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Interpretive Handbook

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Test 89584 :
CD20 on B Cells

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

CD20 is a protein that is expressed on the surface of B cells, starting at the pre-B cell stage and also on mature B cells in the bone marrow and in the periphery. CD20 is not expressed on hematopoietic stem cells, pro-B cells, or normal plasma cells.(1) Plasmablasts and stimulated plasma cells may express CD20.(2) CD20 is generally coexpressed on B cells with CD19, another B-cell differentiation marker. CD20 appears to play a role in B-cell development, differentiation, B-cell receptor (BCR) signaling, and cell-cycle initiation events.(3) CD20 is not shed from the surface of B cells and does not internalize on binding with anti-CD20 antibody, nor is it typically present as a soluble free antigen in circulation.(3) Certain primary humoral immunodeficiencies, such as X-linked agammaglobulinemia and autosomal recessive agammaglobulinemia, are characterized by a complete absence or profound reduction of peripheral B cells, expressing both CD20 and CD19 (another B-cell differentiation marker).

 

Mutations in the CD19 gene have been shown to be associated with a primary humoral immunodeficiency, sometimes classified as common variable immunodeficiency (CVID).(4) This defect accounts for <1% to 2% of CVID patients and appears to be inherited as an autosomal recessive defect.(4) Since these patients have normal numbers of B cells with absent CD19 expression on the cell surface (4), CD20 can be used as a marker to help identify these patients.

 

A contrasting situation exists for patients receiving rituximab, ofatumumab, and other anti-CD20 monoclonal antibodies that are used to treat certain cancers, autoimmune diseases, or for B-cell depletion to prevent humoral rejection in positive crossmatch renal transplantation. These agents block available CD20-binding sites and, therefore, the antibody used for this flow cytometric assay cannot recognize the CD20 molecule on B cells. The concomitant use of the CD19 marker provides information on the extent of B-cell depletion when using this particular treatment strategy.

 

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 am and noon, with no change between noon and afternoon. Natural killer cell counts, on the other hand, are constant throughout the day.(5) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(6-8) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(6) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening (9), and during summer compared to winter.(10) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

Useful For Suggests clinical disorders or settings where the test may be helpful

Evaluation of CD19 deficiency in patients with a suspected CD19 deficiency (humoral immunodeficiency)

 

Confirming complete absence of B cells in suspected primary humoral immunodeficiencies using both CD19 and CD20 markers

 

To assess therapeutic B-cell depletion quantitatively (absolute counts of cells/mcL) in any clinical context, including malignancies, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and membranous glomerulonephritis among others, and treatment or prevention of acute humoral rejection in positive crossmatch renal transplant recipients.

 

This test is not useful for the following applications, instead TAE / Therapeutic Antibody by Flow Cytometry should be ordered in the contexts described below:

-Assessing whether malignant (and nonmalignant) B cells express the target molecule (CD20) of interest in the context of initiating therapeutic monoclonal anti-CD20 antibody therapy (rituximab, ofatumumab, and tositumomab) for any of the hematological malignancies, or in other clinical contexts, such as autoimmunity.

Interpretation Provides information to assist in interpretation of the test results

The presence of CD20+ B cells with corresponding absence of CD19 staining in individuals not receiving anti-CD20 monoclonal antibody treatment or with clinical features of variable primary humoral immunodeficiency may suggest an underlying CD19 deficiency, which should be further evaluated.

 

Absence of both CD20 and CD19 markers on B cells in blood from individuals not on anti-CD20 monoclonal antibody treatment is consistent with complete mature and immature peripheral B-cell depletion, which may be due to an underlying primary immunodeficiency.

 

Patients receiving B-cell depleting therapy with anti-CD20 antibodies can show unusual populations of B cells on reconstitution that express either CD19 or CD20 due to a phenomenon known as trogocytosis.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

This test should be ordered if specifically confirming the absence of B cells due to suspected primary humoral or combined immunodeficiency or evaluating for CD19 deficiency.

 

This test should not be ordered for a comprehensive evaluation of peripheral B-cell subsets. For evaluation of memory B-cell subsets, transitional B cells, mature and immature B cells, order IABCS / B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood.

 

If desirous of only quantitatively measuring total CD19 or CD20+ B cells, order TBBS / T- and B-Cell Quantitation by Flow Cytometry or CD20B / CD20 on B Cells, respectively. Please do not order the detailed analysis of B cell subsets for this purpose.

 

This test should not be used for evaluating presence of CD20 on malignant or nonmalignant B cells. The following test should be used instead, TAE / Therapeutic Antibody by Flow Cytometry, which provides percent of B cells expressing CD19 or CD20 but does not provide absolute cell counts (cells/mcL).

 

Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See Clinical Information.

Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

%CD19 B CELLS

> or =19 years: 4.6-22.1%

 

CD19 ABSOLUTE

> or =19 years: 56.6-417.4 cells/mcL

 

%CD20 B CELLS

> or =19 years: 5.0-22.3%

 

CD20 ABSOLUTE

> or =19 years: 74.4-441.1 cells/mcL

 

CD45 ABSOLUTE

18-55 years: 0.99-3.15 thou/mcL

>55 years: 1.00-3.33 thou/mcL

Clinical References Provides recommendations for further in-depth reading of a clinical nature

1. Nadler LM, Ritz J, Hardy R, et al: A unique cell-surface antigen identifying lymphoid malignancies of B-cell origin. J Clin Invest 1981;67:134

2. Robillard N, Avet-Loiseau H, Garand R, et al: CD20 is associated with a small mature plasma cell morphology and t(11;14) in multiple myeloma. Blood 2003;102(3):1070-1071

3. Pescovitz MD: Rituximab, an anti-CD20 monoclonal antibody: history and mechanism of action. Am J Transplant 2006;6:859-866

4. van Zelm MC, Reisli I, van der Burg M, et al: An antibody-deficiency syndrome due to mutations in the CD19 gene. N Engl J Med 2006;354:1901-1912

5. 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

6. 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

7. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411

8. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Psychosom Med 1997;59:42-50

9. 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 AIDS 1990;3:144-151

10. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516


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