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ZAP 70 Evaluation

Flow Cytometry, ZAP-70, ZAP-70 Evaluation/Chronic Lymphocytic Leukemia (CLL) Prognostic Panel, ZAP70 Evaluation Chronic Lymphocytic Leukemia (CLL) Prognostic Panel

Test Codes

EPIC: LAB6419

Department

Flow Cytometry

Instructions

This test may be ordered to aid in the clinical management of patients with an established diagnosis of CLL. This test should not be ordered on patients without an established diagnosis of CLL, or in patients diagnosed with other types of lymphoproliferative disorders or leukemias other than CLL.

Specimen Collection Criteria

Collect: One Lavender-top EDTA tube or Green-top Sodium Heparin tube.

Ordering Notes

• The initial peripheral blood specimen submitted for the "Hematolymphoid Neoplasm" test may be sub-optimal for ZAP-70 analysis due to time delays that may be encountered prior to establishing a diagnosis of CLL. This delay could potentially reduce ZAP-70 staining and result in a false negative ZAP-70 test, relative to a fresh specimen. Therefore, after the diagnosis of CLL has been definitively established (including clinical and morphologic correlation), it is optimal to order "ZAP-70 Evaluation" test separately if desired. (NOTE: ZAP-70 evaluation is not routinely performed on all new diagnoses of CLL.)
• If there is a concern that a delay could adversely affect patient care or with logistical problems in obtaining a second specimen, the "ZAP-70 Evaluation" and "Hematolymphoid Neoplasm" tests can be ordered on a single blood specimen that will be used to perform both tests concurrently.
• ZAP-70 expression has been shown to be stable in patients with CLL. Therefore, the time between the initial diagnosis of CLL and ZAP-70 evaluation does not appear to be critical, for the purposes of accurate testing.
• A copy of the requisition must be sent with the specimen.

Physician Office/Draw Specimen Preparation

All specimens must be received in the Laboratory within 24 hours of collection. Specimens greater than 24 hours old will be accepted only upon the approval by the Flow Cytometry Laboratory manager or pathologist on service. Maintain whole blood at room temperature (20-26°C or 68-78.8°F) prior to transport.

Preparation for Courier Transport

Transport: Whole blood, at room temperature (20-26°C or 68-78.8°F).

FedEx Shipping Instructions:
Transport 4 mL whole blood at room temperature. Specimen must be received within 24 hours of collection.

Rejection Criteria

• Frozen specimens.
• Clotted specimens or specimens greater than 24 hours old (from the time of collection) acceptable with approval of the Flow Cytometry Laboratory manager or pathologist on service.

In-Lab Processing

Maintain at room temperature (20-26°C or 68-78.8°F) prior to testing. Specimens greater than 24 hours old will be accepted only upon the approval by the Flow Cytometry Laboratory manager or pathologist on service.

Storage

Specimen Stability for Testing:

Room Temperature (20-26°C or 68-78.8°F): 24 hours
Refrigerated (2-8°C or 36-46°F): Only acceptable with the approval of the Flow Cytometry Laboratory manager or pathologist on service.
Frozen (-20°C/-4°F or below): Unacceptable

Specimen Storage in Department Prior to Disposal:

Refrigerated (2-8°C or 36-46°F): 7 days

Laboratory

Royal Oak Flow Cytometry Laboratory

Performed

Monday – Saturday.
Results available in 1-2 days.

Reference Range

Pathologist's interpretation will report results as positive, negative or indeterminate:

Positive: Greater than 30% of CLL cells stain above the control levels.
Indeterminate: 20-30% of CLL cells stain above the control levels.
Negative: Less than 20% of cells stain above the control levels.

Test Methodology

Staining for intracellular ZAP-70 is performed on the specimen and evaluated using mutli-parametric flow cytometric analysis for CD3, CD5, CD19, CD38, CD45 and ZAP-70 with an IgG1 isotype control (Alexa Fluor 488). The level of ZAP-70 expression is determined using matched isotype controls. CD3+ T-cells are used as internal controls.

Interpretation

ZAP-70 expression is evaluated on the CD19+/CD5+ co-expressing lymphocytes. Results are determined based upon the percentage of CD19+/CD5+ cells that show ZAP-70 expression above the level of the matched isotype control. CD38 expression is also reported. The CD3+ T-cells are used as internal positive controls.

Clinical Utility

ZAP-70 may be useful in the clinical management of patients diagnosed with CLL. The results of this test should always be correlated with the morphologic and clinical picture.

Clinical Disease

CLL is a disease in which the clinical course is variable. In patients with aggressive disease, the CLL cells usually express an unmutated immunoglobulin heavy-chain variable region gene and the 70-kD zeta chain associated protein (ZAP-70). Conversely, in patients with more indolent disease the CLL cells usually express the mutated immunoglobulin heavy-chain variable region gene and lack ZAP-70 expression.^1,2 CD38 expression in CLL cells has also been shown to correlate with disease aggression, although it may not be as good a predictor of outcomes as ZAP-70 expression.³ However, the combined use of CD38 and ZAP-70 evaluation has been proven to be useful in providing prognostic information.⁴ (Note: CD38 expression is evaluated and reported as a routine component of the "Hematolymphoid Neoplasm" test performed in our laboratory for the diagnosis of CLL.)

Disease Reporting

The results of this test should always be correlated with the clinical and morphologic information. Patients with CLL who are ZAP-70 positive have been shown to have a worse prognosis compared to patients who are ZAP-70 negative. However, not all patients with ZAP-70 positive CLL have aggressive disease, and not all patients with ZAP-70 negative CLL have indolent disease. Additionally, the significance of ZAP-70 expression in chronic lymphoproliferative disorders other than CLL is unknown.

Reference

1. Crespo M, et al. ZAP-70 expression as a surrogate for immunoglobulin-variable-region mutations in chronic lymphocytic leukemia. N Engl J Med 2003;348:1764-1775.
2. Rassenti LZ, et al. ZAP-70 compared with immunoglobulin heavy-chain gene rearrangement gene mutation status as a predictor of disease progression in chronic lymphocytic leukemia. N Engl J Med 2004;351:893-901.
3. Del Principe MI, et al. Clinical significance of ZAP-70 protein expression in B-cell chronic lymphocytic leukemia. Blood 2006;108:853-861.
4. Schroers R, et al. Combined analysis of ZAP-70 and CD38 expression as a predictor of disease progression in B-cell chronic lymphocytic leukemia. Leukemia 2005;19:750-758.
5. Zucchetto A, et al. ZAP-70 expression in B-cell chronic lymphocytic leukemia: Evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgVH mutations. Cytometry B Clin Cytom 2006;70(4):284-92.
6. Sheridan R, et al. Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia. Cytometry B Clin Cytom 2006;70(4):3320-321.

CPT Codes

88184 – First marker, 88185x5 – each additional marker. 88187 – Interpretation 2-8 markers, 88188 – 9-15 markers, 88189 – 16 or greater markers.

Contacts

Last Updated

8/9/2023