Phase I/II safety study of transfusion of prion-filtered red cell concentrates in transfusion-dependent patients.

BACKGROUND AND OBJECTIVES: Variant Creutzfeldt-Jakob (vCJD) is a fatal transfusion transmissible prion infection. No test for vCJD in the donor population is currently available. Therefore, prion removal by filtration of red cell concentrate (RCC) is an attractive option for prevention. MATERIALS AND METHODS: Twenty patients were recruited with ethical permission, to receive clinically necessary transfusion containing one unit of pfRCC. Follow-up at 24 hours, 6 weeks and 6 months was undertaken. A second pfRCC was administered to 6 patients with similar follow up. pfRCC were prepared using the CE marked P-Capt device by the IBTS. RESULTS: In 20 transfused patients undergoing one exposure to a prion filtered unit, no attributable adverse events were noted. A subset of these (n = 6) underwent re-exposure to a further filtered unit without incident. CONCLUSIONS: This phase 1/11 clinical study provides encouraging data on safety of prion filtration which can be used to plan more extensive studies on the use of filtered blood in adults and children.

Capillary and venous haemoglobin levels in blood donors: a 42-month study of 36,258 paired samples.

BACKGROUND: EU law requires a haemoglobin of > or = 12.5 g/dl for women or > or = 13.5 g/dl for men at the time of donation. As capillary and venous haemoglobin values may differ in the same subject, we examined whether a capillary haemoglobin level of 12.0 g/dl for women or 13.0 g/dl for men, is equivalent to a venous haemoglobin level of > or = 12.5 g/dl and > or = 13.5 g/dl, respectively, to avoid unnecessary loss of blood donations. METHODS: Over a continuous 42-month period, 36 258 paired capillary and venous samples were taken from 25 762 females and 10 496 males, when the capillary haemoglobin was < 12.5 g/dl and < 13.5 g/dl respectively. RESULTS: Venous haemoglobin levels were higher than capillary levels, with a mean difference of 1.07 g/dl (SD 0.68 g/dl), range -2.2 to +3.25 g/dl for men (P < 0.001), and a mean difference of 0.67 g/dl (SD 0.65 g/dl), range -2.5 to +5.4 g/dl for women (P < 0.001). The difference for the three consecutive winters was 0.78 g/dl (SD 0.081 g/dl) for females and 1.26 g/dl (SD 0.162 g/dl) for males and for the three consecutive summers was 0.56 g/dl (SD 0.089 g/dl) for females and 0.88 g/dl (SD 0.134 g/dl) for males: P < 0.001. CONCLUSIONS: Capillary haemoglobin levels of 12.0-12.5 g/dl in healthy females or 13.0-13.5 g/dl in healthy males are substantively equivalent to venous haemoglobin levels of > or = 12.5 and > or = 13.5 g/dl for women and men respectively. This finding has permitted an additional 32 990 blood units to be collected over the period of the study, a gain of 9.4%.

In vitro assessment of red-cell concentrates in SAG-M filtered through the MacoPharma P-CAPT prion-reduction filter.

This study investigated whether filtration of leucodepleted red cells in SAG-M through the P-CAPT filter in order to prevent the potential risk of vCJD infection associated with prion transmission through transfusion has any deleterious effect on red-cell quality. Bottom-and-top SAG-M leucodepleted red-cell concentrates (24 units) were prion-reduction filtered on the day following collection, with half of the units undergoing irradiation on day 14. A control group (12 units) was not prion filtered. Units were sampled at 7-day intervals up to day 35 and tested using standard measures of red-cell quality as well as prothrombin content (to examine prion filter efficacy). Haemoglobin loss per unit was approximately 9 g and in some cases levels were below standard specification (40 g). Haemolysis increased significantly after filtration [0.01 (0.00-0.05) vs. 0.23 (0.07-0.52, p<0.001)]. Prothrombin levels were reduced 41.6-fold compared to leucodepleted red-cell units. Product specifications were within or close to routine acceptable levels. Owing to the reduction in haemoglobin levels below those specified, it may be preferable to reduce haemoglobin specification levels and transfuse more prion-filtered units rather than transfuse potentially unsafe blood product. The risk of transfusing more units with less haemoglobin should be offset against the risk of transfusing unfiltered blood.

Screening platelet concentrates for bacterial contamination: low numbers of bacteria and slow growth in contaminated units mandate an alternative approach to product safety.

BACKGROUND AND OBJECTIVES: We introduced 100% screening of platelets for bacterial contamination in 2005 to reduce the risk of clinical sepsis from platelet transfusion. We test all outdating units again at expiry to assess the sensitivity of the initial test. MATERIALS AND METHODS: We test all platelet concentrates prior to release for clinical use using a large volume automated culture technique on the day after manufacture. All units that expire unused are retested. Platelets still in stock on day 4 of storage may have a repeat culture performed, and are returned to stock with two extra days of shelf life. RESULTS: Of 43,230 platelet units screened, 35 (0.08%) were positive; of 8282 expired unused, 18 (0.22%) were positive; and of 3310 day-4 retests, four (0.12%) were positive. Overall sensitivity of the initial screening test was 29.2% (95% confidence interval 19.4 to 39.1%). Thirteen of the 35 positive screening tests would have been expected to grow in both aerobic and anaerobic bottles; eight grew in aerobic culture only and five grew in anaerobic culture only, indicating that the likely number of bacteria in the contaminated platelet units at the time of sampling was less than 60 colony-forming unit per platelet unit. CONCLUSIONS: Screening platelet concentrates for bacterial contamination using the most sensitive method available has a sensitivity of less than 40% because of the low numbers of bacteria in the initial contamination. Effective resolution of this problem will require a pathogen-inactivation technique.

Methyldopa-induced autoantibodies against red blood cells.

Methyldopa therapy results in the formation of red cell autoantibodies in 10-20% of patients taking the drug for longer than 4 months. These red cell antibodies are true autoantibodies, that is they are directed against an autoantigen on the red blood cell membrane and not against the drug or against a drug-altered antigen. The target membrane antigen is usually within the Rhesus system, although often the antibody specificity cannot be defined. Red cell antibody is usually detectable in the patient's sera as well as on the red cells. The autoantibody is usually a warm reacting IgG antibody. Most patients who develop these autoantibodies do not go on to develop hemolytic anemia in spite of high titres of antibodies on their red cells. In addition, these patients do not tend to develop hemolysis if methyldopa therapy is continued. Rarely patients develop hemolytic anemia which can be severe. Differences in antibody characteristics, including subclass restriction, complement-binding ability, or titre do not explain why some patients with autoantibody hemolyze while most do not. One group of investigators found that hemolyzing patients had IgM on their red cells while those who did not had IgG only. But while this observation could explain why some patients (IgM-sensitized red cells) hemolyze, it does not explain why most patients with IgG-sensitized red cells do not hemolyze. Why the autoantibody forms is not known but some investigators have proposed that the drug may directly affect B or T cells with resulting impairment of immune tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure to kill Yersinia enterocolitica by plasma diluted to the concentration found in red cell units.

The possibility that the use of additive solutions for red cell storage might impair the ability of plasma to kill Yersinia enterocolitica was investigated by studying killing of Y enterocolitica by neat and diluted plasma. The ability of neat citrated plasma to kill complement sensitive organisms was lost at around 26%, the dilution typically found in red cell units. These results should be considered in the light of evidence that killing in plasma is important in the protection of donated blood against growth of Y enterocolitica, and the observation that the increase in frequency of transfusion reactions caused by Y enterocolitica coincided with the widespread introduction of additive solutions. Taken together, these points support the suggestion that the introduction of additive solutions may have precipitated the problem of growth of Y enterocolitica in stored blood.

Neonatal alloimmune thrombocytopenia in the Irish population: a discrepancy between observed and expected cases.

AIMS: To estimate the rate of detection of neonatal alloimmune thrombocytopenia (NAITP) in the Irish population, to investigate clinical presentation and outcome in affected infants, and to determine the extent, if any, to which this condition is underdiagnosed at present. METHODS: Cases were collected in a retrospective fashion from a review of platelet serology laboratory records from January 1992 to December 2000. Clinical data were obtained from hospital records. Testing for maternal antiplatelet antibody was by one or more of the following: the platelet suspension immunofluorescence test, a commercial antigen capture enzyme linked immunosorbent assay (GTI-PakPlus), and the monoclonal antibody immobilisation of platelet antigens assay. Platelet antigen typing was by the polymerase chain reaction technique with sequence specific primers. RESULTS: Twenty seven serologically verified cases of NAITP were identified in 18 families. Maternal antibody to human platelet antigen 1a accounted for 25 of the 27 confirmed cases. Twenty one of 26 infants were born with severe thrombocytopenia. Nineteen of 27 infants had bleeding manifestations at birth. Petechiae and bruising were most commonly observed (n = 17). There were no documented cases of intracranial haemorrhage in this group but systematic cranial ultrasound was not performed. CONCLUSIONS: Screening studies in predominantly white populations have estimated the incidence of NAITP to be between 1 in 1000 and 1 in 2000 live births. With 50 000 births each year in Ireland, these results give a clinical detection rate for NAITP of just 1 case in 16 500 live births, strongly suggesting that NAITP is currently underdiagnosed. Antenatal screening to detect women at risk of having babies with NAITP is now scientifically feasible and should be considered.

Rate of growth of Pseudomonas fluorescens in donated blood.

AIMS: To examine how delayed refrigeration of blood affects the growth of Pseudomonas fluorescens, one of the two most important causes of sepsis resulting from transfusion of contaminated blood. METHODS: Two donations of whole blood were each divided into three aliquots and inoculated with 5-10 cfu/ml of a P fluorescens strain from a case of transfusion associated sepsis. From each donation, one aliquot was placed at 4 degrees C, one was held at 20 degrees C for six hours prior to refrigeration and the third was held at 20 degrees C for 24 hours prior to refrigeration. Samples were aseptically withdrawn over 17 days and bacterial counts were determined using a pour plate technique. RESULTS: The rate of growth of P fluorescens in blood at 20 degrees C was increased compared with blood at 4 degrees C. At 24 hours the aliquots held at 20 degrees C for six and 24 hours had, respectively, 174 and 29,000 cfu/ml compared with 15 cfu/ml in aliquots held at 4 degrees C. There was no evidence of increased killing of P fluorescens at the higher temperature. CONCLUSIONS: These results suggest that blood for transfusion should be refrigerated as soon as possible after collection.

Yersinia enterocolitica septicaemia from transfusion of red cell concentrate stored for 16 days.

Two cases of transfusion transmitted Yersinia enterocolitica biotype 3, serotype 09 infection occurred in south east Scotland within four months of each other. In one case, a 79 year old man died the day after receiving a unit of red cell concentrate that had been stored for 29 days after donation. In the second case a 78 year old man died three days after transfusion of a unit of red cell concentrate that had been collected 16 days before transfusion. The donors of both units had no symptoms attributed to gastrointestinal infection. Early outdating of blood for transfusion after three weeks of storage is unlikely to eradicate Y enterocolitica associated fatalities from blood transfusion, and alternative methods should be considered.

A new competitive binding enzyme-linked immunosorbent assay for glycocalicin in plasma and platelet concentrate supernatants.

A new competitive binding enzyme-linked immunosorbent assay (CB ELISA) for glycocalicin (GC) was developed using GC coated wells and a monoclonal antibody (MAb) to glycoprotein Ib (AN51). The principal stages of the CB ELISA consisted of coating the plate with GC extract overnight, blocking with 3% BSA, incubating the wells with test or standard sample dilution and AN51, and a final incubation with horseradish peroxidase-conjugated goat anti-mouse IgG. Serial dilutions of purified GC, starting in 2% BSA, yielded standard curves which were linear between 10 and 0.4 micrograms/ml. Parallel curves were obtained for platelet concentrate supernatants and for citrated plasma. We used the ELISA to measure GC levels in platelet concentrates during storage. The results indicated that soluble GC increased progressively during storage from 3.3 to 6.7 micrograms/ml, while GC levels in platelet-poor plasma remained at 1.9-2.2 micrograms/ml. These results show that the new CB ELISA is a simple and short assay for the direct measurement of GC in plasma solutions, and may be of use in clinical studies.

Plasma glycocalicin levels are not elevated in patients with a history of transient ischaemic event and are normal in aspirinated normal volunteers.

Glycocalicin (GC) is the soluble portion of platelet membrane protein GP1b, and may be cleaved from the platelet surface during platelet activation. Previous study has indicated that plasma glycocalicin/platelet (GC/plt) levels are elevated in patients presenting with acute stroke. The present study was undertaken to determine the GC/plt levels in patients being treated for transient ischaemic episodes, to assess whether the elevated GC/plt level in acute stroke is due to a detectable, constitutive premorbid state of platelet activation. In sixteen consecutive patients attending a vascular surgery clinic, GC levels were measured on a citrated plasma sample, and corrected for circulating platelet count. Since 15 of 16 patients were taking aspirin when seen at clinic, a control study was undertaken to assess the effect of aspirin on sequential plasma GC/plt levels measured over 10 days--5 pre and post daily aspirin for 5 days, 4 acting as non-aspirinated controls. Plasma GC/plt levels in normal plasma were 21.6 +/- 8.0 fg; mean +/- SD. In the 16 patients the GC/plt levels were 13.1 fg/plt; SD 5.4, range 2.9-24.3. All platelet counts were in the normal range in all patients involved. While a masking effect due to aspirin cannot be completely ruled out, these studies indicate that plasma GC/plt level is not useful as a predictor of acute stroke in the premorbid population.

Monitoring the release of glycocalicin in platelet concentrates by ELISA.

Platelet membrane glycoprotein Ib (GPIb) is one of many GPs on the platelet membrane which contribute to the functional and morphological properties of platelets. A principal function of GPIb is its attachment to von Willebrand Factor (vWF) on injured blood vessels which leads to the adhesion of platelets to these vessels. The binding site to vWF resides on glycocalicin (GC), which is a major segment of GPIb. Glycoprotein Ib is particularly susceptible to centrifugation and storage of platelets. The assessment of GPIb status on platelets, therefore, comprises one of many traditional methods for monitoring the quality of platelets during storage. We have recently developed a novel ELISA to monitor GC levels in the supernatant of platelet concentrates (PCs) during storage. Using this ELISA we observed a progressive rise of GC in PC supernatants during storage. A recent study of citrated PCs with or without EDTA produced similar results, and showed a substantial increase of GC levels in EDTA-treated PCs. The GC ELISA could therefore be used as a novel method to monitor PCs during storage under various conditions.

Glycoproteins Ib and IIb/IIIa in the quality assessment of platelet concentrates during storage.

We have previously shown that levels of soluble glycocalicin (GC) in plasma supernatants derived from units of platelet concentrates (PC) increase progressively during storage. We now report further studies which show that the levels of both microparticle-bound and soluble GC in PC during storage are influenced by exposure of PC samples to EDTA and treatment of PC packs with ultraviolet B (UVB) irradiation. EDTA leads to a significant increase in the release of microvesicle-bound and soluble GC, while UVB irradiation leads to a dose- and rate-dependent increase in GC release. Paradoxically, UVB leads to an unexpected decrease in supernatant levels of von Willebrand factor (vWf) during storage which contrasts with its increase in untreated, stored PC. Moreover, an increase in GC release during storage is associated with a corresponding decrease in platelet size as determined by measurement of mean platelet volume (MPV) in citrated PC. The GC release is significantly correlated with standard platelet functional tests and other new generation tests such as dMPV and supernatant levels of vWf. In addition, preliminary results show the presence of microparticle-bound and soluble glycoprotein (Gp) IIb/IIIa in the supernatant plasma of stored PC. Our results suggest that supernatant levels of GpIb, GpIIb/IIIa, and vWf, together with alteration in MPV, provide essential new informative parameters for quality assessment of PC.

Thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura is an uncommon disorder, but it continues to be of considerable interest. The disease mechanisms are unclear and the aetiology is unknown. Perhaps most enigmatic of all, the mode of action of plasma therapy, which successfully induces remission in about two-thirds of cases, is wholly inexplicable. There are currently several areas of debate on the subject of thrombotic thrombocytopenic purpura. This paper addresses these points of contention: the definition of the disease, its distinction from haemolytic uraemic syndrome, the nature of the platelet aggregating factors in the plasma of patients with acute disease, the importance of the abnormalities of von Willebrand's factor observed in the acute and quiescent phases of the disease, the nature of the factor in normal plasma that induces remission, and the possible causes of the observed superiority of plasma exchange combined with plasma infusion, over plasma infusion alone.

Clinicians' satisfaction with a hospital blood transfusion service: a marketing analysis of a monopoly supplier.

One of the objectives of the NHS reforms is to improve customer focus within the health service. In a study to assess the quality of customer service provided by the Edinburgh and South East Scotland Blood Transfusion Service a 19 item questionnaire survey of the main clinical users of the service was performed to ascertain their satisfaction, measured on a 5 point anchored scale, with important aspects of the service, including medical consultation, diagnostic services, blood and blood components or products and their delivery, and general satisfaction with the service. Of 122 clinicians in medical and surgical disciplines in five hospitals in Edinburgh, 72 (59%) replied. Fourteen (22%) indicated dissatisfaction with any aspect of the medical consultation service, owing to inadequate follow up of clinical contacts and unsatisfactory routing of incoming calls. Diagnostic services were criticised for the presentation, communication, and interpretation of results. The restricted availability of whole blood, the necessity to order platelets and plasma through the duty blood transfusion service doctor, and the use of a group and screen policy, attracted criticism from a small number of clinicians. Ten of 68 respondents expressed dissatisfaction with delivery of blood and components to the wards and theatres. The findings indicate that the clinicians served by this blood transfusion service are largely satisfied with the service. Changes are being implemented to improve reporting of laboratory results and measures taken to improve liaison with clinicians.

Effects of a brief theory-based intervention on the practice of testicular self-examination by high school males.

This preliminary study evaluated a brief intervention, based on the theory of planned behavior, designed to encourage high school students to practice testicular self-examination (TSE). Ninety-nine male ninth and 10th grade students were exposed to a message challenging beliefs about outcomes of performing TSE as suggested by the theory of planned behavior, a message providing information about TSE and testicular cancer, or a control message, after which they completed a questionnaire operationalizing components of the theoretical model. Four weeks later, students reported their practice of TSE. As predicted, students exposed to the theory-based message reported a stronger intention to perform TSE and, at follow-up, were more likely to report having performed the exam than study participants exposed to other messages. Data provide partial support for the utility of the theory of planned behavior as a framework for constructing health-promoting messages. Future research recommendations are offered.

Implementation of the rhesus prevention programme: a prospective study.

All rhesus-negative women who completed a pregnancy between January and August 1992 in two Scottish regions were studied to assess whether the administration of the rhesus prevention programme was complete: 671 rhesus positive (or rhesus type unknown) pregnancies were completed in 1120 Rh D negative women. For eight pregnancies no record of Rh D administration at birth was available. For recorded antenatal events that should have resulted in its administration anti-D was given in 195/280 (69.6%). Kleihauer testing was carried out in 9 of 98 instances of antepartum haemorrhage occurring after 20 weeks gestation. No cases of antenatal immunisation were identified. The study identified a need to increase awareness of the necessity for anti-D administration after potentially immunising events during pregnancy, and for increasing compliance with administration of postnatal anti-D in one of the study regions. Re-evaluation is also required of the recommendation that Kleihauer testing should be done when antenatal anti-D is given following an obstetric event after 20 weeks.

Lessons from the response to the threat of transfusion-transmitted vCJD in Ireland.

By the time vCJD was first described in 1996, it was already far too late to offset further disaster from transmission of the disease by blood transfusion: almost all the humans who would be infected and infectious were already diseased. Nothing done by the blood transfusion services around that time, with the exception of excluding transfusion recipients as blood donors, would have made any useful contribution to containing the extent of the epidemic. The ability to spread emerging diseases before the problem is manifest or understood is a fixed and unavoidable feature of blood transfusion as it is practiced today. A second fixed property of blood transfusion is that the root cause of disaster is not within the control of the blood transfusion universe. Strategies that have emerged to cope with similar threat in other enterprises that also contain these properties comprise the components of robust design: surveillance, preparedness for action, engagement, herding together, evasion or avoidance, early adoption of potentially useful measures, engineered resilience, defence in depth, damage limitation including modularity and removal of feedback loops, and contingency, redundancy and failure management, and ultimately, individual escape. Early adoption of leucodepletion based on the possibility that it might work rather than any hard evidence was a good example of threat management. Exclusion of previously transfused donors is a robust mechanism for containing any future infection; optimal blood use structures that provide a national transfusion rate as low as possible also constitute an effective threat management strategy.

Pathogen reduction: state of reflection in Ireland.

The Irish Blood Transfusion Service is currently assessing the feasibility and affordability of implementing pathogen reduction for platelets in Ireland. Since 2002, almost all plasma transfused in the country has been subjected to a pathogen reduction process in the form of Octaplas™ (or Uniplas™ for group AB recipients), manufactured from plasma from donors at the South Texas Blood and Tissue Center, San Antonio, TX, USA. Pathogen reduction of platelets for Ireland is driven by two major concerns: by the need for robust systems to prevent the transmission of any emerging transfusion transmissible infections or of diseases for which we do not currently test, and by the poor sensitivity and efficacy of even the most sensitive available approaches to bacterial contamination of platelets. While the safety of blood transfusion is a matter of public safety rather than health economics, it is currently the case that money spent in Ireland on pathogen reduction of platelets will result in fewer resources available for public use elsewhere, so that detailed cost balancing is required in deciding whether or not to implement pathogen reduction. Considerations that influence the costs of implementation in our hands include the ability to discontinue platelet irradiation, the ability to maintain a single inventory from the point of view of CMV, extending storage to day 7 of shelf-life as a routine, and avoidance of travel deferrals for platelet donors.