Assessing cytomegalovirus (CMV)-specific immune competence in allo-hematopoietic stem cell transplantation patients who are at risk for developing late CMV disease (beyond day 100 after transplant)
Assessing CMV-specific immune competence in solid organ transplant patients who are at high risk for CMV reactivation posttransplant
Monitoring immune competence in patients post-primary CMV infection after transplant who are at risk for CMV reactivation after the cessation of antiviral prophylaxis
Identifying individuals who are likely to be protected from posttransplant CMV infection and those who are at higher risk of CMV reactivation
The global CD8 T cell immune competence assay is useful for determining over immunosuppression within the CD8 T cell compartment, when used on transplant recipients and patients with autoimmune disorders receiving therapy with immunosuppressant agents
Flow Cytometry
CMV (Cytomegalovirus)
Cytomegalic Inclusion Disease (CMID)
Cytomegalovirus (CMV)
CMID (Cytomegalic Inclusion Disease)
WB Sodium Heparin
Patient must be cytomegalovirus seropositive and have 1 or more of the 5 major histocompatibility complex alleles: HLA A1, A2, B7, B8, or B35 to utilize this assay.
It is important to ascertain the patient's and the donor's cytomegalovirus serostatus, as well as the patient's major histocompatibility complex class I HLA haplotype, before ordering this assay; see 1DIS / Human Leukocyte Antigens (HLA) A-B-C Disease Association Typing Low Resolution, Blood.
Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Collect and package specimen as close to shipping time as possible. Ship specimen overnight in an Ambient Shipping Box-Critical Specimens Only (T668) following the instructions in the box.
It is recommended that specimens arrive within 24 hours of collection.
Samples arriving on the weekend and observed holidays may be canceled.
Ordering physician's name and phone number are required.
Supplies: Ambient Shipping Box-Critical Specimens Only (T668)
Container/Tube: Green top (sodium heparin)
Specimen Volume: 20 mL
Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.
Additional Information: For serial monitoring, it is recommended that specimens are collected at the same time of day.
10 mL
| Gross hemolysis | Reject |
| Gross lipemia | Reject |
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| WB Sodium Heparin | Ambient | 48 hours | GREEN TOP/HEP |
Assessing cytomegalovirus (CMV)-specific immune competence in allo-hematopoietic stem cell transplantation patients who are at risk for developing late CMV disease (beyond day 100 after transplant)
Assessing CMV-specific immune competence in solid organ transplant patients who are at high risk for CMV reactivation posttransplant
Monitoring immune competence in patients post-primary CMV infection after transplant who are at risk for CMV reactivation after the cessation of antiviral prophylaxis
Identifying individuals who are likely to be protected from posttransplant CMV infection and those who are at higher risk of CMV reactivation
The global CD8 T cell immune competence assay is useful for determining over immunosuppression within the CD8 T cell compartment, when used on transplant recipients and patients with autoimmune disorders receiving therapy with immunosuppressant agents
Cytomegalovirus (CMV), a double-stranded DNA virus, belongs to the Herpesviridae family of viruses and is structurally similar to other herpes viruses. Although many human strains of CMV exist, there is little genetic homology between human CMV and CMV of other species. The reported seroprevalence rates of CMV range from 40% to 100% in the general population. In the urban United States, the seroprevalence of CMV has been reported to be 60% to 70%.(1) However, data from Mayo Clinic's laboratory indicate that the seroprevalence in the Midwestern US population is closer to 30% (unpublished observations).
Once CMV infection occurs, the virus spreads hematogenously to almost every organ. After acute infection, the virus enters a latent phase. Activation from this phase can be seen after acute illness, immunosuppression in allogeneic hematopoietic stem cell transplantation (HSCT) or solid organ transplantation, or use of chemotherapy agents. CMV infection or reactivation has been implicated in allograft rejection in renal(2) and cardiac transplantation.(1) In cardiac transplants, CMV infection has been shown to contribute to accelerated development of transplant atherosclerosis (cardiac allograft vasculopathy). CMV remains a significant cause of morbidity and mortality after HSCT. Of allogeneic HSCT patients who are CMV-seropositive, 60% to 70% will experience reactivation and, without ganciclovir or other preemptive therapy, 20% to 30% will develop end-organ disease.(3)
CD8 T cells play a critical role in viral immunity, and CD8 T-cell effector functions include cytotoxicity and cytokine production. Cytotoxicity occurs after CD8 T-cell activation, causing target cell apoptosis. Cytotoxic T-cell responses mediate killing of target cells via 2 major pathways, granule-dependent (perforin and granzymes) and granule-independent (Fas and Fas ligand [FasL]) mechanisms. The granule-dependent pathway does not require the de novo synthesis of proteins by effector CD8 T cells, but rather it utilizes preformed lytic granules located within the cytoplasm. Among the proteins in these preformed lytic granules are the lysosomal-associated membrane proteins (LAMP), including LAMP-1 (CD107a) and LAMP-2 (CD107b). These proteins are not normally found on the surface of T cells. Degranulation of activated CD8 T cells occurs rapidly after T-cell receptor (TCR) stimulation, exposing CD107a and CD107b. The cytokines produced by activated T cells include interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 1 alpha (MIP-1 alpha), macrophage inflammatory protein 1 beta (MIP-1 beta), and interleukin-2 (IL-2). Several studies have shown the importance of cytotoxic T-cell responses to CMV in conferring protection from subsequent CMV disease.
Antiviral drugs have helped reduce the incidence of early CMV infection, and acyclovir and ganciclovir have been the mainstay of antiviral treatment for a number of years, although these drugs have poor bioavailability.(4) The development of the new antiviral drugs valacyclovir and valganciclovir (by the addition of a valine ester) has increased the bioavailability of these drugs by 10-fold.(4) There is some data to suggest valganciclovir prophylaxis may be considered for HSCT patients who are at high risk for infection and disease, though there is a need for further study in this area.(5)
Two main strategies have been used for the prevention of early CMV infection and disease in CMV-seropositive patients and in seronegative recipients who receive a seropositive graft-preemptive therapy:
-Patient monitoring for CMV infection and treatment only when CMV viremia is present.
-Prophylactic management-where all patients receive treatment after transplantation with the goal of preventing CMV disease.(5) The disadvantage of prophylactic therapy is that it requires monitoring for myelosuppression and infections-side effects of antiviral drug therapy. Despite this disadvantage, there are several reasons to consider prophylaxis, including the fact that the incidence of recurrent infections after treatment is 30% to 40%,(5) patients receiving preemptive therapy have a 5% CMV disease break-through, and prophylaxis reduces the risk of viral reactivation.
Late CMV infection occurs 3 months after transplantation and is now recognized as a significant cause of morbidity after allogeneic HSCT.(6) These complications usually occur in the setting of continued immunosuppression for chronic graft-versus-host disease (GVHD). The clinical manifestations of late CMV disease differ slightly from those seen early after transplantation. Within the first 100 days after HSCT, almost all patients with CMV disease have CMV pneumonia or gastrointestinal disease. In late CMV disease, the more unusual manifestations of CMV infection (eg, CMV retinitis, CMV-associated bone marrow failure, or CMV encephalitis) tend to occur.(7) These late manifestations occur in a setting of continued CMV-specific T-cell immunodeficiency. Therefore, it is necessary to monitor CMV-specific CD8 T-cell responses, in addition to viral load, to effectively identify patients at higher risk of CMV disease.
It has been shown that ganciclovir may delay the recovery of the protective CMV-specific T-cell response, which may contribute to the occurrence of late CMV disease.(8) The use of ganciclovir as early treatment after detection of CMV in blood or other body fluid and as a prophylaxis for CMV infection in bone marrow transplant (BMT) and heart transplant recipients has dramatically reduced the incidence of CMV in these immunocompromised hosts. Yet, early treatment and prophylaxis have not been uniformly successful, with up to 15% of BMT recipients developing CMV disease after discontinuation of antiviral therapy. Similarly, patients undergoing lung transplantation have been shown to be only transiently protected with antiviral agents. These data suggest that the CMV-specific responses necessary for protection may not recover during the time the host is receiving antiviral therapy.
Ganciclovir exerts its antiviral effects at the stage of viral DNA replication and, therefore, in the presence of the drug, infected cells may express some of the immediate early and early gene products, but not the full complement of CMV genes required for replication and virion formation. In latently infected CMV-seropositive individuals, the human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocyte response to CMV is predominantly specific for epitopes derived from structural virion proteins. Therefore, in patients receiving ganciclovir, the viral antigens may be inadequate to activate host T-cell responses, resulting in the failure to reconstitute CMV-specific immunity. In fact, a prospective, randomized placebo-controlled study of ganciclovir prophylaxis revealed that when ganciclovir therapy is discontinued, a larger fraction of patients (who received the drug) are deficient in CMV-specific T-cell immunity at day 100 than in the placebo group.(8) That study also showed that bone marrow donor serology has an important influence on the early detection of virus-specific T-cell responses.(8) Not all medical centers use ganciclovir for prophylaxis; some use acyclovir and the same findings may apply in this case as well.
In a more recent study, it was shown that impaired CD8 function was associated with the use of high-dose steroids, bone marrow as a source of stem cells, and CD8 T-cell lymphopenia.(3) In the absence of high-dose steroids, low-level subclinical CMV antigenemia was found to stimulate both CD4 and CD8 functional recovery in recipients of ganciclovir prophylaxis, suggesting that subclinical CMV reactivation while on antiviral therapy can be a potent stimulator of T-cell function.(3) Regardless of antiviral therapy, immunologic reconstitution remains the key element in protection from late-onset CMV disease.
This test assesses the number of CMV-specific CD8 T cells and their function (activation via production of the cytokine IFN-gamma and cytotoxic potential via CD107a and CD107b as markers of degranulation) using a panel of 5 major histocompatibility complex (MHC) class I alleles (HLA A1, A2, B7, B8, and B35) along with their respective immunodominant CMV epitopes. This 3-part assay allows a comprehensive assessment of CMV-specific CD8 T cell immunity and, when combined with evaluation of viremia using molecular analyses, provides a more accurate picture of the nature of CMV reactivation and the corresponding immune response than evaluating viremia alone.
Assessment of Global CD8 T-Cell Function:
CD8 T cell activation occurs either through the TCR-peptide-MHC or by use of a mitogen (eg, phorbol myristate acetate and the calcium ionophore ionomycin). Mitogen-mediated activation is antigen nonspecific.
Impairment of global CD8 T cell activation (due to inherent cellular immunodeficiency or as a consequence of immunosuppression by therapeutic agents) results in reduced production of interferon-gamma and other cytokines, reduced cytotoxic function, and increased risk for developing infectious complications. Agents associated with over-immunosuppression include the calcineurin inhibitors (eg, cyclosporine A, FK506 [Prograf/tacrolimus], and rapamycin [sirolimus]), antimetabolites (eg, mycophenolate mofetil), and thymoglobulin.
The incorporation of global CD8 T cell function in this assay is helpful in determining if the lack of CMV-specific (antigen-specific) response is due to a global impairment in CD8 T cell function, due to immunosuppression or other causes, or whether the lack of CMV CD8 T cell immunity is unrelated to overall CD8 T cell function.
The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon, with no change between noon and afternoon. Natural killer cell counts, on the other hand, are constant throughout the day.(9) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(10-12) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(10) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening(12), and during summer compared to winter.(14) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.
Total CD3 T cells: 884-5830 x 10(3)/mL
Total CD8 T cells: 168-1847 x 10(3)/mL
Total CMV CD8 T cells: 0-115 x 10(3)/mL
The adult reference values were determined for healthy adult controls ages 20 to 80 years (n=94), for HLA A1, A2, B7, B8, and B35 alleles.
Reference values for cytomegalovirus (CMV) specific T cells that are functional (interferon-gamma+, IFN-g+) and have cytotoxic activity (CD107a and CD107b expression, CD107 a/b+):
Total CMV CD8 T-cells IFN-g: 0.028-52.200 x 10(3)/mL
Total CMV CD8 T-cells CD107a/b: 0.252-50.760 x 10(3)/mL
The 95% confidence interval reference values were determined from 102 healthy adult donors:
Interferon-gamma (IFN-gamma) expression (as % CD8 T cells): 10.3-56.0%
CD107a/b expression (as % CD8 T cells): 8.5-49.1%
The reference values were developed for each of the following 4 major histocompatibility complex class I alleles: A1, A2, B7, and B8 (n=45). We were unable to develop ranges for the B35 allele due to a lack of matching donors. The data is expressed as the absolute number of CMV-specific CD8 T cells that are IFN-gamma+ or CD107a/b+.
For allogeneic hematopoietic stem cell transplantation (HSCT) and solid organ transplant patients who are cytomegalovirus (CMV)-seropositive and at risk for CMV reactivation, posttransplant results should be compared to pretransplant (preconditioning/baseline) results.
The report includes absolute CD3 and CD8 T-cell counts as well as a derived CMV-specific CD8 T-cell count (derived from CD3 and CD8 T-cell counts). The absolute count of CMV-CD8 T cells that are activated and have cytotoxic function in response to specific CMV peptide is also provided. The data from the 3 components of this assay should be interpreted together and not individually.
In the setting of CMV viremia, frequent monitoring of CMV-immune competence helps define the evolution of the CMV-immune response. In this clinical context, CMV-immune competence should be measured as frequently as viral load to determine correlation between the 2 parameters. CMV-specific CD8 T-cell counts may show a decline in numbers over time in the absence of active infection or antigenemia.
The absence of CMV-specific CD8 T cells may be a risk factor in the immune-compromised or immune-incompetent transplant patient, who is at risk for CMV reactivation. The presence of CMV-specific CD8 T cells may not be protective in itself. If the CMV-specific CD8 T cells do not show appropriate cytotoxic function (due to the fact that they recognize CMV epitopes that do not effectively induce a cytotoxic response), these patients may be at higher risk of an inadequate immune response to CMV infection.
While the reference values provide a guideline of CMV-specific CD8 T-cell numbers and function in a cohort of healthy individuals, baseline (pretransplant/preconditioning) assessments should be taken into consideration when determining CMV-specific immune competence posttransplant. Correlation between data from multiple post-transplant specimens (if available) and the presence or absence of viremia (or active CMV disease) also are useful in the interpretation of results.
CD8 T cell counts are elevated when the immune system is initially reconstituted post-HSCT, and the CD4 to CD8 ratio can be inverted for about 12 months post-HSCT.
Interferon-gamma (IFN-gamma) and CD107a/b expression below the defined reference range are consistent with a global impairment in CD8 T cell function, most likely due to over-immunosuppression. IFN-gamma and CD107a/b levels greater than the defined reference range are unlikely to have any clinical significance.
The assay is specific for 5 major histocompatibility complex (MHC) class I alleles: HLA A1, A2, B7, B8, and B35, which cover about 70% of the White population and approximately 40% of the Asian population. It is important to ascertain the patient's and the donor's cytomegalovirus (CMV) serostatus, as well as the patient's MHC class I HLA haplotype, before ordering this assay; see 1DIS / Human Leukocyte Antigens (HLA) A-B-C Disease Association Typing Low Resolution, Blood.
Serial assessment of CMV-immune competence posttransplant in the setting of viremia is recommended to establish CMV-specific immune response trends. In the absence of viremia, CMV-immune competence assessment should include a day 100 or later posttransplant specimen or a specimen after the cessation of antiviral prophylaxis in solid organ transplantation patients.
A single measurement of CMV-immune competence is not sufficient to determine immune response to CMV; serial measurements are essential to proper interpretation of the results. Appropriate interpretation of results requires a pretransplant measurement of CMV-specific immunity to assess baseline immune competence.
Since healthy, asymptomatic individuals may not have detectable CMV-specific CD8 T cells, for these individuals, the absence of CMV-specific CD8 T cells alone may not be a risk factor.
The global CD8 T cell immune competence assessment is specific only for CD8 T cells; it does not provide information for overall T cell competence. Further studies are needed to determine if, within the reference range, certain levels of interferon-gamma and CD107a/b expression confer greater or lesser degrees of risk for infectious disease.
Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See data under Clinical Information.
1. Melnick JL, Adam E, Debakey ME: Cytomegalovirus and atherosclerosis. Eur Heart J. 1993 Dec;14 Suppl K:30-38
2. von Willebrand E, Petterson E, Ahnonen J, et al: CMV infection, class II antigen expression, and human kidney allograft rejection. Transplantation. 1986 Oct;42(4):364-367
3. Hakki M, Riddell SR, Storek J, et al: Immune reconstitution to CMV after allogeneic hematopoietic stem cell transplantation: impact of host factors, drug therapy, and subclinical reactivation. Blood. 2003 Oct 15;102(8):3060-3067
4. Baden LR: Pharmacokinetics of valganciclovir in HSCT patients with gastrointestinal GVHD. Biol Blood Marrow Transplant. 2005;15(2):5-7
5. Bachier C, Shaughnessy P, Wall D, et al: Valganciclovir for the prophylaxis of early cytomegalovirus (CMV) infection after allogeneic stem cell transplantation,. Biol Blood Marrow Transplant. 2005;11(2):90-91
6. Annaloro C, Serpenti F, Saporiti G, et al: Viral Infections in HSCT: Detection, Monitoring, Clinical Management, and Immunologic Implications. Front Immunol. 2021 Jan 20;11:569381. doi:10.3389/fimmu.2020.569381
7. Boeckh M: Prevention of late CMV infection in HSCT. Biol Blood Marrow Transplant. 2005;15(2):9-11
8. Li CR, Greenberg PD, Gilbert MJ, et al: Recovery of HLA-restricted CMV-specific T cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis. Blood. 1994 Apr 1;83(7):1971-1979
9. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment.15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052
10. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood. 2009 May 21;113(21):5134-5143
11. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep. 2007 Apr;30(4):401-411
12. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Pyschosom Med. Jan-Feb 1997;59:42-50
13. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J Acquir Immune Defic Syndr (1988). 1990;3(2):144-151
14. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion. 1994 June;34(6):512-516
Assessment of cytomegalovirus (CMV)-immune competence measures 3 different components of the immune response: 1) numerical analysis of the CMV-specific CD8 T-cell population (enumeration), which provides absolute counts of the antigen-specific CD8 T cells, 2) functional, and 3) cytotoxic analysis of the CMV-specific CD8 T cells (functionality and cytotoxic activity).
Enumeration:
Enumeration of CD8 T cells is performed on whole blood using Beckman Coulter MHC class I tetramers, specific for 5 major histocompatibility complex (MHC) class I alleles-A1, A2, B7, B8, and B35.
Panel I consists of whole blood stained with CD3 PE-Cy5 (PhycoErythrin-Cy5), CD8 fluorescein isothiocyanate (FITC), and CD4 PhycoErythrin (PE) followed by a 20-minute incubation at ambient temperature in the dark. The sample is then treated with the tetramer lyse reagent for 10 minutes at ambient temperature in the dark. The flow count bead solution is then thoroughly mixed and added at a volume exactly equal to the volume of whole blood added. After the addition of beads, the sample is analyzed on a BD FACS Canto flow cytometer within 1 hour and exactly 3000 bead events are counted.(Package insert: Flow-Count Fluorospheres. Beckman Coulter, Inc.; 12/2018)
The number of CD3 and CD8 cells per mcL of whole blood is counted using the following formula:
No. of CD3+cells/mcL = No. of events in CD3+ gate X concentration of beads/mcL*
No. of events in the bead capture gate (3000)
Multiply by 1000 to obtain results as cells x 10(3)/mL
No. of CD8+cells/mcL = No. of events in CD8+ gate X concentration of beads/mcL*
No. of events in the bead capture gate (3000)
Multiply by 1000 to obtain results as cells x 10(3)/mL
*This number is provided by the manufacturer for each lot.
In Panel II, whole blood is stained with CD3 (PE-Cy5), CD8 (FITC) and incubated with the relevant MHC class I tetramer-PE for 20 minutes at ambient temperature in the dark. The whole blood is then lysed using the tetramer lyse reagent and washed with the wash buffer. The sample is then run on the flow cytometer and the CMV-specific CD8 T cells are analyzed as a percentage of the total CD8+ T cells. The absolute count of the CMV-specific CD8 T cells is obtained using the following formula:
Absolute count of CMV-specific CD8 T cells =
% of CMV specific CD8 T cells X absolute count of CD8 T cells/mcL**
100
Multiply by 1000 to obtain results as cells x 10(3)/mL
**This number will be obtained from Panel I.
The final absolute counts for CD3 T cells, CD8 T cells, and CMV-specific CD8 T cells are expressed as cells/mL of whole blood.
Functionality (Intracellular Interferon-gamma: IFN-gamma) and Cytotoxic Activity (Surface CD107a/b):
Peripheral blood mononuclear cells (PBMC) are isolated from the same sample of whole blood used for enumeration. PBMC are stimulated with human leukocyte antigen (HLA) allele-specific CMV peptides and costimulatory molecules (CD28/CD49d) in a polypropylene tube. Antihuman CD107a/b conjugated with FITC is added along with the peptides to capture the transient expression of the CD107a and CD107b, which are markers for cytotoxic activity. A mixture of Brefeldin A (BFA) and monensin is also added during the stimulation to facilitate the intracellular accumulation of IFN-gamma, which is detected using the antihuman IFN-gamma antibody conjugated with allophycocyanin (APC). A similar tube is prepared for the patient sample with the absence of exogenous CMV peptide and this tube serves as the unstimulated control (back-ground stimulation). Thus, there are 2 tubes for every HLA allele. After peptide stimulation for 5 hours, EDTA is added to the sample to arrest activation and to remove adherent cells from the activation tube. Antihuman CD8 antibody and the relevant MCH-class I tetramer are added and incubated at ambient temperature for 20 minutes in the dark. This step is followed by a simultaneous lysis and fixation of the cells to prepare them for permeabilization. Cells are then washed and permeabilized with BD FACS Perm II solution. The antihuman IFN-gamma antibody is added, and the sample is incubated for 30 minutes at ambient temperature in the dark. Finally, the cells are washed and analyzed by flow cytometry. The delta percent (stimulation in the presence of CMV-specific peptide stimulation in the absence of specific peptide) CMV-specific CD8 T cells expressing IFN-gamma and CD107a/b is used to calculate the absolute count of activated and functionally cytotoxic CMV-specific CD8 T cells.(Functionality: IFN-gamma assay, Unpublished Mayo method; Cytotoxic Activity: Betts MR, Brenchley JM, Price DA, et al: Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods. 2003 Oct 1;281 (102):65-78; Betts MR, Price DA, Brenchley JM, et al: The functional profile of primary human antiviral CD8 T cell effector activity is dictated by cognate peptide concentration. J Immunol. 2004 May 15;172(10):6407-6417)
Global CD8 T-Cell Immune Competence:
Peripheral blood mononuclear cells (PBMC), which contain CD8 T cells, are stimulated with a mixture of phorbol myristate acetate (PMA) and ionomycin, and with stimulatory signals derived using antibodies against the costimulatory molecules CD28/CD49d. The cells are simultaneously treated with a mixture of BFA and monensin, which blocks extracellular secretion of IFN-gamma, enabling intracellular retention and detection of the protein. PBMCs that have not been stimulated are used as a control to determine the background levels of IFN-gamma and CD107a and CD107b. The cells are analyzed on the BD FACS Canto flow cytometer and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma and CD107a and CD107b signals are determined within the "gated" CD8 T cell population. Global CD8 T cell immune competence is measured by the amount of IFN-gamma produced (CD8 T-cell functional activity) and surface expression of CD107a/b (cytotoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)
Quantitive Lymphocyte Subsets: T, B, and Natural Killer:
The T, B, and natural killer NK-cell surface marker assay uses monoclonal antibodies to identify the various membrane antigens, and flow cytometry to enumerate the number of cells expressing these differentiation antigens. CD14 is used to exclude monocytes, thereby improving accuracy and enhancing the purity of the lymphocyte population. The results are reported as the percent of lymphocytes that are total T cells (CD3+), CD3+CD4+ T cells, CD3+CD8+ T cells, natural killer (CD16+56+, CD3-), and B-lymphocytes (CD19+), and the absolute number of each cell type per mL of blood. The assay is a 7-color no-wash procedure and the absolute counts are calculated from internal bead standards. In addition, the total lymphocyte count and the CD4:CD8 ratio are reported.(Hoffman RA, Kung PC, Hansen WP, Goedstien G: Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc Natl Acad Sci USA. 1980 Aug:77(8):4914-4917; Mandy FF, Nicholson JK, McDougal JS; CDC. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR Recomm Rep. 2003 Jan 31;52(RR-2):1-13)
Monday through Friday
This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.
86356 x 6
86359
86352
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| CMVC8 | CMV CD8 T-Cell QN by Flow Cytometry | 95184-8 |
| Result Id | Test Result Name |
Result LOINC Value
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
|---|---|---|
| 28157 | CMV CD8 T cells CD107 A1 | 95191-3 |
| 28158 | CMV CD8 T cells CD107 A2 | 95190-5 |
| 28159 | CMV CD8 T cells CD107 B7 | 95189-7 |
| 28160 | CMV CD8 T cells CD107 B8 | 95188-9 |
| 28161 | CMV CD8 T cells CD107 B35 | 95187-1 |
| 30694 | CD107a/b expression | 95203-6 |
| 28155 | CMV CD8 T cells IFN-g B35 | 95193-9 |
| 28151 | CMV CD8 T cells IFN-g A1 | 95197-0 |
| 28152 | CMV CD8 T cells IFN-g A2 | 95196-2 |
| 28153 | CMV CD8 T cells IFN-g B7 | 95195-4 |
| 28154 | CMV CD8 T cells IFN-g B8 | 95194-7 |
| 30693 | IFN-gamma expression | 95204-4 |
| 28162 | Total CMV CD8 T cells CD107a/b | 95186-3 |
| 28156 | Total CMV CD8 T cells IFN-g | 95192-1 |
| 28163 | Interpretation | 69052-9 |
| 28145 | CMV CD8 T cells A1 | 95185-5 |
| 28146 | CMV CD8 T cells A2 | 95202-8 |
| 28147 | CMV CD8 T cells B7 | 95201-0 |
| 28148 | CMV CD8 T cells B8 | 95200-2 |
| 28149 | CMV CD8 T cells B35 | 95199-6 |
| 28150 | Total CMV CD8 T cells | 95198-8 |
| 28143 | Total CD3 T cells | 8122-4 |
| 28144 | Total CD8 T cells | 14135-8 |