Test Catalog

Test Id : BTKFP

Bruton Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry, Blood

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

Preferred test for confirming a diagnosis of X-linked agammaglobulinemia (XLA) in male patients with a history of recurrent sinopulmonary infections, profound hypogammaglobulinemia, and below 1% peripheral B cells

 

Identifying female carriers of XLA.

 

Providing a comprehensive assessment and enabling appropriate genotype-phenotype correlations due to the inclusion of both protein and gene analyses

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

The Bruton tyrosine kinase (BTK) gene is present on the long arm of the X-chromosome and encodes the intracellular signaling protein BTK critical for B-lymphocyte development and function. Loss of function variants in this gene cause X-linked agammaglobulinemia in male patients.

Profile Information
A profile is a group of laboratory tests that are ordered and performed together under a single Mayo Test ID. Profile information lists the test performed, inclusive of the test fee, when a profile is ordered and includes reporting names and individual availability.

Test Id Reporting Name Available Separately Always Performed
BTKSP BTK, Full Gene Sequence Yes, (order BTKS) Yes
BTKSQ BTK, Full Gene Sequencing No Yes
BTK Btk Protein Flow, B Yes Yes

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

 

Special Instructions
Library of PDFs including pertinent information and forms related to the test

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

BTKSP: Polymerase Chain Reaction (PCR) Followed by DNA Sequence Analysis

BTK: 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 Full-Gene Panel, B

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

B-Cell Progenitor Kinase

Tyrosine Kinase

Tyrosine-Protein Kinase BTK (Bruton's Tyrosine Kinase)

Agammaglobulinemia Tyrosine Kinase

BTK (Bruton's Tyrosine Kinase)

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

 

Specimen Type
Describes the specimen type validated for testing

Whole Blood EDTA

Ordering Guidance

New York Clients: A portion of this profile, BTK / Bruton Tyrosine Kinase, Protein Expression, Flow Cytometry, Blood is not New York State approved. Order BTKS / Bruton Tyrosine Kinase (BTK) Genotype, Full Gene Sequence, 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, phone number, and patient information (Bruton Tyrosine Kinase [BTK] Genotype Patient Information [T620]) are required.

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

Two separate whole blood specimens are required.

 

Specimen Type: Whole blood for BTKSP / Bruton Tyrosine Kinase (BTK) Genotype, Full Gene Sequence

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions:

1. Send whole blood specimen in original tube. Do not aliquot.

2. Label as BTKSP.

Specimen Stability Information: Refrigerated (preferred)/Ambient

 

Specimen Type: Whole blood for BTK / Bruton Tyrosine Kinase, Protein Expression, Flow Cytometry, Blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions:

1. Send whole blood specimen in original tube. Do not aliquot.

2. Ship at ambient temperature only.

3. Label as BTK.

Specimen Stability Information: Ambient 72 hours

Special Instructions
Library of PDFs including pertinent information and forms related to the test

Forms

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

BTKSP: 0.35 mL

BTK: 2 mL

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

No specimen should be rejected.

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 Varies (preferred) 72 hours

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

Preferred test for confirming a diagnosis of X-linked agammaglobulinemia (XLA) in male patients with a history of recurrent sinopulmonary infections, profound hypogammaglobulinemia, and below 1% peripheral B cells

 

Identifying female carriers of XLA.

 

Providing a comprehensive assessment and enabling appropriate genotype-phenotype correlations due to the inclusion of both protein and gene analyses

Genetics Test Information
Provides information that may help with selection of the correct genetic test or proper submission of the test request

The Bruton tyrosine kinase (BTK) gene is present on the long arm of the X-chromosome and encodes the intracellular signaling protein BTK critical for B-lymphocyte development and function. Loss of function variants in this gene cause X-linked agammaglobulinemia in male patients.

Testing Algorithm
Delineates situations when tests are added to the initial order. This includes reflex and additional tests.

 

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

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency affecting male patients in approximately 1 in 200,000 live births. XLA is caused by variants in the Bruton tyrosine kinase gene (BTK),(1) which results in a profound block in B-cell development within the bone marrow and a significant reduction, or complete absence, of mature B cells in peripheral blood.(2) Approximately 85% of male patients with defects in early B-cell development have XLA.(3) Due to the lack of mature B cells, XLA patients have markedly reduced levels of all major classes of immunoglobulins in the serum and are, therefore, susceptible to severe and recurrent bacterial infections. Pneumonia, otitis media, enteritis, and recurrent sinopulmonary infections are among the key clinical diagnostic characteristics of the disease. The spectrum of infectious complications also includes enteroviral meningitis, septic arthritis, cellulitis, and empyema, among others. The disease typically manifests in boys younger than 1 year of age.

 

BTK, the only gene associated with XLA, maps to the X-chromosome at Xq21.3-Xq22 and consists of 19 exons spanning 37.5 kb genomic DNA.(4) BTK encodes a nonreceptor tyrosine kinase of the BTK/Tec family. The BTK protein consists of 5 structural domains (PH, TH, SH3, SH2, and TK). Variants causing XLA have been found in all domains of the BTK gene, as well as noncoding regions of the gene. Missense variants account for 40% of all variants, while nonsense variants account for 17%, deletions 20%, insertions 7%, and splice-site variants 16%. Over 900 unique variants in the BTK gene have been detected by full gene sequencing and are listed in BTKbase, a database for BTK variants (https://databases.lovd.nl/shared/genes/BTK).(5) Genotype-phenotype correlations have not been completely defined for BTK, but it is clear that nonsense variants are overrepresented 4-fold compared to substitutions, which indicates that the latter may be tolerated without causing a phenotype. The type and location of the variant in the gene clearly affect the severity of the clinical phenotype. Some variants manifest within the first year or 2 of life, enabling an early diagnosis. Others present with milder phenotypes, resulting in diagnosis later in childhood or in adulthood.(5) Delayed diagnoses can be partly explained by the variable severity of XLA, even within families in which the same variant is present. While the disease is considered fully penetrant, the clinical phenotype can vary considerably depending on the nature of the specific BTK variant.(5) Lyonization of this gene is not typical and only 1 case of XLA in a female patient has been reported so far due to skewed lyonization in a carrier female patient. Therefore, female patients with clinical features that are identical to XLA should be evaluated for autosomal recessive agammaglobulinemia when deemed clinically appropriate,(6) and for XLA, if a male parent is affected with the disease.

 

A flow cytometry test for intracellular BTK in monocytes using an anti-BTK monoclonal antibody was developed, which was used to evaluate both XLA patients and carriers.(7) In this study, 41 unrelated XLA families were studied and deficient BTK protein expression was seen in 40 of these 41 patients, with complete BTK deficiency in 35 patients and partial BTK deficiency in 5 patients. One patient had a normal level of BTK protein expression. The 6 patients with partial or normal BTK expression had missense BTK variants. Additionally, the flow cytometry assay detected carrier status in the mothers of 35 of the 41 patients (approximately 85%). In the 6 patients where the BTK expression was normal in the mothers of XLA patients, it was noted that all these patients were sporadic cases without previous family history of the disease.(7) 

It appears, therefore, that most BTK variants result in deficient expression of BTK protein, which can be detected by flow cytometry in monocytes.(7,8) Also, the mosaic expression of BTK protein in the monocytes by flow cytometry is potentially useful in the diagnosis of female carriers.(8) The flow cytometry test therefore provides a convenient screening tool for the diagnosis of XLA with confirmation of the diagnosis by BTK genotyping.(7,8) In the rare patient with the clinical features of XLA but normal BTK protein expression, BTK genotyping must be performed to determine the presence of a variant.

 

A diagnosis of XLA should be suspected in male patients with early-onset bacterial infections, marked reduction in all classes of serum immunoglobulins, and absent B cells (CD19+ cells). The decrease in numbers of peripheral B cells is a key feature, though this also can be seen in a small subset of patients with common variable immunodeficiency. As well, some BTK variants can preserve small numbers of circulating B cells and, therefore, all the 3 criteria mentioned above need to be evaluated. Patients should be assessed for the presence of BTK protein by flow cytometry, although normal results by flow cytometry do not rule out the presence of a BTK variant with aberrant protein function (despite normal protein expression). The diagnosis is established or confirmed only in those individuals who have a variant identified in the BTK gene by gene sequencing or who have male family members with hypogammaglobulinemia with absent or low B cells. Appropriate clinical history is required with or without abnormal BTK protein results by flow cytometry.

 

It was shown that there are patients with XLA whose mothers have normal BTK protein expression by flow cytometry and normal BTK genotyping, and that the variant in the patient is a result of de novo variants in the maternal germline. In the same study, it was shown that there can be female carriers who have normal BTK protein expression but are genetically heterozygous, and they do not show abnormal protein expression due to extremely skewed inactivation of the altered X-chromosome.(6) Also, the presence of one copy of the normal BTK gene and associated normal BTK protein can stabilize altered protein abrogating the typical bimodal pattern of protein expression seen in female carriers. Therefore, female carrier status can only conclusively be determined by genetic testing, especially if the BTK protein flow test is normal.

 

It is important to keep in mind that the mere presence of BTK gene variants does not necessarily correlate with a diagnosis of XLA unless the appropriate clinical and immunological features are present.(9,10)

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.

BTKSP: An interpretive report will be provided.

BTK: 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

A patient-specific interpretive report is provided.

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

Some patients with X-linked agammaglobulinemia (XLA) may have a higher level of B cells than 1% but still be below the reference value for their age, and  their immunoglobulin concentrations may initially be low or normal. If family history, age, or clinical and immunological history raise the suspicion for XLA, this test can be ordered.

 

Patients who have received a heterologous blood transfusion within the preceding 6 weeks, or who have received allogeneic blood or marrow transplant, can have inaccurate genetic test results due to presence of donor DNA.

Rare variants could potentially lead to false-negative or false-positive results. If results obtained do not match clinical findings, additional testing should be considered. Any error in the diagnosis or in the pedigree provided to the laboratory could lead to an erroneous interpretation of results.

 

This method will not detect variants that occur in intronic (other than exon-intron boundaries) and regulatory regions of the gene or large rearrangement-type variants (which could cause a false-negative result). Bruton tyrosine kinase

(BTK) protein and genetic tests are not meant 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 BTK gene.

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

1. Tsukada S, Saffran DC, Rawlings DJ, et al: Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell. 1993 Jan;72(2):279-290. doi: 10.1016/0092-8674(93)90667-f

2. Noordzij JG, de Bruin-Versteeg S, Comans-Bitter WM, et al: Composition of precursor B-cell compartment in bone marrow from patients with X-linked agammaglobulinemia compared with health children. Pediatr Res. 2002 Feb;2:159-168. doi: 10.1203/00006450-200202000-00007

3. Conley ME, Broides A, Hernandez-Trujillo V, et al: Genetic analysis of patients with defects in early B-cell development. Immunol Rev. 2005 Feb;203:216-234. doi: 10.1111/j.0105-2896.2005.00233.x

4. Lindvall JM, Blomberg KEM, Vargas L, et al: Bruton's tyrosine kinase: cell biology, sequence conservation, mutation spectrum, siRNA modifications, and expression profiling. Immunol Rev. 2005 Feb;203:200-215. doi: 10.1111/j.0105-2896.2005.00225.x

5. Valiaho J, Smith CI, Vihinen M: BTKbase: the mutation database for X-linked agammaglobulinemia. Hum Mutat. 2006 Dec;27(12):1209-1217. doi: 10.1002/humu.20410

6. Takada H, Kanegane H, Nomura A, et al: Female agammaglobulinemia due to the Bruton's tyrosine kinase deficiency caused by extremely skewed X-chromosome inactivation. Blood. 2004 Jan;103(1):185-187. doi: 10.1182/blood-2003-06-1964

7. 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. doi: 10.1067/mai.2001.120133

8. 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

9. Graziani S, Di Matteo G, Benini L, et al: Identification of a BTK mutation in a dysgammaglobulinemia patient with reduced B cells: XLA or not? Clin Immunol. 2008 Sept;128(3):322-328. doi: 10.1016/j.clim.2008.05.012

10. Fleisher TA, Notarangelo LD: What does it take to call it a pathogenic mutation? Clin Immunol 2008 Sep;128(3):285-286

11. 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

Special Instructions
Library of PDFs including pertinent information and forms related to the test

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

Genomic DNA is first extracted from whole blood, followed by Bruton tyrosine kinase (BTK) gene amplification by polymerase chain reaction (PCR). The PCR product is purified from unincorporated primers and nucleotides by enzymatic digestion and sequenced in both directions using sequencing primers and fluorescent dye-terminator chemistry. Sequencing products are separated on an automated sequencer and trace files are analyzed for variations in the exons and intron/exon boundaries of all 19 exons using specialized variant detection software and visual inspection.(Unpublished Mayo method)

 

The BTK protein expression flow cytometry 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, 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 an antihuman BTK-fluorescent pre-conjugated antibody from BD Biosciences. After the staining and wash process, the cells are resuspended in 500 mcL of BD FACS stain buffer in the final step of the assay and the sample is analyzed by multiparametric flow cytometry.(Unpublished Mayo method; 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))

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

No

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.

4 to 6 weeks

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.

  • Authorized users can sign in to Test Prices for detailed fee information.
  • Clients without access to Test Prices can contact Customer Service 24 hours a day, seven days a week.
  • Prospective clients should contact their Regional Manager. For assistance, contact Customer Service.

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.

81406-Bruton Tyrosine Kinase (BTK) Genotype, Full Gene Sequence

88184-Bruton Tyrosine Kinase (BTK), Protein Expression, Flow Cytometry, Blood

LOINC® Information
Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the order and results codes of this test. LOINC values are provided by the performing laboratory.

Test Id Test Order Name Order LOINC Value
BTKFP BTK Full-Gene Panel, B 94241-7
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.
89011 Btk Protein Flow, B 75708-8
BTKSQ BTK, Full Gene Sequencing Bill only; no result
29305 BTK Full Gene Result 82939-0
45486 BTK Full Gene Interpretation 69047-9
45487 Reviewed By 18771-6

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