Assessment of a New Centrifugal Plasmapheresis Machine: The SPC-600

An approach to the full assessment of new plasmapheresis machines is outlined. This involves testing for changes in donated blood, donors and blood components using fresh donor blood and during full procedures in an animal model prior to performing full procedures in human volunteers. Using this approach, a new centrifugal plasmapheresis machine, the SPC-600, was assessed. No adverse effects associated with the use of the machine were detected in the initial studies. When used for plasmapheresis of volunteers, 500 ml of plasma was obtained in 43 +/- 4 min without adverse effects on donors. The plasma product contained normal levels of plasma proteins, including factor VIII, and 20 +/- 6 X 10(9)/1 platelets. There was no evidence of activation of complement or coagulation systems, and the factor VIII yield in cryoprecipitate prepared from single donations of plasma was equivalent to that observed for standard whole blood donations.     

Microwave-Thawed Plasma for Cryoprecipitate Production

A microwave oven has been used to obtain rapid and controlled thawing of frozen plasma packs for cryoprecipitate production. The resulting factor VIII yields were higher than the average of 77.8 IU (in a volume of 17.2 ml) obtained by the slow overnight thaw procedures in routine use. Assays on 10 individual packs showed mean results of 142 +/- 24.5 IU factor VIII procoagulant activity, 188.6 +/- 51.68 IU factor VIII-related antigen, 0.152 +/- 0.05 g fibrinogen and 218.9 +/- 66.5 IU fibronectin in a volume of 23.2 +/- 6.6 ml. The results of this preliminary study indicate that microwave thawing of plasma is worthy of further investigation and should be reconsidered for routine production of cryoprecipitate and high yield fibronectin.     

Treatment of von Willebrand's disease and hypofibrinogenemia with single donor cryoprecipitate from plasma exchange donation

Conventional replacement therapy for hypofibrinogenemia and von Willebrand's disease requires multiple donor exposures and a correspondingly high risk of blood-borne infection. We describe the collection and successful use of cryoprecipitate derived from a single donor by plasma exchange donation to support such patients through major hemostatic stresses. The father of an epileptic patient with von Willebrand's disease produced cryoprecipitate containing 23,546 units of von Willebrand factor (vWF) in nine desmopressin-stimulated donations; this provided total factor replacement for neurosurgery to remove a seizure focus. The average yield was 2,616 units per donation and the average VWF concentration in cryoprecipitate was 17.7 units/ml. The husband of a hypofibrinogenemic patient with a history of postpartum hemorrhage provided cryoprecipitate containing 13.4 g of fibrinogen in five donations; this supported his wife through parturition without recourse to other blood products. The average yield was 2.7 g per donation, and the average fibrinogen concentration was 15.3 g/liter. Plasma exchange donation is a practical alternative source for cryoprecipitate. It can provide vWF and fibrinogen that carry a reduced risk of infectious disease transmission.     

Subsequent donation requests among 2472 unrelated hematopoietic progenitor cell donors are associated with bone marrow harvest

Approximately 1 in 20 unrelated donors are asked to make a second donation of hematopoietic progenitor cells, the majority for the same patient. Anthony Nolan undertook a study of subsequent hematopoietic progenitor cell donations made by its donors from 2005 to 2011, with the aims of predicting those donors more likely to be called for a second donation, assessing rates of serious adverse reactions and examining harvest yields. This was not a study of factors predictive of second allografts. During the study period 2591 donations were made, of which 120 (4.6%) were subsequent donations. The median time between donations was 179 days (range, 21–4016). Indications for a second allogeneic transplant included primary graft failure (11.7%), secondary graft failure (53.2%), relapse (30.6%) and others (1.8%). On multivariate analysis, bone marrow harvest at first donation was associated with subsequent donation requests (odds ratio 2.00, P=0.001). The rate of serious adverse reactions in donors making a subsequent donation appeared greater than the rate in those making a first donation (relative risk=3.29, P=0.005). Harvest yields per kilogram recipient body weight were equivalent between donations, although females appeared to have a lower yield at the subsequent donation. Knowledge of these factors will help unrelated donor registries to counsel their donors.     

Serum Ferritin Levels in Male Blood Donors

Serum ferritin estimation has been shown to be a reliable test to reveal iron deficiency. Such estimations have been made in groups of male blood donors with a varying number of previous phlebotomies and a mean interval between donations of 9.9 +/- 1.7 SD weeks. It was found that the mean ferritin level was significantly (p less than 0.001) lower in the blood donors than in nondonors. After 6-8 phlebotomies it was about 40% lower. Subnormal ferritin values were found in 10% of the donors, almost exclusively among those who had taken less than 1,000 mg of iron supplementation since the last donation. It is concluded that with a donation interval of about 10 weeks, there is a considerable risk for iron deficiency after about 6 donations. This risk is far less if more than 1,000 mg of iron supplementation is taken between phlebotomies. A role for serum ferritin estimation in monitoring donation intervals and/or iron therapy is suggested.     

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.     

HIV Transmission by Seronegative Blood Components: Report of 2 Probable Cases

HIV seroconversion was reported in 2 haemophiliacs after having corrective orthopaedic surgery. They received solvent-detergent/heat-treated factor VIII concentrate, HIV-seronegative cryoprecipitate and fresh frozen plasma during the course of surgery. HIV seroconversion was found on days 31 and 71 after surgery. It is highly probable that the infections were acquired by transfusions of seronegative blood components. In countries with a relatively low prevalence of HIV infection, transmission of HIV by transfusion of derivatives of seronegative blood is occasionally reported as a rare complication of blood transfusion [1–3]. In Thailand the prevalence of HIV infection and the incidence of new infections in the general population and in blood donors has recently increased dramatically (fig. 1) [4–5]. As a result of these components prepared from HIV-seronegative blood donations pose a significant hazard to recipients because of the risk of viraemia during the ‘window period’ of HIV infection. Here we report HIV infection in 2 haemophilia patients treated with HIV-seronegative (using Fujirebio agglutination or second-generation Abbott ELISA) cryoprecipitate and fresh frozen plasma in 1991, prepared locally from single-unit donations. All donors were voluntary. Anti-HIV was tested in every unit of donor blood before processing to blood components. Although not proven, it is highly probable that the infections were acquired by transfusions of seronegative blood components.     

WMDA guidelines for subsequent donations following initial BM or PBSCs

Unrelated donor SCT activity is increasing, and in 5-10% of cases a subsequent donation of stem cells or donor lymphocytes may be requested. Second donations of stem cells are not associated with an increased chance of donor complications, but the yield of CD34+ cells may be lower in some donors. It is acceptable practice for any registry to request subsequent donations and it is recommended that donors should be counselled about this possibility before their first donation. Guidance is provided on the requirements for further medical assessment, the procedures used to agree requests, frequency and timing of donation and timing and duration of donor follow up.     

A study of serum ferritin levels among male blood donors in Hospital Universiti sains Malaysia

Iron deficiency is the commonest cause of anemia worldwide and healthy blood donors are estimated to lose about 236 mg of iron with each donation. The objective of this study was to determine the serum ferritin levels among first time and regular male blood donors, and also to correlate the serum ferritin levels with the number of donations and hemoglobin levels. Hemoglobin levels and serum ferritin were measured in three groups of donors divided into first time donors; (n = 92), donors with 2-4 donations (n = 41), and regular donors (n = 78). The mean hemoglobins in the first time donors, second group and regular blood donor group were 14.95 +/- 1.08, 15.12 +/- 1.44 and 15.56 +/- 1.48, respectively. The serum ferritin level were found to be significantly lower among the regular donors (62.0 +/- 39.78 ng/ml) compared to first time donors (90.7 +/- 66.63) and second group donors (114.12 +/- 66.97). The serum ferritin levels gradually decrease according to the number of donations and there was a significant correlation between frequency of donations and the serum ferritin level (r2 = 0.082). Significant correlation between the number of donations and hemoglobin level r2 = 0.061) was noted. However, there was no significant correlation between hemoglobin and serum ferritin levels (r2 = 0.015). Eleven percent of regular donors had depleted iron stores. This was not noted in donors who donated less than 5 times within 2 years.     

Evidence that anti-HBc but not HBV DNA testing may prevent some HBV transmission by transfusion

Blood donor screening for antibody to hepatitis B core antigen (anti-HBc) implemented in some countries as a surrogate marker for non-A, non-B hepatitis has been superseded by anti-HCV screening. To assess the value of anti-HBc screening for the detection of hepatitis B surface antigen-negative blood donations that might contain infectious HBV, HBV genomic detection and recipient testing were used. Blood donations were screened and confirmed by multiple anti-HBc assays. Donations containing isolated anti-HBc and those with anti-hepatitis B surface antigen (anti-HBs) level < 0.1 IU/ml were tested for the presence of HBV DNA. Recipients of previous donations from the corresponding donors during the previous 5 years were traced and tested for markers of HBV infection. Of 103 869 donations screened, 586 (0.56%) were anti-HBc positive, two of which contained HBsAg, and 413 (0.4%) had protective (>/= 0.1 IU/ml) levels of anti-HBs. Anti-HBs < 0.1 IU/ml was found in 102 of these donations (0.1%) and isolated anti-HBc in 69 (0.07%). No donations with isolated anti-HBc were HBV DNA confirmed positive. Of 278 recipients of previous donations from 171 donors at risk of HBV carriage, 12 had markers of HBV infection. Six recipients had other identified risk factors. An association with blood transfusion was considered probable in two and possible in four recipients. None of the six corresponding donors had detectable HBV DNA 6-40 months after the implicated donation. The frequency of HBV transmission by chronic carriers negative for hepatitis B surface antigen was estimated in this study to be 1 in 52,000 donations (CI 0.3-7.8/100,000) from HBsAg-negative donors. Such HBV infectious donations may not be detected by DNA amplification.     

Frequency of inhibitor development in severe haemophilia A children treated with cryoprecipitate and low-dose immune tolerance induction.

The frequency of factor VIII inhibitor development was evaluated in a hundred severe haemophilia A patients < 18 years of age (mean 10.4 +/- 5.1 years); 25 were previously untreated patients (PUPs), with a mean age of 11.2 +/- 2.9 months. All were followed up for 3 years from December 1996. Immune tolerance (IT) was induced with low-dose factor VIII (FVIII); 25-50 IU kg(-1) every other day for the 10 haemophiliacs who developed persistent inhibitors. The incidence of inhibitors for PUPs was 3/25 (12%; 95% confidence interval [CI], 0. 7-24.7%) and were detected after 4, 15 and 20 exposure days (mean 13 +/- 8.2 days; 95% CI, 3.7-22.2%). Children with maximum inhibitor levels of > 40 Bethesda units (BU) per mL (n=4) received IT therapy as 25 U kg(-1) FVIII in the form of cryoprecipitate every other day for 1-4 months (mean 2.4 +/- 1.6 months; 95% CI, 0.8-3.9%), which was successful in all of them. FVIII (50 U kg(-1)) was given every other day for six patients with maximum inhibitor level > 40 BU mL(-1) for 3-9 months (mean 5.4 +/- 3.2 months; 95% CI, 2.9 -7.9%) with success in 4/6 (66.6%; 95% CI, 28.8-104.3%). Patients who showed a good IT response had an inhibitor level < or = 30 BU mL(-1), were < or = 9 years of age at inhibitor development with few exposure days to FVIII and had an early immune tolerance. In conclusion, inhibitor development in severe haemophilia A children exclusively treated with cryoprecipitate is low. Early low-dose IT induction for high responders may be achieved successfully if inhibitor level is < or = 50 BU mL(-1).     

Increased factor VIII/von Willebrand factor antigen and von Willebrand factor activity in renal failure.

Factor VIII/von Willebrand factor antigen and von Willebrand factor activity (ristocetin assay) were studied in 12 patients in renal failure. A dramatic increase in both activities was observed (antigen 315 +/- 30 per cent in patients verus 104 +/- 9 per cent in control subjects; activity 402 +/- 48 per cent in patients versus 111 +/- 5 per cent in control subjects; p less than 0.001 for both). Since von Willebrand factor is thought to play at least a facilitative role in the development of arteriosclerosis, these increased activities may contribute to the premature arteriosclerosis reported in patients with chronic renal failure undergoing dialysis.     

Tumor necrosis factor alpha production by peripheral blood mononuclear cells of patients with chronic liver disease

We investigated the production of tumor necrosis factor alpha by peripheral blood mononuclear cells of patients with chronic liver disease and its association with hepatitis activity. Tumor necrosis factor alpha production was measured with an enzyme-linked immunosorbent assay. Tumor necrosis factor alpha production by peripheral blood mononuclear cells stimulated with recombinant gamma-interferon of patients with chronic active hepatitis (5.8 +/- 4.0 units per ml, p less than 0.05) and patients with cirrhosis (4.1 +/- 2.1 units per ml, p less than 0.05) was significantly increased when compared with controls (2.5 +/- 1.6 units per ml). Tumor necrosis factor alpha production by peripheral blood mononuclear cells stimulated with a combination of recombinant gamma-interferon and recombinant interleukin 2 of patients with chronic persistent hepatitis (5.8 +/- 3.8 units per ml, p less than 0.05), patients with chronic active hepatitis (8.9 +/- 3.0 units per ml, p less than 0.001) and patients with cirrhosis (6.7 +/- 3.2 units per ml, p less than 0.05) was significantly increased in comparison with controls (3.3 +/- 1.8 units per ml). Tumor necrosis factor alpha production of patients with chronic active hepatitis was significantly higher than that of patients with chronic persistent hepatitis (p less than 0.05). There was a significant correlation (r = 0.5699, p less than 0.005) between tumor necrosis factor alpha production and histologic activity index in patients with chronic persistent hepatitis or chronic active hepatitis. These findings show that tumor necrosis factor alpha production is increased in chronic liver disease and that the increased tumor necrosis factor alpha production is related to hepatitis activity.     

Quantitating Donor Behaviour to Model the Effect of Changes in Donor Management on Sufficiency in the Blood Service

BACKGROUND AND OBJECTIVE: To describe donor behaviour quantitatively and apply the information on actual donations derived during observed changes in blood collection in Melbourne between July 1990 and June 1996 to construct a logical model to examine the effects of changes in donor management on the sufficiency of the blood supply. METHOD: Computerised donation data files were searched to determine time to next or previous donation for donors giving blood between 1990 and 1996 stratified by age, donation history and venue. Actual numbers of whole blood donation given by new and repeat donors, July 1990 to June 1996, and numbers of donors refused were used to construct and test a logical predictive model. RESULTS: 558,682 donation intervals were analysed. Donor return rates at 2 years were shown to increase with donor donation history and donor age. The prediction model showed that a 25% decline in whole blood collections over 2 years could be explained by the cumulative effect of a decrease in donor return rates of 2-4%. Between 1990 and 1996 many donors moved from static city collection sites to suburban mobiles. New donor attendance correlated with repeat donor attendance. Donor complaints correlated with donor deferral numbers. CONCLUSIONS: The model showed that large shifts in nett blood collections can be explained by relatively small shifts in donor return rates at 2 years.     

Influence of factor VIII/von Willebrand complex on the activated protein C-resistance phenotype and on the risk for venous thromboembolism in heterozygous carriers of the factor V Leiden mutation.

High factor VIII plasma levels have been shown to represent a common increased risk for venous thromboembolism (VTE) and may cause an activated protein C (APC) resistance in the absence of the factor V Leiden mutation, but there are no studies specifically aimed to establish if high factor VIII and von Willebrand factor (vWF) concentrations may influence the APC sensitivity ratio (APC-SR) and increase the risk for VTE in the presence of the factor V Leiden mutation. For this purpose, we performed a retrospective case-control study to investigate the influence of the procoagulant factor VIII (VIII:C) and the antigen of vWF (vWF:Ag) on the normalized APC-SR (n-APC-SR) and on the risk for VTE, in two selected groups of 30 symptomatic (Group I) and 32 asymptomatic (Group II) related heterozygotes for the factor V Leiden mutation. Differences between the two groups (Group I versus Group II) were: n-APC-SR, 0.57+/-0.06 versus 0.63+/-0.08, P = 0.001; factor VIII:C, 1.49+/-0.42 versus 1.13+/-0.28 IU/ml, P<0.001; vWF:Ag, 1.46+/-0.53 versus 1.26+/-0.32 IU/ml, NS. As a whole (Group I + Group II), Pearson correlation coefficients were: n-APC-SR versus factor VIII:C, r = -0.410, P = 0.001; n-APC-SR versus vWF:Ag, r = -0.309, P = 0.01; factor VIII:C versus vWF:Ag, r = +0.640, P<0.0001. The relative risk for VTE in individuals with the factor VIII:C concentration > 1.5 IU/ml was 2.5 (95% confidence interval 1.6-3.9). We concluded that high factor VIII:C levels, probably in the effect of vWF, play a determinant role in worsening the APC-resistance phenotype and represent a common additional risk factor for VTE in heterozygous carriers of the factor V Leiden mutation.     

Collection of Heparinized Plasma by Plasmapheresis

BACKGROUND AND OBJECTIVES: Heparinized plasma can be used for exchange transfusions in neonates and is usually collected by drawing whole blood using heparin as anticoagulant. The heparinized red blood cells and buffy coat cannot be used and are therefore discarded. To collect heparinized plasma more efficiently, a method was developed using an apheresis machine. MATERIALS AND METHODS: With an MCS3p apheresis machine (Haemonetics), plasma was collected from volunteer donors as anticoagulant, heparin in saline (30,000 IU/l) was added in a 1:9 ratio. The activated partial thromboplastin time (APTT) of the donors was measured before and immediately after the procedure, and various parameters were determined in the collected plasma. RESULTS: In 2 collection cycles, an average of 456+/-52 ml (mean +/- SD; n = 20) of heparinized plasma was collected, and 504+/-57 ml (n = 2; donors with a high hemoglobin level) when 3 cycles were performed. The leukocyte and platelet contamination in the plasma (n = 22) was 1.11+/-0.92x10(6) and 0. 05+/-0.22x10(9) per unit, respectively, which conformed to national specifications. Sodium levels were normal, but due to dilution of the plasma with heparin solution, potassium and calcium levels were about 20% lower than the serum levels in the donors. The donor APTT values were slightly longer after the procedure than before, but remained all within normal values. CONCLUSION: For the collection of heparinized plasma, apheresis has the advantage that (1) high-quality heparinized plasma can be harvested; (2) no blood components need to be discarded; (3) more plasma can be harvested with each donation, and (4) these procedures can be performed more often than whole blood donations.     

Effect of endotoxin on digestive enzyme and superoxide dismutase in mouse pancreas

In order to clarify the effect of endotoxin on pancreatic lesions, changes in the level of activity of pancreatic proteolytic enzymes and superoxide dismutase (SOD) were measured following the intraperitoneal administration of Escherichia coli endotoxin in mice. Microscopic examination revealed that congestion of zymogen granules in acinar cells appeared in the early stages. The accumulation increased gradually and was widespread at 24 h after E. coli endotoxin administration. The serum amylase level increased significantly from its basal level of 1,630 +/- 128 SU to 2,395 +/- 42 SU at 4 h (p less than 0.05), 3,172 +/- 513 SU at 8 h (p less than 0.05), and 2,572 +/- 229 SU at 12 h (p less than 0.05) after E. coli endotoxin administration. The serum lipase level also increased significantly from its basal level of 37.6 +/- 1.6 IU/l to 65.8 +/- 8.9 IU/l at 12 h (p less than 0.05). The level of trypsinogen in the pancreas increased markedly from its basal level of 161.8 +/- 15.7 U/g tissue to 270.0 +/- 9.6 U/g tissue at 2 h (p less than 0.001) after E. coli endotoxin administration and showed continued increases at 8 h (313.3 +/- 10 U/g tissue, p less than 0.001) and 24 h (352.9 +/- 19.3 U/g tissue, p less than 0.001); however, the level of net stimulated secretion of trypsinogen in the medium decreased significantly at 4 h (p less than 0.05) and 8 h (p less than 0.01) after E. coli endotoxin administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevalence and Consistency of ALT Elevation in Plasmapheresis Donors: Implications for the Assessment of Blood Product Infectivity

Alanine aminotransferase (ALT) levels were determined in 8,420 plasmapheresis donations obtained from 431 donors over a period of 18 months. Using sex-differentiated normal ranges 2.5% of donations but 23% of donors exhibited elevated ALT levels on at least 1 occasion. Amongst the donors with elevated ALT this was only seen on 1 occasion in one third, while a quarter had elevations in consecutive donations. No donors with consecutive elevations above 100IU/1 were detected. The results are discussed in terms of the guidelines currently recommended for assessing post-transfusion hepatitis infectivity of blood products, such as factor VIII. It is concluded that the current allowance for infection acquired from sources other than blood products under consideration may be over-generous, leading to a potential underestimate of the true rate of infection.     

Altered Red Cell Indices in Repeat Blood Donors: Experience of a North Indian Blood Bank

Subclinical Iron deficiency appearing in blood donors after blood donation is a recognized problem. Donors at an increased risk of iron deficiency need to be identified. Blood donors meeting national selection criteria were included in the study. Complete blood counts were done using Sysmex XP-100 three part hematology analyzer. Differences in RBC indices among donor groups defined by previous donations were then analyzed statistically. Six hundred and seventy three males and ninety six females were studied. In males, Kruskal–Wallis test showed significant differences between groups defined by number of donations for MCH and MCV (P value?<?0.001), but not for MCHC (P value?=?0.09) and RDW (P value?=?0.6). Post-hoc tests showed statistically significant difference between donors having six or more donations compared to donors having no previous donations for both MCH as well as MCV. No significant difference was found between donor subgroups in females for any of the indices; however, no female donated blood more than three times in the study. There is increased risk for iron deficiency in donors having six or more previous donations, and evidence for starting an iron screening and supplementation programme for these donors.