Mast cells in the GI tract: How many is too many? (Part eight)

One study assessed whether mast cell count would be influenced depending on which part of the organ biopsies were taken from. While the difference in count was not large, it is worth considering that these counts all straddle the cut off of 20 mast cells/hpf.  This means that patients with the same GI symptoms could have biopsies with over or under 20/hpf depending on the site of the biopsy.  See Table 24 for details.

Table 24: Effect of sampling site on mast cell count/hpf in colon of chronic diarrhea patients
Zare-Mirzaie A, et al. Analysis of colonic mucosa mast cell count in patients with chronic diarrhea. Saudi J Gastroenterol 2012; 18 (5): 322-326.
Microscopy method: 400x magnification, mast cells counted in 5 hpf and averaged
Visualization: Tryptase (IHC), toluidine blue
Rectum Sigmoid Descending colon Transverse colon Ascending colon Cecum
20.5±5 18.3±3.5 22.6±3.9 20.7±4.9 25.5±6.7 22.1±4.9

 

The same paper also looked at effect of season on mast cell count.  There was no significant difference, but again, the range of biopsies in each season straddles the 20/hpf line. See Table 25 for details.

Table 25: Effect of season on mast cell count in colon of diarrhea patients
Zare-Mirzaie A, et al. Analysis of colonic mucosa mast cell count in patients with chronic diarrhea. Saudi J Gastroenterol 2012; 18 (5): 322-326.
Microscopy method: 400x magnification, mast cells counted in 5 hpf and averaged
Visualization: Tryptase (IHC), toluidine blue
Spring Summer Fall Winter
20.6±4.7 24.2±4.9 19.5±3.9 20.3±4.9

 

The most telling portion of this study compared mast cell counts when using a simple stain (toluidine blue) and when using IHC (antibody for tryptase) to find mast cells in biopsies.  Mast cells are not easy to see on biopsy.  They require special stains, and even then, they are hard to see.  Immunohistochemistry (IHC) uses antibodies to identify markers on cells that are easier to see with a microscope.  It is not uncommon for unfamiliar doctors to refuse the use of IHC testing (which usually includes CD117, CD25, CD2 or tryptase) in lieu of commonly available stains in the lab.  However, even stains that visualize mast cells are inferior to IHC methods.  In biopsies taken from all parts of the colon, toluidine blue staining showed less than half of the mast cells visualized using IHC for tryptase.  This means that when IHC testing isn’t ordered, counts reported by simple staining are much lower than the true count. See Table 26 for details.

Table 26: Comparison of mast cell count in biopsies stained with toluidine blue and with tryptase antibody (IHC)
Zare-Mirzaie A, et al. Analysis of colonic mucosa mast cell count in patients with chronic diarrhea. Saudi J Gastroenterol 2012; 18 (5): 322-326.
Microscopy method: 400x magnification, mast cells counted in 5 hpf and averaged
Visualization: Tryptase (IHC) and toluidine blue
Staining method Rectum Sigmoid Descending colon Transverse colon Ascending colon Cecum
IHC 20.5±5 18.3±3.5 22.6±3.9 20.7±4.9 25.5±6.7 22.1±4.9
Toluidine blue 8.5±0.7 6.8±1.2 10.3±4.2 10.3±3.5 12.5±5 8.1±2.9
% of cells identified by IHC seen by toluidine blue staining 41% 37% 46% 50% 49% 37%

 

There are other factors that contribute to lack of consensus in mast cell counts in GI tissue. One of the biggest causes is that not all labs use standard size high powered fields.  HPF is usually 0.25mm2, but it is not uniform throughout the research world.  Many papers don’t even provide the size of their high powered fields.  More than that, many papers report mast counts per mm2 without providing conversion factors so it’s not always possible to compare results from one paper to another.  There were some papers I wanted to use for this series that I couldn’t because I couldn’t be sure that I could convert their mast cells/mm2 confidently to mast cells/hpf.

Together with the fact that number of hpf counted, methods of biopsy slide preparation, stains and IHC antibodies are variable, it is hard to get a real understanding of whether the cut off of 20 mast cells/hpf is meaningful.  It is my finding that there are a number of conditions that cause mast cells/hpf to be higher than controls in an experiment.  It is also my finding that in some experiments, control subjects have baseline mast cell counts over 20 mast cells/hpf. It is reasonable to assume that inflammatory GI conditions can cause mast cell hyperplasia.  But the fact that chronic urticaria patients often have mast cell counts higher than control subjects is also telling.  It speaks to the fact that an allergic process can elevate mast cell counts in a space where there is no appreciable symptomology. If patients have reactions to “pseudoallergens” as described in that paper, then it is possible that these reactions could drive the increase in mast cell count in the GI tract.  If this is true, then the many mast cell patients who have “pseudoallergen” responses could see an increase in GI mast cell burden as a result of their mast cell disease.

References:

Jakate S, et al. Mastocytic enterocolitis: Increased mucosal mast cells in chronic intractable diarrhea.  Arch Pathol Lab Med 2006; 130 (3): 362-367.

Akhavein AM, et al. Allergic mastocytic gastroenteritis and colitis: An unexplained etiology in chronic abdominal pain and gastrointestinal dysmotility. Gastroenterology Research and Practice (2012): Article ID 950582.

Martinez C, et al. Diarrhoea-predominant irritable bowel syndrome: an organic disorder with structural abnormalities in the jejunal epithelial barrier. Gut 2013; 62: 1160-1168,

Sethi A, et al. Performing colonic mast cell counts in patients with chronic diarrhea of unknown etiology has limited diagnostic use. Arch Pathol Lab Med 2015; 139 (2): 225-232.

Doyle LA, et al. A clinicopathologic study of 24 cases of systemic mastocytosis involving the gastrointestinal tract and assessment of mucosal mast cell density in irritable bowel syndrome and asymptomatic patients. Am J Surg Pathol 2014; 38 (6): 832-843.

Ramsay DB, et al. Mast cells in gastrointestinal disease. Gastroenterology & Hepatology 2010; 6 (12): 772-777.

Zare-Mirzaie A, et al. Analysis of colonic mucosa mast cell count in patients with chronic diarrhea. Saudi J Gatroenterol 2012; 18 (5): 322-326.

Walker MM, et al. Duodenal mastocytosis, eosinophilia and intraepithelial lymphocytosis as possible disease markers in the irritable bowel syndrome and functional dyspepsia. Aliment Pharmacol Ther 2009; 29 (7): 765-773.

Hahn HP, Hornick JL. Immunoreactivity for CD25 in Gastrointestinal Mucosal Mast Cells is Specific for Systemic Mastocytosis. American Journal of Surgical Pathology 2007; 31(11): 1669-1676.

Vivinus-Nebot M, et al. Functional bowel symptoms in quiescent inflammatory bowel diseases : role of epithelial barrier disruption and low-grade inflammation. Gut 2014; 63: 744-752.

Minnei F, et al. Chronic urticaria is associated with mast cell infiltration in the gastroduodenal mucosa. Virchows Arch 2006; 448(3): 262-8.

Hamilton MJ, et al. Mast cell activation syndrome: A newly recognized disorder with systemic clinical manifestations. J Allergy Clin Immunol 2011; 128: 147-152.

Barbara G, et al. Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome. Gastroenterology 2004; 126(3): 693-702.

Guilarte M, et al. Diarrhoea-predominant IBS patients show mast cell activation and hyperplasia in the jejunum. Gut 2007; 56: 203-209.

Dunlop SP, et al.  Age related decline in rectal mucosal lymphocytes and mast cells. European Journal of Gastroenterology and Hepatology 2004; 16(10): 1011-1015.

Afrin LB, Molderings GJ. A concise, practical guide to diagnostic assessment for mast cell activation disease. World J Hematol 2014; 3 (1): 1-17.

Molderings GJ, et al. Mast cell activation disease: a concise, practical guide to diagnostic workup and therapeutic options. J Hematol Oncol 2011; 4 (10).

Akin C, et al. Mast cell activation syndrome: proposed diagnostic criteria. J Allergy Clin Immunol 2010; 126 (6): 1099-1104.

Valent P, et al. Definitions, criteria and global classification of mast cell disorders with special reference to mast cell activation syndromes: a consensus proposal. Int Arch Allergy Immunol 2012: 157 (3): 215-225.

4 Responses

  1. Mark July 10, 2016 / 4:25 am

    Appreciate much of your incisive analysis, Lisa. I’m nonetheless left w/ a few Qs/comments.

    1. The NIH authority/Office of Rare Diseases HAS embraced the 20 MCs/high power field as its decisive consensus criterion for Mastocytic Enterocolitis. Whatever persisting debate is challenging our reigning authority on this disease.

    2. >”chronic urticaria patients often have mast cell counts higher than control subjects is also telling. It speaks to the fact that an allergic process can elevate mast cell counts in a space where there is no appreciable symptomology. . . . []The many mast cell patients who have “pseudoallergen” responses could see an increase in GI mast cell burden as a result of their mast cell disease.”

    Right, Lisa. So doesn’t this suggest CU often IS a manifestation of a Systemic Mast Cell Disorder? Many CU patients can develop related mediator symptoms outside the skin and GI tract — even if not all such data get published. I was told by an impeccable source, for example, that low psuedoallergen diets DO typically help the substantial minority of responsive CU patients w/ any GI, asthma symptoms related to their CU. Alas, I’m afraid these data will never be published.

    FWIW, I have read many patient accounts besides mine consistent w/ these unpublished data. Conversely, I don’t recall accounts of CU patients w/ related mediator symptoms that did NOT improve when dietary measures substantially benefitted their urticaria. FWIW.

    3. The NIH’s (Indolent) SM protocols have typically turned on “increased mast cell number” in BMB, or some such. This, of course utterly dispenses w/ the WHO criteria: none can be satisfied or ruled out by sheer MC numbers. Consistent, though w/ Dr. Akin presenting / a slide picturing BM tissue stained for tryptase that he said was “diagnostic” for mastocytosis. This despite satisfying only a single “minor” WHO criterion — >25% spindle-shaped MCs. Lots, many, degranulated. But no clusters; yet “diagnostic” for SM, TMS conference attendees were told, no matter what analysis for other WHO minor criteria would/would not detect.

    I have encountered no authority establishing a threshold for an “increased number” of MCs in BMB. Have you? Anyone? Certainly not in any NIH protocol SM I’ve seen despite turning on some such criterion w/ no regard for any WHO ISM criterion.

    4. >”an allergic process can elevate mast cell counts in a space where there is no appreciable symptomology”. Indeed. Albeit my understanding is that “pseudoallergens” can trigger gut MCs typically absent any apparent IgE-mediated “true” allergy. Again, however, is this not common w/ SM patients. How many manifest marrow symptoms, notwithstanding BM MC pathology?

    • Lisa Klimas July 10, 2016 / 1:56 pm

      Thanks for the comments, I will come back to them when I can carve out some time for in depth answers.

      Regarding the 20 mc/hpf cutoff, if the NIH has decided that is indeed high, I suggest they publish on it. I also suggest they publish the standard size for a hpf because having no enforced definition for that term is part of why we are in this pickle. Until it is published upon, it is just speculation. If NIH publishes their support for this idea, I am more than open to having my mind changed. Unity is always helpful.

      • Mark July 10, 2016 / 6:12 pm

        Largely agreed, Lisa, as far as it goes. I wasn’t aware of the issue w/ HPF standardization or lack thereof. Again, though, how have this and some other aspects of your critique been addressed — let alone adequately — w/ BMBs?

        Sorry I somehow blundered in leaving out the link to the NIH’s Mastocytic Enterocolitis webpage.
        https://rarediseases.info.nih.gov/gard/10176/mastocytic-enterocolitis/resources/1

        While I surmise Congress has not have appropriated the Office of Rare Diseases funding adequate to conduct or sponsor further research itself on ME, you will see they DO cite authority. Perhaps nothing you don’t address in your critique, other than their definitive assessment, interpretation. Not sure to what

        Given those, however, why shouldn’t the burden fall on other researchers, orgs, to definitely refute the NIH’s established criteria for ME w/ the decisive 20 MCs/HPF criterion. I don’t recall whether/what you discuss with similar patients diagnosed w/ diarrhea-predominent Irritable-Bowel Syndrome. An additional/overrlapping body of research including one of the 2 publicastions cited on the NIH webpage. How they would each stack up to your critique I don’t know.

        On the other hand, my impression is that MC lamina propia elevations are most likely to be detected in the ileum: the vey part of the GI tract especially clinicians, even funded researchers, are least likely to biopsy. Plus, seem to be next to no data how many MCs may be missed by any stain — if degranulated, no tryptase , no CD117/KIT (receptor). Or varieties of MCs that can be found, can differ depending on ?? in different people/creatures, different organs.

        • Lisa Klimas July 10, 2016 / 8:08 pm

          But where is the supporting data?

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