Effect of microaggregate blood filtration on granulocyte concentrates in vitro

To determine the effect of transfusing granulocyte concentrates through microaggregate blood filters, granulocytes prepared with a cell processor were passed through screen and depth microaggregate filters. Pre- and postfiltration evaluations were made of total granulocyte count, levels of muramidase, granulocyte viability, motility, phagocytosis, bactericidal activity, and hydrogen peroxide-forming capacity. Compared to prefiltration levels, a significant (p less than 0.05) decrease in postfiltration granulocyte counts was seen for all the depth filters studied but not for the standard 170 microns (control) or the 40 microns screen filter. For the various tests of granulocyte function evaluated prefiltration, no significant postfiltration differences (p greater than 0.05) were seen for any of the filters studied. Screen microaggregate filters retained only 1 to 3 percent of granulocytes contained in the concentrates, and thus appear satisfactory for use in clinical transfusions. The large percentage of neutrophils retained by the depth filters (20–62%), however, precludes their use for transfusion of granulocyte concentrates.     

Effects of in vitro adult platelet transfusions on neonatal hemostasis

Summary. Background: Thrombocytopenia is frequent among neonates, and 20–25% of affected infants are treated with platelet transfusions. These are frequently given for mild thrombocytopenia (platelets: 50–100 × 10⁹ L^?1), largely because of the known hyporeactivity of neonatal platelets. In tests of primary hemostasis, however, neonates have shorter bleeding and closure times (CTs) than adults. This has been attributed to their higher hematocrits, higher von Willebrand factor (VWF) concentrations, and predominance of longer VWF polymers. Objective: To determine whether the ‘transfusion’ of adult (relatively hyperreactive) platelets into neonatal blood results in a hypercoagulable profile. Methods: Cord blood (CB) and adult peripheral blood (PB) were separated (with a modified buffy coat method) to generate miniaturized platelet concentrates (PCs) and thrombocytopenic blood. PB‐derived and CB‐derived PCs (n = 7 per group) were then ‘transfused’in vitro into thrombocytopenic CB and PB. The effects of autologous vs. allogeneic (developmentally mismatched) ‘transfusions’ were evaluated with whole blood aggregometry, a platelet function analyzer (PFA‐100), and thromboelastography (TEG). Results: Adult platelets aggregated significantly better than neonatal platelets in response to thrombin receptor‐activating peptide, ADP, and collagen, regardless of the blood into which they were transfused. The ‘transfusion’ of adult platelets into thrombocytopenic CB resulted in shorter CTs‐EPI (PFA‐100) and higher clot strength and firmness (TEG) than ‘transfusion’ of neonatal autologous platelets. Conclusions: In vitro‘transfusion’ of adult platelets into neonatal blood results in shorter CTs than ‘transfusion’ with neonatal platelets. Our findings should raise awareness of the differences between the neonatal and adult hemostatic system and the potential ‘developmental mismatch’ associated with platelet transfusions for neonatal hemostasis.     

The in vitro evaluation of two filters (Erypur and Imugard IG 500) for white cell-poor platelet concentrates

White cell-poor blood components are useful in patients with white cell antibodies. White cells are efficiently removed by two different filters, Imugard and Erypur, which have used saline as the filter solution. This study evaluated these filters as to their production of white cell-poor platelets. Pools of random-donor platelet concentrates were filtered. Prefiltration and postfiltration samples were evaluated for percentages of platelet recovery, white cell (WBC) removal, and platelet function. The two filter solutions tested were normal-strength saline (NSS) and fresh-frozen plasma (FFP). Postfiltration samples using NSS showed no measurable platelet aggregation with ADP, epinephrine, or collagen. However, with FFP, both filters showed 100 percent platelet aggregation with ADP, epinephrine, and collagen. The FFP filter solution provided excellent white cell removal in both filters (Imugard: 100% WBC removal or less than 1.0 X 10(6) residual WBC; Erypur: 99.5% removal or greater than 1.0 X 10(7) residual WBC); however, platelet recovery was better with Imugard (95%) than with Erypur (55%). The filtration procedure is an excellent method for the preparation of white cell-poor platelets; however, the quantity of the saline solution recommended for the filtering of red cells must be minimized for platelets.     

Platelet filters--an evaluation

An in vitro study was made to evaluate the effects of three different infusion sets [HB 182 (Fenwal); Abbott 4594 (Metal filter); and HE 792 (Fenwal)] on the recovery of platelets. The results indicate that platelets are not lost during the filtration process. Platelet recoveries were approximately the same with all three filters under study. The volume of platelet concentrates varies significantly. The loss of platelets in infusion sets and plastic bags is relatively large if small volume concentrates are used.     

Detection and significance of alpha granule membrane protein 140 expression on platelets collected by apheresis

Activation of platelets may be measured by using the monoclonal antibody S12 to detect alpha granule membrane protein 140 (GMP-140) antigen expressed only on activated platelets. S12 was used to measure activation of apheresis platelet concentrates by flow cytometry. Eleven platelet concentrates obtained by one method and nine obtained by another were analyzed 4 hours after collection. Means +/- 1SD of 25.3 +/- 14.8 percent and 28.9 +/- 11.6 percent, respectively, of platelets were found to be activated (range, 4.8-53.1%). Six of the platelet concentrates obtained by the second method were filtered. There was a drop of 17.8 percent (range, 10-25%) in the mean total number of platelets recovered after filtration, but there was no difference in the proportion of activated platelets measured immediately before and after filtration. The 1-hour posttransfusion platelet count increment was obtained after 12 of these apheresis platelet concentrates were transfused to eight patients who were not refractory from either a clinical or alloimmune standpoint. There was a significant inverse correlation between the platelet count increment and the proportion of activated platelets in the component (p less than 0.05, r = -.58). These data suggest that apheresis platelet concentrates with more activated platelets have a reduced 1-hour recovery. Filtration did not enhance activation or selectively remove activated platelets. Expression of GMP-140 may serve as a useful quality control measurement.     

Removal by white cell-reduction filters of activated platelets expressing CD62

White cell (WBC)-reduction filters that remove more than 99 percent of the WBCs from platelet concentrates are rapidly being introduced into routine use. Using activation-dependent monoclonal antibodies and flow cytometry, platelet activation was evaluated before and after WBC reduction in 10 platelet concentrates prepared manually from whole blood obtained from five male and five female regular volunteer blood donors. In general no significant increases were found in platelet activation markers after WBC reduction using filters. However, if platelets were activated during preparation, increased numbers of platelets were found expressing the activation marker CD62, and this correlated with the decrease in the platelet count after WBC reduction. These observations may explain increased platelet loss following WBC reduction in some platelet components.     

Preparation, storage and quality control of platelet concentrates

Patients with thrombocytopaenia need transfusions of platelet concentrates to prevent or stop bleeding. A platelet transfusion should provide platelets with good functionality. The quality of platelet concentrates (PCs) is affected by the preparation method and the storage conditions including duration of storage, type of storage container, and storage solution (plasma or an additive solution). Different in vivo and in vitro techniques can be used to analyse PCs. Platelets can be collected by apheresis technique, and from whole blood using either the buffy-coat or the platelet-rich plasma method. PCs can be gamma irradiated to prevent occurrence of graft-versus-host disease in the recipient. Pathogen inactivation procedures have been developed to prevent transmission of bacteraemia.     

改良白膜法制备浓缩血小板及回收率的影响因素

背景：白膜法和富含血浆法制备的浓缩血小板有无效输注发生率高和不良反应发生率高的缺点。 目的：观察改良白膜法制备浓缩血小板的实验研究,分析制备浓缩血小板回收率的影响因素。 方法：随机抽取126例站内采集后4-6 h的400 mL血液,随机分成改良白膜法组、白膜法组和富含血浆法组。改良白膜法采用3步离心,第1次采用次重离心,离心转速2300 r/min,离心时间12 min,降速5,离心温度（22±2）℃;第2次采用轻离心,离心转速910 r/min,离心时间10 min,离心温度（22±2）℃;第3次离心转速2800 r/min,离心时间12 min,离心温度（22±2）℃,离心后,挤去上层含血小板较少的血浆,袋中留30 mL血浆悬浮血小板,即为浓缩血小板。通过数据库文献检索的方法分析制备浓缩血小板回收率的影响因素。 结果与结论：改良白膜法、白膜法以及富含血浆法制备的手工浓缩血小板中,制备前各组血小板总数差别无统计学意义（P 〉0.05）;富含血浆法组和改良白膜法组较白膜法组血小板回收率高,差异有显著性意义（P 0.05）;白膜法组和改良白膜法组较富含血浆法组残留红细胞和残留白细胞的量少,差异有显著性意义（P0.05）。制备浓缩血小板的回收率受到全血量、离心转速、离心时间、离心方法等因素的影响。改良白膜法制备浓缩血小板减少红细胞和白细胞的残留量,提高了血小板的回收率,可在血液中心或中心血站推广应用。     

Transfusion of Platelet Concentrates Prepared from Acidified Platelet-Rich Plasma*

With the use of Aster's and excess ACD solutions, the recovery of chromium⁵¹-labeled platelets was greater, and the sequestration phenomenon decreased in patients without hematologic disease, compared with preparations collected in the normal amounts of ACD as the anticoagulant. Analysis of platelet counts following transfusions of platelet concentrates demonstrated a significant increase of platelets in transfused leukemic patients using acidified preparations as compared with those collected in normal amounts of ACD. The use of additional ACD solution which is routinely available in the blood bank appears to be a satisfactory method to prepare the acidified platelet concentrates.     

冰冻血小板低温损伤及防护机制的研究进展

血小板作为人体血液中的重要组分,在生理性止血中起着非常重要的作用。血小板的输注主要用于特发性血小板减少性紫癜、放化疗病人的血小板减少以及其他原因导致的出血性血小板减少症。室温保存时间（5 d）限制了血小板的临床应用,导致血小板的废弃率不断升高,因此延长血小板的保存时间对血小板的临床应用意义重大。目前-80℃冻存血小板是一种较为理想的长期保存方法,本文就血小板冰冻保存过程中损伤及对其防护机制的研究进展作一综述。     

In vivo survival of platelets prepared in CPD anticoagulant

Autologous platelet concentrates (PC) were prepared from 480 ml of whole blood collected into citrate-phosphate-dextrose (CPD) anticoagulant in order to evaluate the latter with regard to (1) efficiency of platelet harvest in the PC; (2) in vivo platelet recovery; and (3) platelet survival after transfusion. Platelets were labeled with ⁵¹Cr. In consecutive trials an average of 0.81 ± 0.07 × 10¹¹ (mean ± SE) platelets were harvested per unit of PC. This represented recovery of 72.4 ± 7.5 per cent of the calculated number of platelets originally present per donor unit. After auto-transfusion, the in vivo recovery of labeled platelets was 46.3 ± 4.6 per cent, per donor unit. The disappearance of the labeled platelets from the circulation was linear between 6 and 144 hours after transfusion, with a mean T½ of 4.36 days. A method of calculating in vivo recovery and of estimating blood volume is suggested to simplify comparisons among the works of various investigators. CPD is a suitable anticoagulant for the preparation of platelet concentrates.     

Use of an electromechanical infusion pump for transfusion of platelet concentrates

To determine whether platelet concentrates can be administered safely through electromechanical infusion devices, we studied stored platelet concentrates passed through one pump system (Abbott). We measured in vitro changes in platelet count and lactic dehydrogenase (LDH) and beta- thromboglobulin (beta-TG) release which occurred after passing the concentrates through the pump system. To compare in vivo survival, five normal volunteers were given an injection of autologous Indium-111- labeled platelet concentrates at two different times, once using platelets which had been passed through the pump system (test group) and once using platelet concentrates which had not (control group). In vitro studies showed no significant changes (p greater than 0.05) in platelet count, or in LDH or beta-TG release after passage through the pump system. In vivo platelet recovery at 2 hours was 39.8 +/− 4.7 percent (mean +/− 1 SD) for the control platelets and 40.7 +/− 9.3 percent for the platelets passed through the pump system (p greater than 0.05; n = 5). There was no significant difference in platelet survival measured in days between the control group and the test group using a linear (8.0 +/− 0.9 vs. 7.2 +/− 0.3), exponential (3.7 +/− 0.7 vs. 3.1 +/− 0.5), or multiple hit (5.4 +/− 2.3 vs. 4.8 +/− 1.0) (p greater than 0.05; n = 5) model. We conclude that this pump system is acceptable for use in clinical practice when control over volume and rate of platelet transfusion is important.     

Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22 degrees C. Importance of storage time

We studied the transfusion response from random donor platelet concentrates in 15 stable multitransfused, thrombocytopenic patients by comparing the platelet counts measured before and 20 hours after transfusion. The observed platelet increments were corrected (corrected increment, C.I.) for the number of units of platelet concentrate transfused and the patient's body surface area in square meters (platelets/microliter per unit/m2). Using platelet concentrates stored for less than 24 hours, the patients achieved a median C.I. of 9500 (range: 5000–18,000). When platelet concentrates stored for 24 to 48 hours or 48 to 72 hours were given, the median C.I. markedly decreased to 1000 (range: 0–4800) and 0 (range: 0–5100), respectively (p less than 0.001). These differences could not be explained by further recipient alloimmunization. Transfusion with platelet concentrates less than 24 hours old on a second occasion, bracketing the transfusions of older platelet concentrates, resulted in a median C.I. of 7200 (range: 5400–14,500). Similar results were obtained in three patients when HLA- identical sibling platelet concentrates were employed. In vitro tests, including pH, morphology, and aggregation, demonstrated no statistically significant differences among the platelet concentrates stored for less than 24 hours, 24 to 48 hours, and 48 to 72 hours. These studies suggest that, although platelet concentrates can be stored for 72 hours without loss of in vitro function, the in vivo recovery is significantly diminished after 24 hours of storage, and preferably patients should not be transfused prophylactically with platelet concentrates greater than 24 hours old.     

利用SCID小鼠模型和流式细胞术体内评估血小板制品质量

目的建立一种血小板制品体内质量评价的动物实验方法。方法用带有超细针头的胰导素注射器将人血小板经尾静脉注入BALB／c、FVB、SCID小鼠体内，于30min及2、4、6、8、12和24h采集小鼠全血，肝素抗凝，用CD61-PE标记后，流式细胞术计数人血小板，以30min的人血小板计数为100％，计算人血小板的存活率。结果新鲜人血小板在SCID小鼠体内的存活时间与BALB／c、FVB相比明显延长，其输注4h血小板存活率：BALB／c为29．9％±6．5％（n=8）、FVB为28．1％±5．5％（n=8）、SCID为68．6％±8．1％（n=10）；推算半寿期（T1／2）分别为2．5、2、8h。由化学药物及不正确储存所致损伤的血小板在SCID小鼠体内的存活时间明显缩短。结论利用流式细胞术分析人血小板在SCID小鼠模型中的存活率可以评价血小板制品体内质量。     

Biocompatibility of white cell filters as evaluated by complement activation

The biocompatibility of nine different white cell filters was examined by analysis of complement activation in plasma specimens obtained from blood components before and after filtration. Filters for both red cell (RBC) concentrates and platelet concentrates (PCs) were tested. It was found in all of the filters tested that the postfiltration levels of complement activation products were not higher than the prefiltration levels in RBC concentrates and PCs. One exception was the filtration of multiple PCs with Imugard IG-500, in which case a rise in C3 activation products was seen. Moreover, there was a significant rise in C3 activation products, but not the terminal complement complex, when plasma was filtered through Imugard E, which contrasted with results with the other filters. High initial and storage time-dependent levels, especially of C3 activation products, were observed in the PCs, probably due to their processing at room temperature. It can be concluded that the majority of the filters tested do not activate complement.     

Circulation and function of human platelets isolated from units of CPDA- 1, CPDA-2, and CPDA-3 anticoagulated blood and frozen with DMSO

Platelet concentrates (PC) were isolated by serial differential centrifugation from units of blood anticoagulated with one of the citrate-phosphate-dextrose-adenine solutions (CPDA-1, CPDA-2, CPDA-2). The platelet concentrates were frozen with six percent dimethylsulfoxide at 2–3 degrees C per minute and stored in a -80 degrees C mechanical freezer in polyvinyl chloride or polyolefin plastic containers. After frozen storage at -80 degrees C for up to three months, the concentrates were thawed at 42 degrees C within 2.5 to 4.0 minutes, washed with autologous plasma, two percent dimethylsulfoxide and 10 percent acid-citrate-dextrose solution, and then resuspended in plasma. The washed platelets were labeled with 51Cr and transfused back to the donor from whom they had been obtained. In vitro recovery from whole blood to platelet concentrate was 70.5 ± 17 percent (mean ± one SD). In vitro freeze-thaw-wash recovery determined by phase microscopy was 78.5 ± 12.8 percent, in vivo 51Cr platelet recovery two hours after transfusion was 41.3 ± 13.5 percent, and the platelets had a linear lifespan of about eight days. A single unit of previously frozen platelets shortened an aspirin- prolonged bleeding time two and 24 hours after infusion. Results were similar with platelets isolated from all three anticoagulants and stored in both plastics. The results also were comparable to previous findings in this laboratory with platelets isolated from ACD and CPD anticoagulated blood.     

In vitro assessment of platelet function

With the realization that the skin bleeding time is often an unreliable measure of platelet function, efforts have been made to identify ways to assess qualitative platelet dysfunction. Currently available techniques measure platelet adhesion, platelet aggregation, the ability of platelets to retard or stop flow through filters, and the contribution of platelets to in vitro clot formation. Glass bead adhesion, which continues to be performed in some laboratories, is gradually being replaced by measures of platelet adhesion to filters composed of glass fibers, Dacron fibers, or collagen. In each instance, anticoagulated platelet-rich plasma or whole blood flows through the filter under a regulated pressure gradient. The amount of blood flowing through the filter versus time and/or the time to filter occlusion are measured. Recent developments in platelet aggregation have focused on whole blood and stagnation point flow aggregation techniques. Whole blood aggregation does not require blood sample processing and accommodates blood obtained from citrated vacutainer tubes. Stagnation point flow measures both platelet adhesion and aggregation and may be able to detect pathologically-enhanced platelet function. Global measures of hemostasis attempt to simultaneously evaluate the adequacy of fluid phase coagulation and platelet function. Currently available techniques include Thromboelastography, SonoClot Analyzer, Hemodyne Hemostasis Analyzer, PITT, and Hemostatometry. Although each of these technologies have been shown to provide interesting data in the research setting, the ability of any of these techniques to detect abnormal or clinical inadequate platelet function remains to be established.     

Administration of microaggregate filtered blood using a manual infusion pump

Administration sets containing in-line plastic blood pumps are commonly used to transfuse blood. Flow rates were measured while six units of whole blood and red blood cells were infused through a blood pump administration set coupled to either a large pore 260-micron filter or a 20-micron microaggregate blood filter. Using the same type of blood pump administration system and three units of whole blood, the flow rates achieved by the currently available microaggregate blood filters were compared. Results showed that despite a smaller pore size the 20- micron microaggregate blood filter achieved flow rates that were faster than or equal to those recorded for the larger pore (260-micron) filter. This was attributed to the larger filtration surface area possessed by the smaller pore filter, 140 cm2 versus only 30 cm2 for the 260-micron filter. All of the microaggregate filters studied were able to filter three units of whole blood at flow rates in excess of 80 ml/minute. There was no evidence of blood pump induced hemolysis. We concluded that a manual infusion pump can be used to transfuse microaggregate filtered blood rapidly enough to be acceptable for routine clinical or intraoperative use.     

Reduction of febrile but not allergic reactions to RBCs and platelets after conversion to universal prestorage leukoreduction

BACKGROUND: Between January 1995 and November 1998, at Yale-New Haven Hospital, 25 percent of RBCs transfused were processed through prestorage or bedside leukoreduction filters, chosen on a per patient basis (selective leukoreduction [SLR]). Between January 1995 and July 1999, 30 percent of platelet concentrates (PCs) were infused through bedside leukoreduction filters. In an attempt to decrease febrile nonhemolytic transfusion reactions (FNHTR), a change was made from SLR to universal prestorage leukoreduction (UPL) for RBCs between November 1998 and December 1999 and for random donor PCs between July 1999 and January 2000. FNHTR and allergic transfusion reactions (ATR) reported from January 1995 through December 2002 were reviewed. STUDY DESIGN AND METHODS: For retrospective observational analysis, blood bank data were available on the number of RBCs and PCs transfused, percent products leukoreduced, and rate of FNHTR and ATR from 1995 through December 2002. After dividing this time period into three phases (SLR, transition, and UPL), these data were evaluated using odds ratio (ORs) and Student's t tests. RESULTS: A total of 145,369 RBCs and 137,982 PCs (29,487 PC pools) transfused between January 1995 and December 2002 were evaluated. For RBCs, the relative FNHTR rate decreased 47.1 percent, from 0.34 percent (SLR) to 0.18 percent (UPL) (p < 0.0001). ATR rates for RBCs showed 0.09 percent for both SLR and UPL groups (p > 0.05, NS). For PCs, the FNHTR relative rate decreased 93.1 percent, from 2.18 percent for SLR to 0.15 percent for UPL (p < 0.0001). Rates for ATR were 0.49 percent (SLR) and 0.35 percent (UPL) (p > 0.05, NS). CONCLUSIONS: A significant decrease in the frequency of posttransfusion FNHTR, but not ATR, for RBCs and PCs followed introduction of 100-percent UPL. The data support the hypothesis that the practice of UPL of RBCs and PCs decreases the frequency of FNHTR and thus improves patient care over the practice of selective leukoreduction.     

Preparation of leukocyte-poor platelets by filtration

There is evidence that leukocyte contaminating red blood cells and platelet concentrates are responsible for refractoriness to platelet transfusions. The efficacy of a cotton-wool filter to remove leukocytes from red blood cells has been documented previously. The present study was designed to evaluate whether the cotton-wool filters can effectively remove leukocytes from platelet concentrates. Sixty pools of random-donor platelets and single-donor plateletpheresis products were filtered through a cotton-wool filter. The efficacy of filtration was determined by measuring the absolute numbers of leukocytes and platelets and subpopulations of mononuclear cells. The average platelet loss was 8% per pool of random platelets and 10% per plateletpheresis product. The average leukocyte removal was 99% from a pool of random platelets and plateletpheresis concentrates collected by CS-3000 and 90% from plateletpheresis concentrates harvested by single-stage COBE/IBM-2997. The filtration removed 100% of granulocytes, 95% of monocytes, 90% of B-lymphocytes, and 85% of T-lymphocytes. We conclude that filtration through a cotton-wool filter is an efficient and cost-effective method for preparation of leukocyte-poor platelets.