Auteur Topic: Artikel over interpretatie Marsh classificatie (hoofdauteur M.J. Marsh)  (gelezen 640 keer)


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Volledig artikel - hoofdauteur is Michael N. Marsh.

Published by MDPI AG, Basel, Switzerland
MDPI – Open Access
Nutrients 2017, 9(3), 213; doi:10.3390/nu9030213

Evolutionary Developments in Interpreting the Gluten-Induced Mucosal Celiac Lesion:
An Archimedian Heuristic

Michael N. Marsh 1,* and Calvin J. Heal 2
1  Luton and Dunstable Hospitals University NHS Trust, and Wolfson College, University of Oxford, Linton Road, Oxford OX2 6UD, UK
2 Centre for Biostatistics, Faculty of Biology, Academic Health Science Centre, University of Manchester, Manchester M13 9PL, UK
* Correspondence: Tel.: +44-1608-8100-29
   Received: 19 December 2016 / Accepted: 22 February 2017 / Published: 28 February 2017

The evolving history of the small intestinal biopsy and its interpretation—and misinterpretations—are described in this paper.
Certain interpretative errors in the technical approaches to histological assessment are highlighted—even though we may never be rid of them.

For example, mucosal “flattening” does not reduce individual villi to their cores, as still seems to be widely believed. Neither is the mucosa undergoing an atrophic process—since it can recover structurally.
Rather, the intestinal mucosa manifests a vast hypertrophic response resulting in the formation of large plateaus formed from partially reduced villi and their amalgamation with the now increased height and width of the inter-villous ridges: this is associated with considerable increases in crypt volumes.
Sections through mosaic plateaus gives an erroneous impression of the presence of stunted, flat-topped villi which continues to encourage both the continued use of irrelevant “atrophy” terminologies and a marked failure to perceive what random sections through mosaic plateaus actually look like.

While reviewing the extensive 40+ year literature on mucosal analysis, we extracted data on intraepithelial lymphocytes (IEL) counts from 607 biopsies, and applied receiver-operating characteristic (ROC)-curve analysis.
From that perspective, it appears that counting IEL/100 enterocyte nuclei in routine haematoxylin and eosin (H&E) sections provides the most useful discriminator of celiac mucosae at histological level, with an effective cut-off of 27 IEL, and offering a very high sensitivity with few false negatives.
ROC-curve analysis also revealed the somewhat lesser accuracies of either CD3+ or γδ+ IEL counts.

Current official guidelines seem to be somewhat inadequate in clearly defining the spectrum of gluten-induced mucosal pathologies and how they could be optimally interpreted, as well as in promoting the ideal manner for physicians and pathologists to interact in interpreting intestinal mucosae submitted for analysis.
Future trends should incorporate 3-D printing and computerised modelling in order to exemplify the subtle micro-anatomical features associated with the crypt-villus interzone.
The latter needs precise delineation with use of mRNA in-section assays for brush border enzymes such as alkaline phosphate and esterase.

Other additional approaches are needed to facilitate recognition and interpretation of the features of this important inter-zone, such as wells, basins and hypertrophic alterations in the size of inter-villous ridges.
The 3-D computerised models could considerably expand our understandings of the microvasculature and its changes—in relation both to crypt hypertrophy, in addition to the partial attrition and subsequent regrowth of villi from the inter-villous ridges during the flattening and recovery processes, respectively.

1. Introduction

2. Early (Mis-) Interpretations of Intestinal Biopsies

3. The Immunological Functions of Intestinal Mucosa

4. Re-Evaluating Intraepithelial Lymphocyte (IEL) Counts Derived from the Existing Literature

5. Objective (Computerised) Measurements of Intestinal Mucosa

6. Classification of Mucosal Remodelling: A Major Hypertrophic Process

7. Interpreting the Marsh Classification
The changes noted in this diagram (Figure 5) as the mucosa progresses from villous infiltration to flattening is illustrated (Figure 5H) by appropriate diagrams (Marsh Stages I through III).
It is to this classification of the mucosal changes that we now pass.

7.1. So-Called “Non-Specificity” of the Marsh I and II Lesions
Many have dismissed early Marsh I and II lesion as ‘non-specific’ [69,70].
On the other hand, there are those who have understood that Marsh I/II lesions should be investigated prospectively [31], thus to exclude true glutensensitivity: as these authors summarise—’a raised IEL count with normal villous architecture is of sufficient clinical importance to be highlighted in routine duodenal biopsy reports’.

To clarify this position for histopathologists, a series of differential diagnoses has been set out by the Bucharest Consensus [71], under the terminological umbrella of “microscopic enteritis”.
It is to be hoped that these widelyagreed guidelines will be recognised and employed. And within a family setting and DQ 2/8 haplotypes, the possibility of celiac disease remains a high probability.
Individuals with these mucosal changes should be closely followed up, or even treated [72], particularly if they have disabling symptoms associated with malabsorption of important nutrients.

Despite a lesser mucosal involvement there is often considerable abdominal symptomatology and pain, osteoporosis and iron deficiency anemia, features surely necessitating a gluten-free diet—even only if a defined, agreed, short-term trial to monitor clinical response and reversal of malabsorptive defects is undertaken.
Given the growing literature, it is now unacceptable to refuse a diet on the grounds that the mucosa is not flat.
By now, it should be widely recognised that there is neither a specific, nor certainly a uniquely related diagnostic mucosal change.

7.2. Irrelevance of the Marsh III Sub-Classification
The division [73] of the Marsh III lesion into three subdivisions (a, b, c), as a “guide to histopathologists” has been widely, but surprisingly uncritically, employed.
This proposed analytical system is a failure because of the following flaws:
Absence of appropriate criteria: these subdivisions were never precisely defined morphologically as verification of the proposed subdivisions. It is interesting to envisage how (and why) so many histologists thought they were identifying real structures.
Even the micrographs illustrated in a later publication [74] written by histopathologists, for the help of other histologists, failed to correspond to the originals, again demonstrative of the subjective nature of the whole scheme.

Oberhuber’s approach has now been further degraded by additional studies:
morphological—which highlight the misinterpretations of sectioned mosaic plateaus as supposedly representing ‘blunted’, ‘degenerate’ ‘villi’ [75];

immunohistochemical—demonstrating that varied sub-immunophenotype IEL are equally represented in each subdivision, when their density should have increased with the worsening histological picture alleged to represent each successive stage: a, b, c [76];

mathematical—the regression equations employed by Charlesworth and colleagues failed to identify the a,b,c subgrades as valid entities for improved pathological recognition [77];

clinical—there appear to be no published accounts in which a gastroenterologist necessarily had to rely, ultimately and crucially, on the pathologist’s sub-classification of the relevant mucosal biopsy in order to facilitate diagnosis, treatment, or offer a prognosis for the patients concerned;

generalised usage—finally, given the failure of this attempted reclassification, it seems to follow that more recently revised classifications of Marsh were based, however, on these sub-divisions, offering no further decisive clarity.
In fact, they could be said to increase complexity and interpretational difficulties. For example, from a review of relevant papers published over the last decade, it is abundantly clear that these recent contenders for the job have not surfaced either as being more useful, more acceptable, or more easily employed. The original classification is as simple as could be.

7.3. The “Normal” Mucosa
Finally, we come to the interpretation of the ‘normal’ (Marsh Stage 0) mucosa.
One problem concerns origins of specimens—from referred, symptomatic patients or apparently healthy individuals.
There are differences—but which nowadays are rarely considered or explored (see last paragraph: Immunological Function of Mucosa, above). Second, ‘normality’ is no longer defined, although from early times, villi were seen as long, pencil-shaped structures 350–600 μm in height [9].

Overriding those relevant considerations, however, is the recognition that ‘normal’ mucosae, viewed histologically, may be consistent with gluten sensitivity, harbouring abnormalities requiring additional but difficult technologies for detection, including immunofluorescence of anti-TG antibodies on epithelial and microvascular basement membranes [78]; transmission EM detection of necrotic enterocytes [79,80], or assays of fatty acid binding protein as presumptive indicator of cell death [72].

7.4. Failures in Understanding the Marked Hypertrophic Remodelling Response
The problems arising from the sub-classification of the Marsh III lesion stem from the continuing belief that mucosal flattening strips every single villus down to the crypt-villus border, supposedly considered the end-stage of a progressive, atrophic process.
There is no morphologic evidence for that presumption. The changes that involve most of the mucosa (excepting epithelium) represent the effects of considerable remodelling, embodying a vast hypertrophic response in terms of the upward growth and enlargement of the inter-villous ridges, and their amalgamation with partially reduced villi to create irregular mosaic plateaus over the mucosal surface, with height elevations of ~200 μm.

The hypertrophic response is further exemplified by the vast increase in the size of the crypts, their infiltration by a population of large IEL, and the increased dynamic of the ascending enterocyte column in its movement towards the surface. The lamina also swells to twice its volume due to extravasation of plasma fluid through the inflamed capillaries, and great increases in the bulk of infiltrating cells.
This is a complex epithelial-mesenchymal response indeed, and a markedly dynamic hypertrophic response to gluten.

It seems that this end-phase of mucosal flattening is not generally wellunderstood. As a result, random sections through the mosaic plateaus create a variety of appearances which histologically are invariably taken to represent stunted or branched villi. Surface microscopy, however, does not reveal the presence of any villi, so these structures seen two-dimensionally merely reflect the many possibilities on offer when a mosaic plateau is observed in any random section.

This state-of-affairs is scarcely helped by current, expert guidelines [81,82] whose authors collectively provide no incisive practical outcomes from the literature. The guidelines signally fail to engender the vital cooperative understandings required between pathologist and clinician regarding mucosal interpretation.
In fact, these guidelines do not confidently explore the full spectrum of mucosal abnormalities of gluten-induced mucosal change, being more at ease with “atrophy” and the flat lesion.
As a result (a) they tend to dismiss all other preliminary phase transitions as “non-specific”; (b) rely on traditional definitional criteria—that is, ”atrophy”—resulting in a flat mucosa and (c) are hesitant to recommend a gluten-free diet without that latter criterion, despite a very large literature to the contrary [31,32,69,70,71,72,80,83].

There is a pressing need to reconstruct biopsies with computerised programmes, using either the systems of indices and matrices employed in computer-assisted design, or by employing 3-D printing.
Such approaches would further expand our understandings of the mucosa, and its internal changes, especially where the remodelled microvasculature is concerned.

If we knew more about the effects of gluten on the small vessels and how they influence the hypertrophic responses throughout the mucosa, we might be in a more enviable position to understand how these changes come about—both in their association with flattening as much as with regrowth. There is much to be re-remembered from the past, organised from the present, and planned for the future [15].

8. Afterword
Ptolemy may have been a little disgruntled when his geocentric theory was overtaken by the more ambitious heliocentric-based Copernican view of the universe. Yet it hardly seems time to declare that celiac disease has become so universalised that the intestinal tract has been side-stepped and no longer plays a central role in furthering insights into the disease: that seems to us to be a misleading—if not premature—conclusion.

From all this it should be clearly understood that:
1.   there are no (immuno)histologically unique diagnostic features for celiac disease that “absolutely” distinguish it from other mucosal enteropathies or more importantly, disease-control biopsies;

2.   the spectre of the “normal” mucosa, but which is consistent with true gluten sensitivity, remains a difficult problem to deal with, including its redefinition;

3.   there is considerable overlap between the populations of celiac intraepithelial lymphocyte (IEL) and controls (Figure 2)—regardless of the identifying technique used;

4.   IEL populations do not comprise two separate populations (bimodal), but represent graded biological outcomes (to luminal antigens), analogous to height, weight, blood pressure or acid secretion (Figure 3);

5.   additionally detailed studies of the dose-response characteristics of the CD3− innate pool of IEL, and their CD127+ and CD127− components may bring new insights to diagnosis and mucosal interpretation;

6.   ROC curve analysis (Figure 4 and Table 2) provides usable answers which overcome the immense numerical overlapping between IEL populations, including CD3+ and γδ+ cells, and removes to a great extent the inherent uncertainty, engendered with numerical counts, as to where to draw the cut-off;

7.   log-transformation of the skewed celiac data does not produce means which materially differ from the numerical means (data not shown). Together, these results confirm that histopathologists do not need to log-transform their numerical counts, and that IEL counts in routine hematoxylin and eosin (H&E)sections can now be seen as a very easy and resourceful way of defining one’s cut-off, provided receiver-operating characteristic (ROC) curve analysis is additionally carried out;

8.   there is a vast cavern between high-level research still needed in continued interrogations of the mucosal response to gluten ingestion, and the somewhat more unsophisticated approaches deployable at histopathological level during routine diagnostic service work.
Notwithstanding those difficulties, the tracing of the historical development of our understandings of the structure and functioning of the small intestinal mucosa is a truly fascinating story.
Our own view is that the mucosa still occupies a very central role in diagnosis and, together with related research, into its response to gluten peptides.

There is a long list of historic figures who have welded the story of the intestinal mucosa into one which still causes dissent, re-evaluation, and the pull of additional research initiatives. That is the true nature of investigative science, and there will surely be more advances to clarify, and to strengthen our grasp on this important field of gluten-induced hypersensitivity reactions within the intestinal mucosa.

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