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Test ID: BTKMP Bruton Tyrosine Kinase (BTK) Genotype and Protein Analysis, Known Mutation Sequencing and Flow Cytometry

Reporting Name

BTK Known Mutation Panel, B

Useful For

Preferred test for confirming a diagnosis of X-linked agammaglobulinemia (XLA) in male family members of affected individuals with known BTK variants


Preferred test for determining carrier status of female relatives of male XLA patients with known BTK variants


By including protein and gene analysis, this test provides a comprehensive assessment and enables appropriate genotype-phenotype correlations.

Profile Information

Test ID Reporting Name Available Separately Always Performed
BTKKM BTK Gene, Known Mutation Yes, (order BTKK) Yes
BTKKQ BTK Gene, Known Mutation Sequencing No Yes
BTK Btk Protein Flow, B Yes Yes

Specimen Type

Whole Blood EDTA

Advisory Information

New York Clients: The BTK / Btk Protein Flow portion of this test is not New York State approved. Order BTKK / Bruton Tyrosine Kinase (BTK) Genotype, Known Mutation.

Shipping Instructions

Specimens are required to be received in the laboratory weekdays and by 4 p.m. on Friday. Draw and package specimen as close to shipping time as possible.


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


Specimens arriving on the weekend may be canceled.

Necessary Information

Ordering physician's name, phone number, and patient information are required.

Specimen Required

Two separate EDTA specimens and the patient information sheet are required.


Specimen Type: Blood for BTKKM / Bruton Tyrosine Kinase (BTK) Genotype, Known Mutation Sequence

Container/Tube: Lavender top (EDTA)

Specimen Volume: 3 mL

Collection Instructions:

1. Send specimen in original tube.

2. Label as BTKKM.

Specimen Stability Information: Refrigerated (preferred) 14 days/Ambient 4 days


Specimen Type: Blood for BTK / Bruton Tyrosine Kinase (Btk), Protein Expression, Flow Cytometry, Blood

Container/Tube: Lavender top (EDTA)

Specimen Volume: 4 mL

Collection Instructions:

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

2. Ship at ambient temperature only.

3. Label as BTK.

Specimen Stability Information: Ambient 72 hours

Additional Information: For flow cytometry serial monitoring, we recommend that specimen draws be performed at the same time of day.

Specimen Minimum Volume

BTKKM: 0.35 mL
BTK: 2 mL

Specimen Stability Information

Specimen Type Temperature Time
Whole Blood EDTA Varies 72 hours

Reference Values


An interpretive report will be provided.



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

Day(s) and Time(s) Performed

Specimens are required to be received in the laboratory on weekdays and by 4 p.m. on Friday. No weekend processing.

CPT Code Information

Bruton Tyrosine Kinase (BTK) Genotype, Known Mutation Sequence

81403-Known familial variant


Bruton Tyrosine Kinase (Btk), Protein Expression, Flow Cytometry, Blood


LOINC Code Information

Test ID Test Order Name Order LOINC Value
BTKMP BTK Known Mutation Panel, B 69479-4


Result ID Test Result Name Result LOINC Value
89011 Btk Protein Flow, B 75708-8
BTKKQ BTK Gene, Known Mutation Sequencing No LOINC Needed
29304 BTK Known Mut Result 69479-4
45477 BTK Known Mut Interpretation 69047-9
45478 Reviewed By 18771-6

Clinical Information

X-linked agammaglobulinemia (XLA) is a humoral primary immunodeficiency affecting males 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 male children 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 Bruton tyrosine kinase (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 600 unique variants in the BTK gene have been detected by full gene sequencing and are listed in BTKbase, a database for BTK variants ( 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 affects 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 has been reported so far due to skewed lyonization in a carrier female. Therefore, females 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 by Futatani et al, 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 males with 1) early-onset bacterial infections, 2) marked reduction in all classes of serum immunoglobulins, and 3) 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 (CVID). 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 XLA patients with mothers who 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 extreme skewed inactivation of the mutant X chromosome.(6) Also, the presence of 1 copy of the normal BTK gene and associated normal Btk protein can stabilize mutant 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)


A patient-specific interpretive report is provided.

Clinical Reference

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;72:279-290

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;2:159-168

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

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;203:200-215

5. Valiaho J, Smith CI, Vihinen M: BTKbase: The mutation database for X-linked agammaglobulinemia. Hum Mutat 2006;27:1209-1217

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;103:185-187

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;91(2):595-602

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;108:1012-1020

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;128:322-328

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

Analytic Time

4 weeks

Method Name

BTKKM: Polymerase Chain Reaction (PCR) followed by DNA Sequence Analysis

BTK: Flow Cytometry

Test Classification

See Individual Test IDs
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