A Simple Protocol for Identification of Heterophile Antibody Interferences in

the Abbott AxSYM hCG Assay.

P Giannopoulos, G Jones, E-M LimDepartment of Chemical Pathology

St Vincent’s Hospital, Sydney

Heterophile Antibodies?

• Immunoglobulins found in some patient’s serum that bind antibodies from other species

• in 2 site immunoassays Heterophilic Antibodies can mimic the presence of analyte by binding the two antibodies in the assay

• produce false results

Why?

• Interference with Abbott AxSYM hCG assay

• Dr Laurence Cole, USA hCG Reference Laboratory– over 40 cases false positives– Average “hCG” 81 U/L– Laparoscopy, surgery, chemotherapy

• Consistent with heterophile antibodies

Abbott?

• Letter from Worldwide Marketing Manager

• January 2001– Urine hCG– Re-measure serum on alternate analyser– Identify clinical inconsistency

Why are we telling you?

• RCPA AACB Quality Assurance Program

-37% use AxSYM hCG assay

How did we do it?

AxSYM:

How do we do it now?

Sydpath protocol

• hCG levels: 10 and 100U/L manual 1 in 2

• hCG levels: 100 and 1000U/L auto dilution

1 in 10

• Dilution performed in hCG specific diluent

What is the Rationale for the Sydpath Protocol?

•Increased amounts of animal proteins may block heterophile antibody

interference

Diluent 11 uL

Sample 52 uL

What we found?

• 2400 hCG measurements

• 133 dilution protocol (5.6%)

• 2 patients with discrepant results on dilution (0.08%)

Discrepant results?

• Patient A ?Pregnant

hCG = 87 U/L (46-122)

1 in 2 = 0 U/L

• Patient B ? Pregnant

hCG = 83 U/L (52-121)

1 in 2 = 0 U/L

What we did next?

• Obtained urine samples– Negative on Hybritech Icon and AxSYM

• Measured serum on another analyser– hCG not detected

• Issued results: <2 U/L hCG

Further Investigation?

• Other analysers

• Protein G agarose precipitation

• Serial dilution

Summary of Measurements

NegNegHybritech Icon

-NDDPC Immulite

NDNDAbbott i2000

NDNDRoche E170

NDNDBayer Centaur

8387AxSYM (neat)

Patient BPatient A

Protein G Agarose

Ptn A Ptn B Control

hCG-pre 46 122 64

hCG-post 0 0 66

Protein G: An Immunoglobulin G binding protein

Dilutions in buffer

0

10

20

30

40

50

60

0 0.2 0.4 0.6 0.8 1

Dilution

hC

G (

U/L

)

Protein-free buffer

Abbott Letter

• Discrepant results with clinical findings

• Urine HCG measurement

• Measure on another analyser

What we learnt?

• The Abbott recommendation is limited for identifying false positive results.

• SydPath Protocol: a fast, simple, cost-effective solution.

• We prevented two incorrect results from being issued

• Acknowledge Dr Laurence Cole.

These powerpoint slides will be available at www.sydpath.stvincents.com.au after the conference.