Point-of-Care Test in Adult Case-Finding in Celiac Disease
Point-of-Care Test in Adult Case-Finding in Celiac Disease
170 first-degree healthy relatives of 70 index cases (77 children and 15 adults) were invited over a five years period (2007–2012) to be evaluated for the presence of coeliac-autoantibodies. The inclusion criterion for the index cases was biopsy-proven CD (Marsh 3 mucosal lesion). Of the 170 subjects, 148 (18 children, 130 adults; median age 36 years, range 1.2–77) agreed to participate. The remaining 22 could not been reached or declined. At the time of the evaluation, all participants confirmed that they were consuming a regular gluten-containing diet.
The screening for the presence of CD autoantibodies in the healthy relatives took place on the same day as the diagnostic examinations of the index patients or in certain cases later, during regular follow-up visits. All centralized laboratory serologic determinations were made in blinded fashion without knowledge of the on-site fingertip whole blood POCT results.
The Biocard Celiac Test, AniBiotech, Vantaa, Finland, was selected as the point-of-care biomarker test for CD case-finding. This rapid whole blood self TG2-based IgA-class fingertip test uses the patient's own endogenous TG2 found in the erythrocytes of a whole blood sample. When the finger-tip blood is haemolyzed, the liberated TG2 forms a complex with circulating autoantibodies, if present in the same sample. Blood erythrocyte self-TG2-autoantibody complexes are captured from the haemolyzed sample by TG2-binding proteins onto a solid phase and the presence of autoantibodies is measured by labeled antihuman IgA. The results of the POCT were evaluated visually on site after five minutes, but no later than ten minutes, according to the manufacturer's recommendations (line visible in test window – positive result; no line in test window – negative result).
On the same day when the POCT was performed, venous blood was drawn for laboratory serum autoantibody measurements. Serum IgA-class antiendomysial antibodies (EMA) were determined using an indirect immunofluorescence method (Nova Lite, Inova Diagnostics, CA, USA) with cut-off for positivity at a serum dilution of 1:5. Further serum dilutions (1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:4000) were made for initial positive samples till the highest positive titer was obtained for each sample. In the present series, no cases of selective IgA deficiency were found.
Serum samples showing positivity in the EMA test were further evaluated for TG2-IgA (Celikey, Phadia GmbH, Freiburg, Germany) using an enzyme-linked immunosorbent assay (ELISA) with a positivity cut-off at 5 U/ml, as recommended by the product manufacturer.
Upper gastrointestinal endoscopy with small-bowel mucosal biopsies was recommended for all subjects with positive POCT or EMA tests. During endoscopy, up to 5 duodenal mucosal specimens were taken. The biopsy specimens were fixed in formalin, embedded in paraffin, cut, stained with haematoxylin-eosin and scored by pathologists (AE and FV) according to the Marsh-Oberhuber classification (Marsh 0, 1, 2, 3a, 3b, or 3c).
The study was approved by the Ethical Committee of the University and Pharmacy "Carol Davila" and Ethical Committee of the Institute for Mother and Child Care "Alfred Rusescu" Bucharest, Romania. The antibody-positive subjects or their parents gave written informed consent for upper gastrointestinal endoscopy.
Methods
Patients
170 first-degree healthy relatives of 70 index cases (77 children and 15 adults) were invited over a five years period (2007–2012) to be evaluated for the presence of coeliac-autoantibodies. The inclusion criterion for the index cases was biopsy-proven CD (Marsh 3 mucosal lesion). Of the 170 subjects, 148 (18 children, 130 adults; median age 36 years, range 1.2–77) agreed to participate. The remaining 22 could not been reached or declined. At the time of the evaluation, all participants confirmed that they were consuming a regular gluten-containing diet.
Whole Blood and Serum Case-finding Biomarkers
The screening for the presence of CD autoantibodies in the healthy relatives took place on the same day as the diagnostic examinations of the index patients or in certain cases later, during regular follow-up visits. All centralized laboratory serologic determinations were made in blinded fashion without knowledge of the on-site fingertip whole blood POCT results.
The Biocard Celiac Test, AniBiotech, Vantaa, Finland, was selected as the point-of-care biomarker test for CD case-finding. This rapid whole blood self TG2-based IgA-class fingertip test uses the patient's own endogenous TG2 found in the erythrocytes of a whole blood sample. When the finger-tip blood is haemolyzed, the liberated TG2 forms a complex with circulating autoantibodies, if present in the same sample. Blood erythrocyte self-TG2-autoantibody complexes are captured from the haemolyzed sample by TG2-binding proteins onto a solid phase and the presence of autoantibodies is measured by labeled antihuman IgA. The results of the POCT were evaluated visually on site after five minutes, but no later than ten minutes, according to the manufacturer's recommendations (line visible in test window – positive result; no line in test window – negative result).
On the same day when the POCT was performed, venous blood was drawn for laboratory serum autoantibody measurements. Serum IgA-class antiendomysial antibodies (EMA) were determined using an indirect immunofluorescence method (Nova Lite, Inova Diagnostics, CA, USA) with cut-off for positivity at a serum dilution of 1:5. Further serum dilutions (1:50, 1:100, 1:200, 1:500, 1:1000, 1:2000, 1:4000) were made for initial positive samples till the highest positive titer was obtained for each sample. In the present series, no cases of selective IgA deficiency were found.
Serum samples showing positivity in the EMA test were further evaluated for TG2-IgA (Celikey, Phadia GmbH, Freiburg, Germany) using an enzyme-linked immunosorbent assay (ELISA) with a positivity cut-off at 5 U/ml, as recommended by the product manufacturer.
Endoscopy and Biopsies
Upper gastrointestinal endoscopy with small-bowel mucosal biopsies was recommended for all subjects with positive POCT or EMA tests. During endoscopy, up to 5 duodenal mucosal specimens were taken. The biopsy specimens were fixed in formalin, embedded in paraffin, cut, stained with haematoxylin-eosin and scored by pathologists (AE and FV) according to the Marsh-Oberhuber classification (Marsh 0, 1, 2, 3a, 3b, or 3c).
Ethics Approval
The study was approved by the Ethical Committee of the University and Pharmacy "Carol Davila" and Ethical Committee of the Institute for Mother and Child Care "Alfred Rusescu" Bucharest, Romania. The antibody-positive subjects or their parents gave written informed consent for upper gastrointestinal endoscopy.