Using Phadia 2500 for EliA GPC and IFAB. Rachel Farrah, SNP, Immunology

Transcription

1 Using Phadia 2500 for EliA GPC and IFAB Rachel Farrah, SNP, Immunology

2 GPC and IFAB clinical significance GPC 84% Pernicious Anaemia 47% Atrophic Gastritis 33% Thyroid Diseases 7-20% of general healthy adult population IFAB 50-70% Pernicious Anaemia patients Positive result not found in otherwise healthy adults as of yet

3 Dilution Slides Loading Wash Dilute serum and titre if required. See IFA preparation and reporting folder for dilutions. Remove slides form fridge and bring to room temperature. Remove FITC from fridge Label slides and place in humidified chamber Place enough diluted serum on wells to cover approx. 25uL Place lid on humidified chamber and incubate at room temperature for 30 5mins Rinse serum off the tissue slides with PBS, taking care not to squirt the PBS directly at the sections. Place slides into the metal slide holder / rack that are sitting in the glass-staining dish filled with fresh PBS. Wash twice for 5mins IFA vs FEIA Antigen binds to surface Antibody-enzyme conjugate attaches to antigen Conjugate Drop sufficient FITC for appropriate tissue to cover each well. Incubate at room temperature for 30 5mins. Wash Wash as above Mount slides with buffered glycerol Read Read using 100x & 400x Fluorescent Microscope. Table 1: IFA method description Substrate and enzyme interaction creates colour change detection Figure 1: FEIA method description

4 Two discrepancies Table 2: Comparison of Gastric Antibody Results from the Phadia 2500 FEIA and IFA. The highlighted are the two discrepancies that were found and investigated

5 Possible GPC misinterpretations Figure 2: GPC on rat tissue (left) and mouse tissue (right) Figure 3: Heterophile on rat tissue (left) and mouse tissue (right) Figure 4: Illustration of Chief cells compared to Parietal cells Figure 5: AMA on rat liver and kidney

6 Phadia 2500/250 comparison Table 3: Comparison of Gastric Antibody FEIA Results performed on the Phadia 2500 and Phadia 250. The highlighted is the outlier that was removed from the further evaluation

7 Phadia 2500/250 comparison Figure 6: Scatterplot of GPC FEIA Results performed on the Phadia 2500 compared to results the Phadia 250 Figure 7: Scatterplot of GPC FEIA Results performed on the Phadia 2500 compared to results from the Phadia 250. Adjusted for bias caused by outlier

8 Orgentec anti-intrinsic factor ELISA workflow Figure 8: ELISA method description

9 Comparison of IFAB from Phadia 2500 and ELISA assay Table 4: Comparison of Intrinsic Factor Antibody Results from the Phadia 2500 FEIA and ELISA assay. The cut off for declaring a positive result on the FEIA were values 10 U/mL. The cut off for declaring a positive result on the ELISA assay is 6 U/mL. Values that were >6 U/mL and <22 U/mL are further classified as Low positive results. Yellow Highlighted Rows: Low positive ELIA that were called negative by FEIA. Red Highlighted Rows: Positive Results that were called negative by FEIA.

10 ELISA assay: 64.2% sensitivity and 95% specificity FEIA Phadia 2500: sensitivity 73% with 100% specificity Comparison of the two assays show a 35.95% agreement. Table 5: Continuation of the comparison of Intrinsic Factor Antibody Results from the Phadia 2500 FEIA and ELISA assay. The cut off for declaring a positive result on the FEIA were values 10 U/mL. The cut off for declaring a positive result on the ELISA assay is 6 U/mL. Values that were >6 U/mL and <22 U/mL are further classified as Low positive results. Yellow Highlighted Rows: Low positive ELIA that were called negative by FEIA. Red Highlighted Rows: Positive Results that were called negative by FEIA.

11 Phadia 2500 (U/mL) FEIA IFAB Phadia 2500 vs % correlation IFAB FEIA: Phadia 2500 Vs R² = Phadia 250 (U/mL) Figure 9: Scatterplot of Intrinsic Factor Antibody FEIA Results performed on the Phadia 2500 compared to results from the Phadia 250.

12 Workflow Workflow with FEIA Received in Specimen Reception Area (SRA) Aliquoted Put on ICAP Result/Authorised within hours Workflow with KLS Received in SRA Aliquoted Put in rack to await morning s run of KLS Result/Authorised by end of day Workflow with ELISA Received in SRA Aliquoted Put on Tecan to be dropped out procedure as normal Result/Authorised by end of day

13 Noticeable differences in the lab ~1000 IFAB samples/per month ~650 GPC samples/per month by 63% for IFAB since change by 85% for GPC since change in KSL slide numbers

14 Noticeable differences in the lab Two labs test set because one lab test set Less aliquots No need to find samples risk of losing samples

15 Why GPC on Phadia 2500/250? Rodent Tissue KLS identifies several antibodies Different clinical notes from doc When GPC is detected, patient sample is put on ICAP More reliable value

16 Summary GPC KLS vs FEIA with 96.6% correleation IFAB ELISA vs FEIA with 35.95% with outliers and then 97.7% without Excellent correlation between Phadia 2500/250 with both tests Increase in both GPC and IFAB test numbers FEIA = more user friendly