Thirty-one consecutive patients undergoing AHSCT were evaluated between August 2007 and May 2010. Patients agreed to take part in the study after receiving the necessary explanations and signing an informed consent. The patients were admitted, evaluated and followed under the routines of the Transplantation Unit.

A standard protocol was followed by all the patients for clinical evaluation and performance of complementary tests, according to the following steps:

• 1

  On admission to the Transplant Unit, forms were filled out with socio-demographic, clinical and laboratory data. An intestinal permeability study was performed preferentially before or during the conditioning regime.

• 2

  Patients with no symptoms of GI tract impairment: clinical evaluation, laboratory data and intestinal permeability studies at or around D + 14 and D + 30 post-transplant.

• 3

  Patients with GI tract impairment symptoms (nauseas/vomiting, pronounced hyporexia and diarrhea) at any moment between D + 14 and D + 90 post-transplant: clinical GVHD evaluation and classification, clinical evaluation, laboratory data, performance of an upper digestive endoscopy with stomach and duodenum biopsies. The biopsy samples were sent to the Pathology Laboratory of the Hospital.

All patients included in the study were clinically monitored from admission until hospital discharge or D + 90 post-transplantation, whichever came first, and clinical diagnosis of GVHD was performed according to the criteria defined by Ferrara,14 starting on D + 14 post-transplantation. Criteria used for staging acute clinical GVHD were those revised by the Consensus Conference on Acute GVHD Grading.15 Patients were separated into two groups: those with mild to moderate acute clinical GVHD (final staging grades I or II) and those with severe acute clinical GVHD (final staging grades III or IV).

The intestinal permeability tests were performed in accordance with a standardized technique,16 using a meal of a standard solution containing 6.25 g of 95% PA crystalline lactulose, Sigma–Aldrich® and 3.0 g of crystalline mannitol PA, Sigma–Aldrich®, after a 6 h fast and followed by a 5 h urine collection. A 50 ml aliquot of urine was separated off for analysis of urinary lactulose and mannitol concentrations by High Performance Liquid Chromatography (HPLC), followed by the calculation of their excretion rates (in relation to the amount of sugar ingested). Finally, the ratio between the urine excretion rates of the two substances was obtained (lactulose/mannitol excretion rates).

Upper digestive endoscopy examinations were performed according to service routines. Biopsies were performed only when the platelet count was equal to or above 50,000 mm³. Patients with lower counts received transfusion of platelet concentrates prior to the procedure.

The samples of gastric and duodenal tissue stored in 10% formalin were sent to the Pathology Laboratory to be processed in paraffin and stained with Hematoxylin-eosin (HE); the stomach biopsy samples were also stained for Helicobacter pylori with Giemsa.

All biopsies were evaluated by two experienced pathologists specialized in the upper GI tract, with research on apoptosis, using Sidney’s modified histological classification.17 Alterations compatible with intestinal GVHD were evaluated according to the NIH Consensus5 and described separately. Edema was evaluated according to intensity: none, moderate or severe.

The Apoptotic Index (AI) was obtained in a blind fashion on terminal uridine nick-end labeling (TUNEL)-stained laminae.18 Apoptotic cell quantification was made by a single observer. As an inclusion criterion, the simultaneous presence of at least three of the typical morphological characteristics was adopted: anoikia—cell retraction and loss of adhesion between cells and basal membrane; cytoplasmic and nuclear condensation—compaction of the nuclear chromatin into dense uniform masses, aligned on the inner side of the nuclear membrane, sometimes forming crescent images; nuclear fragmentation—convolution and fragmentation of the nuclear membrane—without karyorrhexis or rupture, and; cellular fragmentation—with formation of the apoptotic bodies.

The AI was determined by the following formula: (AI) = (Σ no. of apoptotic cells/Σ no. of total cells) ×100.

To validate the morphological criteria in order to quantify the AI and the TUNEL technique18 (Apoptag® Peroxidase in situ, Apoptosis Detection Kit, Cat S7100, Chemicon), immunohistochemistry was also performed for Bax (Mouse x Bax, Invitogen, Cat.#18-0218).

For the morphometrics study, digital images of the histological lamina were acquired utilizing the video-camera system, coupled to the CH30 Olympus light microscope with a 40× objective to make the apoptosis cell count and determine the AI. The images were transferred by the video-camera system to a computer and captured through Honestech TVR2.5 Software. The acquisition of images was performed at the Morphometrics Laboratory of the General Pathology Department at the University. The IA Quantification was made using the Media Cybernetics Image Pro-Plus, version 4.0, image analyzer.

The collected data were stored in a data bank created in the EpiData 3.1 software. The data for the intestinal permeability study were analyzed utilizing the SSPS version 13.0 software and the apoptosis index data were analyzed utilizing the Graph Pad Prisma version 5.0 software.

The study was approved by the Research Ethics Committee (UFMG, ETIC 092/07).

Thirty-one patients admitted for AHSCT were included in the study. There were no statistical differences of sex, age, diagnosis, type of transplantation, conditioning regime, cell source and GVHD prophylaxis in the two groups analyzed, as Table 1 shows.

Eleven patients were diagnosed with acute GVHD on clinical grounds (35.5%), while the remaining 20 patients (64.5%) were considered free of GVHD. All 11 patients with acute GVHD were considered to have gastrointestinal GVHD. Three of them also had acute skin and liver GVDH, two had skin GVDH and one had liver GVDH.

Thirteen of the 31 patients (41.9%) died during the study period. Deaths occurred between D + 8 and D + 66 post-transplantation with a 37-day median. Four of the deaths (36.4%) were in Group 1 (n = 11), three were from acute grade IV GI tract GVHD and one from infection, the latter with a severe acute GVHD diagnosis too. Nine deaths (45.0%) occurred in group 2 (n = 20), four of them from sinusoidal obstruction syndrome, four from respiratory insufficiency and one from infection/sepsis.

Fifteen patients with GI tract symptoms underwent upper digestive endoscopy in the period up to D + 90 post-transplantation. In addition to the suspicion of GVHD, endoscopy was performed to dismiss esophageal candida, cytomegalovirus (CMV) infection according to clinical and/or serological evidence, as well as other gastroduodenal pathologies. Five of the patients underwent more than one endoscopy. There were visible endoscopic alterations in 11 of the 21 (52.4%) procedures performed. The most commonly found mucosa abnormalities were esophageal erosions and ulcers, moderately intense enanthematous gastritis, subepithelial hemorrhage areas in the body, antral erosions and pronounced pangastritis with micronodularity, erosive duodenitis, enanthematous duodenitis, serrated appearance of the mucosa and duodenal petechiae. Esophageal moniliasis was also found in the histology sample of one patient.

Matching was positive between clinical suspicion of GVHD at the time of the examination and the suggestive endoscopic observations (Table 2) in 10 of the 21 (47.6%), and negative in six of the 21 (28.6%) procedures performed, with no statistical significance.

Biopsies were not performed in two procedures due to evident signs of bleeding. Esophagus biopsies were only performed when viral or fungal infection was suspected.

The histological alterations suggestive of GVHD found in the stomach (n = 3), were a predominantly leukocyte inflammatory infiltration, necrosis of the glandular epithelium, increased apoptosis and presence of apoptotic corpuscles. In the duodenum (n = 3) atrophy and ulceration of the mucosa, flattened villi with a decreased villous to crypt ratio, cell vacuolization, eroded areas, regenerative atypias in the crypts and frequent nuclear alterations with increased apoptosis, as well as a granulomononuclear inflammatory infiltrate, congestion and areas of hemorrhage. Stomach and duodenum GVDH was found simultaneously in two patients.

Endoscopic alterations suggestive of GVHD only matched the histological confirmation in one of the 19 endoscopies, as shown in Table 3. Only four of the 19 biopsy samples (21%) had a histological GVHD diagnosis, also suspected on clinical grounds. Signs of mild unspecific chronic gastritis or active chronic duodenitis were seen in five patients with clinical suspicion of GVHD, none of whose etiology was determined by histology.

Other histological alterations other than signs suggestive of GVHD were esophageal moniliasis (n = 1), Helicobacter pylori in the stomach (n = 1) and inclusions suggestive of cytomegalovirus infection in stomach (n = 2) and duodenum (n = 1) biopsies.

The apoptotic index (AI) analysis and immunohistochemistry studies were performed on only nine patients because the biopsy samples did not display a sufficient number of fields for statistical analysis, chiefly due to the presence of artifacts and fragmentation. The AI was calculated in 10 fields for each patient. AI values ranged from 3.791% to 24.51%. The mean AI of the 10 fields analyzed for each patient ranged from 6.503% to 11.92% and the median from 6.198% to 12.43%.

The median of the AI values and its relation to deaths from GVHD or TRM (transplant-related mortality), GVHD staging, endoscopic diagnosis and histological diagnosis (Table 4) were evaluated. Higher AIs were observed in the worst outcomes, without statistical significance. In relation to the severity of GVHD, the difference among AIs was close to statistical significance. A higher AI was significantly correlated with endoscopy classified as suggestive of GVHD.

Of the 31 patients included in the study, 25 (80.64%) underwent intestinal permeability evaluation in the pre-transplant phase, 21 (67.74%) around D + 14 post-transplantation and 11 (35.48%) around D + 30 post-transplantation, while only nine (29.0%) were able to perform the examination in the three periods, as initially scheduled.

Paired Student’s t-tests comparing the different intestinal permeability collection times did not reveal statistical significance. The several losses between study phases may have impaired analysis. The graph in Fig. 1 shows a considerable increase in the separate lactulose/mannitol excretion rates (L/M R) means (non-paired), rising from 0.0127 in the pre-transplant phase to 0.092 and 0.141 on days +14 and +30, respectively. However, these were not accompanied by similar alterations in the median, which showed a rise of 0.007 to 0.022 between the pre-transplant phase and D + 14, dropping to 0.005 on D + 30. Two cases outside the curve, with a L/M R of 0.860 on D + 14 and of 1.142 on D + 30, not represented in Fig. 1 to avoid affecting the graph’s scale, helped increase the separate means on those dates.

Fig. 1.

L/M ratios in different phases of the study.

(0.18MB).

Twenty four percent of the cases in the pre-transplant period, 60% on D + 14 and 27% on D + 30 had an R L/M value higher than 0.012, corresponding to the mean +2 standard deviations of a control sample of 16 healthy people of both sexes, previously evaluated at the intestinal permeability laboratory. There was no relation between the R L/M values observed and clinical outcomes, such as the development of clinical GVHD and death.