Dextran sedimentation in a semi-closed system for the clinical banking of umbilical cord blood

BACKGROUND: The results of current processing procedures for reducing volume and recovering HPCs from umbilical cord blood (UCB) before cryopreservation vary. STUDY DESIGN AND METHODS: Dextran was added to bags containing UCB, followed by sedimentation for 30 minutes. The processed UCB was then frozen. RBCs, nucleated cells, MNCs, CD34+ cells, CFUs and long-term culture-initiating cells (LTC-ICs), viability, and sterility were evaluated. Fractionations in ficoll-hypaque and hydroxyethyl starch (HES) were also run in parallel for comparison. RESULTS: The nucleated cell (NC) recovery and RBC depletion were 86.1 percent and 94.3 percent, respectively (n = 50). Sedimentation with dextran also enabled the recovery of 80.7 percent MNCs and 82.6 percent CD34+ cells (n = 30). Postsedimentation samples displayed no impairment of CFU growth (n = 42, 108.7% CFU-C, 104.6% CFU-GEMM, 107% CFU-GM, and 95.7% BFU-E). Long-term cultures on five paired samples before and after sedimentation generated similar numbers of CFU-C each week (p = 0.88). Limiting dilution analysis of 12 paired pre/postsedimentation samples showed comparable median proportions of LTC-ICs (1/6494 vs. 1/5236; p = 0.18). The cell viability of 24 samples of thawed UCB after sedimentation was 90.3 percent (77.5-96%) and the recovery of CFU-C, CFU-GEMM, CFU-GM, and BFU-E of 11 postsedimentation samples was 93.4 percent, 84.9 percent, 92.3 percent, and 83.4 percent, respectively. NC recovery was significantly higher after treatment with dextran than with ficoll-hypaque (n = 30; 88.5% vs. 29.1%; p<0.005) and HES treatment (n = 21; 88.5% vs. 76.4%; p = 0.004). However, MNCs, CD34+ cells, CFUs, LTC-ICs, and RBCs were comparable. Two cycles of dextran sedimentation recovered 93.9 percent of NCs with cell viability of 98.6 percent (96.5-100%), whereas 11.7 percent of RBCs were retained (n = 20). The final yield volume was 33.5 (28-41) mL. CONCLUSION: In a semi-closed system, dextran sedimentation enabled volume reduction of UCB without significant quantitative and qualitative losses of HPCs.     

Efficacy of leucocyte-poor red blood cell suspensions prepared by sedimentation in hydroxyethyl starch

Hydroxyethyl starch (HES), which is licensed for human use as a plasma volume expander, has red blood cell sedimenting properties similar to high molecular weight dextran (HMWD). The present studies document the effectiveness of commercially available HES in preparing leukocyte-poor red blood cell suspensions (L-PRBCs) from donor whole blood and packed red blood cells stored in CPD up to 120 hours prior to processing. The effect of HES sedimentation on final white blood cell count, on erythrocyte p50 and 2,3-DPG concentration, and on short- and long-term in vivo survival of 72-hour stored donor red cells are described, along with favorable clinical experience with over 300 transfusions of HES- sedimented L-PRBCs. Since the method requires no special equipment, it is immediately available for widespread use.     

应用干细胞分离仪分离脐血与传统手工分离法的效果比较

目的:脐血处理的关键问题是提高干细胞的回收率及实现处理过程的标准化和可重复化,实验对此进行探讨,比较干细胞分离仪与传统羟乙基淀粉手工法分离脐血的效果。方法:实验于2006-12/2007-05在广州医学院附属市一人民医院完成。①脐血来源:39份脐血采自广州医学院附属市一人命医院妇产科健康顺产新生儿脐带,产妇均知情同意。随机数字表法分为仪器分离组17份、手工分离组22份。②实验方法:仪器分离组收集脐血称质量,计算体积,在开始处理前20min缓慢加入相当于20%脐血体积的60g/L羟乙基淀粉。仪器分离组按仪器要求自动分离,分离终体积20mL。手工分离组50g离心5min,压浆板压出全部血浆以及18mL红细胞移至无菌空袋,500g离心13min,自动压浆板压出血浆,保留20mL终体积样本。③实验评估:采用全自动计数仪进行检测有核细胞(白细胞)、红细胞数量。流式细胞仪分析CD34 含量。结果:采用干细胞分离仪处理浓缩脐血,有核细胞回收率为(89.7±3.4)%,CD34 细胞回收率为(98.8±5.1)%,红细胞去除率为(55.2±16.7)%,均比手工分离组分离效果好,差异有显著性意义(P<0.05或0.01)。同时,仪器分离组有核细胞回收率、CD34 细胞回收率的标准差均明显低于手工分离组(3.4vs.15.3;5.1vs.10.3)。结论:相比传统的羟乙基淀粉手工分离法,干细胞分离仪脐血分离浓缩效果理想,且结果标准误小,数据稳定。     

Related cord blood banking for haematopoietic stem cell transplantation

The aims of this single centre study were to assess the feasibility of related cord blood collecting, the appropriateness of storage and the final suitability for transplantation. Since September 1994, 63 families were enrolled in this study. Families were eligible if they were caring for a patient with a disorder treatable by haematopoietic stem cell transplantation and were experiencing a pregnancy. A total of 72 cord blood units were collected and stored for 64 patients (both siblings and parents). We focussed on human leucocyte antigen (HLA) compatibility and cell content as critical requirements to unit's suitability for transplantation. HLA‐typing was carried out for 34 donor–recipient couples and most units (72%) mismatched with the related patients. About 60% of collections had a minimum cell dose considered acceptable for transplantation. Only 21% of units had both compatibility degree and cell content suitable for transplantation. When applicable, information on the compatibility degree between the foetus and the patient should be obtained during pregnancy. Appropriateness of related cord blood banking for parents should be further investigated and cost‐effective guidelines policies should be provided. Finally, as banking of related cord blood units is an important resource then, this public service should be supported and enhanced.     

Gravity sedimentation of granulocytapheresis concentrates with hydroxyethyl starch efficiently removes red blood cells and retains neutrophils

BACKGROUND: Transfusion of granulocytapheresis concentrates can be limited by the volume of incompatible donor red blood cells (RBCs) in the component. Efficient reduction of RBCs in granulocyte units would result in safe transfusion of RBC‐incompatible units. STUDY DESIGN AND METHODS: Granulocyte concentrates were collected by continuous‐flow apheresis from granulocyte–colony‐stimulating factor (G‐CSF) and dexamethasone‐stimulated volunteer donors, with 6% hydroxyethyl starch (HES) added continuously during apheresis as a RBC sedimenting agent to enhance granulocyte collection efficiency. After collection, the component was placed in a plasma extractor for 4 hours. A sharp line of demarcation between the starch‐sedimented RBCs and the granulocyte‐rich supernatant developed, and the supernatant was transferred to a sterilely docked transfer pack. RBC reduction and white blood cell recovery were determined. RESULTS: Gravity sedimentation was performed on 165 granulocyte concentrates. Mean sedimentation time was 267 minutes (range, 150‐440 min). RBC depletion was 92% (range, 71%‐99%) with mean residual RBC content of 3.2 ± 1.4 mL. Twelve percent of components contained less than 2 mL of RBCs. Mean granulocyte and platelet (PLT) recoveries were 80 and 81%, respectively. There were no transfusion reactions or signs of hemolysis after transfusion of 66 RBC‐incompatible granulocyte concentrates (RBC volume, 1.6‐8.2 mL). The remaining concentrates were used for topical or intrapleural applications. CONCLUSIONS: RBCs were significantly reduced and granulocytes and PLTs effectively retained in G‐CSF/steroid–mobilized granulocyte components collected with HES and processed by gravity sedimentation. This procedure allows safe transfusion of RBC‐incompatible sedimented granulocyte units and may be used to expand the pool of available granulocyte donors for specific recipients.     

Human red blood cell behaviour in hydroxyethyl starch: probed by single cell spectroscopy

Hydroxyethyl starch (HES) is a commonly used intravenous fluid in hospital settings. The merits and demerits of its application is still a debatable topic. Investigating the interaction of external agents like intravenous fluids with blood cells is of great significance in clinical environments. Micro-Raman spectroscopy combined with an optical tweezers technique has been utilized for conducting systematic investigations of single live red blood cells (RBCs) under the influence of external stress agents. The present work deals with a detailed biophysical study on the response of human live red blood cells in hydroxyethyl starch using optical techniques. Morphological changes in red blood cells were monitored using quantitate phase imaging techniques. Micro-Raman studies suggest that there is a significant reduction in the oxy-haemoglobin level in red blood cells suspended in HES. The spectra recorded by using different probe laser powers has shown that the cells are more vulnerable in HES under the influence of externally induced stress than in blood plasma. In addition, the spectral results support the possibility of heme aggregation and membrane damage for red blood cells in HES under externally induced stress. Principle component analysis performed on the Raman spectra were able to effectively discriminate between red blood cells in HES and in blood plasma. The use of Raman tweezers can be highly beneficial in elucidating biochemical alterations happening in live, human red blood cell.

Hydroxyethyl starch (HES) is a commonly used intravenous fluid in hospital settings.     

Target inventory levels for a hospital blood bank or a decentralized regional blood banking system

For any blood type, there is a complex interaction among the optimal inventory level, daily demand level, the transfusion to crossmatch ratio, the crossmatch release period and the age of arriving units that determine the shortage and outdate rate. The blood bank administrator should establish optimal target inventory levels based on a simple equation (decision rule) relating these factors. Evaluation of this rule indicates that its implementation can lead to a very low shortage rate and a reasonable low outdate rate if the blood bank administrator makes efforts to control the crossmatch release period and the average transfusion to crossmatch ratio.     

Blood regeneration after blood substitution with dextran 60 or hydroxyethyl starch of an identical volume]

Male rats of the Wistar strain survived an isovolaemic blood volume replacement down to a haematocrit value of 20% using commercial dextran 60 or hydroxyethyl starch. No significant changes of the blood volume have been observed. 14 days after treatment, the blood values were found to be practically within the normal range. As early as 3 days after the blood exchange, a significant increase in reticulocytes has been noted which reached its maximum on the 7th day. In both series the blood regeneration was identical.     

Ethical issues in umbilical cord blood banking: a comparative analysis of documents from national and international institutions

The issues of collection, storage, and use of cord blood (CB) stem cells have been addressed extensively in national and international guidelines, policies, and regulations. Many of these documents are not binding, but are nonetheless accorded considerable respect on account of the authority of the issuing organizations. Most discussion has to date focused on two topics: informed consent for collection, banking, and use and the debate between those who favor public storage for altruistic purposes and those who advocate private storage for autologous use. There is generally agreement or consensus in the guidelines that public storage for allogeneic transplants is preferable and that private storage should be discouraged. Given the consensus in national and international guidance on these two issues, it is time for other ethical issues to be examined in greater detail. These include additional uses of CB samples, for example, for research or for the production of blood‐derived drugs, and the economic implications arising from the extensive international network for the exchange of CB for transplantation.     

Vortex pumping for routine cardiac surgery: a comparative study

Vortex pumping has become popular for mechanical assist applications, but has only recently received serious consideration for use as the arterial pump of choice for routine open-heart surgery. We report on a prospective randomized study designed to evaluate this pump in routine use. Sixteen patients undergoing routine coronary artery surgery were randomized into two groups in which the only difference in equipment and technique was the use of a Stockert roller pump in group S and a Biomedicus vortex pump in group B. The groups were compared with respect to haematology, perioperative fluid balance, transfusion requirements, complement activation, haemolysis and microbubble transmission. The groups were reasonably well matched, with slightly longer mean bypass and ischaemic times in group S (94.8 vs 105.5 minutes and 75.7 and 83.8 minutes respectively). Group B patients showed significant improvements over group S patients with respect to preservation of platelet numbers, decreased complement activation and reduced microbubble transmission. The afterload sensitivity of the vortex pump did not present the perfusionist with any practical problems although a different technique is required for initiating and terminating bypass. We conclude that vortex pumping would seem to offer better blood handling for routine use. More extensive testing is required to establish whether or not this would be reflected in clinically measurable improved patient outcome.     

Transfusion reactions: a comparative observational study of blood components produced before and after implementation of semiautomated production from whole blood

BACKGROUND: A semiautomated method of component production from whole blood was implemented at Canadian Blood Services. To assess safety of the new components, the frequency of adverse transfusion events (ATEs) to platelet components (PCs) and red blood cell (RBCs) produced before and after implementation of the new method was surveyed and compared. STUDY DESIGN AND METHODS: This retrospective, observational, noninferiority study was conducted in 12 sentinel hospitals across Canada. The control group received RBCs in additive solution‐3 (AS‐3) and platelet‐rich plasma (PRP)‐produced platelets (PLTs) for 3 to 11 months before implementation of semiautomated production, and the study group received RBCs in saline‐adenine‐glucose‐mannitol (SAGM) and buffy coat (BC)‐produced PLTs for 3 to 11 months after implementation. ATE definitions at each hospital and standard practice for reporting did not change between control and study periods. Data for analysis were obtained from databases and original report forms. RESULTS: The pooled risk ratio of a reaction to SAGM versus AS‐3 RBCs was 0.77 (95% confidence interval [CI], 0.66‐0.90), suggesting that SAGM products had significantly lower reaction rates than AS‐3 products (p < 0.01). Reported allergic reactions to RBCs decreased from 0.07% (AS‐3) to 0.04% (SAGM). For PLTs, the difference in reaction rates between BC and PRP was not significant (p = 0.37), and the pooled risk ratio of BC versus PRP was 1.14 (95% CI, 0.86‐1.50). CONCLUSION: The change in manufacturing method was associated with lower reaction rates to SAGM RBCs than to AS‐3 RBCs. Pooled BC PLTs were noninferior to random‐donor PRP PLTs with respect to ATEs.     

SCID-repopulating activity of human umbilical cord blood-derived hematopoietic stem and/or progenitor cells in a nonobese diabetic/Shi-SCID mice serial xenotransplantation model and immune cell activities in vitro: a comparative study of the filter method and the hydroxyethyl starch method

BACKGROUND: A novel filter system was developed for umbilical cord blood (UCB) volume reduction. The aim of this study was to compare the functions of cryopreserved UCB cells processed by the filter and by the hydroxyethyl starch (HES) sedimentation method from the aspect of the graft quality. STUDY DESIGN AND METHODS: UCB specimens were divided into two portions, processed in parallel by the filter or HES, and then cryopreserved in the clinical setting. The thawed UCB specimens containing 1 x 10(5) CD34+ cells were injected into nonobese diabetic/Shi-SCID mice, and the engraftment capacity in primary and secondary transplants was assessed. The functions of natural killer (NK) cells and monocyte-derived dendritic cells (DCs) were also assayed in vitro. RESULTS: The percentage of recovery of CD34+ cells by both methods was equivalent. In the marrow of the primary transplant recipients, the percentage of hCD45+ cells in the filter group and HES group was 58.2 +/- 31.6 and 46.5 +/- 28.4 percent, respectively (p = 0.016). The engraftment capacity and multilineage differentiation in the secondary transplantations were equal in both groups. The cytotoxic activity of the NK cells and phagocytosis activity of the DCs from both the groups were similar. CONCLUSION: The filter yielded a desirable percentage of recovery of hematopoietic cells with engraftment ability in the clinical setting. Thus, it is considered that the filter system may be useful for UCB banking for cord blood transplantation.     

A method for the cryopreservation of red blood cells using hydroxyethyl starch as a cryoprotectant

A simple, quick, inexpensive and long term method of cryopreserving human red cells using hydroxyethyl starch (HES) as a cryoprotectant is described. No sophisticated equipment or skilled labour is required. As HES is a plasma expander, it does not have to be removed prior to transfusion, thereby obviating the requirement for a washing stage. This allows 60 units/h to be prepared. As the microbiological integrity of the system is maintained, there is potential for prolonged storage post-thaw at 4 degrees C. Recoveries of 99% and 30 min saline stabilities in excess of 91% have been consistently achieved. P50O2 and 2,3DPG values are not significantly decreased.     

Resuscitation in hypovolemia and shock: a prospective study of hydroxyethyl starch and albumin

In a prospective study comprising 50 patients, we evaluated the hemodynamic, pulmonary, renal and coagulation changes after resuscitation with 2 colloidal fluids, 6% hydroxyethyl starch (HES) and 5% albumin (ALB). Twenty-five patients studied in each group were well matched for age, clinical presentation, presence of shock and type of surgical procedures. A standard fluid challenge with 500 ml of either solution significantly (p less than 0.01) increased pulmonary artery wedge pressure (WP), mean arterial pressure (MAP) and left ventricular stroke work index (LVSWI) and decreased systemic vascular resistance index (SVRI). Increases in LVSWI of 43% to 68% over baseline in HES patients compared favorably to 15-22% increases in ALB patients. The cardiopulmonary variables at 12 and 24 h were comparable in both groups. Improvement in cardiac function was also reflected by better tissue perfusion as judged by reduction in arterial lactate from 2.9 to 1.5 mM/L (ALB) and 2.6 to 1.4 mM/L (HES). Increased O2 delivery and reduced O2 extraction without significant deterioration of PaO2 or alveolar-arterial O2 gradient were observed with stabilization of circulation. Clinical bleeding due to colloid resuscitation was not documented and renal function was not affected significantly by either fluid. It seems that HES may offer a cost-effective alternative to ALB for patients requiring colloid resuscitation.