Test ID: BTK
Bruton Tyrosine Kinase (Btk), Protein Expression, Flow Cytometry, Blood
Secondary ID 
A test code used for billing and in test definitions created prior to November 2011
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
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)
Because genotype-phenotype correlations are important, the preferred test for confirming a diagnosis of XLA in males and identifying carrier females is:
-BTKFP/89742 Bruton's Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry
-In families where a BTK mutation has already been identified, order BTKMP/89740 Bruton's Tyrosine Kinase (BTK) Genotype and Protein Analysis, Known Mutation Sequencing and Flow Cytometry
Method Name A short description of the method used to perform the test
Flow Cytometry
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Aliases Lists additional common names for a test, as an aid in searching
Tyrosine Kinase
X-Linked Agammaglobulinemia (XLA)
XLA (X-Linked Agammaglobulinemia)
Specimen Type Describes the specimen type needed for testing
Specimen Required Defines the optimal specimen. This field describes the type of specimen required to perform the test and the preferred volume to complete testing. The volume allows automated processing, fastest throughput and, when indicated, repeat or reflex testing.
Specimen must arrive within 72 hours of draw. Send specimen Sunday through Thursday only. Collect and package specimen as close to shipping time as possible. Ship specimen overnight.
Container/Tube: Lavender top (EDTA)
Specimen Volume: 5 mL
Collection Instructions: Send specimen in original tube. Do not aliquot.
Additional Information:
1. Ordering physician name and phone number are required.
2. For serial monitoring, we recommend that specimen draws be performed at the same time of day.
Specimen Minimum Volume Defines the amount of specimen required to perform an assay once, including instrument and container dead space. Submitting the minimum specimen volume makes it impossible to repeat the test or perform confirmatory or perform reflex testing. In some situations, a minimum specimen volume may result in a QNS (quantity not sufficient) result, requiring a second specimen to be collected.
Reject Due To Identifies specimen types and conditions that may cause the specimen to be rejected
| Hemolysis | Mild OK; Gross reject |
| Lipemia | Mild OK; Gross reject |
| Icterus | NA |
| Other | NA |
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
| Specimen Type | Temperature | Time |
|---|---|---|
| Whole Blood EDTA | Refrigerated (preferred) | 72 hours |
| Ambient | 72 hours |
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, adult male patients 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 mutations 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 <1% B cells. 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 XLA patients 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 mutations in the BTK gene, which encodes Bruton tyrosine kinase (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 mutations 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 XLA patients 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 mutations within the gene. Females are typically carriers and asymptomatic. Testing in adult females 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 mutations.
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 mutation within the BTK gene have normal protein expression (again related to the position of the mutation 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/89742 Bruton's Tyrosine Kinase (BTK) Genotype and Protein Analysis, Full Gene Sequence and Flow Cytometry, which includes both flow cytometry and gene sequencing to confirm the presence of a BTK mutation. If a familial mutation has already been identified, then BTKMP/89740 Bruton's Tyrosine Kinase (BTK) Genotype and Protein Analysis, Known Mutation Sequencing and Flow Cytometry 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
Btk 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. 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/89307 Bruton's Tyrosine Kinase (BTK) Genotype, Full Gene Sequence or BTKK/89306 Bruton's Tyrosine Kinase (BTK) Genotype, Known Mutation) 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 (XLA), 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 mutant X-chromosome inactivation) or there is dominant expression of the normal protein in the presence of 1 copy of a mutation
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 adult males (>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 females beyond child-bearing age. For questions about appropriate test selection, contact Mayo Medical Laboratories.
The flow cytometry screening assay is likely to detect the majority of X-linked agammaglobulinemia (XLA) patients with 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 which can only be detected by BTK gene sequencing. The ability to identify carrier females 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 females.
It is also important to note that there are XLA patients with mothers who have normal Btk protein expression by flow cytometry and normal BTK genotyping, and the mutation in the patient is a result of de novo mutations 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 mutant X chromosome.
Clinical Reference Provides recommendations for further 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;108:1012-1020
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;120:512-517
3. Stewart DM, Tian L, Nelson DL: A case of X-linked agammaglobulinemia diagnosed in adulthood. Clin Immunol 2001;99(1):94-99
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;91(2):595-602
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 preconjugated antibody (BD Biosciences). After the staining and wash process, the cells are resuspended in 500 mcL of 1% paraformaldehyde 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;91[2]:595-602)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Do not send specimen after Thursday. Specimen must be received by 10 a.m. on Friday.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test
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's instructions, or as products that do not undergo full FDA review and approval, and are then labeled as an Analyte Specific Reagent (ASR), Investigation Use Only (IUO) product, or a Research Use Only (RUO) product.
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 Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
88184
LOINC® Code Information Provides guidance in determining the Logical Observation Identifiers Names and Codes (LOINC) values for the result codes returned for this test or profile.
| Result ID | Reporting Name | LOINC Code |
|---|---|---|
| 89011 | Btk Protein Flow, B | In Process |
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