Assessment of removal of human cytomegalovirus from blood components by leukocyte depletion filters using real-time quantitative PCR

Abstract

To assess removal of cytomegalovirus (CMV) by leukocyte depletion (LD) filters, we developed a spiking model of latent virus using peripheral blood mononuclear cells (PBMCs) infected by coculture with CMV-infected human fibroblasts. Infected PBMCs were purified by dual magnetic column selection and then spiked into whole blood units or buffy coat pools prior to LD by filtration. CMV load and fibroblast contamination were assessed using quantitative CMV DNA real-time PCR and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA encoding the fibroblast-specific splice variant of prolyl-4-hydroxylase, respectively. After correcting for fibroblast-associated CMV, the mean CMV load was reduced in whole blood by LD from 7.42 10 2 to 1.13 copies per microliter (2.81 10 log reduction) and from 3.8 10 2 to 4.77 copies per microliter (1.9 10 log reduction) in platelets. These results suggest that LD by filtration reduces viral burden but does not completely remove CMV from blood components. (Blood. Introduction Primary cytomegalovirus (CMV) infection results in lifelong carriage of latent virus, notably in CD14 monocytes. 1 Reactivation of donor CMV following transfusion 2 is avoided by selecting CMV-seronegative donors for vulnerable patients. Because universal leukocyte depletion (LD) has been widely implemented, it is debated whether LD alone could provide equivalent protection. 3,4 Eight studies have shown no transmissions following prestorage LD, 5,6 but a recent study reports increased transmission from LD CMV-seropositive components. 7 Quantitative real-time polymerase chain reaction (PCR) has the dynamic range to assess CMV removal by LD, 8,9 but due to low copy number, we and others 9 have failed to detect viremia in seropositive donors, and even optimal CMV PCR would require virtually all leukocytes in an LD component (less than 10 6). 10 We found techniques reported to increase CMV copy number (pollen, 11-interferon, hydrocortisone, granulocyte-macrophage colony-stimulating factor [GM-CSF], and allogeneic cells 2) to be insufficiently robust for assessment of LD. We have previously used infected T lymphocytes and real-time PCR to assess human T-cell leukemia virus-I (HTLV-I) removal by LD. 12 A CMV-infected T-cell line has also been described to measure CMV removal, 13 but T cells are not a major physiological reservoir of CMV. We have therefore modified a previously described system 14 to generate CMV-infected mononuclear cells, including CD14 monocytes, for spiking into blood donations prior to LD. Study design With ethics committee approval and donor consent, 450-mL blood donations from 6 CMV-seronegative donors were collected into citrate phosphate dextrose. Peripheral blood mononuclear cells (PBMCs) were prepared from a 24 mL aliquot by density gradient separation (mean yield, 1.52 10 7 0.27 10 7) and cultured for 12 hours with 2 10 6 human embryonic fibroblasts infected with human CMV strain AD169. PBMCs were separated from fibroblasts by double selection using magnetic beads coate...

Humanes Cytomegalievirus (HCMV)

“…Daraus folgt auch, dass die Infektion von der Mehrheit der seropositiven Spender nicht übertragen wird [11,64,112]. Durch HCMV-seronegative zelluläre Blutkomponenten wurde das Risiko einer HCMV-Infektion bei der Verabreichung nicht leukozytendepletierter Blutkomponenten verringert [113,114]. Die generelle Verwendung von leukozytendepletierten Blutkomponenten hat das Risiko ebenfalls gesenkt.…”

“…Experimentell wurde in leukozytendepletierten Blutkomponenten eine Reduktion der zellgebundenen HCMV-DNA um ca. 3 log 10 -Stufen gezeigt [114,115]. Die minimale infektiö-se Dosis für eine HCMV-Infektion beim Menschen konnte bisher nicht definiert werden.…”

Human Cytomegalovirus (HCMV) – Revised

“…The general use of leucocytedepleted blood components has also reduced this risk. Spiking experiments have shown a reduction in leucocyte-depleted blood components of about 3 log levels with regard to cellular HCMV DNA [84,85].…”

“…It can be concluded that infection is not transmitted from the majority of seropositive donors [8,48,82]. As long as non-leucocytereduced blood components were administered, the risk of HCMV infection was reduced by HCMV-seronegative cellular blood components [83,84]. The general use of leucocytedepleted blood components has also reduced this risk.…”

Concerning Caspari et al: Pathogen Inactivation of Cellular Blood Products – More Security for the Patient or Less? Transfus Med Hemother 2003;30:261–263

“…Other pathogens, for example Trypanosoma cruzi, may be highly prevalent in certain immigrant populations, and there may be no routine tests in place to identify infected donors [6]. Additional highly prevalent latent pathogens such as cytomegalovirus (CMV) may not be completely eliminated by current methods [7][8][9]. The risk of a transfusion-transmitted bacterial contamination is even more compelling.…”