Stephen J. Wagner

Bacterial screening of apheresis platelets and the residual risk of septic transfusion reactions: the American Red Cross experience (2004‐2006)

BACKGROUND: The American Red Cross initiated systemwide bacterial testing of all apheresis platelet (PLT) collections in March 2004, yet continues to receive reports of septic reactions after transfusion of screened components.

Transfusion-associated bacterial sepsis.

The incidence of sepsis caused by transfusion of bacterially contaminated blood components is similar to or less than that of transfusion-transmitted hepatitis C virus infection, yet significantly exceeds those currently estimated for transfusion-associated human immunodeficiency and hepatitis B viruses. Outcomes are serious and may be fatal. In addition, transfusion of sterile allogenic blood can have generalized immunosuppressive effects on recipients, resulting in increased susceptibility to postoperative infection. This review examines the frequency of occurrence of transfusion-associated sepsis, the organisms implicated, and potential sources of bacteria. Approaches to minimize the frequency of sepsis are discussed, including the benefits and disadvantages of altering the storage conditions for blood. In addition, the impact of high levels of bacteria on the gross characteristics of erythrocyte and platelet concentrates is described. The potentials and limitations of current tests for detecting bacteria in blood are also discussed.

Factors affecting virus photoinactivation by a series of phenothiazine dyes.

A series of four phenothiazine dyes, including methylene blue (MB), were previously tested for their ability to photoinactivate viruses in red cell suspensions. One of the dyes, 1,9‐dimethyl‐3‐dimethylamino‐7‐dimethylaminophenothiazine (1,9‐dimethylmethylene blue), exhibited good intracellular and extracellular virucidal activity for several RNA and DNA viruses under conditions that minimally affected red cell properties. In order to understand why the virucidal specificity of 1, 9‐dhnethylmethylene blue was greater than other phenothiazines tested, the physical and chemical properties of the dye were compared to three other closely related analogues (MB, 1,9‐dimethyl‐3‐diethylamino‐7‐dlbutylaminophenothiazine [compound 4‐140], 1,9‐dimethyl‐3‐dimethylamino‐7‐diethylaminophenothiazine [compound 6‐136]). All compounds required light and oxygen for virucidal activity and had relatively high singlet oxygen yields (>0.5), but 1,9‐dimethylmethylene blue had a singlet oxygen yield approximately 50% greater than that of MB. In addition, the hydrophobicity/hydophilicity of the compounds varied, with the partition coefficients (2‐octanol : water) ranging from 0.11 for MB to 3560 for compound 4‐140. The dyes had the following affinities for DNA: 1,9‐dimethylmethylene blue > compound 6‐136 > MB ∼ compound 4‐140. This order was similar to the order of activities for photoinactivation of the nonenveloped bacteriophage, R17, by the four compounds. Results with the most hydrophobic compound, 4‐140, contrasted with those obtained with 1,9‐dimethylmethylene blue. Compound 4‐140 had a high affinity for protein and a low affinity for DNA. Although compound 4‐140 and light inactivated the nonenveloped bacteriophage R17 poorly, the dye readily photoinactivated enveloped viruses in buffer. However, unlike results with 1,9‐dimethylmethylene blue, viral inactivation of enveloped viruses by compound 4‐140 was completely inhibited by the presence of red cells and plasma. Thus, the high affinity of 1,9‐di‐methyymethylene blue for DNA and the dyes efficient singlet oxygen yield suggest viral nucleic acid as a potential target, which could explain the photosensitizers ability to inactivate viruses without adversely affecting anuclete red cells.

The residual risk of sepsis: modeling the effect of concentration on bacterial detection in two‐bottle culture systems and an estimation of false‐negative culture rates

BACKGROUND: Septic reactions continue to be reported with culture‐tested platelet (PLT) products, probably due to false‐negative results associated with inadequate sampling of low‐concentration bacteria. The mechanism of test failure was modeled and false‐negative rates were estimated utilizing published data.

Limiting and detecting bacterial contamination of apheresis platelets: inlet-line diversion and increased culture volume improve component safety

BACKGROUND: Septic transfusion reactions to apheresis platelets (PLTs) continue to occur despite preventive measures. This study evaluated the effect of two operational changes designed to reduce bacterial risk: 1) introducing inlet‐line sample diversion on two‐arm procedures and 2) increasing the sample volume cultured from 4 to 8 mL from all donations.

Diversion of initial blood flow to prevent whole‐blood contamination by skin surface bacteria: an in vitro model

BACKGROUND: Sepsis arising from the transfusion of bacterially contaminated platelet components continues to be an infrequent, yet serious transfusion complication. Skin organisms are implicated in a number of these septic episodes. A model system was used to investigate if a skin organisms bioburden in blood components could be reduced by diverting the first few mL of whole blood away from the primary container.

Preservation of red cell properties after virucidal phototreatment with dimethylmethylene blue

BACKGROUND: All published reports have described methods for virus photoinactivation which significantly alter red cell (RBC) properties during storage. In order to improve virucidal activity and reduce damage to RBCs, a series of phenothiazine derivatives were either synthesized or purified and screened for bacteriophage inactivation and red cell potassium efflux. One compound, 1,9‐dimethylmethylene blue (dimethyl‐methylene blue), had superior screening results and was chosen for further characterization.

Analysis of viral DNA, protein and envelope damage after methylene blue, phthalocyanine derivative or merocyanine 540 photosensitization.

Abstract— Although numerous photosensitizers have been used experimentally to decontaminate viruses in cellular blood components, little is known about their mechanisms of photoinactivation. Using M13 bacteriophage and vesicular stomatitis virus (VSV) as model viruses, we have investigated alteration of the viral genome, protein and envelope after phototreatment. Methylene blue (MB) and aluminum phthalocyanine tetrasulfonate (AlPcS4) phototreatment inactivated bacteriophage M13 and decreased the fraction of single‐stranded circular genomic DNA (sc‐DNA) by converting it to linear form. This conversion was enhanced by treating the extracted DNA with piperidine at 55°C. Piperidine‐labile breaks were well correlated to phage survival (5.1% sc‐DNA at 1.7% phage survival for MB) under conditions where only minor differences were seen in the relative abundance of M13 coat protein on sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS‐PAGE). Neither aluminum phthalocyanine (AlPc) nor merocyanine 540 (MC540) inactivated M13 nor were there significant changes observed in DNA and coat protein. Methylene blue, AlPcS4 and AlPc inactivated VSV and inhibited fusion of the virus envelope to Vero cells at pH 5.7 (i.e. with plasma membrane). However, the degree of this inhibition was small compared to the extent of virus inactivation (43% inhibition vs 4.7 log10 or 99.998% inactivation, for MB). In contrast, an antibody to VSV G‐spike protein inhibited fusion at pH 5.7 by 52% with a concomitant decline in VSV infectivity of 0.15 log10 (30%). Few changes were observed in the relative abundance of G protein for MB and AlPcS4 phototreated samples and no additional protein bands were observed on SDS‐PAGE. Phototreatment did not appreciably change the relative fusion ability at pH 7.2 (via the endocytotic pathway). These results collectively suggest that nucleic acid may be an important target for photoinactivation of these model viruses by MB and AlPcS4.

Comparison of Methylene Blue and Methylene Violet for Photoinactivation of Intracellular and Extracellular Virus in Red Cell Suspensions

Previous studies with methylene blue (MB) in red cell suspensions have demonstrated that extracellular, but not intracellular, virus can be readily photoinactivated. To test if the resistance of intracellular virus to inactivation is related to the permanent positive charge of the phenothiazine, a series of uncharged phenothiazine dyes, methylene violet (MV), monodemethylated MV and didemethylated MV, were studied. Values of the sensitivity of intracellular relative to extracellular vesicular stomatitis virus (VSV) inactivation for the three dyes (D10 extracellular/D10 intracellular) in buffer were 1.0, 0.60 and 0.33, respectively. In contrast, intracellular virus was resistant to inactivation with MB, with a D10 extracellular/D10 intracellular of 0.05 in buffer. Because virucidal activity of MV was inhibited by the presence of plasma, the red cells (30% hematocrit) were repeatedly washed prior to photoinactivation and storage. Under conditions where MB and MV inactivated approximately 5 log10 of extracellular VSV, intracellular VSV was inactivated by more than 4 log10 with MV compared to 0.88 log10 with MB. These phototreatment conditions did not significantly affect red cell morphology, extracellular pH, ATP or 2,3-diphosphoglycerol levels during 42 days of 1-6 degrees C storage. There was enhanced potassium efflux and hemolysis over values obtained from untreated control; the extent of change from controls was comparable for each phototreatment. These results indicate that the uncharged phenothiazine dye, MV, can inactivate both intracellular and extracellular virus yet exhibit similar in vitro red cell storage properties as MB phototreatment.

Platelet products prepared by different methods of sedimentation undergo platelet activation differently during storage

BACKGROUND: The activation marker CD40 ligand (CD40L) has recently been demonstrated to be released from the cytoplasm of platelets (PLTs) during storage. CD40L may be associated with some adverse transfusion reactions including febrile responses and transfusion‐related acute lung injury. CD62P has been traditionally measured to assess PLT activation. This study compares the surface levels of CD40L and CD62P and accumulation of the soluble forms of these activation markers in the plasma of stored PLTs, prepared by PLT‐rich plasma (PRP) or buffy coat (BC) methods and with two apheresis instruments.