Combined Use Of Laser Microdissection Proteomic Mass Spectrometry To Identify Systemic Amyloidosis Subtype

Background and objectivesAmyloidosis is a heterogenous disease, which is caused by extracellular deposition of proteins with an insoluble β-pleated conformation. Systemic amyloidosis can involve various visceral organs, resulting in one organ or multiorgan dysfunction. Diagnoses of different subtypes are based on the identification of amyloidogenic proteins. The most common forms of systemic amyloidosis include immunoglobuiin light-chain (AL) amyloidosis, serum amyloid A protein (AA) amyloidosis, transthyretin (ATTR) amyloidosis, apolipoprotein Al (AApoAl) amyloidosis, fibrinogen A-a chain (AFib) amyloidosis and β2-microglobulin (Aβ2M) amyloidosis. Accurate typing of amyloid deposits is of paramount importance because prognosis and treatment is depended on subtype diagnosis. Heart and kidney involvements are common in AL amyloidosis, and effective treatments include chemotherapy as well as stem cell transplantation. Hereditary or familial ATTR amyloidosis is also known of amyloid neuropathy, for which the first choice is liver transplantation. AA amyloidosis is secondary to chronic inflammatory or infectious diseases, so treatment of primary disease is the key point.Traditional antibody-based diagnostic methods such as immunohistochemistry and immunofluorescence are now widely used. However, drawbacks of those approaches including limited antibody availability and loss of epitopes recognized by the antibody impair sensitivity and specificity of diagnosis. Progresses in amyloidosis treatment have been hindered because of deficiencies of disease typing in our country.Recently, several studies showed that proteomic approaches aided identification of amyloidosis subtypes. Laser microdissection followed by mass spectrometry (LMD/MS) is a highly specific and sensitive novel test for disease typing, with great potential of describing the composition of amyloid deposits, which will provide valuable insights into disease mechanism. So far no relevant research has been reported in China.The objectives of this study are to establish a novel approach for amyloid typing by LMD/MS and to identify the subtypes of systemic amyloidosis diagnosed in Peking Union Medical College Hospital (PUMCH) during the past thirteen years. Materials and methods72specimens from different tissues in patients who were diagnosed as systemic amyloidosis in PUMCH from2000to2013were analyzed. The Institutional Review Board at PUMCH approved the study. Briefly, for each case,10μm-thick sections of formalin-fixed paraffin-embedded tissues were collected, then paraffin was removed from each sample and tissues were stained with Congo Red. Congo Red-positive areas were indentified under fluorescent light and microdissected with LMD. Through standard process proteins extracted from target area were digested into tryptic peptides and analyzed by mass spectrometry. MS raw data files were queried with Mascot algorithm and results were assigned peptide and protein probability scores in Scaffold (Proteome Software, Portland, OR). Peptide identifications were accepted if they could be established at>95%probability. A higher value of emPAl is indicative of greater protein abundance. Typing of amyloidosis is then based on the greatest abundance of protein that corresponds to the specific type of amyloid.ResultsAmyloid types of65cases were identified by LMD/MS method with sensitivity of90.3%(65/72cases).7cases failed in LMD/MS typing because of microdissection failure. The diagnosis of amyloidosis was excluded for one patient by LMD/MS for the absence of serum amyloid protein P. Diagnostic coincidence rate of LMD/MS method with immunofluorescence technique was100%(4/4cases). For amyloidosis along with multiple myeloma and macroglobulinemia, the coincidence rate of LMD/MS subtype with serum monoclonal immunoglobulin light chain was also100%(12/12cases). In this study,24cases of AL-κ (37.5%),33cases of AL-λ(51.6%),4cases of ATTR (6.2%),2cases of AA (3.1%) and1case of AH (1.6%) amyloidosis were diagnosed. Compared with proteomic approach, immunohistochemistry showed lower diagnostic accuracy of12.5%in amyloid typing. All the patients with serum monoclonal immunoglobulin were diagnosed as AL subtype through LMD/MS approach. None of the AA or ATTR cases were found in patients with M protein. Serum free light chain analysis was more sensitive than serum protein electrophoresis and immunofixation electrophoresis in M protein detection and more effective for the diagnosis of AL amyloidosis. ConclusionThis study showed that LMD and MS-based proteomic analysis was a useful and powerful technique for accurate typing of amyloidosis, with higher sentivity and specificity than traditional immunohistochemistry method. It’s an ideal diagnostic tool for amyloidosis and with the most potential ability to be a gold standard for amyloid typing.