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Using Phadia 2500 for EliA GPC
and IFABRachel Farrah, SNP, Immunology
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
IFA vs FEIA
Dilution Dilute serum and titre if required. See IFA preparation and
reporting folder for dilutions.
Slides Remove slides form fridge and bring to room temperature.
Remove FITC from fridge
Label slides and place in humidified chamber
Loading Place enough diluted serum on wells to cover approx. 25uL
Place lid on humidified chamber and incubate at room
temperature for 30 5mins
Wash 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
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.
Antigen binds to surface
Antibody-enzyme conjugate attaches to
antigen
Substrate and enzyme interaction creates
colour change detection
Table 1: IFA method description
Figure 1: FEIA method description
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
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
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
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
Orgentec anti-intrinsic factor ELISA workflow
Figure 8: ELISA method description
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.
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.
• 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.
FEIA IFAB Phadia 2500 vs 250
100% correlation
R² = 0.9881
0
50
100
150
200
250
300
350
400
450
500
0 100 200 300 400 500 600
Ph
adia
250
0 (
U/m
L)
Phadia 250 (U/mL)
IFAB FEIA: Phadia 2500 Vs 250
Figure 9: Scatterplot of Intrinsic Factor Antibody FEIA Results performed on the Phadia 2500 compared to results from the Phadia 250.
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
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
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
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
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