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.

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.

Isolated valvular amyloid.

Two cases are described of grossly evident amyloid infiltration of the cardiac valves, while only minor deposits were found in other locations. The right-sided valves were more heavily involved than the left. No pre-existing disease of the valves was found, and the lesions appeared to have no adverse effect upon the subjects. Only one other similar case was found in the literature. The only consistently associated condition among the recognized cases was advancing age. The name proposed for the condition is isolated valvular amyloid.

Prevention of monocyte adhesion and inflammatory cytokine production during blood platelet storage: an in vitro model with implications for transfusion practice.

A novel platelet additive solution [ThromboSoltrade mark (TS)] was designed to allow extended refrigerated platelet storage. It has been shown to preserve platelet function and prevent cytokine accumulation in platelet concentrates stored for up to 9 days. It consists of amiloride, adenosine, sodium nitroprusside, dipyridamole, quinacrine, and ticlopidine. We hypothesized that the cytokine inhibition may be due to prevention of monocyte (MC) adhesion and activation on the surfaces of platelet storage bag plastic polymers. In an in vitro model, we incubated purified peripheral blood MCs on discs of polyolefin and polyvinylchloride from platelet storage bags, and on polystyrene, in the presence of TS for up to 7 days. We found that after incubation with TS, adherent MC numbers were decreased by >80-95% compared with controls on all surfaces examined. Levels of cytokines [interleukin (IL)-1beta, IL-1RA, IL-6, IL-8, and tumor necrosis factor-alpha] were low in wells with TS but rose progressively in the controls during incubation. Amiloride alone had similar effects on adhesion and cytokine release as the complete TS preparation. Removing amiloride from TS abrogated these effects. These findings suggest an important role for TS and amiloride in monocyte function, and have implications for the development of agents designed for prolonged platelet storage.

Antibodies to hepatitis C virus in autologous blood donors.

Hepatitis C virus (HCV) is the major cause of posttransfusion hepatitis. Two anti-HCV enzyme immunoassay (EIA) kits and one recombinant immunoblot assay (RIBA) were used to test serum samples of 1476 donations from 692 autologous blood donors to assess the prevalence of anti-HCV and its relationship to transfusion history. Of all autologous blood donations, 23 (1.6%) reacted when tested with one EIA kit and 29 (2.0%) reacted when tested by the other EIA kit. Of the autologous donors, 12 (1.78%) reacted by the first EIA kit and 14 (2.02%) by the second. Discrepancies in the EIA results from different donations by the same donor were seen in seven donors. The RIBA was positive or indeterminate in 33 percent of the EIA-reactive donations and in 41 percent of EIA-reactive donors. All RIBA-positive and -indeterminate samples reacted with both EIA kits. There was no significant difference in the EIA-reactive rates of autologous and first-time homologous blood donors. Previously transfused autologous blood donors had a higher anti-HCV EIA-reactive rate than nontransfused autologous donors, but the difference was not significant. In regard to hepatitis C, the use of autologous blood for homologous transfusion appears to be as safe as the use of blood from first-time homologous donors. Universal testing of previously transfused patients for hepatitis C appears premature at this time. Discrepant anti-HCV EIA results from different donations from the same individual have implications regarding donor deferral.

Reliability of automated platelet counts: comparison with manual method and utility for prediction of clinical bleeding.

The 20 x 10(9)/L (20,000/microliters) threshold for prophylactic platelet transfusion may be unnecessarily high. The widespread use of this threshold may reflect lack of confidence in the reliability of low platelet counts. We evaluated the performance of automated platelet counts and their relation to clinical bleeding. First, we prepared serial blood dilutions with "target" platelet counts from 2 to 40 x 10(9)/L. For the 48 measurements on 2 x 10(9)/L "target" dilutions, values of 1 or 2 x 10(9)/L were obtained with the Sysmex NE-8000 analyzer (mean 1.44 x 10(9)/L; SD 0.31 x 10(9)/L). Similarly, for 5 x 10(9)/L "target" counts, automated counts were 3-6 x 10(9)/L (mean 4.42 x 10(9)/L; SD 0.18 x 10(9)/L). Similar results were observed with all other "target" levels, with coefficients of variation (CV) from 6.39% to 7.71% with 10-40 x 10(9)/L "target" values. Secondly, we compared triplicate automated and manual platelet counts on thrombocytopenic patients with platelet counts from 4-30 x 10(9)/L. The triplicate automated platelet counts differed by no more than 5 x 10(9)/L among themselves, whereas the manual counts varied by as much as 30 x 10(9)/L. Mean platelet counts: automated, 14.40 x 10(9)/L (CV 10.12%); manual, 16.48 x 10(9)/L (CV 30.39%) (P = 0.038 for counts; P < 0.001 for CV). Finally, we prospectively evaluated bleeding in thrombocytopenic patients (1,809 patient-days of observation). Univariate and multivariate logistic regression analysis revealed highly significant correlations between the automated platelet count and major and minor bleeding manifestations. Thus, automated platelet counts are highly reliable and accurately predict clinical bleeding. The use of automated analyzers should facilitate improved prophylactic platelet transfusion protocols.