Residual subset population analysis in WBC‐reduced blood components using real‐time PCR quantitation of specific mRNA

Pennington, J. H.; Garner, Stephen F.; Sutherland, Janet; Williamson, Lorna M.

doi:10.1046/j.1537-2995.2001.41121591.x

Cited by 29 publications

(28 citation statements)

References 19 publications

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“…In line with our previous study of random blood donors, 24 we found that CD3 signal reduction in asymptomatic carriers was similar to that of Tax, suggesting that removal of HTLV-I-infected T lymphocytes by the filter was equivalent to that of overall Tlymphocyte removal. By contrast, in the MT-2 model system, provirus removal was approximately 1 log 10 lower than removal of T lymphocytes as measured by CD3 mRNA.…”

Section: Htlv-i Removal By Leukocyte Depletion 679supporting

confidence: 91%

“…We have previously shown this to be specific for T cells linear over 5 log 10 concentration range, with a sensitivity limit of 30 leukocytes/mL. 24 Here we observed a mean 4.71 log 10 reduction in CD3 signal after whole blood filtration, indicating that overall T-lymphocyte reduction was around 1 log 10 greater than that of MT-2 removal measured by HTLV-I provirus detection.…”

Section: Removal Of Mt-2 Cells and T Lymphocytes By Filtration Of Whosupporting

confidence: 50%

“…cDNA synthesis was performed using MultiScribe Reverse Transcriptase (PE Biosystems, Foster City, CA) as previously described. 24 Real-time PCR and data analysis was performed on the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and probes were designed to detect HTLV-I Tax DNA and CD3 mRNA using Primer Express software (Applied Biosystems).…”

Section: Quantification Of Htlv-i Tax and Cd3 Mrna In Blood Componentmentioning

confidence: 99%

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Persistence of HTLV-I in blood components after leukocyte depletion

Pennington

Taylor²,

Sutherland³

et al. 2002

Blood

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The human T-cell leukemia virus HTLV-I is a transfusion-transmissible retrovirus targeting T lymphocytes for which screening is not currently undertaken in United Kingdom blood donors. The introduction of universal leukocyte depletion (LD) of the United Kingdom blood supply raises the question as to the degree of protection afforded by this procedure against HTLV-I transmission by blood components. HTLV-I viral DNA removal by leukocyte-depleting filters was assessed in units of whole blood and platelets by real-time quantitative polymerase chain reaction (PCR) and by nested PCR for HTLV-I Tax DNA. We examined HTLV-I removal by LD filters using a model system of blood units containing exogenous spiked HTLV-I-positive MT-2 cells at a relevant concentration and whole blood donations from asymptomatic HTLV-I carriers. T-lymphocyte removal was assessed in parallel by measurement of endogenous subset-specific CD3 mRNA. In the MT-2 model system we observed 3.5 log 10 to 4 log 10 removal of HTLV-I Tax DNA by filtration of whole blood and 2 log 10 to 3 log 10 removal across platelet filters with 13 of 16 whole blood and 8 of 8 platelet units still positive after filtration.Despite 3 log 10 to 4 log 10 viral removal, HTLV-I Tax DNA could be detected after whole blood filtration in asymptomatic carriers with viral loads above 10 8 proviral DNA copies/L. T-lymphocyte removal was also between 3.5 log 10 and 4.5 log 10 . HTLV-I provirus removal was incomplete in the model system and in asymptomatic carriers with viral loads greater than 10 8 copies/L. These results suggest that LD alone may not provide complete protection from HTLV-I transmission by transfusion. (Blood. 2002;100:677-681)

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Section: Htlv-i Removal By Leukocyte Depletion 679supporting

confidence: 91%

Section: Removal Of Mt-2 Cells and T Lymphocytes By Filtration Of Whosupporting

confidence: 50%

Section: Quantification Of Htlv-i Tax and Cd3 Mrna In Blood Componentmentioning

confidence: 99%

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Persistence of HTLV-I in blood components after leukocyte depletion

Pennington

Taylor²,

Sutherland³

et al. 2002

Blood

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“…Quantities of the various HIV-1 RNA species were determined in reference to a standard curve prepared by amplification of serial dilutions of in vitro-transcribed RNA (RiboMAX large-scale RNA production system; Promega, Charbonnières, France) containing matching sequences into 10 ng of bacteriophage MS2 RNA (Roche Diagnostics)/l. Determination of cell equivalents in CEM RNA samples was performed by measuring levels of CD3ε mRNA, which were reported to be unaffected by HIV-1 infection, in a lymphoid cell line (38) and using previously described primers and probes (30). Determination of cell equivalents in HeLa RNA samples was performed by measuring levels of cyclophilin A mRNA.…”

Section: Vol 78 2004mentioning

confidence: 99%

Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

Brussel

Sonigo

2004

J Virol

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The integrated form of human immunodeficiency virus type 1 (HIV-1) DNA is classically considered to be the sole template for viral gene expression. However, several studies have suggested that unintegrated viral DNA species could also support transcription. To determine the contribution of the different species of HIV-1 DNA to viral expression, we first monitored intracellular levels of various HIV-1 DNA and RNA species in a single-round infection assay. We observed that, in comparison to the precocity of HIV-1 DNA synthesis, viral expression was delayed, suggesting that only the HIV-1 DNA species that persist for a sufficient period of time would be transcribed efficiently. We next evaluated the transcriptional activity of the circular forms of HIV-1 DNA bearing two long terminal repeats, since these episomes were reported to exhibit an intrinsic molecular stability. Our results support the notion that these circular species of HIV-1 DNA are naturally transcribed during HIV-1 infection, thereby participating in virus replication.

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“…11,17 We and others have previously reported more than 4 10 log monocyte reduction by whole blood LD. 18,19 The cause of the discrepancy between monocyte and CMV removal is not clear, but with HTLV-infected T cells and similar filters, overall T-cell reduction was also 1 log greater than HTLV Tax removal. 12 It may be that monocytes activated by CMV infection are removed less well by filters than noninfected cells.…”

mentioning

confidence: 99%

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

Visconti

Pennington

Garner

et al. 2004

Blood

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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...

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Residual subset population analysis in WBC‐reduced blood components using real‐time PCR quantitation of specific mRNA

Abstract: Real-time RT-PCR assays are suitable for analysis of subset removal during WBC reduction. There was no significant difference between the two whole-blood filters or between platelet filtration and apheresis in the removal of any WBC subset.

Cited by 29 publications

References 19 publications

Persistence of HTLV-I in blood components after leukocyte depletion

Persistence of HTLV-I in blood components after leukocyte depletion

Evidence for Gene Expression by Unintegrated Human Immunodeficiency Virus Type 1 DNA Species

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

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