Test Catalog

Test Id : BTK

Bruton Tyrosine Kinase, Protein Expression, Flow Cytometry, Blood

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

Preliminary screening for X-linked agammaglobulinemia (XLA), primarily in male patients (<65 years of age) or female carriers (child-bearing age: <45 years)

Method Name
A short description of the method used to perform the test

Flow Cytometry

NY State Available
Indicates the status of NY State approval and if the test is orderable for NY State clients.

No

Reporting Name
Lists a shorter or abbreviated version of the Published Name for a test

Btk Protein Flow, B

Aliases
Lists additional common names for a test, as an aid in searching

Tyrosine Kinase

X-Linked Agammaglobulinemia (XLA)

XLA (X-Linked Agammaglobulinemia)

BTK (Bruton's Tyrosine Kinase)

Specimen Type
Describes the specimen type validated for testing

Whole Blood EDTA

Ordering Guidance

Bruton tyrosine kinase (BTK) protein and genetic tests are not mean for patients with hematological neoplasias on kinase inhibitor therapy, including but not restricted to the selective BTK inhibitor, ibrutinib. This test is only meant for the assessment of patients with a suspected monogenic primary immunodeficiency, X-linked agammaglobulinemia, caused by germline variants in the Bruton tyrosine kinase gene.

 

The preferred test for confirming a diagnosis of X-linked agammaglobulinemia in male patients and identifying carrier female patients is BTKFP / Bruton Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry, Blood.

 

In families where a BTK variant has already been identified, order FMTT / Familial Mutation, Targeted Testing, Varies.

Shipping Instructions

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.

 

It is recommended that specimens arrive within 24 hours of collection.

 

Samples arriving on the weekend and observed holidays may be canceled.

Necessary Information

Ordering physician's name and phone number are required.

Specimen Required
Defines the optimal specimen required to perform the test and the preferred volume to complete testing

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.

Specimen Minimum Volume
Defines the amount of sample necessary to provide a clinically relevant result as determined by the Testing Laboratory

2 mL

Reject Due To
Identifies specimen types and conditions that may cause the specimen to be rejected

Gross hemolysis Reject
Gross lipemia Reject

Specimen Stability Information
Provides a description of the temperatures required to transport a specimen to the performing laboratory, alternate acceptable temperatures are also included

Specimen Type Temperature Time Special Container
Whole Blood EDTA Ambient (preferred) 48 hours PURPLE OR PINK TOP/EDTA

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

Preliminary screening for X-linked agammaglobulinemia (XLA), primarily in male patients (<65 years of age) or female carriers (child-bearing age: <45 years)

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

The differential diagnosis for patients with primary hypogammaglobulinemia of unclear etiology (after other secondary causes of hypogammaglobulinemia have been ruled out) includes common variable immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA). CVID is the most common diagnosis of humoral immunodeficiency, particularly in adults but also in children over 4 years of age. However, men with XLA may be misdiagnosed with CVID. XLA is an independent humoral immunodeficiency and should not be regarded as a subset of CVID.

 

The BTK gene is present on the long arm of the X-chromosome and encodes for a cytoplasmic tyrosine kinase with 5 distinct structural domains. While BTK gene sequencing is the gold standard for definitively identifying variants and confirming a diagnosis of XLA, it is labor intensive and expensive. Flow cytometry is a screening test for XLA and should be included in the evaluation of patients with possible CVID, particularly in male patients with less than 1% B cells. Bruton tyrosine kinase (BTK) is an intracellular protein and absence of the BTK protein by flow cytometry provides a strong rationale for performing a BTK gene-sequencing test. However, 20% to 30% of patients with XLA may have intact or truncated BTK protein with abnormal function; therefore, genetic analysis remains the more definitive test for diagnosing XLA (besides other clinical and immunological parameters).

 

XLA is a prototypical humoral immunodeficiency caused by variants in the BTK gene, which encodes BTK, a hematopoietic-specific tyrosine kinase. XLA is characterized by normal, reduced, or absent BTK expression in monocytes and platelets, a significant reduction or absence of circulating B cells in blood, and profound hypogammaglobulinemia of all isotypes (IgG, IgA, IgM, and IgE). The clinical presentation includes early onset of recurrent bacterial infections and absent lymph nodes and tonsils. BTK plays a critical role in B-cell differentiation. The defect in BTK may be "leaky" in some patients (ie, a consequence of variants in the gene that result in a milder clinical and laboratory phenotype), such that these patients may have some levels of IgG and/or IgM and a small number of B cells in blood.(1)

 

The vast majority of patients with XLA are diagnosed in childhood (median age of diagnosis in patients with sporadic XLA is 26 months), although some patients are recognized in early adulthood or later in life. The diagnosis of XLA in both children and adults indicates that the disorder demonstrates considerable clinical phenotypic heterogeneity, depending on the position of the variants within the gene. Female patients are typically carriers and asymptomatic. Testing in women should be limited to those in their child-bearing years (<45 years). Carrier testing ideally should be confirmed by genetic testing since it is possible to have a normal flow cytometry test for protein expression in the presence of heterozygous (carrier) BTK gene variants.

 

Flow cytometry is a preliminary screening test for XLA. It is important to keep in mind that this flow cytometry test is only a screening tool and approximately 20% to 30% of patients who have a variant within the BTK gene have normal protein expression (again related to the position of the variant in the gene and the antibody used for flow cytometric analysis). Therefore, in addition to clinical correlation, genetic testing is recommended to confirm a diagnosis of XLA. Furthermore, it is helpful to correlate gene and protein data with clinical history (genotype-phenotype correlation) in making a final diagnosis of XLA. Consequently, the preferred test for XLA is BTKFP / Bruton Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry, Blood, which includes both flow cytometry and gene sequencing to confirm the presence of a BTK variant. If a familial variant has already been identified, then FMTT / Familial Mutation, Targeted Testing, Varies should be ordered.

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.

Present

Bruton tyrosine kinase expression will be reported as present, absent, partial deficiency, or mosaic (carrier).

Interpretation
Provides information to assist in interpretation of the test results

Results are reported as Bruton tyrosine kinase (BTK) protein expression present (normal) or absent (abnormal) in monocytes and B cells if present. Additionally, mosaic BTK expression (indicative of a carrier) and reduced BTK expression (consistent with partial BTK protein deficiency) are reported when present and correlated with a healthy experimental control.

 

BTK genotyping (BTKS / Bruton Tyrosine Kinase (BTK) Genotype, Full Gene Sequence, Blood or FMTT / Familial Mutation, Targeted Testing, Varies) should be performed in the following situations:

-To confirm any abnormal flow cytometry result

-In the rare patient with the clinical features of X-linked agammaglobulinemia, but normal BTK protein expression

-In mothers of patients who do not show the classic carrier pattern of bimodal protein expression (to determine if there is maternal germinal mosaicism or skewed altered X-chromosome inactivation), or there is dominant expression of the normal protein in the presence of one copy of a genetic variant.

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

This test is typically not indicated in men (>65 years, unless there is a strong clinical and family history, and the patient has not received a formal diagnosis and may or may not be on replacement immunoglobulin therapy) or women beyond child-bearing age. For questions about appropriate test selection, call 800-533-1710.

 

The flow cytometry screening assay is likely to detect the majority of patients with X-linked agammaglobulinemia (XLA) and completely or partially deficient Bruton tyrosine kinase (BTK) protein expression. However, approximately 20% to 30% of male patients may have normal BTK protein expression with aberrant function that can only be detected by BTK gene sequencing. The ability to identify carrier female patients by the flow cytometry assay is largely dependent on the BTK -specific antibodies used for flow detection. In general, genetic testing is preferable and more definitive to flow cytometry for identification of carrier female patients.

 

It is also important to note that there are patients with XLA whose mothers have normal BTK protein expression by flow cytometry and normal BTK genotyping, and the genetic variant in the patient is a result of de novo variants in the germline BTK gene.(1) In the same study, it has been shown that there can be female carriers who have normal BTK protein expression, but who are genetically heterozygous and do not show abnormal protein expression due to extreme skewed inactivation of the altered X chromosome.

Clinical Reference
Recommendations for in-depth reading of a clinical nature

1. Kanegane H, Futatani T, Wang Y, et al: Clinical and mutational characteristics of X-linked agammaglobulinemia and its carrier identified by flow cytometric assessment combined with genetic analysis. J Allergy Clin Immunol. 2001 Dec;108(6):1012-1020. doi: 10.1067/mai.2001.120133

2. Kanegane H, Tsukada S, Iwata T, et al: Detection of Bruton's tyrosine kinase mutations in hypogammaglobulinemic males registered as common variable immunodeficiency (CVID) in the Japanese Immunodeficiency Registry. Clin Exp Immunol. 2000 Jun;120(3):512-517. doi: 10.1046/j.1365-2249.2000.01244.x

3. Stewart DM, Tian L, Nelson DL: A case of X-linked agammaglobulinemia diagnosed in adulthood. Clin Immunol. 2001 Apr;99(1):94-99. doi: 10.1006/clim.2001.5024

4. Futatani T, Miyawaki T, Tsukada S, et al: Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as evaluated by a flow cytometric analysis and its clinical application to carrier detection. Blood. 1998 Jan;91(2):595-602

5. Kraft MT, Pyle R, Dong X, et al: Identification of 22 novel BTK gene variants in B cell deficiency with hypogammaglobulinemia. Clin Immunol. 2021 Aug;229:108788. doi: 10.1016/j.clim.2021.108788

6. Chear CT, Ripen AM, Mohamed SA, Dhaliwal JS: A novel BTK gene mutation creates a de-novo splice site in an X-linked agammaglobulinemia patient. Gene. 2015 Apr 15;560(2):245-8. doi: 10.1016/j.gene.2015.02.019

Method Description
Describes how the test is performed and provides a method-specific reference

The Bruton tyrosine kinase (BTK protein expression screening assay is carried out with a whole blood sample. The cells in the blood are stained with antihuman CD20 (B cells) and CD14 (monocytes) antibodies, which is followed by red blood cell lysis (using a premade Lysis buffer), cell fixation, and permeabilization to prepare the cell membrane for the antihuman BTK antibody. After the permeabilization step, the cells are stained for intracellular BTK using antihuman BTK-fluorescent pre-conjugated antibody (BD Biosciences). After the staining and wash process, the cells are fixed and analyzed by multiparametric flow cytometry.(Unpublished Mayo method)

PDF Report
Indicates whether the report includes an additional document with charts, images or other enriched information

No

Day(s) Performed
Outlines the days the test is performed. This field reflects the day that the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time before the test is performed. Some tests are listed as continuously performed, which means that assays are performed multiple times during the day.

Monday through Friday

Report Available
The interval of time (receipt of sample at Mayo Clinic Laboratories to results available) taking into account standard setup days and weekends. The first day is the time that it typically takes for a result to be available. The last day is the time it might take, accounting for any necessary repeated testing.

3 to 4 days

Specimen Retention Time
Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded

4 days

Performing Laboratory Location
Indicates the location of the laboratory that performs the test

Rochester

Fees
Several factors determine the fee charged to perform a test. Contact your U.S. or International Regional Manager for information about establishing a fee schedule or to learn more about resources to optimize test selection.

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Test Classification
Provides information regarding the medical device classification for laboratory test kits and reagents. Tests may be classified as cleared or approved by the US Food and Drug Administration (FDA) and used per manufacturer instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR) product.

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.

CPT Code Information
Provides guidance in determining the appropriate Current Procedural Terminology (CPT) code(s) information for each test or profile. The listed CPT codes reflect Mayo Clinic Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.

CPT codes are provided by the performing laboratory.

88184

Test Setup Resources

Setup Files
Test setup information contains test file definition details to support order and result interfacing between Mayo Clinic Laboratories and your Laboratory Information System.

Excel | Pdf

Sample Reports
Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports
International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports