Real-Time Polymerase Chain Reaction (RQ-PCR) for the Monitoring of Epstein-Barr Virus (EBV) Load in Peripheral Blood Mononuclear Cells

Abstract:

Measurement of Epstein-Barr virus (EBV) load is useful in peripheral blood for detecting primary and reactivated EBV-infections especially in immunosuppressed patients being at high risk for developing posttransplant lymphoproliferative disorder. For quantification of EBV DNA in peripheral blood of patients two real time polymerase chain reaction (RQ-PCR) assays were developed detecting sequences specific for the BAM HI-W and BAM HI-K region of EBV. In order to determine the optimal material of peripheral blood for RQ PCR analysis, DNA preparations of whole blood, peripheral blood mononuclear cells (PBMC) and B cells from 11 healthy, EBV-seropositive individuals were analysed in parallel and compared with regard to efficiency and sensitivity. While in whole blood preparations inhibitors of RQ PCR were detected influencing sensitivity, analysis of B cells being most sensitive is limited by being too labour intensive. In contrast, analysis of DNA preparations of PBMCs is sensitive enough to frequently detect EBV-specific sequences in all individuals tested and the preparation of PBMCs itself needs only a reasonable time. Thus, longitudinal monitoring of EBV load in peripheral blood of patients is possible by RQ-PCR, the optimal material for analysis being PBMCs.

Die Epstein-Barr-Virus (EBV)-Lastmessung im peripheren Blut ist sinnvoll zum Nachweis primärer und reaktivierter EBV-Infektionen, insbesondere bei immunsupprimierten Patienten, die ein hohes Risiko haben, eine lymphoproliferative Erkrankung nach Transplantation zu entwickeln. Um EBV-DNA im peripheren Blut zu quantifizieren, wurden zwei „Real-Time”-Polymerase-Kettenreaktionsverfahren (RQ-PCR) entwickelt, die spezifisch Sequenzen der BAM HI-W- und BAM HI-K-Region des EBV nachweisen. Um das optimale Analysenmaterial aus dem peripheren Blut zu ermitteln, wurden DNA-Präparationen aus Vollblut, mononukleäre Zellen des peripheren Blutes (PBMC) und B-Zellen von 11 gesunden, EBV-seropositiven Probanden im Hinblick auf Effizienz und Sensitivität parallel untersucht. Während in Vollblutpräparationen Inhibitoren des RQ-PCR nachweisbar waren, die die Sensitivität beeinträchtigten, war die Analyse der B-Zellen zwar am sensitivsten, jedoch am arbeitsaufwendigsten. Im Gegensatz hierzu konnten in DNA-Präparationen von PBMCs bei allen Patienten EBV-spezifische Sequenzen bei einem tolerablen Zeitaufwand für die Zellpräparation nachgewiesen werden. Somit ist eine longitudinale Verlaufskontrolle der EBV-Last im peripheren Blut von Patienten möglich, als optimales Material hierfür bieten sich PBMCs an.

Key words:

Epstein-Barr virus - posttransplant lymphoproliferative disorder (PTLD) - real time quantitative polymerase chain reaction - patient monitoring

Schlüsselwörter:

Epstein-Barr-Virus - Lymphoproliferation nach Transplantation (PTLD)„Real-Time”-Polymerase-Kettenreaktion - Patienten-Monitoring

References

• 01 Babcock  G J, Decker  L L, Freeman  R B, Thorley-Lawson  D A. Epstein-Barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients.  J Exp Med. 1999;  190 567-576
• 02 Ehlin-Henriksson  B, Zou  J-Z, Klein  G, Ernberg  I. Epstein-Barr virus genomes are found predominantly in IgA-positive B cells in the blood of healthy carriers.  Int J Cancer. 1999;  83 50-54
• 03 Green  M, Cacciarelli  T V, Mazariegos  G V, Sigurdsson  L, Qu  L, Rowe  D T, Reyes  J. Serial measurement of Epstein-Barr viral load in peripheral blood in pediatric liver transplant recipients during treatment for posttransplant lymphoproliferative disease.  Transplantation. 1998;  66 1641-1644
• 04 Heid  C A, Stevens  J, Livak  K J, Williams  P M. Real time quantitative PCR.  Genome Res . 1996;  6 986-994
• 05 Lucas  K G, Burton  R L, Zimmerman  S E, Wang  J, Cornetta  K G, Robertson  K A, Lee  C H, Emanuel  D J. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation.  Blood. 1998;  91 3654-3661
• 06 Lucas  K G, Pollock  K E, Emanuel  D J. Post-transplant EBV induced lymphoproliferative disorders.  Leuk Lymphoma. 1998;  25 1-8
• 07 Miyashita  E M, Yang  B, Lam  K M, Crawford  D H, Thorley-Lawson  D A. A novel form of Epstein-Barr virus latency in normal B cells in vivo.  Cell. 1995;  80 593-601
• 08 Okano  M. Epstein-Barr virus and its role in the expanding spectrum of human diseases.  Acta Paediatr. 1998;  87 11-18
• 09 Riddler  S A, Breinig  M C, McKnight  J LC. Increased levels of circulating Epstein-Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients.  Blood. 1994;  84 972-984
• 10 Rooney  C M, Loftin  S K, Holladay  M S, Brenner  M K, Krance  R A, Heslop  H. Early identification of Epstein-Barr virus-associated post-transplantation lymphoproliferative disease.  Br J Haematol. 1995;  89 98-103
• 11 Rowe  D T, Qu  L, Reyes  J, Jabbour  N, Yunis  E, Putnam  P, Todo  S, Green  M. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.  J Clin Microbiol. 1997;  35 1612-1615
• 12 Smets  F, Bodeus  M, Goubau  P, Reding  R, Otte  J-B, Buts  J-B, Sokal  E M. Characteristics of Epstein-Barr virus primary infection in pediatric liver transplant recipients.  J Hepatol. 2000;  32 100-105
• 13 Stevens  S JC, Verhoort  M BHJ, van den Brule  A JC, Meenhorst  P L, Meijer  C JLM, Middeldorp  J M. Monitoring of Epstein-Barr virus load in peripheral blood by quantitative competitive PCR.  J Clin Microbiol. 1999;  37 2852-2857
• 14 Wagner  H J, Bein  G, Bitsch  A, Kirchner  H. Detection and quantification of latently infected B lymphocytes in Epstein-Barr virus-seropositive, healthy individuals by polymerase chain reaction.  J Clin Microbiol. 1992;  30 2826-2929