Lisa J. Cardo

Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light

Background and Objectives  Leishmania is transmitted by the bite of the phlebotomine sandfly or by transfusion of infected blood products. Leishmaniasis currently poses a significant problem in several parts of the world, and is an emerging problem in others. The Mirasol PRT technology is based on the use of riboflavin and ultraviolet light to generate chemical reactions in the nucleic acids of pathogens, which prevents replication and leads to inactivation. The intent of this study was to examine the ability of the Mirasol PRT System to kill the Leishmania parasite in human plasma and platelet concentrates.

Leishmania: risk to the blood supply.

Leishmania infection is most often transmitted to humans via the bite of the phlebotomine sandfly, but transmission of Leishmania by transfusion has also been reported. There has been a huge increase in the incidence of cutaneous and visceral leishmaniasis in Iraq and Afghanistan. The deployment of US troops to these countries and published case reports of transmission to soldiers in endemic areas, by transfusion to infants with immature immune systems, and to individuals immunocompromised by disease or immunosuppressive therapy beckon a reexamination of blood donor deferral procedures. The length of the ongoing military conflict and the nature of exposure indicate that prior decisions regarding blood donor deferral made during the first Gulf War may no longer apply. Operation Iraqi Freedom and Operation Enduring Freedom present a much greater Leishmania threat than did Operation Desert Storm. Because most transmission by transfusion occurs in endemic areas, and visceral infection is asymptomatic in healthy individuals such as blood donors, it is difficult to determine the absolute risk of transmission by transfusion, but review of the literature provides many clues as to the appropriate measures to be taken for blood donor deferral.

Neutrophil priming, caused by cell membranes and microvesicles in packed red blood cell units, is abrogated by leukocyte depletion at collection

UNLABELLED BACKGROUND AND OBJECTS: Lipids with platelet activating factor (PAF)-like activity in supernatant of packed red blood cells (PRBC) cause priming of the neutrophil respiratory burst. This effect increases with length of storage. Washing of PRBC has been considered as a means to eliminate this effect; however, the role of the cellular component was not evaluated independently of the supernatant. The source of the inflammatory lipids of the supernatant is likely to be cell membranes altered during ageing in storage and therefore, washing will not eliminate neutrophil priming caused by transfusion of aged PRBC units. The ability of washed PRBC to prime mononuclear cells for another known effect of PAF, the production of IL-8, and the probability that this lipid activity is present on microparticles in PRBC supernatant were also investigated. MATERIALS AND METHODS At collection 10 units of whole blood were split into two equal aliquots one filtered and one unfiltered. PRBC were prepared and stored at 4 degrees C in CPD-AS5. Each week, fresh neutrophils were incubated with samples of washed PRBC and fixed. Change in CD11b, a marker known to increase on the surface of primed neutrophils, was determined by flow cytometry. To determine whether neutrophil priming ability of PRBC supernatant is contained on microvesicles, centrifuged and uncentrifuged supernatant samples were incubated with fresh neutrophils and change in CD11b expression was determined. Plasma IL-8 levels were also measured after exposure of monocytes from fresh whole blood to filtered and unfiltered washed PRBC with and without the addition of fMLP. RESULTS Washed PRBC caused a 50-116% increase in CD11b neutrophil surface expression over baseline expression. Filtration of whole blood at collection reduced this CD11b up-regulation by 25-34%. Reduction of priming ability by filtration began on the day of collection and persisted for the storage life of the units. Centrifugation resulted in a reduction of CD11b up-regulation of 11-28% compared with unspun supernatant. Incubation of unfiltered PRBC resulted in priming of mononuclear leukocytes for IL-8 production with a 73-109% increase over baseline, but no increase over baseline was seen for incubation with filtered blood. CONCLUSION Washing does not eliminate the ability of PRBC units to prime neutrophils and mononuclear cells, because the cellular component of PRBC, in addition to the supernatant, induces priming. Leukodepletion filters significantly reduce these effects compared with unfiltered PRBC. The in vitro beneficial effect of filtration lasts for the shelf life of 42 day units. The ability of PRBC supernatant to prime neutrophils is present on microvesicles.

Stored packed red blood cells contain a procoagulant phospholipid reducible by leukodepletion filters and washing

BACKGROUND Ageing RBC gradually increase the exposure of phosphatidylserine (PS) on their surface, due to loss of membrane asymmetry. PS expression on red cells is not normally a significant factor in the hemostatic process, because aged RBC are rapidly cleared from the circulation. We propose that the presence of many altered red cells during massive transfusion can lead to increased procoagulant activity similar to what is seen in disease states where it is known to play a pathophysiologic role in microvascular disease. STUDY DESIGN AND METHODS Procoagulant activity of phospholipid generated during storage of PRBC was evaluated using PRBCs as the only source of phospholipid in the determination of modified Russells viper venom times of 10 PRBC units in which half of each unit was left unfiltered and half of each unit filtered. Florescent labeled annexin V binding by PRBC was also assessed by flow cytometry over time in storage. The effect of washing and filtration on these parameters was also determined. RESULTS As time of storage increased, the Russells viper venom time of both the unfiltered and filtered units shortened (p<0.01). There was a significant lengthening of the Russells viper venom time at all time points measured when unfiltered units were compared to filtered units (p<0.01). In both unfiltered and filtered units, with increased length of storage, there was a gradual increase in the percentage of cells or particles binding annexin V (p<0.01). Filtration resulted in a significant reduction in the percentage of cells or particles binding annexin V at all time points measured (p<0.01). The effect of washing of PRBC units on the RVVT was assessed for unfiltered and filtered units on day 42. Washing resulted in a significant reduction of the RVVT in both unfiltered and filtered groups (p<0.01). CONCLUSIONS Levels of annexin V binding and procoagulant phospholipid activity similar to levels seen in disease states associated with significant vasoocclusive pathophysiology were found toward the end of the storage period of PRBC units. It was possible to reduce both of these parameters by leukodepletion at collection, and with washing of PRBC at the end of storage. Filtration at collection resulted in a 67% increase in RVVT over unfiltered units by day 42 of storage. On day 42 of storage, washing of filtered units resulted in a 21% increase in RVVT, and washing of unfiltered units resulted in a 34% increase in RVVT. The effects seen with filtration and washing were additive suggesting that in spite of filtration at collection, deterioration of cells continues based on age since further removal of phospholipid can be induced with washing of filtered units on day 42.

Laboratory evaluation of rapid test kits to detect hepatitis C antibody for use in predonation screening in emergency settings.

BACKGROUND: Emergency whole blood transfusion is a lifesaving procedure employed on modern battlefields. Rapid device tests (RDTs) are frequently used to mitigate transfusion‐transmitted infection risks.

A Leishmania minicircle DNA footprint assay for sensitive detection and rapid speciation of clinical isolates

BACKGROUND: Diversity in clinical outcome, due to different species of Leishmania, and its presence in asymptomatic blood donors in endemic areas warrant development of methods that are sensitive and can rapidly identify infecting species.

Leukodepletion filters reduce Leishmania in blood products when used at collection or at the bedside

BACKGROUND:  Leishmania is an intracellular parasite of monocytes transmissible by transfusion. The feasibility of reducing Leishmania with leukodepletion filters was studied. At collection, infected blood contains the amastigote form of Leishmania within monocytes. Amastigotes cause the rupture of monocytes releasing free amastigotes that convert to promastigotes, which exist extracellularly at blood storage temperatures. Leukodepletion filters were tested at various time points in this process.

Photoinactivation of Leishmania donovani infantum in red cell suspensions by a flexible thiopyrylium sensitizer

Background and Objectives  Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania, which are intracellular parasites of monocytes and macrophages. Transmission of the organism has been observed by transfusion of infected blood from asymptomatic donors to immunocompromised recipients, leading to clinically apparent disease. There is no licensed Leishmania screening test currently available.

Removal of Plasmodium falciparum–infected red blood cells from whole blood by leukoreduction filters

BACKGROUND: There has been an unexplained decrease in the incidence of transfusion‐transmitted malaria in recent years. The decrease in incidence has paralleled the increasing use of leukoreduction filters. Malaria‐infected red blood cells (RBCs) share surface characteristics of hemoglobin S–containing cells. Because units collected from donors with sickle trait do not filter optimally due to adherence of RBCs to the filters, the possibility that malaria‐infected RBCs may also adhere to filters was investigated.

Inactivation of Leishmania donovani infantum and Trypanosoma cruzi in red cell suspensions with thiazole orange

BACKGROUND: Methods for pathogen inactivation are currently available in some European countries for treatment of plasma and platelet (PLT) components; no approved method for treatment of red cells (RBCs) or whole blood is ready for implementation. In a previous study, thiazole orange (TO), a dye commonly used to count reticulated RBCs and PLTs, exhibited potent photoactivity against human immunodeficiency virus‐1 and several model viruses in RBC suspensions. The aim of this study is to further evaluate the ability of TO to inactivate pathogens by measuring its activity against the protozoa Leishmania donovani infantum and Trypanosoma cruzi.