Enlargement of the WHO international repository for platelet transfusion-relevant bacteria reference strains.

BACKGROUND AND OBJECTIVES: Interventions to prevent and detect bacterial contamination of platelet concentrates (PCs) have reduced, but not eliminated the sepsis risk. Standardized bacterial strains are needed to validate detection and pathogen reduction technologies in PCs. Following the establishment of the First International Reference Repository of Platelet Transfusion-Relevant Bacterial Reference Strains (the 'repository'), the World Health Organization (WHO) Expert Committee on Biological Standardisation (ECBS) endorsed further repository expansion. MATERIALS AND METHODS: Sixteen bacterial strains, including the four repository strains, were distributed from the Paul-Ehrlich-Institut (PEI) to 14 laboratories in 10 countries for enumeration, identification and growth measurement on days 2, 4 and 7 after low spiking levels [10-25 colony-forming units (CFU)/PC bag]. Spore-forming (Bacillus cereusPEI-B-P-07-S, Bacillus thuringiensisPEI-B-P-57-S), Gram-negative (Enterobacter cloacaePEI-B-P-43, Morganella morganiiPEI-B-P-74, PEI-B-P-91, Proteus mirabilisPEI-B-P-55, Pseudomonas fluorescensPEI-B-P-77, Salmonella choleraesuisPEI-B-P-78, Serratia marcescensPEI-B-P-56) and Gram-positive (Staphylococcus aureusPEI-B-P-63, Streptococcus dysgalactiaePEI-B-P-71, Streptococcus bovisPEI-B-P-61) strains were evaluated. RESULTS: Bacterial viability was conserved after transport to the participating laboratories with one exception (M. morganiiPEI-B-P-74). All other strains showed moderate-to-excellent growth. Bacillus cereus, B. thuringiensis, E. coli, K. pneumoniae, P. fluorescens, S. marcescens, S. aureus and S. dysgalactiae grew to >106 CFU/ml by day 2. Enterobacter cloacae, P. mirabilis, S. epidermidis, S. bovis and S. pyogenes achieved >106 CFU/ml at day 4. Growth of S. choleraesuis was lower and highly variable. CONCLUSION: The WHO ECBS approved all bacterial strains (except M. morganiiPEI-B-P-74 and S. choleraesuisPEI-B-P-78) for repository enlargement. The strains were stable, suitable for spiking with low CFU numbers, and proliferation was independent of the PC donor.

Serratia marcescens strains implicated in adverse transfusion reactions form biofilms in platelet concentrates and demonstrate reduced detection by automated culture.

BACKGROUND AND OBJECTIVES: Serratia marcescens is a gram-negative bacterium that has been implicated in adverse transfusion reactions associated with contaminated platelet concentrates. The aim of this study was to investigate whether the ability of S. marcescens to form surface-attached aggregates (biofilms) could account for contaminated platelet units being missed during screening by the BacT/ALERT automated culture system. MATERIALS AND METHODS: Seven S. marcescens strains, including biofilm-positive and biofilm-negative control strains and five isolates recovered from contaminated platelet concentrates, were grown in enriched Luria-Bertani medium and in platelets. Biofilm formation was examined by staining assay, dislodging experiments and scanning electron microscopy. Clinical strains were also analysed for their ability to evade detection by the BacT/ALERT system. RESULTS: All strains exhibited similar growth in medium and platelets. While only the biofilm-positive control strain formed biofilms in medium, this strain and three clinical isolates associated with transfusion reactions formed biofilms in platelet concentrates. The other two clinical strains, which had been captured during platelet screening by BacT/ALERT, failed to form biofilms in platelets. Biofilm-forming clinical isolates were approximately three times (P<0·05) more likely to be missed by BacT/ALERT screening than biofilm-negative strains. CONCLUSION: S. marcescens strains associated with transfusion reactions form biofilms under platelet storage conditions, and initial biofilm formation correlates with missed detection of contaminated platelet concentrates by the BacT/ALERT system.

Establishment of the first international repository for transfusion-relevant bacteria reference strains: ISBT working party transfusion-transmitted infectious diseases (WP-TTID), subgroup on bacteria.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) still remains a significant problem in transfusion with potential important clinical consequences, including death. The International Society of Blood Transfusion Working Party on Transfusion-Transmitted Infectious Diseases, Subgroup on Bacteria, organised an international study on Transfusion-Relevant Bacteria References to be used as a tool for development, validation and comparison of both bacterial screening and pathogen reduction methods. MATERIAL AND METHODS: Four Bacteria References (Staphylococcus epidermidis PEI-B-06, Streptococcus pyogenes PEI-B-20, Klebsiella pneumoniae PEI-B-08 and Escherichia coli PEI-B-19) were selected regarding their ability to proliferate to high counts in PCs and distributed anonymised to 14 laboratories in 10 countries for identification, enumeration and bacterial proliferation in PCs after low spiking (0·3 and 0·03 CFU/ml), to simulate contamination occurring during blood donation. RESULTS: Bacteria References were correctly identified in 98% of all 52 identifications. S. pyogenes and E. coli grew in PCs in 11 out of 12 laboratories, and K. pneumoniae and S. epidermidis replicated in all participating laboratories. The results of bacterial counts were very consistent between laboratories: the 95% confidence intervals were for S. epidermidis: 1·19-1·32 × 10(7) CFU/ml, S. pyogenes: 0·58-0·69 × 10(7) CFU/ml, K. pneumoniae: 18·71-20·26 × 10(7) CFU/ml and E. coli: 1·78-2·10 × 10(7) CFU/ml. CONCLUSION: The study was undertaken as a proof of principle with the aim to demonstrate (i) the quality, stability and suitability of the bacterial strains for low-titre spiking of blood components, (ii) the property of donor-independent proliferation in PCs, and (iii) their suitability for worldwide shipping of deep frozen, blinded pathogenic bacteria. These aims were successfully fulfilled. The WHO Expert Committee Biological Standardisation has approved the adoption of these four bacteria strains as the first Repository for Transfusion-Relevant Bacteria Reference Strains and, additionally, endorsed as a project the addition of six further bacteria strain preparations suitable for control of platelet contamination as the next step of enlargement of the repository.

A confusion in antibody identification: anti-D production after anti-hrB.

It is well known that certain combinations of alloantibodies are frequently found together. Patients with sickle cell disease (SCD) are mostly ofAfrican ancestry,and they may make anti-hrB. A transfusion of hrB- blood is often achieved by using e- (R2R2) RBCs; it is generally believed that hrB- patients readily make anti-E or a"broad-spectrum" anti-Rh34 (-HrB). We describe two multiply transfused D+ patients with SCD and a history of anti-hrB who subsequently produced anti- D. This raises the question whether anti-hrB together with anti-D is a more common antibody combination than anti-hrB with anti-E or anti-Rh34.

Lack of isohemagglutinin production following minor ABO incompatible unrelated HLA mismatched umbilical cord blood transplantation.

While immune hemolysis due to donor isohemagglutinin (IH) production often complicates minor ABO incompatible peripheral blood hematopoietic stem cell transplantation (PBSCT), it is not known if this occurs with umbilical cord blood transplantation (UCBT). We compared IH production and hemolysis following minor ABO allogeneic PBSCT and UCBT. We reviewed 24 ABO minor incompatible allogeneic PBSCTs and 14 ABO minor incompatible UCBTs. Patients were evaluated for donor-derived IH by reverse ABO grouping. Evaluation of hemolysis was based on clinical and laboratory findings of anemia associated with increased RBC transfusion need, concomitant with the appearance of donor-derived IH. Of the 24 ABO minor incompatible allogeneic PBSCTs, 15 produced donor-derived IH from 6 to 88 days following transplantation, with seven of 15 patients exhibiting clinically evident hemolysis. There was no significant difference in days to leukocyte engraftment or infused CD34 cells in patients with or without donor-derived IH. None of the 14 patients receiving ABO incompatible UCBTs showed evidence of donor-derived IH following transplantation with a median follow-up of 60 days. We conclude that donor IHs are not produced in patients undergoing minor ABO incompatible UCBTs suggesting fundamental immunologic differences between allogeneic PBSCT and UCBT.

Prolonged isolated thrombocytopenia after hematopoietic stem cell transplantation: morphologic correlation.

Prolonged isolated thrombocytopenia, defined as recovery of other cell counts with continuous dependence on platelet transfusions for greater than 90 days after hematopoietic stem cell transplantation (HSCT), develops in approximately 5% of patients who undergo HSCT. Although the clinical conditions associated with prolonged isolated thrombocytopenia have been studied, a systematic review of bone marrow biopsies has not been performed and the pathophysiologic basis has not been defined. We reviewed all HSCT at one center from 1990 to 1995 (n = 454) and found 12 cases that met criteria for prolonged isolated thrombocytopenia (incidence = 12/454 or 3%). Bone marrow core biopsies from 12 patients with prolonged isolated thrombocytopenia were reviewed to determine cellularity, numbers of megakaryocytes, the presence of atypical forms, and clusters of megakaryocytes. These marrow megakaryocyte counts were compared to age and disease matched controls, and 11 normal donors. Patients (aged 1-56 years, mean 32 years) who underwent HSCT (four sibling HLA-identical, five autologous bone marrow, three autologous peripheral stem cell) with prolonged isolated thrombocytopenia had a statistically significant lower absolute megakaryocyte count in bone marrow biopsies performed before transplantation and more than 30 days after transplantation compared to control patients (aged 4 months to 50 years, mean 31 years) who underwent HSCT (four sibling HLA-identical, four autologous bone marrow, four autologous peripheral stem cell) for similar conditions. No apparent differences were seen in size of megakaryocytes, nuclear-cytoplasmic ratios, or clustering of megakaryocytes. Overall marrow cellularities were similar in the three groups. These findings suggest that decreased differentiation of megakaryocytes from stem cells, rather than ineffective platelet production or peripheral destruction of platelets, causes prolonged isolated thrombocytopenia in HSCT patients. Low megakaryocyte counts prior to HSCT may be a useful prognostic indicator, as this feature was associated with the development of prolonged isolated thrombocytopenia.

Successful large volume leukapheresis on a small infant allogeneic donor.

We successfully performed a hematopoietic stem cell apheresis on the smallest allogeneic donor reported to date, a 6.1 kg female. After placement of a dialysis catheter in the left femoral vein, the COBE Spectra was primed with one unit of paternal whole blood. Heparin and anticoagulant citrate dextrose, solution A (ACD-A) were slowly administered to the patient. Ionized calcium levels were checked hourly and calcium gluconate was given for hypocalcemia. Coagulation parameters were checked throughout the procedure. We collected 4.4 x 10(6) CD34+ cells/kg (recipient). The donor tolerated the procedure well and was discharged the following day. Five months later, the child manifests no obvious late effects.

Bacterial contamination of platelets at a university hospital: increased identification due to intensified surveillance.

BACKGROUND: A cluster of bacterial contamination of platelets occurred at a university hospital in a one-month period. This unusual clustering allowed us to examine the likely mechanism of contamination and clinical sequelae. METHODS: We reviewed medical records of patients receiving random donor platelet transfusions to determine numbers of platelets transfused, reactions reported, and episodes of bacterial contamination. We also reviewed procedures at the collecting blood agencies and the hospital blood bank. RESULTS: Four patients received bacterially contaminated platelets during June and July 1991. The rates of reported platelet transfusion reactions increased significantly (P < 0.001) from September 1989 through July 1991 (study period); in addition, the rate of contamination of platelets during June and July 1991 was 23-fold higher than during the previous 21 months (P < 0.001). Surveillance methodology changed dramatically during the study period, contributing to the recognition of the current cluster. Pathogens isolated from the contaminated platelet pools were Bacillus cereus, Staphylococcus epidermidis, or Pseudomonas aeruginosa in titers ranging from 10(6) to 10(8) colony forming units/mL. Four constituent individual platelet units identified as the probable cause of the outbreak (including one postepidemic episode) were significantly older (mean age, 4.8 days) than 106 randomly selected individual platelet units (mean age, 3.7 days; P = 0.04). Platelet pools were transfused an average of 2.5 hours after pooling. Review of blood collection and platelet preparation practices did not identify breaks in procedure or technique that could have caused contamination. CONCLUSIONS: Increased awareness of platelet transfusion reactions by clinical staff and routine culturing of all platelets associated with transfusion reactions will identify contaminated platelets. Identification of contaminated platelets is necessary to treat affected patients appropriately and to determine the prevalence of and risk factors for contaminated platelets (Infect Control Hosp Epidemiol 1994;15:82-87).

Practical aspects of preoperative autologous transfusion.

Autologous transfusion, which is widely endorsed as the safest transfusion practice, has undergone rapid-and often unchecked-growth since the mid-1980s, primarily in response to the recognition of transfusion-associated HIV. Substantial improvements in blood transfusion safety, combined with the increasing emergence of managed care during the past decade, have spurred a reexamination of autologous transfusion and its ability to provide increased transfusion safety in a cost-effective manner. The ultimate utility of preoperative autologous donation and transfusion depends on whether (1) waste is limited by the development of meticulously crafted benchmarks for appropriate surgical applications, (2) donor-patient risk is limited by careful donor selection and scheduling and scrupulous attention to iron supplementation, (3) strategies are identified to enhance cost-effectiveness, and (4) policies and procedures are developed to reduce or prevent accidents and errors.

Munchausen syndrome by proxy documented by discrepant blood typing.

The authors describe a case of Munchausen syndrome by proxy, a form of child abuse, documented by use of routine blood bank serologic procedures. While immunohematologic testing has been used in the legal arena to resolve issues of disputed paternity and to investigate instances of criminal acts, the authors believe this to be the first documented application to this clinical disorder.

Lowering the prophylactic platelet transfusion threshold: a prospective analysis.

The 20 x 10(9) /L threshold for prophylactic platelet transfusion may be unnecessarily high. Few prospective studies, however, in which other trigger values were tested have been published. In this study all hospitalized, thrombocytopenic adult hematology-oncology patients in our institution were prospectively evaluated daily for hemorrhage and platelet transfusion during a one year period; no patients were excluded for bleeding or infectious problems. By design, during the initial six-months (baseline period), the prophylactic platelet transfusion trigger was 20 x 10(9) /L; for the second six-months (study period) this threshold was changed to 10 x 10(9) /L. Patients studied during the two periods did not differ significantly in age, gender, diagnosis, blood or marrow transplant status, and duration of neutropenia. Compliance with the thresholds was 95.6% (baseline period) and 93.5% (study period). For patients with platelet counts under 20 x 10(9) /L, the mean use of platelet transfusions per patient per day was significantly lower in the study period (4.47) than in the baseline period (6.48; p<0.001). Both mean prophylactic (1.54/patient-day) and therapeutic (2.93/patient-day) platelet transfusions were reduced in the study period compared with the baseline period (2.26 and 4.22/patient-day, respectively). Hemorrhage was slightly reduced in the study period compared with the baseline period: major hemorrhage, 15.2% vs. 18.4% (p=0.014); minor hemorrhage, 63.6% vs. 70.1% (p<0.001). Thus, hemorrhage was not increased with the lower trigger level. A 10 x 10(9) /L prophylactic platelet transfusion threshold value is safe and effective.

Is directed blood transfusion a good idea?

The appearance of transfusion-transmitted HIV infection has heightened awareness of dangers that have always been present. Directed donation, while no panacea, may provide one alternative.