Skip to main content

EBV inSIGHT™ T Cell Immunity Testing

Test Code: 33217

EBV Epstein–Barr virus

Cell Function Testing requires special attention to specimen collection and shipping in order to ensure the integrity of the sample.  Please watch Viracor’s video for complete specimen collection and shipping instructions.

WATCH VIDEO

Expand All Collapse All
Clinical and Procedure
Clinical Utility

The Epstein-Barr Virus (EBV) inSIGHT™ T Cell Immunity test measures the strength of T cell responses to Epstein-Barr (EBV) specific antigens , BZLF1 and EBNA3. It evaluates and reports the activity of CD4 and CD8 T cell responses independently.

Background

Epstein-Barr Virus is a member of the herpes virus family, also known as human herpesvirus 4 (HHV-4). Approximately 90-95% of people have been infected with the virus by the time they reach adulthood. The virus is predominantly spread through bodily fluids, like saliva.

A primary infection with EBV can cause infectious mononucleosis (mono), as well as, other illnesses. EBV can affect patients with weakened immune systems and is a common risk factor in patients following solid organ (SOT) or hematopoietic stem cell transplant (HSCT). In the transplant setting, post-transplant lymphoproliferative disorders (PTLD) can arise as a consequence of the immunosuppression used and the loss of T cells that typically control reactivation of EBV. Rapidly rising EBV viral loads can indicate patients at risk for PTLD, a critical cause of morbidity and mortality in , SOT and HSCT patients. The overall incidence of PTLD ranges from 1 – 20% depending on the type of transplant. There is evidence which suggests there is higher incidence associated with multi-organ transplants, high intensity immunosuppression and in EBV naïve hosts.1 For this reason PTLD is a serious concern in children, due to the lower EBV infection rate at an early age.

Often, reduction in immunosuppression (RIS) is used to allow immune recovery to occur as part of treatment for reactivated EBV to help prevent progression to PTLD. When RIS is ineffective or fails to resolve the PTLD, EBV specific immune cell therapy or Rituximab (Brand Name: RITUXAN®), or chemotherapy have been found to be effective treatment for PTLD.2

Cell mediated immunity is a component of EBV immune control. Monitoring EBV-specific T cell responses utilizing intracellular cytokine staining may aid in the detection of patients with an increased risk of EBV DNAemia and may be useful for identifying patients that could benefit from reduced immunosuppression if their viral load should start to increase. T cell immunity testing may be useful for monitoring immunity to EBV pre- and post-transplant and may be useful when used with monitoring low level viral load while reducing -immunosuppression (RIS), or in administration of preemptive therapy in tandem with RIS.

This assay was developed to include reporting for Interferon-γ (IFN-gamma) producing T cells, as well as polyfunctional CD4 and CD8 T cells. The presence of virus specific polyfunctional T cells has been found to be an indicator of highly functional T cells. The Polyfunctional Index (PFI) represents the proportion of IFN-gamma cells that also express Tumor Necrosis Factor alpha and/or Interleukin.2The lytic phase antigen (BZLF1) and latent phase antigens (EBNA3) were selected to detect CD8 T cell responses to both phases of a virus infection. An EBV cell lysate was selected to contain multiple proteins to better stimulate a CD4 T cell response (which is often reported to be much lower than the CD8 counterparts). Research has shown that healthy individuals who carry EBV mount a significant polyfunctional T cell response to EBV lytic and latent proteins; conversely patients with PTLD have shown impaired T cell immunity and a low proportion of polyfunctional T Cells. Additionally, in the case of EBV induced PTLD, research has also found that highly functional EBV Specific CD8 T Cells are required to control an infection.1,2,3,4

“…Results inferred that EBV-specific PFCs were functionally superior to their single functional counterparts because they have greater production of cytokines and degranulation capacity.4

Learn more about the assay design here.

Quantitative EBV DNA PCR can also be used to monitor the course of the disease, aid in the early diagnosis of PTLD , and monitor response to treatment.

Procedure

Whole blood is stimulated with the positive control SEB, EBV specific antigens (EBV lysate, BZLF1 and EBNA 3) or left unstimulated as a negative control and incubated to 37ºC. During the incubation Brefeldin A is added causing the cytokines (Interferon – gamma (IFN-γ) Tumor Necrosis Factor – alpha (TNF-α) and/or Interleukin 2 (IL-2)) to be retained inside the cell. Following the stimulation phase, cells are recovered, stained for cellular markers (CD3, CD4, CD8, CD69), the cytokines above and analyzed by flow cytometry. This test was developed and its performance characteristics determined by Eurofins Viracor. This test has not been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.

Specificity

Test will measure the % Interferon-gamma and polyfunctional index responses to the EBV lytic antigen BZLF1, EBV latent antigens EBNA 3A/3C and an EBV infected cell lysate. Polyfunctional index is the proportion of interferon -gamma expressing cells that also express Tumor necrosis factor-alpha and/or interleukin-2. All EBV seronegative donors tested did not have T cell responses to any of the EBV antigens.

Turnaround Time

3-4 business days from receipt of specimen.

Specimen Information
Specimen Type Order Code CPT Code NY Approved Volume Assay Range Special Instructions
whole blood 33217 86352 x 4 Yes

10 mL

Quantitative. The percent of CD4 and CD8 cells that are activated after stimulation with either EBV antigens or mitogen are reported quantitatively.

 

  • Collect 10 mL (preferred) whole blood in a sodium heparin tube. Tube must be at least ¾ full (7.5 mL) to maintain proper ratio of blood to anticoagulant. Please see table below specimen information table for other sodium heparin tubes that can also be accepted.
  • Blood must be drawn Monday through Friday after 7:00 AM CST and shipped the same day.
  • DO NOT SHIP on days when a holiday follows within 2 days of the shipping day.
  • Ship samples priority overnight Monday through Friday, at ambient temperature on the same day as collection.
  • Please contact Client Services at 1-800-305-5198 if it is your first time sending Viracor a live cell test. Client Services will assist in setting up live cell shippers with you, or your courier.

For Pediatric Patients

  • Collect whole blood in a sodium heparin tube. Minimum volume required is 4 mL. Tube must be at least ¾ full to maintain proper ratio of blood to anticoagulant. Please see table below specimen information table for other sodium heparin tubes that can also be accepted.
  • Blood must be drawn Monday through Friday after 7:00 AM CST and shipped the same day.
  • DO NOT SHIP on days when a holiday follows within 2 days of the shipping day.
  • Ship samples priority overnight Monday through Friday, at ambient temperature on the same day as collection.
  • Please contact Client Services at 1-800-305-5198 if it is your first time sending Viracor a live cell test.

Client Services will assist in setting up live cell shippers with you, or your courier.

ADULT Acceptable Alternate Sodium Heparin Tubes

Sodium Heparin Tube Volume Minimum Volume
10 mL 7.5 mL
6.0 mL 4.5 mL
4.0 mL 4.0 mL

PEDIATRIC Acceptable Alternate Sodium Heparin Tubes

Sodium Heparin
Tube Volume
Quantity of
Tubes
Minimum Volume
within each Tube
Tube Fill Volume
based on Min. Volume
10.0 mL 1 7.5 mL 3/4 full
6.0 mL 1 4.5 mL 3/4 full
4.0 mL 1 4.0 mL completely full
2.0 mL 2  4.0 mL completely full

 

Shipping

Ship Monday through Friday. All specimens must be labeled with patient's name and collection date. A Eurofins Viracor test requisition form must accompany each specimen. Ship specimens FedEx Priority Overnight® to: Eurofins Viracor Laboratories, 18000 W 99th St., Lenexa, KS 66219.

Collection time requirements for EBV T cell samples

Time Zone Earliest collection time for same day shipping
Eastern 8:00 a.m.
Central 7:00 a.m.
Mountain 6:00 a.m.
Pacific 5:00 a.m.

 

Causes for Rejection

• Whole blood outside stability (more than 32 hours after collection at set up)
• Whole blood received cold or frozen
• Tubes received less than 3/4 full
• Specimens received in lithium heparin, ACD tubes or EDTA anticoagulants

Disclaimer

Specimens are approved for testing in New York only when indicated in the Specimen Information field above. The CPT codes provided are based on Eurofins Viracor’s interpretation of the American Medical Association's Current Procedural Terminology (CPT) codes and are provided for informational purposes only. CPT coding is the sole responsibility of the billing party. Questions regarding coding should be addressed to your local Medicare carrier. Eurofins Viracor assumes no responsibility for billing errors due to reliance on the CPT codes illustrated in this material.

 

References

1. Guppy, A. E., Rawlings, E., Madrigal, J. A., Amlot, P. L., & Barber, L. D. (2007). A quantitative assay for Epstein-Barr Virus-specific immunity shows interferon-gamma producing CD8+ T cells increase during immunosuppression reduction to treat posttransplant lymphoproliferative disease. Transplantation, 84(11), 1534–1539. https://doi.org/10.1097/01.tp.0000290232.65830.e7


2. Elstrom, R. L., Andreadis, C., Aqui, N. A., Ahya, V. N., Bloom, R. D., Brozena, S. C., Olthoff, K. M., Schuster, S. J., Nasta, S. D., Stadtmauer, E. A., & Tsai, D. E. (2006). Treatment of PTLD with rituximab or chemotherapy. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 6(3), 569–576. https://doi.org/10.1111/j.1600-6143.2005.01211.x


3. Lam JKP, Hui KF, Ning RJ, Xu XQ, Chan KH and Chiang AKS (2018) Emergence of CD4+ and CD8+ Polyfunctional T Cell Responses Against Immunodominant Lytic and Latent EBV Antigens in Children With Primary EBV Infection. Front. Microbiol. 9:416. doi: 10.3389/fmicb.2018.00416


4. Ning, R. J., Xu, X. Q., Chan, K. H., & Chiang, A. K. (2011). Long-term carriers generate Epstein-Barr virus (EBV)-specific CD4(+) and CD8(+) polyfunctional T-cell responses which show immunodominance hierarchies of EBV proteins. Immunology, 134(2), 161–171. https://doi.org/10.1111/j.1365-2567.2011.03476.


5. Taylor, G. S., Long, H. M., Brooks, J. M., Rickinson, A. B., & Hislop, A. D. (2015). The immunology of Epstein-Barr virus-induced disease. Annual review of immunology, 33, 787–821. https://doi.org/10.1146/annurev-immunol-032414-112326


6. Gil L, Styczyński J, Komarnicki M. Strategy of pre-emptive management of Epstein-Barr virus post-transplant lymphoproliferative disorder after stem cell transplantation: results of European transplant centers survey. Contemp Oncol (Pozn). 2012;16(4):338-40. doi: 10.5114/wo.2012.30064. Epub 2012 Sep 29. PMID: 23788905; PMCID: PMC3687436.


7. Can Liu, Ren Lin, Yu Wang, Fen Huang, Zhiping Fan, Shen Zhang, Yajing Xu, Ting Yang, Li Xuan, Na Xu, Jieyu Ye, Jing Sun, Xiaojun Huang, Qifa Liu; Preemptive Therapy Based on the Load and Time of EBV-Emia for Preventing EBV Related PTLD after Haploidentical Hematopoietic Stem Cell Transplantation: A Prospective Multicenter Study. Blood 2019; 134 (Supplement_1): 5693. doi: https://doi.org/10.1182/blood-2019-132106

Back to top