Comparison of blood component preparation methods from whole blood bags based on buffy coat extraction

We compared the data of our quality control laboratory of the blood components according to the blood component preparation method that we used. We prepared blood components from top and top whole blood (WB) bags and manual pooling of buffy coats (BCs) (method I) or from top and bottom WB bags and automated pooling of BCs with OrbiSac (method II). Pooled platelet concentrates (PC) obtained with method II had higher platelet content when compared with pooled PCs obtained with method I (3.5 ± 0.7 × 10¹¹ vs. 2.6 ± 0.8 × 10¹¹; p < 0.001). The hemoglobin content in the RBCs obtained with method I was higher when compared with method II (55 ± 7 g vs. 52.5 ± 6.6 g; p < 0.001).     

P04In Vitro Evaluation of Buffy Coat Derived Platelet Concentrates in SSP+ Platelet Storage Medium

Introduction  As part of the process to improve the safety and quality of the platelet supply, the NBS is evaluating the use of platelet storage media (PSM). The objective of this study was to produce a platelet concentrate (PC) containing 70% SSP+ and 30% plasma, whilst maintaining platelet yield, and to evaluate their in vitro function. Study Design. Buffy coats were prepared using the standard NBS method and four (ABO matched) were pooled together with 250 mL of SSP+. Centrifugation was performed using optimised conditions for PSM and an Optipress used to express the PC through a Pall Autostop LD filter into a Pall ELX storage bag. Ten PCs were prepared, and platelet quality was assessed using in vitro assays for functionality and activation on days 1, 5, 7 and 9 of storage.
Results   

     

Red blood cell aggregability is increased by aspirin and flow stress, whereas dipyridamole induces cell shape alterations: measurements by digital image analysis

We evaluated the influence of pretreatment with aspirin in vitro , alone or combined with dipyridamole, on red cell aggregability. Samples were tested before and after being exposed to well-defined flow conditions. An aggregation rate was estimated through digital analysis of light microscopy images. Red cells of untreated blood that had been exposed to flow showed a higher aggregation rate (38.54 ± 1.63 vs. 27.52 ± 1.36; P  < 0.05). This effect was not observed in the absence of platelets. Treatment with aspirin induced a high aggregation rate, with or without exposure to flow conditions. Dipyridamole alone or combined with aspirin provoked echinocytosis, disturbing the rouleaux arrangement (rates ranging from 11.25 ± 1.91–13.62 ± 1.62). Washing red cells after treatment restored about 90% of echinocytes to their biconcave shape, but aggregation rate did not recover in parallel. These results highlight the influence of red cell–platelet interactions in the regulation of haemostasis and show how therapeutic agents can interfere with rheological phenomena.     

Mini buffy coat photopheresis for children and critically ill patients with extracorporeal photopheresis contraindications

BACKGROUND: Conventional extracorporeal photopheresis (ECP) has proven efficacy for the treatment of several diseases but is limited to patients with sufficient body weight. A novel simplified mini buffy coat ECP technique that allows treatment of small children and patients with apheresis contraindications has been developed.
STUDY DESIGN AND METHODS: White blood cell (WBC)-rich buffy coat fractions were prepared from 5 to 8 mL/kg whole blood in a closed system, diluted, and ultraviolet A (UVA)-irradiated after addition of 8-methoxypsoralen (8-MOP). Apoptosis and cell death were analyzed by annexin V and 7-aminoactinomycin staining. Lymphocyte proliferation was measured after CD3/CD28 and phytohemagglutinin (PHA) stimulation. Autologous residual blood and UVA-irradiated buffy coat were returned to the patients. Fifty-six mini buffy coat ECP procedures were applied to three children with acute steroid-refractory skin graft-versus-host disease and apheresis contraindications.
RESULTS: Mean whole blood and buffy coat volumes were 166 (±61.8) and 8 (±1.6) mL, respectively, and resulted in a hematocrit of 2.2% (±0.4) after saline dilution (median ± SD). UVA irradiation of 8-MOP buffy coat preparations resulted in significant induction of WBC apoptosis at 48-72 hours (p ≤ 0.006). WBC proliferation was significantly inhibited both after CD3/CD28 stimulation and after PHA stimulation when compared to controls (p ≤ 0.001). No clinical or laboratory side effects were observed during mini ECP procedures and the three patients responded to the therapy.
CONCLUSION: Mini buffy coat ECP induces apoptosis and lymphocyte proliferation inhibition, both of which occur after standard ECP. This study proposes that mini buffy coat ECP be used as a simple and inexpensive alternative to classical ECP in children and adult patients with apheresis contraindications.     

Red blood cell depletion with a semiautomated system or hydroxyethyl starch sedimentation for routine cord blood banking: a comparative study

BACKGROUND: The major problem with long-term cord blood (CB) banking is the required storage space. In this sense, many studies have been performed to establish techniques for volume reduction of CB units. STUDY DESIGN AND METHODS: We compared two different methods for CB volume reduction in both development and routine phases: hydroxyethyl starch (HES) sedimentation and top-and-bottom fractionation with the Optipress II (Baxter Healthcare). Monitoring the total nucleated cell (TNC) count, lymphocytes, CD34+ cells, and colony-forming unit (CFU) content in both preprocess and postprocess CB units assessed the volume reduction process. RESULTS: The CB units processed in both groups had comparable volume and cells counts before and after volume reduction, except for number of red blood cells (RBCs), which was significantly greater for the Optipress II group. Recoveries of CD34+ and RBC depletion were significantly better for the HES group. For routine processing, TNC and lymphocyte recoveries were significantly better for CB units processed by the Optipress II system. There was, however, significantly less depletion of RBCs for this group. The time required for CB processing with the Optipress II was significantly shorter than the time needed for volume reduction by addition of HES (25+/-5 min vs. 55+/-10 min). CONCLUSION: The volume reduction method with the Optipress II is a closed time-saving system that allows good cell recoveries. In contrast, the main advantage of the HES method is the higher RBC depletion that influences CFU content. Reducing RBC content must be the object of further improvements for volume reduction using the Optipress II method.     

Effects of RBC removal and TRIzol of peripheral blood samples on RNA stability

BackgroundPurification of mRNA from stored specimens is very important because results from RT-PCR and microarray analyses are largely affected by the quality of mRNA. Moreover, many preanalytical factors during collection, processing, and storage may affect mRNA quality and the expression of peripheral blood mononuclear cells (PBMC). In this study, we evaluate the effects of RBC removal techniques and TRIzol on RNA quality in blood samples.MethodsWe obtained EDTA-blood samples from 50 adult volunteers, and made 10 pools of buffy coats for comparison between protocols and also evaluated RNA quality of clinical samples in biobank. Use of TRIzol and RBC removal (RBC lysis or cell separation) were evaluated their effect on the quality of mRNA from the stored blood samples.ResultsRNA integrity with TRIzol was significantly better than that without TRIzol (RIN 4.5 vs. 9.2, respectively; P = 0.002). The change in RIN of the PBMC separation method was equivalent to that of the RBC lysis method. After 12 months, IL6 mRNA expression from stored clinical samples in cell separation/TRIzol was stable.ConclusionsThe blood samples frozen in TRIzol after RBC removal preserved RNA quality well. PBMC/TRIzol preservation for storage of blood samples could be a simple protocol for rapid, low-cost biobanking.     

A prospective randomized study of three types of platelet concentrates in patients with haematological malignancy: corrected platelet count increments and frequency of nonhaemolytic febrile transfusion reactions

We prospectively randomized 51 patients with haematological malignancy requiring platelet concentrates (PCs) to receive either single donor plateletpheresis products (SD-PC), PCs made from pooled buffy coats (BC-PC) or pooled units of platelets made by the platelet-rich plasma method (PRP-PC). The leucocyte content of each type of PC was 0.33 (0.03–13.5), 5.68 (0.19–99.0) and 365 (65–910) × 10⁶; median (range), respectively; P  < 0.0001. All red cell transfusions were leucodepleted by filtration. Statistical comparison of the probability of the occurrence of a nonhaemolytic febrile transfusion reaction (NHFTR) following transfusion of PCs in patients in each group showed a significant decrease for the SD-PC and BC-PC groups (0.031 and 0.038, respectively) when compared with PRP-PC (0.171); P  =0.001. The actual corrected platelet count increments (CCI) at 1–6 and 18–24 h post-transfusion for all three types of PC did not differ significantly. We conclude that transfusion of PRP-PC is associated with a significant increase in NHFTR.     

Cord blood (CB) stem cells for wound repair. Preliminary report of 2 cases.

In 2 patients, to promote skin wound/lesion repair we used fibrin-platelet glue combined with HLA compatible (2 mismatches accepted) buffy coats containing CD 34+ cord blood cells. The fibrin platelet glue was prepared with autologous apheresis platelets and cryoprecipitate. The original product was divided into 3 and 4 aliquots respectively for a correspondent number of applications. At each application, the margins of the lesion were infiltrated with 3 ml of cord blood buffy coat, containing 30 x 10(3) CD 34+ cells. No graft versus tissue reaction was seen in our patients in a follow-up of 3-7 months. The level of improvement, scored arbitrarily from 0 to 4, was 3 and 4, respectively. Our conclusion is that the use of cord blood cells along with fibrin platelet glue is of clinical interest.     

Elimination and multiplication of bacteria during preparation and storage of buffy coat-derived platelet concentrates

BACKGROUND: The prevalence of bacterial contamination of random-donor platelet concentrates (PCs) is considerably lower than that of blood donations. Which key steps of the preparation procedure contribute to the elimination of bacteria was investigated. STUDY DESIGN AND METHODS: Ten bacteria species were used. Blood donations were spiked with bacteria and stored at 22 degrees C for 8 hours. The buffy coats were kept for 6 hours. PCs were prepared from pools of 4 buffy coats. At each preparation step and during PC storage, bacteria contents were measured. In additional experiments, the titers of spiked blood and buffy coats were determined after storage at 20, 22, or 24 degrees C for 8 and up to 24 hours, respectively. RESULTS: Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Yersinia enterocolitica were completely inactivated during storage in blood or buffy coats. Titer reduction was between 3.32 and 4.62 log. Bacillus cereus, Propionibacterium acnes, Staphylococcus aureus, and Staphylococcus epidermidis did not multiply. Compared with their values in spiked blood the titers in the PCs were reduced by 1.7 to 2.8 log. Klebsiella pneumoniae was the only species that grew in blood. With the exception of P. acnes, those species that were not removed by the preparation process multiplied in the PCs. Remarkable donor-to-donor variations of the bactericidal activities of buffy coats were detected when the storage time was prolonged to 24 hours. CONCLUSIONS: Bacteria are significantly eliminated by the preparation procedure for random donor PCs. Also, blood and buffy coats are bactericidal for most species. When buffy-coat storage is prolonged, it cannot, however, be predicted whether specific strains vanish or multiply.     

Ten-year quality control of a semiautomated procedure of cord blood unit volume reduction

BACKGROUND: Volume reduction of cord blood units decreases the cost of cryogenic storage. This study reports the analysis of a 10-year quality control program of a semiautomated cord blood volume reduction procedure.
STUDY DESIGN AND METHODS: Cord blood was collected in a plastic bag containing 29 mL citrate-phosphate-dextrose, centrifuged at 2124 ×  g for 12 minutes, and processed with a semiautomated device. The procedure was aimed at removing most red blood cells and plasma and concentrating hematopoietic progenitors in the buffy coat (BC), thus reducing the unit volume and saving cryogenic space. Finally, the BC was cryopreserved with an equal volume of 20 percent dimethyl sulfoxide. Total nucleated cells (TNCs) were counted before and after processing in the 4311 units banked from 1998 through 2007, whereas CD34+ cells and colony-forming units–granulocyte-macrophage (CFU-GM) were counted in 420 random units from 2001 through 2007.
RESULTS: Mean postvolume reduction annual recoveries of TNCs, CD34+ cells, and CFU-GM ranged from 82.8 ± 12.3 (standard deviation) to 91.4 ± 6.4 percent, from 87.8 ± 14.1 to 95.2 ± 23.8 percent, and from 101.5 ± 51.4 to 117.8 ± 59.5 percent, respectively. Very strong correlations were found (r > 0.87) between postprocessing versus preprocessing TNCs, CD34+ cells, and CFU-GM; a moderate correlation between initial TNC count and unit's volume (r = 0.51); and no correlation between TNC percentage of recovery in the BC and initial unit's volume. The latter data indicate that most TNCs concentrate in the BC.
CONCLUSIONS: The semiautomated procedure of cord blood unit volume reduction used in this study provides high and stable cellular recoveries during several years of routine cord blood banking.     

In vitro evaluation of platelet concentrates, prepared from pooled buffy coats, stored for 8 days after filtration

BACKGROUND: Posttransfusion complications can be prevented by pretransfusion removal of donor white cells from platelet concentrate. The filtration used for this removal seems to have little effect on platelet function and activation, but more information is needed on its effect on function during subsequent long-term storage of concentrate. STUDY DESIGN AND METHODS: The effect of prestorage filtration of buffy coat-prepared platelet concentrates (PCs) on platelet function, metabolism, and activation was investigated. A pool of three PCs, each made of four buffy coats, was split into three equal volumes; two were filtered over two different filters and the third served as a control. Variables monitored immediately after filtration and during the subsequent 8-day storage period at 22 degrees C included aggregation upon stimulation with collagen and/or ADP, platelet adhesion capacity to collagen and fibrinogen in flowing blood, nucleotide content of and nucleobase release by the platelets, expression of activation-dependent antigens, and beta-thromboglobulin release by the platelets. RESULTS: No differences were observed between the PCs filtered over two different filters and the nonfiltered control PCs immediately after filtration and during storage, except for a selective removal (20%) of beta-thromboglobulin by one filter. CONCLUSION: PCs prepared from a pool of four buffy coats can be filtered and subsequently stored for 8 days (starting +/− 24 hours after whole blood collection) without detriment to platelet function, metabolism, or activation.     

序列特异性引物PCR法检测MN血型基因型

目的　建立MN血型系统基因分型的方法 ,检测人群中MN基因的频率。方法　用快速盐析法抽提样本DNA ,序列特异性引物PCR法检测MN基因。结果　 115例汉族人群中MM基因型为 2 5例 ,MN基因型为 6 0例 ,NN基因型为 30例。M基因频率为 0 .4 78,N基因频率为 0 .5 2 2。结论　该方法可以检测MN血型基因型。浙江汉族中N基因频率大于M基因频率     

Universal adoption of pathogen inactivation of platelet components: impact on platelet and red blood cell component use

BACKGROUND: Pathogen inactivation of platelet (PLT) components (INTERCEPT Blood System, Cerus Europe) was implemented into routine practice at a blood center supporting a tertiary care hospital. Utilization of platelet components (PCs) and red blood cell (RBC) components was analyzed for 3 years before and 3 years after introduction of pathogen inactivation to assess the impact of pathogen inactivation on component use.
STUDY DESIGN AND METHODS: This was a retrospective analysis of prospectively collected data. An electronic database used in routine blood bank hemovigilance to monitor production and use of blood components was analyzed to assess clinical outcomes.
RESULTS: Transfusion records were analyzed for 688 patients supported with conventional PCs and 795 patients supported with pathogen inactivation PCs. Additional analyses were conducted for intensively transfused hematology patients. Patient demographics (age category, sex, and diagnostic category) were not different in the two observation periods. For all patients, mean numbers of PC per patient were not different for conventional PCs and pathogen inactivation PCs (9.9 ± 19.5 vs. 10.1 ± 20.9, p = 0.88). Data for hematology patients (272 conventional PCs and 276 pathogen inactivation PCs) confirmed that days of PLT support were not different (31.6 ± 42.6 vs. 33.1 ± 47.9, p = 0.70) nor was total PLT dose (10¹¹) per patient (87.3 ± 115.4 vs. 88.1 ± 111.6, p = 0.93). RBC use, for all patients and hematology patients, was not different in the two observation periods, either during periods of PLT support or outside periods of PLT transfusion support.
CONCLUSION: Pathogen inactivation of PCs had no adverse impact on component use during a 3-year observation period of routine practice.     

Ambulatory blood pressure monitoring and cardiovascular function tests in multiple system atrophy

Summary— Cardiovascular tests (CT) of autonomic function and non-invasive ambulatory blood pressure (BP) and heart rate (HR) monitoring were performed in 17 patients with multiple system atrophy (MSA) (mean age 61 ± 9 years) and in 12 healthy subjects matched for sex and age. CT showed severe autonomic dysfunction with orthostatic hypotension (OH) in eight patients with MSA (47%) (Group I). The remaining nine out of the 17 patients didn't show BP abnormalities during CT but an impaired HR reflex response was found (Group II). BP monitoring showed a reversed circadian BP rhythm in Group I with higher night-time than day-time values, a blunted circadian BP pattern in Group II and a normal day-night BP reduction in controls. Day-night HR reduction was poor in Group II and absent in Group I. Post-prandial hypotension was evaluated after a standard meal. In Group I systolic/diastolic BP fell within 30 minutes after meal (from 135 ± 16/89 ± 13 to 118 ± 17/73 ± 12 mmHg; p < 0.05) and after two hours had not returned to basal levels. In Group II a reduction of only systolic BP was found within 45 minutes after meal and persisted for one hour. OH clinically identifies a subgroup of MSA patients with a more severe BP dysregulation characterized by severe post-prandial hypotension and reversed circadian BP rhythm. CT and ambulatory BP monitoring are useful tools in identifying early stage of cardiovascular autonomic impairment.     

医用电解质溶液作添加液制备汇集少白细胞血小板

目的采用医用电解质溶液作添加液(PAS),建立1种白膜法制备添加液汇集少白细胞血小板(PAS汇集BC-PCs)的方法。方法从400ml全血中分离白膜层,容量35—40ml,放(22±2)℃静置过夜,将ABO同型的6袋白膜汇集,加220g添加液(90%复方电解质注射液、8%ACD-A血液保养液和2%含量为50g/L的碳酸氢钠注射液的混合液)稀释白膜,汇集后的白膜在(22±2)℃中,以300×g离心10min,将上层的富含血小板悬液再经白细胞滤除器过滤去除白细胞,并转移到血小板保存袋内,即制备成1个成人治疗量的PAS汇集BC-PCs,制备过程在一个特制的密闭系统内完成。结果共制备30个成人治疗量的PAS汇集BC-PCs,其容量为(270±32)ml、血小板含量为(2.96±0.31)×1011、WBC混入量为(1.3±0.2)×106、RBC混入量为(5.8±1.1)×109、CD62P表达率为(22.5±10.6)%。保存8d后的pH为7.14±0.04、低渗休克反应率(HSR)为(54.0±8.2)%、CD62P表达率为(45.7±13.8)%。结论由复方电解质注射液、ACD-A保养液和碳酸氢钠注射液的混合液为添加液汇集血小板的方法可行。     

Comparison of in-vitro and in-vivo parameters of whole blood derived random donor platelets (RDP) after over-night hold with RDP prepared after 2-h hold: Single centre report from India

BackgroundRandom Donor Platelet (RDP) derived from whole blood is the major source of platelets in India. At our centre, we prepare RDPs by buffy coat method after a holding period of 2-hours (THRDP) as per current regulatory guidelines.Overnight hold of buffy coats before RDP preparation (OHRDP) would logistically optimise the manpower usage at our centre. The aim of this study was to compare both in-vitro as well as in-vivo parameters of OHRDPs with THRDPs.MethodologyHematological (Platelet, leucocyte counts), physical (pH and Swirling) and biochemical parameters (pO2, pCO2, lactate, bicarbonate and glucose) as well as platelet activation markers were tested in THRDPs and OHRDPs each at Day-1 and Day-5 as in-vitro studies. Separately, in-vivo study was done where Corrected count increment (CCI) and percentage platelet recovery (PPR) were considered. All parameters were expressed as Mean ± Standard deviation and were analysed using paired t-test with level of significance, p < 0.05.ResultsOHRDPs had higher platelet counts and lower leucocytes and CD62 P expression than THRDPs. All other markers were well within the quality control range in both groups. No significant differences were seen in the two groups when comparing CCI and PPR.ConclusionOHRDPs were found to be as good or better as compared to the THRDPs in the in-vitro part of our study. Additionally, there were no significant differences between the two groups when they were compared in vivo. This makes us conclude that overnight hold of buffy coats may be implemented at our center.     

YS05In Vitro Evaluation of Platelet Concentrates Prepared Using the Automated Gambro OrbiSac System

Introduction  The Gambro OrbiSac device is designed to automate the preparation of platelet concentrates (PC) from buffy coats (BC). The aim of this study was to evaluate the in vitro function of PC produced using the OrbiSac system and stored in plasma.
Study design  Ten PC were produced by automated pooling of four ABO matched BC and a single unit of plasma. The ring-shaped pooling bag was centrifuged and the platelet rich supernatant automatically expressed, through a leucodepletion filter, into a Gambro ELP storage bag. All PC were stored at 22°C with agitation and sampled on days 1, 5, 7 and 9. Samples were evaluated for platelet count and standard markers of platelet quality.
 

     

Density gradient separation of peripheral blood stem cells: comparison of an automated cell processing device and manual methods

Peripheral blood stem cells were collected from normal donors by leukapheresis on a cell separator. The leukapheresis product contained 1.5 x 10(10) mononuclear cells (MNCs) and was divided into two aliquots that underwent either automated or manual density gradient separation with ficoll-hypaque and subsequent washing. In the automated process, recovery of MNCs was 85 percent, reduction in platelet content was 64 percent, and the final hematocrit (Hct) was less than 1 percent. The manual separation resulted in 76-percent MNC recovery, a 79-percent reduction in platelet content, and a final Hct of less than 1 percent. The purified MNCs were then placed in methylcellulose culture at a concentration of 4 x 10(5) MNCs per mL. Quadruplicate 1-mL aliquots were cultured, and colonies were counted and classified on Day 14. Comparison of automated and manual ficoll-hypaque separations demonstrated no differences in the total, erythroid, or granulocyte-macrophage colony numbers. The cell processor used is fast, reliable, uncomplicated, and provides a sterile product containing progenitor cells that are not adversely affected by the automated ficoll-hypaque separation.     

Preparation and in vitro function of granulocyte concentrates for transfusion to neonates using the IBM 2991 blood processor

Clinical studies have suggested that granulocyte transfusions may be of value in the treatment of septic neonatal patients who present with severe granulocytopenia. We have developed a protocol for the preparation of granulocyte concentrates from freshly collected units of whole blood, using an automated blood cell processor. The red cells were washed with saline. Then, the buffy coats were collected from the washed red cells and studied for their suitability as granulocyte concentrates for neonatal transfusion. The mean number of granulocytes per concentrate was 1.6 × 10(9) in a mean volume of 25 ml. Studies of granulocyte function, including viability, random mobility, chemotaxis, phagocytosis and nitro-blue tetrazolium reduction, demonstrated that the granulocytes were functionally unimpaired following preparation of the concentrates. These studies suggest that concentrates of functional granulocytes, suitable for transfusion to neonatal patients, can be prepared from fresh units of whole blood, using a cell processor. This procedure is more cost-effective than leukapheresis and allows for delivery of granulocytes for transfusion in a more timely fashion.     

Low cytokine contamination in buffy coat-derived platelet concentrates without filtration

BACKGROUND: Cytokines (interleukin [IL]-1 beta, IL-6, and tumor necrosis factor [TNF]) generated by white cells during the storage of platelet concentrates can cause febrile nonhemolytic transfusion reactions. The high rate of febrile reactions reported in other studies was not observed in the patients in the authors' center. This discrepancy prompted the determination of cytokine levels in buffy coat- derived platelet concentrates. STUDY DESIGN AND METHODS: Platelet concentrates were produced from buffy coats by a standard large-scale production process. Buffy coats were separated from the red cell and plasma components, and then platelets were recovered from the buffy coats by a soft-spin procedure. Levels of cytokines (IL-1 beta, IL-6, IL-8, and TNF) were determined with commercial enzyme-linked immunosorbent assays. RESULTS: In platelet concentrates produced by the buffy coat method, IL-1 beta, IL-6, IL-8, and TNF were observed at or below the detection limit of current enzyme-linked immunosorbent assays after 5 days' storage at 22 +/− 2 degrees C. Therefore, prestorage filtration had no measurable effect on cytokine levels. In controls, IL- 1 beta, IL-6, IL-8, and TNF were quantitatively detected after exogenous addition of recombinant cytokines or exposure to lipopolysaccharide. CONCLUSION: Platelet concentrates prepared from buffy coats may be virtually free of cytokines (IL-1 beta, IL-6, IL-8, and TNF) during 5 days of storage. Filtration is not required to reduce the recipient's cytokine exposure via such platelet concentrates.