title: Histological and immunohistochemical characterisation of conjunctival graft vs host disease following haematopoietic stem cell transplantation.
abstract: BACKGROUND
  Conjunctival graft vs host disease (cnGvHD) is a complication of haematopoietic stem cell transplantation, in most cases as part of systemic GvHD. Diagnostic biopsies are commonly collected from bulbar conjunctiva only. The aims of our study were to evaluate whether additional biopsies from the tarsal conjunctiva increase sensitivity upon histopathologic evaluation and to investigate the staining profile for common immunohistochemical markers in cnGvHD. We additionally propose an adaptive histological classification for cnGvHD analogous to Lerner's GvHD skin classification for predicting patient survival.
  
  
  METHODS
  Formalin-fixed and paraffin-embedded conjunctival specimens from 23 post-mortem control eyes and 42 patients after haematopoietic stem cell transplantation (HSCT) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) stain and with antibodies against CD1a, CD4, CD8, CD25, CD45RO, CD68, Fas ligand, TIA-1, HLA-DRalpha by means of immunohistochemistry. Cell counting took place in ten representative fields at 64.4 microm (length) x 21.2 microm (width). Multifactorial analysis of variance was performed to assess any influence of cnGvHD on the staining pattern for the immunohistochemical markers. Survival times were estimated by the Kaplan-Meier method.
  
  
  RESULTS
  All 42 specimens and none of the controls were diagnosed as cnGvHD. The bulbar specimens were staged according to the modified Lerner classification: grade (G) I: 0; G II: 17 (tarsal with G<or=II, 2; G>II, 8); G III: 12 (tarsal with G<or=III: 2; G>III: 1); G IV: 12 (tarsal with G<or=IV: 6); G V: 1. The number of pairs with either the tarsal or bulbar counterpart being more severely affected was almost equal (10 vs 9). A tendency towards shorter survival in advanced bulbar cnGvHD was demonstrated (G III-V vs G I-II, p =0.06). Staining for the immunohistochemical markers in cnGvHD differed significantly from that in controls (p<0.01). Proposed markers for cnGvHD (e = epithelium, s = stroma; mean cell counts +/- SD; cnGvHD vs controls) are: CD8 s (15.7 +/- 18.4 vs 6 +/- 5.6), CD25 s (2.6 +/- 2.8 vs 0.7 +/- 1.6), CD68 s (8 +/- 9 vs 3.9 +/- 3.5) at the bulbar site and CD1a e (1.2 +/- 1.6 vs 0.3 +/- 0.6) and TIA-1 e (2.2 +/- 2.2 vs 1.1 +/- 1.3) at the tarsal site.
  
  
  CONCLUSIONS
  Additional tarsal biopsy does not seem to add relevant diagnostic sensitivity for cnGvHD when the modified Lerner classification is applied. The modified Lerner classification of the bulbar cnGvHD seems to be of prognostic value.
authors.affiliation: Eye Hospital, Albert-Ludwigs University, Killianstrasse 5, 79106 Freiburg, Germany. claudia.auw-haedrich@uniklinik-freiburg.de
authors.forename: Claudia
authors.initials: C
authors.lastname: Auw-Haedrich
authors.forename: Christian
authors.initials: C
authors.lastname: Potsch
authors.forename: Daniel
authors.initials: D
authors.lastname: Böhringer
authors.forename: Hans
authors.initials: H
authors.lastname: Mittelviefhaus
authors.forename: Philip
authors.initials: P
authors.lastname: Maier
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinhard
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Freudenberg
authors.forename: Jürgen
authors.initials: J
authors.lastname: Finke
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Coupland
mesh_terms: D000328:Adult; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001706:Biopsy; D002452:Cell Count; D003229:Conjunctival Diseases / Q000145:classification / Q000175:diagnosis* / Q000209:etiology / Q000378:metabolism; D015352:Dry Eye Syndromes / Q000209:etiology; D005260:Female; D006086:Graft vs Host Disease / Q000145:classification / Q000175:diagnosis* / Q000209:etiology / Q000378:metabolism; D006684:HLA-DR Antigens / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation / Q000009:adverse effects*; D006801:Humans; D007124:Immunoenzyme Techniques; D007938:Leukemia / Q000601:surgery; D008223:Lymphoma / Q000601:surgery; D008297:Male; D039102:Poly(A)-Binding Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D000074019:T-Cell Intracellular Antigen-1
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D006684:HLA-DR Antigens; D039102:Poly(A)-Binding Proteins; D000074019:T-Cell Intracellular Antigen-1; C071558:TIA1 protein, human