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