Bruton Tyrosine Kinase (BTK) Genotype and Protein Analysis, Known Mutation Sequencing and Flow Cytometry
Method Description Describes how the test is performed and provides a method-specific reference
Genomic DNA is first extracted from whole blood, followed by BTK gene amplification by PCR. Only the specific familial mutation will be evaluated in this genetic test. 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 will be analyzed for the known familial mutation in the appropriate exons and/or intron/exon boundaries using specialized mutation detection software (Phredphrap/Consed/Polyphred) and visual inspection.(Unpublished Mayo method)
The Bruton tyrosine kinase (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 RBC 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 preconjugated 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of BD FACS stain buffer in the final step of the assay and the sample is analyzed by multiparametric flow cytometry.(Unpublished Mayo method; Futatani T, Miyawaki T, Tsukada S, et al: Deficient expression of Bruton's tyrosine kinase in monocytes from X-linked agammaglobulinemia as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;91:595-602)
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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.
Specimens are required to be received in the laboratory on weekdays and by 4 p.m. on Friday. No weekend processing.
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
Whole Blood: 4 days Extracted DNA: 2 months
Performing Laboratory Location The location of the laboratory that performs the test