Friday, July 1, 2016

Article Reviews (2012 and 2013) - Laser microdissection and mass spectrometry-based proteomics

Today's post will be two article reviews. These two articles are very similar and also quite technical, so there is no need to cover them in separate posts.

Those of you following the always exciting monthly blog stats might like to know that May of 2016 had more new countries visit the blog than in all of 2015, and it also had the most new countries in one month since May of 2013. I do not think this sudden uptick is due to increased global awareness of fibrinogen amyloidosis, but is instead due to a more global infiltration of spammers and automated programs that register as visits. In any case, it's fun to keep track of it. On to the articles . . .

First article

Title: Laser microdissection and mass spectrometry-based proteomics aids the diagnosis and typing of renal amyloidosis

Authors: Sanjeev Sethi, Julie A. Vrana, Jason D. Theis, Nelson Leung, Anjali Sethi, Samih H. Nasr, Fernando C. Fervenza, Lynn D. Cornell, Mary E. Fidler and Ahmet Dogan (Mayo Clinic, Minnesota, USA and Drexel University College of Medicine, Pennsylvania, USA)

Journal: Kidney International (July 2012)

Accurate diagnosis and typing of renal amyloidosis is critical for prognosis, genetic counseling, and treatment. Laser microdissection and mass spectrometry are emerging techniques for the analysis and diagnosis of many renal diseases. Here we present the results of laser microdissection and mass spectrometry performed on 127 cases of renal amyloidosis during 2008–2010. We found the following proteins in the amyloid deposits: immunoglobulin light and heavy chains, secondary reactive serum amyloid A protein, leukocyte cell-derived chemotaxin-2, fibrinogen-a chain, transthyretin, apolipoprotein A-I and A-IV, gelsolin, and b-2 microglobulin. Thus, laser microdissection of affected areas within the kidney followed by mass spectrometry provides a direct test of the composition of the deposit and forms a useful ancillary technique for the accurate diagnosis and typing of renal amyloidosis in a single procedure.

Second article

Title: Mass Spectrometry Based Proteomics in the Diagnosis of Kidney Disease

Authors: Sanjeev Sethi, Julie A. Vrana, Jason D. Theis, and Ahmet Dogan (Mayo Clinic, Minnesota, USA)

Journal: Current Opinion in Nephrology and Hypertension (May 2013)

Purpose of review: Laser microdissection (LMD) and mass spectrometry (MS) is a new technique that consists of dissection of glomeruli, tryptic digestion of dissected material, analysis by MS and generation of a protein profile using different algorithms. The review focuses on the use of this methodology as an ancillary technique in a clinical laboratory for the diagnosis of kidney diseases.
Recent findings: LMD/MS is used in the diagnosis and typing of kidney diseases with organized deposits such as amyloidosis. Uncommon and familial forms of renal amyloidosis are diagnosed and typed on the basis of the presence of specific amyloidogenic proteins. LMD/MS is used to confirm and identify immunoglobulins and complement factors in immune complex mediated and complement-mediated proliferative glomerulonephritis, respectively. In particular, LMD/MS can detect monoclonal immunoglobulins in cases of equivocal immunofluorescence studies in monoclonal immunoglobulins-associated glomerulonephritis. LMD/MS can detect specific complement factors of the alternative pathway and terminal pathway in complement-mediated glomerulonephritis.
Summary: LMD/MS is currently used for diagnosis and typing of amyloidosis. In addition, LMD/MS is useful in determining the type of immunoglobulins and complement factors in immune complex and complement-mediated glomerulonephritis, respectively.

Here are the online links to these two articles if you would like to follow along: nephrolhypertens/Abstract/2013/05000/Mass_spectrometry_based_proteomics_in_the.5.aspx

As you can probably tell from the titles alone, and definitely from the abstracts, these two articles are a bit more technical in nature than most of the previously reviewed articles on the blog. Both articles go into quite a bit of detail on two relatively new techniques for analyzing tissue samples to determine the type of amyloidosis and, as discussed in the second article, to diagnose other kidney diseases. I will not be covering these two articles in any detail, but I will instead focus on the sections that are relevant to the diagnosis of fibrinogen amyloidosis.

First, some definitions of a few words in the titles:

Proteomics is the study of the entire complement of proteins produced by an organism. For the purposes of this discussion, humans are the organism.

Laser Microdissection is a technique for isolating and separating a specific portion of biopsy tissue. We have seen photos of biopsies stained with Congo Red, for instance, in which a well-defined portion of the tissue is that apple green color. Laser microdissection cuts out just that portion so it can be analyzed further.

Mass Spectrometry, in oversimplified terms, is a method of breaking down and analyzing a tissue sample to determine the protein components that are present. In the typical process of staining a biopsy with various stains (immunohistochemistry), each stain will react to a different protein. So if the pathologist is looking for 20 different proteins, 20 different stains must be applied. Mass spectrometry, on the other hand (often referred to a mass spec), is a process to identify all of the proteins with one process. It is a complicated, multi-step process that involves some sophisticated software to analyze the results. It is not an exact, precise method, however, as the results are given in terms of probabilities that each protein is present, and only those with a greater than 90% probability are considered to be identified.

The 2012 article is a retrospective analysis of 127 cases of renal amyloidosis analyzed by laser microdissection and mass spectrometry between 2008 and 2010 at Mayo Clinic. Of those 127 cases, seven were identified as fibrinogen amyloidosis. In the analysis of the fibrinogen cases, it was noted that both mutated and wild-type (non-mutated) fibrinogen were detected in the samples. If I understand that correctly, that means the amyloid deposits consist of both types of fibrinogen molecules. That is interesting because the data on AFib patients who have had liver transplants indicates that the wild-type fibrinogen does not add to the existing amyloid deposits (unlike in the case of ATTR). So maybe the presence of mutated fibrinogen allows for both mutated and wild-type fibrinogen to contribute to the buildup of amyloid deposits. But once there is no more circulating mutated fibrinogen, the wild-type fibrinogen can no longer attach to the existing deposits.

The Discussion section of the 2012 article lists several advantages of laser microdissection/mass spectrometry over the conventional methods of amyloid typing:

1. It is one test to identify the protein vs. a series of tests, as in immunohistochemistry.
2. It is performed on only the involved tissue (as identified by Congo red staining).
3. It is performed on paraffin-embedded material and does not require frozen material.
4. It is better at typing problematic cases, such as heavy-chain (AH) amyloidosis.
5. It is useful for typing familial types of amyloidosis.
6. Again regarding the familial types, it can identify the specific genetic mutation.
7. It can detect amyloid even before a tissue sample is positive for Congo red staining.
8. It can prevent misdiagnosis of familial cases of amyloid as AL or AH amyloid, especially in those cases where the familial amyloidosis patient also has a monoclonal gammopathy.

The 2013 article has much of the same information regarding laser microdissection and mass spectrometry. Since amyloidosis is only one of the five or six kidney diseases discussed in this article, it does not have as much detail on the various types of amyloidosis. But it does specifically mention fibrinogen amyloidosis as one of the hereditary types for which laser microdissection/mass spectrometry is very useful for determining the type.


At the familial amyloidosis meeting in Chicago last October, one of the doctors mentioned that Mayo Clinic does mass spec on all kidney biopsies. That seems like a good indicator that the doctors at Mayo are confident in the results of mass spec. I am sure it reduces the chances of misdiagnosis of amyloid type, and for the rare types it helps reach a diagnosis more quickly than would otherwise occur if they only used the older techniques.

The question for someone like me who has not yet had a kidney biopsy is this: If and when I do develop symptoms that warrant having a kidney biopsy, should I request that it be sent to Mayo Clinic for analysis by mass spec? I am leaning toward answering that question with a "yes," given how rare this disease is. Plus, it makes Mayo Clinic aware of the patient, which may help establish a dialog between Mayo Clinic and the patient's local doctors for future consultation.

In the next blog post I might have the results of my capsule endoscopy. Or maybe not.

=====Monthly Blog Status Update=====

As of May 31, 2016:

Total posts: 167 (1 in May)

Total pageviews: 41,300 (~1500 in May)

Email subscribers: 14 (unchanged)

Total number of countries that have viewed the blog: 123

Seven new countries viewed the blog in May:

Congo (DRC)
French Polynesia

(1) Sethi S, Vrana JA, Theis JD, et al. Laser microdissection and mass spectrometry-based proteomics aids the diagnosis and typing of renal amyloidosis. Kidney Int. 2012;82(2):226-234.

(2) Sethi S, Vrana JA, Theis JD, Dogan A. Mass spectrometry based proteomics in the diagnosis of kidney disease. Curr Opin Nephrol Hypertens. 2013.

Edit 7-1-16: Corrected country count in blog stats