Rectal forceps biopsy procedure in cystic fibrosis: technical aspects and patients perspective for clinical trials feasibility

Abstract

Background: Measurements of CFTR function in rectal biopsies ex vivo have been used for diagnosis and prognosis of Cystic Fibrosis (CF) disease. Here, we aimed to evaluate this procedure regarding: i) viability of the rectal specimens obtained by biopsy forceps for ex vivo bioelectrical and biochemical laboratory analyses; and ii) overall assessment (comfort, invasiveness, pain, sedation requirement, etc.) of the rectal forceps biopsy procedure from the patients perspective to assess its feasibility as an outcome measure in clinical trials.

Methods: We compared three bowel preparation solutions (NaCl 0.9%, glycerol 12%, mannitol), and two biopsy forceps (standard and jumbo) in 580 rectal specimens from 132 individuals (CF and non-CF). Assessment of the overall rectal biopsy procedure (obtained by biopsy forceps) by patients was carried out by telephone surveys to 75 individuals who underwent the sigmoidoscopy procedure.

Results: Integrity and friability of the tissue specimens correlate with their transepithelial resistance (r = -0.438 and -0.305, respectively) and are influenced by the bowel preparation solution and biopsy forceps used, being NaCl and jumbo forceps the most compatible methods with the electrophysiological analysis. The great majority of the individuals (76%) did not report major discomfort due to the short procedure time (max 15 min) and considered it relatively painless (79%). Importantly, most (88%) accept repeating it at least for one more time and 53% for more than 4 times.

Conclusions: Obtaining rectal biopsies with a flexible endoscope and jumbo forceps after bowel preparation with NaCl solution is a safe procedure that can be adopted for both adults and children of any age, yielding viable specimens for CFTR bioelectrical/biochemical analyses. The procedure is well tolerated by patients, demonstrating its feasibility as an outcome measure in clinical trials.

Figure 1

Flow-chart of the technical biopsing aspects assessed in the present study. Bioelectrical measurements were performed for rectal biopsies (n = 580) from all the individuals enrolled in the study (n = 132) to assess tissue viability [14]. Bowel preparation included enemas of either NaCl 0.9%, glycerol 12% (v/v) or oral mannitol 20% (w/v) solutions. Two different biopsy forceps were tested, namely jumbo (3.4 mm Ø) and standard (2.5 mm Ø), independently of bowel preparation. Macroscopic and histologic evaluation of rectal biopsies was achieved for 107 and 78 individuals, respectively. Patient assessment surveys were carried out for 75 individuals undergoing sigmoidoscopy with rectal biopsy collection by biopsy forceps, divided into 4 age groups, namely (yrs): 0–9; 10–9; 20–29; ≥30.

Figure 2

Correlations between tissue transepithelial resistance (R_te) and macroscopic descriptors (tissue integrity, friability, bleeding and mucus) according to A) biopsy forceps and B) bowel preparation (n = 107 individuals).

Figure 3

Histological and macroscopic evaluation of rectal tissues and biochemical analysis. A) Rectal biopsies (longitudinal cuts) were histologically evaluated by Hematoxilin-Eosin (HE) and Masson’s Tricome stainings in non-CF and CF tissues. Images show healthy epithelia, with no fibrotic processes and only some inflammatory cells were detected. Images shown are representative of the total and correspond to biopsies from a non-CF individual (left) and a CF patient (right) performed with jumbo (3.4 mm Ø) forceps after bowel preparation with glycerol (non-CF) or isotonic saline (CF). In HE-stained sections (top), nuclei are stained in blue and cytoplasm in red. In Tricome’s Masson-stained sections (bottom) collagen (fibrotic biomarker) is stained in blue, nuclei in black, and muscle and cytoplasm in red. Black scale bar represents 250 μM. B) Immunohistofluorescence of rectal biopsies showing nuclei in blue (DAPI staining) and CFTR in green. Images evidence CFTR at the membrane in a non-CF tissue (top panels) and also, albeit weaker, in a biopsy from a CF patient with the F508del/P205S-CFTR genotype (bottom panels). In contrast, a biopsy from a F508del-homozygous CF patient evidences intracellular CFTR staining (middle panels). A negative control (no primary antibody n.c.) was also performed. Scale bar represents 25 μm. C) Western blot of a single rectal biopsy from non-CF individuals (wt-CFTR, lanes 1–2) and from a CF patient with F508del/R334W-CFTR genotype (lane 3) evidence the presence of both immature and mature forms of CFTR (bands B and C, respectively; and from a CF patient with the F508del/F508del-CFTR genotype (lane 4) evidencing only immature form (band B) which is characteristic of the endoplasmic reticulum (ER) and thus corroborating the traffic defect associated with this mutation.