been helpful to perform Leishmaniaand T. cruzi– specific PCRs on the renal biopsy since it is difficult to differentiate the amastigotes from these two genera by direct microscopic examination. This observation has implications related to the article being commented on because it would explain the patient’s clinical symptoms showing after the renal transplant and immediately before initiating the transfusions. Nifurtimox was given to treat Chagas disease. This may have also contributed to delaying the diagnosis of visceral leishmaniasis, since this medication is partially effective against Leishmania spp. as in vivo studies have shown. All these aspects could also be compatible with the reactivation of a latent Leishmania infection by immunosuppression. We believe that this is a very interesting report concerning a case of visceral leishmaniasis caused by L. (L.) mexicana in an immunocompromised patient in Colombia. This is already in itself a very remarkable contribution made by the authors because, as mentioned in the article, L. (L.) mexicana is a species that has been associated mainly with cutaneous leishmaniasis. There are, however, various aspects that might support the fact that this case was due to the reactivation of a latent Leishmania infection, that is, the patient’s clinical symptoms, the lack of data related to the patient’s infection status before the transfusion, the blood components transfused, and their donors. Similarly, other aspects, which are primarily based on ruling out the transmission of visceral leishmaniasis through solid-organ transplantation, and the fact that the patient’s medical interview did not reveal any prior exposure to Leishmania, all support the hypothesis that the visceral leishmaniasis was transmitted through blood transfusion. Since there are arguments both for and against the two options, we would suggest that this report be considered as a possible case of transfusion-transmitted visceral leishmaniasis.

The effect of irradiation on platelets in M‐sol additive solution with 30 percent residual plasma

Jr is a high-prevalence red blood cell (RBC) antigen. The clinical significance of anti-Jr is still not well established because it occurs rarely and has only been evaluated in few cases, but generally is thought to be of little relevance in the development of hemolytic disease of the fetus and newborn (HDFN). However, anti-Jr has been reported as a cause of mild HDFN and, recently, a case of fatal HDFN associated with anti-Jr was published. We describe a fatal immune HDF possibly due to anti-Jr. A 39-year-old Caucasian woman was referred to our hospital in the 12th week of pregnancy. A routine ultrasonography examination showed hidrops fetalis. The obstetric history of this patient included four pregnancies before the current one. The first pregnancy was in 1997 and ended in abortion, but the antibody screening test (AST) was not performed. In 1998 and with the second pregnancy, an AST revealed an antibody of a high-incidence RBC antigen (Makropanel 16, Amsterdam, Netherlands; Panocell10, Immucor Gamma, Norcross, CA; DiaMed, Morat, Switzerland) by column agglutination Diana Gel (Group Grifols, Barcelona, Spain). This antibody was studied in a reference laboratory (American Red Cross, Los Angeles, CA) and an anti-Jr (titer <8) was identified. The antibody subclass was immunoglobulin G (IgG)4 and the monocyte monolayer assay (MMA) was normal (7%) by the method described by Nance and coworkers. Beside this pregnancy, she had two more in 2000 and 2004, but none of the three babies had anemia, even if all had positive DAT (2+) and the elution showed anti-Jr (Gamma Elu-Kit II, Immucor Gamma; titer <16). The IgG subclass and MMA were not performed at that time. In the fifth pregnancy (2008), the ultrasound examination showed hidrops fetalis with no congenital anomaly. The titer of anti-Jr had increased to less than 128 and the IgG predominant subclasses were IgG2 and IgG3, performed with murine monoclonal antibodies (Peli Class human IgG subclass kit, Sanquin Reagents, Amsterdam, Netherlands). The result of the MMA with maternal serum and Jr(a+) RBC was 15 percent (control value, 7%). Serologic tests to rule out infectious causes of anemia were all negative (IgM parvovirus B19, IgM varicela-zoster, IgM rubeola, IgM Epstein-Barr, IgM toxoplasma, VHC, and VHB). The anti-phospholipid, anti-DNA, anti-nuclear, and anti-cardiolipin antibodies were also negative. The pregnancy ended in a voluntary abortion because of the little viability of the fetus. Although anti-Jr is not a common cause of HDFN, there are many data that suggest its involvement in this case, such as previous pregnancies, positive DAT in the newborns, identification of anti-Jr in their eluates, increased titer of anti-Jr, positive MMA, IgG subclasses, and the absence of other causes that could explain hidrops fetalis. A review of the literature indicates that anti-Jr may be clinically significant, because it has been related to some cases of HDFN. Most cases are mild or moderate, but some severe and fatal cases have been recently reported. This case provides more information about the clinical significance of anti-Jr as a cause of HDFN, which can have a fatal outcome. Therefore, and according to other authors, we recommend close monitoring of pregnant women with high or increasing titers of anti-Jr, especially those with previous pregnancies. Francisco Arriaga, MD e-mail: arriaga_fra@gva.es Ines Gomez Maria Dolores Linares Adriana Gascon Nelly Carpio Blood Bank Hematology Department Alfredo Perales Obstetric Service Hospital Universitario La Fe Valencia, Spain

Evaluation of ADAM-rWBC for counting residual leucocytes in leucocyte-reduced whole blood and apheresis platelet concentrates

Dear Sir, Universal leucocyte reduction of blood components has been widely implemented to reduce the risk of transfusion reactions (Beckman et al., 2004). The residual numbers of leucocytes are required to be <1× 106 cells per blood product and <5× 106 cells per blood product in the European and American guidelines, respectively (U.S. Food and Drug Administration, 2012, Council of Europe Publishing, 2013). In Japan, 1% of manufactured leucocyte-reduced (LR) products are quality control (QC) tested and residual leucocytes in ≥95% of those blood products should be ≤1× 106 cells bag−1. The leucocyte concentrations in LR-blood products tend to be <1 cells μL−1 (Seghatchian et al., 2001; Strobel et al., 2014). Therefore, accurate and easy methods of counting low concentrations of leucocytes are essential for QC of LR-blood products to ensure compliance with the guidelines. The flow cytometry method (FCM) has the advantages of good reproducibility and high throughput for counting the low concentration of leucocytes in LR-blood products (Dzik et al., 2000; van der Meer et al., 2001). Recently, ADAM-rWBC (NanoEnTek, Seoul, Korea), a new system to count residual leucocytes in blood components for transfusion was introduced (Bae et al., 2007; Strobel et al., 2014). ADAM-rWBC uses fluorescence microscopy for cell counting. The blood sample is stained with a solution containing fluorescent dye (propidium iodide, PI) and detergent. PI only stains cells containing DNA. Because PI can permeate through cell membranes due to the detergent, both viable and dead cells are stained. The stained sample is applied onto a disposable plastic slide and inserted into the ADAM-rWBC device. The light source for the sample excitation is a green light emission diode (LED). The emission images of the cells are detected by charge coupled device (CCD) camera. The pictures of 203 fields on the slide taken by the camera are recorded and the fluorescence spots are counted as cells using analysis software. The concentration of the cell is calculated by the analysis software and displayed. ADAM-rWBC is thought to be more convenient than FCM (Kline et al., 2012; Strobel et al., 2014). In the study by Kline et al., counting the number of leucocytes in red blood cell (RBC) products and platelet concentrate (PC)s, obtained values

The effect of 30-Gy X-ray irradiation on platelets washed with M-sol additive solution.

(15 of 75 donors) of paid, source plasma donations in the United States tested positive for marijuana. At present, the Universal Donor Health Questionnaire (UDHQ) does not query volunteer donors about the use of marijuana and there is no federal requirement for testing of blood donors for any illicit drugs, including marijuana. Although only two states currently have legalized recreational marijuana, blood products are routinely shipped across state lines and blood donors may choose to donate while visiting another state. As a result, the blood supply in all 50 states is theoretically impacted by the legalization of recreational marijuana in Colorado and Washington. In humans, the psychoactive component of marijuana, Δ-THC, is mainly bound to low-density lipoproteins and albumin. Ten percent or less of blood cannabinoids are bound to red blood cells. Δ-THC has been shown to be present in the plasma of chronic marijuana users even after several days of continuous, monitored abstinence. However, given the large volume of distribution of Δ-THC, we believe that it would be unlikely for a transfusion-associated marijuana exposure to be detected in the urine of a transfusion recipient. Nonetheless, physicians should be aware that blood transfusion, particularly transfusion with plasma or platelets, is a potential cause of Δ-THC exposure. At present, the significance of transfusion-associated Δ-THC exposure is not known. However the effects of this drug on special populations, such as premature neonates, have not been established. There are many substances that donors may be exposed to but are not tested for or queried about on the UDHQ. Some relevant substances include methotrexate, cyclophosphamide, and heavy metals. We believe that the legalization of recreational marijuana, in the context of its recent history as an illegal drug, is fairly unique among substances. This event could be interpreted to indicate that marijuana use has been determined to be “safe.” In addition, the potential medicinal uses of marijuana are well publicized. Overall, we are concerned that these events may create a perception of social and medical approval of marijuana use among blood donors, which may lead to blood donor confidence that it is safe to donate blood, even after recent marijuana use. We feel that it is not yet known whether or not exposure to a small quantity of marijuana metabolites in the context of a blood transfusion is safe. Until further evidence is available, particularly regarding the effect of marijuana metabolites on premature infants, we feel that steps should be taken to limit the number of active marijuana drug users in the volunteer blood donor pool. Due to the fact that urine is the usual matrix for marijuana drug testing, and urine is not collected from blood donors, we favor screening for active marijuana use by means of the UDHQ rather than by laboratory testing. Based on available data, we feel that including the question “Have you used marijuana in the past 2 weeks?” on the UDHQ and instituting a 2-week deferral for donors who answer “yes” would likely eliminate marijuana metabolites from the blood supply. Given that the UDHQ already asks questions about sexual history and intravenous drug use, we find it unlikely that donors would be reluctant to answer a single question about recent marijuana use.