Amyloidosis, Transthyretin-Associated Familial, Reflex, Blood
NY State Approved Indicates the status of NY State approval and if the test is orderable for NY State clients.
Diagnosis of adult individuals suspected of having transthyretin-associated familial amyloidosis
Genetics Test Information Provides information that may help with selection of the correct test or proper submission of the test request
Mass spectrometry to evaluate transthyretin (TTR) protein structure is performed first. In all cases demonstrating a structural change, the TTR gene will be further analyzed by DNA sequence analysis. If no alterations are detected, subsequent gene analysis will not be performed unless a request for AMYL/83667 Amyloidosis, Transthyretin-Associated Familial, DNA Sequence, Blood is submitted by the ordering physician or client.
Reflex Tests Lists test(s) that may or may not be performed, at an additional charge, depending on the result and interpretation of the initial test(s)
|Test ID||Reporting Name||Available Separately||Always Performed|
|AMYL||Familial Amyloidosis, DNA Sequence||Yes||No|
Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.
If familial amyloidosis by liquid chromatography-mass spectrometry is abnormal, DNA sequence will be performed and charged separately.
See Amyloidosis (Familial) Test Algorithm in Special Instructions.
Special Instructions and Forms Describes specimen collection and preparation information, test algorithms, and other information pertinent to test. Also includes pertinent information and consent forms to be used when requesting a particular test
Liquid Chromatography-Mass Spectrometry (LC-MS)
Reporting Name A shorter/abbreviated version of the Published Name for a test; an abbreviated test name
Familial Amyloidosis Reflex
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 96 hours of draw.
Preferred: Lavender top (EDTA)
Specimen Volume: 3 mL
1. Invert several times to mix blood.
2. Send specimen in original tube.
1. 1. New York Clients-Informed consent is required. Please document on the request form or electronic order that a copy is on file. An Informed Consent for Genetic Testing (Supply T576) is available in Special Instructions.
2. 2. If not ordering electronically, submit a Biochemical Genetics Request Form (Supply T439) with the specimen.
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.
Mild OK; Gross OK
Mild OK; Gross OK
Mild OK; Gross OK
Specimen Stability Information Provides a description of the temperatures required to transport a specimen to the laboratory. Alternate acceptable temperature(s) are also included.
|Whole blood||Refrigerated (preferred)||4 days|
Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test
The systemic amyloidoses are a group of diseases that result from the abnormal deposition of amyloid in various tissues of the body. They have been classified into 3 major types: primary, secondary, and hereditary. The most common form of amyloidosis (AL) is a disease of the bone marrow called primary systemic AL (immunoglobulin light chain). Secondary AL usually occurs in tandem with chronic infectious or inflammatory diseases, such as rheumatoid arthritis, tuberculosis, or osteomyelitis. Familial or hereditary AL is the least common form. Determining the specific type of AL is imperative in order to provide both an accurate prognosis and appropriate therapies.
Familial or hereditary transthyretin AL is an autosomal dominant disorder caused by mutations in the transthyretin gene (TTR). The resulting amino acid substitutions lead to a relatively unstable, amyloidogenic transthyretin (TTR) protein. Most individuals begin to exhibit clinical symptoms between the third and seventh decades of life. TTR-associated AL is progressive over a course of 5 to 15 years and usually ends in death from cardiac or renal failure or malnutrition. Affected individuals may present with a variety of symptoms including peripheral neuropathy, blindness, cardiomyopathy, nephropathy, autonomic nervous dysfunction, and bowel dysfunction.
More than 90 mutations that cause TTR-associated familial AL have now been identified within the TTR gene. Most of the mutations described to date are single base pair changes that result in an amino acid substitution. Some of these mutations correlate with the clinical presentation of AL.
The Division of Laboratory Genetics recommends a testing strategy that includes both protein analysis by mass spectrometry (MS) and TTR gene analysis by DNA sequencing (AMYL/83667 Amyloidosis, Transthyretin-Associated Familial, DNA Sequence, Blood) for patients in whom TTR-associated familial AL is suspected. The structure of TTR protein in plasma is first determined by MS. Only the transthyretin (also known as prealbumin) is analyzed for amino acid substitutions. Other proteins known to be involved in other less common forms of familial amyloidosis are not examined. If no alterations are detected, gene analysis will not be performed unless requested by the provider (ie, when the diagnosis is still strongly suspected; to rule-out the possibility of a false-negative by MS). In all cases demonstrating a structural change by MS, the entire TTR gene will be analyzed by DNA sequence analysis to identify and characterize the observed alteration (gene mutation or benign polymorphism).
For predictive testing in cases where a familial mutation is known, testing for the specific mutation by DNA sequence analysis (AMYKM/83705 Amyloidosis, Transthyretin-Associated Familial, Known Mutation) is recommended. These assays do not detect mutations associated with non-TTR forms of familial AL. Therefore, it is important to first test an affected family member to determine if TTR is involved and to document a specific mutation in the family before testing at risk individuals.
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.
An interpretive report will be provided.
The presence of a structural change in transthyretin (TTR) is suggestive of a gene mutation that requires confirmation by DNA sequence analysis. A negative result by mass spectrometry does not rule-out a TTR mutation. Mass spectrometric (MS) results are falsely negative if the amino acid substitution does not produce a measurable mass shift for the mutation transthyretin. Approximately 90% of the TTR mutations are positive by MS (see Cautions).
After identification of the mutation at the DNA level, predictive testing for at-risk family members can be performed by molecular analysis (AMYKM/83705 Amyloidosis, Transthyretin-Associated Familial, Known Mutation).
Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances
There are 3 circumstances where testing by mass spectrometry will not identify amyloid-causing mutations:
-If the amino acid change results in a protein different by <10 atomic mass units (amu), the mutation will not be reliably detected.
-If an amino acid change results from a frequent nondisease-causing mutation (+30 amu). Since over 12% of the population has this innocuous polymorphism, it is an instance in which molecular testing must be done.
-Coinheritance of the polymorphism with a -30 amu mutation would also result in a transthyretin mass indistinguishable from normal.
Mass spectrometry was performed on 107 control specimens along with 18 specimens with known TTR mutations. Of the 18 mutants detected, 11 corresponded to a common benign polymorphism and 7 corresponded to a pathogenic mutation.
Clinical Reference Provides recommendations for further in-depth reading of a clinical nature
1. Benson MD: The hereditary amyloidoses. Best Pract Res Clin Rheumatol 2003;17:909-927
2. Shimizu A, Nakanishi T, Kishikawa M, et al: Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis. J Chromatogr B Analyt Technol Biomed Life Sci 2002 Aug 25;776(1):15-30
3. Lim A, Prokaeva T, McComb ME, et al: Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis. Anal Chem 2002 Feb 15;74(4):741-751
4. Boston University School of Medicine, BUSM. Mass Spectrometry Resource. Available from URL: bumc.bu.edu/Dept/Home.aspx?DepartmentID=354
5. Eneqvist T, Sauer-Eriksson AE: Structural distribution of mutations associated with familial amyloidotic polyneuropathy in human transthyretin. Amyloid 2001;8:149-168
Method Description Describes how the test is performed and provides a method-specific reference
Familial Amyloidosis, Mass Spectrometry (MS):
Transthyretin (TTR) is purified from plasma using affinity chromatography. The chromatography is done using an antihuman-TTR antibody that has been coupled to POROS-aldehyde media. Plasma is reduced with 12.5 mM dithiothreitol (DTT) to simplify the mass spectra by removing Cys10 adducted species. The plasma:DTT solution is then injected onto the affinity column, which sequesters TTR using pH 7.4 phosphate buffer saline. TTR is then eluted from the affinity column with pH 2.5 100 mM glycine:2% acetic acid buffer and concentrated on a C4 column, which is then washed with acetic acid:methanol:water (1:2:97) to remove excess phosphate and other buffer components that suppress MS response. TTR is then eluted from the C4 column and introduced to the MS using methanol:water:glacial acetic acid:TFA (94.5:5:0.5:0.04). The acquired ion spectra are deconvoluted and reviewed for TTR variants. After deconvolution, normal patients present with a single peak corresponding to wild-type (wt) TTR, which serves as a reference. When positive, amyloid patients are typically heterozygous and are detected by the presence of 2 peaks (ie, wt TTR and mutant TTR) differing in mass.(Bergen HR 3rd, Zeldenrust SR, Butz ML, et al: Identification of transthyretin variants by sequential proteomic and genomic analysis. Clin Chem 2004 Sep;50:1544-1552)
Familial Amyloidosis, DNA Sequence:
All 4 exons of the TTR gene are amplified by PCR and then subjected to direct DNA sequence analysis.(Bergen RH 3rd, Zeldenrust SR, Butz ML, et al: Identification of transthyretin variants by sequential proteomic and genomic analysis. Clin Chem 2004;50:1544-1552)
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; 8 a.m.
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.
This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the U.S. 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 Medical Laboratories interpretation of CPT coding requirements. It is the responsibility of each laboratory to determine correct CPT codes to use for billing.
83788-Mass spectrometry-tandem mass spectrometry (MS-MS/MS)
81404-TTR (transthyretin) (eg, familial transthyretin amyolidosis), full gene sequence (if appropriate)
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|
|22668||Wild Type Mass||In Process|
|22669||Wild Type Width at Half Height||In Process|
|22670||Second Mass||In Process|
|22671||Mass Difference||In Process|
|22673||Abnormal result||In Process|