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ImmuKnow®*

Test Code: 9000
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Clinical Utility

ImmuKnow is an immune cell function assay that detects cell-mediated immunity in an immunosuppressed population. The assay detects cell mediated immunity by measuring the concentration of ATP from CD4 cells following stimulation.

About ImmuKnow

CMI is mediated by T lymphocytes in the acquired immune response, monocytes in the innate immune response, and their effector interactions with other cells in the immune system. The measurement of CMI is valuable in a variety of applications including transplantation. Traditionally, in vivo methods such as the skin test have been used to measure CMI, but there is a need for in vitro methods to rapidly assess CMI. Current in vitro methods for assessing CMI include measuring cell activation signals, lymphoproliferation, cytotoxicity, and cytokine production. Most methods currently used for investigating immune function focus on lymphocytes.1

The patented ImmuKnow technology combines cell stimulation, cell selection, and quantification of metabolic markers (e.g., ATP) to measure the global cell-mediated immune response of CD4 cells.2,3 ImmuKnow measures the early response to stimulation by detecting intracellular ATP synthesis in CD4 cells selected from whole blood by monoclonal antibody coated magnetic beads.4,5 The amount of ATP present in stimulated whole blood specimens is a measure of immune cell activity.6,7,8 Because the CD4 lymphocytes orchestrate CMI responses through immunoregulatory signaling, the measurement of CD4 activation reflects the degree of immune cell function.1.9 As response to immunosuppressive therapy varies among individuals, assessment of a patient’s immune cell function may provide useful information to the clinician in the course of individual patient management.10,11,12

Procedure

ImmuKnow technology combines cell stimulation, cell selection, and quantification of metabolic markers (ATP) to measure cell-mediated immunity. ImmuKnow measures early response to stimulation by detecting intracellular ATP synthesis in CD4 cells selected from blood by monoclonal antibody-coated magnetic beads. The amount of ATP present in stimulated blood specimens is a measure of lymphocyte activity. Since the CD4 lymphocytes orchestrate cell-mediated immunity responses through immunoregulatory signaling, the measurement of CD4 activation reflects the degree of immune function. This test has been cleared or approved for diagnostic use by the U.S. Food and Drug Administration.

Specificity

Sensitivity: The limit of ATP detection of ImmuKnow is 1 ng/mL.

36 – 48 hours from receipt of specimen.

Specimen Type Order Code CPT Code NY Approved Volume Assay Range Special Instructions
whole blood 9016 86352 Yes 2 mL (min. 0.5 mL)

Low immune response: ATP level ≤225 ng/mL.

Moderate immune response: ATP level 226-524 ng/mL.

Strong immune response: ATP level ≥525 ng/mL.

  • 2 to 3 mL whole blood collected and shipped in original sodium heparin (green top) tube.
  • Ship at ambient temperature, priority overnight Monday through Friday.
  • Specimen must be drawn after 4:00 A.M. (CST) and shipped on the same day as collection to meet the 30 hour requirement.

The ATP level ranges for ImmuKnow were established by testing 155 apparently healthy adults and 127 transplant recipients. A cumulative frequency of differences was used to select the ATP levels that give the best balance of results between immunosuppressed and non-immunosuppressed individuals. The cutoffs for the ATP level ranges are 225 and 525 ng/mL.

Interpretation of Results

Low immune response: ATP level ≤225 ng/mL.

Moderate immune response: ATP level 226-524 ng/mL.

Strong immune response: ATP level ≥525 ng/mL.

Results of the ImmuKnow assay should be used in conjunction with clinical presentation, medical history, and other clinical indicators when establishing the immune status of a patient. This is a qualitative assay; therefore, the result does not quantify the level of immunosuppression.

Shipping

Ship Monday through Friday. Friday shipments must be labeled for Saturday delivery or specimens will not meet the 30 hour time requirement. All specimens must be labeled with patient's name, collection date and time. A Viracor Eurofins test requisition form must accompany each specimen. Multiple tests can be run on one specimen. Ship specimens FedEx Priority Overnight® to: Viracor Eurofins, 1001 NW Technology Drive, Lee's Summit, MO 64086

Causes for Rejection

Whole blood frozen, refrigerated, clotted or received in transfer tube. Specimen will be rejected if greater than 30 hours old. Specimen type other than heparinized whole blood collected in sodium heparin tube.

Specimens are approved for testing in New York only when indicated in the Specimen Information field above.

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

*ImmuKnow is a registered trademark. ImmuKnow technology is patented by Viracor Eurofins' Laboratories. Use of intellectual property owned by Viracor Eurofins' to recreate or replicate the ImmuKnow® assay is strictly prohibited and may be considered infringement of U.S. Patent numbers 5, 773, 232, 6, 630, 316, 7, 169, 571, 7, 476, 514, and all applicable international patents. Further, use or past use of ImmuKnow kits and/or information provided within the kits to reverse engineer or replicate the ImmuKnow test, or use of ImmuKnow test results to evaluate, validate or certify a competing assay, may violate the terms under which the kits or results were supplied. Please contact Viracor Eurofins' at 800-305-5198 with any questions regarding its ImmuKnow patents, or visit www.uspto.gov for general information on patent and IP law policy.

References

1 Roitt, I, et al. Immunology, 5th edition. Mosby. 1998.

2 Wier, ML. Methods for Measurement of Lymphocyte Function. United States Patent 5,773,232. 1998.

3 Sottong, PR, et al. Measurement of T-Lymphocyte Responses in Whole-Blood Cultures Using Newly Synthesized DNA and ATP. Clin and Diagn Lab Immunol. 2000 Mar;7:307-311.

4 Brinchmann, JE, et al. Quantification of Lymphocyte Subsets Based on Positive Immunogenetic Selection of Cells Directly from Blood. J Immunogenetics. 1989 Apr;16:177-183.

5 DelGratta, C, et al. Detection and Counting of Specific Cell Populations by Means of Magnetic Markers Linked to Monoclonal Antibodies. Phys Med Biol. 1995 Apr;40: 671-681.

6 Buttgereit, F, et al. Bioenergetics of Immune Functions: Fundamental and Therapeutic Aspects. Immunology Today. 2000 Apr;21(4):192-199.

7 Andreotti, PE, et al. ATP Lymphocyte Activation Assay Application for Lymphokines and Cytokines. Bioluminescence and Chemiluminescence. John Wiley & Sons. 1993;257.

8 Crouch, SPM, et al. The Use of ATP Bioluminescence as a Measure of Cell Proliferation and Cytotoxicity. J Immunol Methods. 1993 Mar 15;160:81-88.

9 Shearer, GM, Clerici, M. In Vitro Analysis of Cell-Mediated Immunity: Clinical Relevance. Clin Chem. 1994 Nov; 40(11 Pt2):2162-2165.

10 Ahmed, M, et al. Quantitation of Immunosuppression by Tacrolimus Using Flow Cytometric Analysis of Interleukin-2 and Interferon-y Inhibition in CD8- and CD8+ Peripheral Blood T-cells. Therapeutic Drug Monitoring. 2001 Aug;23(4):354-362.

11 Schulick, RD, et al. Longitudinal Study of In Vitro CD4 T Helper Cell Function in Recently Transplanted Renal Allograft Patients Undergoing Tapering of Their Immunosuppressive Drugs. Transplantation. 1993 Sep;56(3):590-596.

12 Kowalski, R, et al. Immune Cell Function Testing: An Adjunct to Therapeutic Drug Monitoring in Transplant Patient Management. Clin Transplantation. 2003 Apr;17(2):77-88.

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