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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)
TTR Gene, Full Gene Analysis:
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)
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