Background/Purpose:

IgG4-related disease (IgG4-RD) is frequently associated with elevations in serum IgG4 concentration. However, the frequency of serum IgG4 elevation varies in published series from 44%-100%. The prozone effect, also known as the “hook” effect, occurs when antigen excess interferes with antibody-based assay methods that require immune complex formation for detection, and can lead to spuriously low results. Additional sample dilutions are the solution to the prozone effect.  After identifying the prozone effect in one patient with IgG4-RD whose serum IgG4 corrected following appropriate dilutions from 17 to 1850 mg/dL (nl: 2.4–121 mg/dL), we examined additional samples to determine the frequency of this problem.

Methods:

We re-tested 38 serum samples from patients with the diagnosis of IgG4-RD whose original serum IgG4 results had been reported earlier. The IgG subclasses were measured by nephelometry in dilutions up to 1:160,000, using two different commercially-available reagents (Siemens; The Binding Site).  The testing laboratory was blinded to the patients’ clinical history and previous values. The serum IgG4 concentrations from these assays were compared to the original results.

Results:

Falsely low IgG4 values were reported in 10/38 patients (26%) using The Binding Site assay   (Table). The prozone effect was identified as the cause of 8 incorrect values (21% of all samples tested). Correction of the prozone effect by sample dilution until the concentration reported was stable, led to a revision of the mean IgG4 result from 21.6 to 2440. In contrast, samples measured by the Siemens reagent were checked automatically for antigen excess as part of the testing method; the appropriate numbers of dilutions were performed either automatically by the instrument or manually as the result of a flag associated with the value, thereby avoiding the prozone effect.  The Binding Site assay gave no indication that antigen excess might be present.

All 8 patients whose samples were affected by the prozone effect had active IgG4-RD, and 6 had multi-organ disease. Medical record review indicated that the original clinical decision regarding further evaluation (e.g., tissue biopsy) or treatment might have been different had the correct value been known to the clinician. 

Conclusion:

We have identified a major issue in the serological measurement of IgG4 concentrations. The prozone effect which led to substantial underestimations of IgG4 concentrations in 21% of the samples, offers potential explanations for the poor correlation observed between disease activity and serum IgG4 level in some patients.  This phenomenon should be considered when the serum IgG4 measurement appears discordant with the clinician’s assessment of disease activity.

Table. Characteristics of patients with IgG4-RD with significant differences in their retested IgG4 results

 

Case #	* Original reported IgG4 values  (mg/dL)	** Retested IgG4 values (mg/dL)	X-fold increase after dilution	Number of affected organs	Active disease	Presence of prozone effect
1	10.3	2470	247	1	Yes	Yes
2	28.4	941	33	3	Yes	Yes
3	29.3	219	7.5	1	Yes	No
4	59.8	337	5.6	1	Yes	No
5	12.7	5340	420	3	Yes	Yes
6	17.6	1850	105	4	Yes	Yes
7	8.0	5160	645	7	Yes	Yes
8	43.9	1030	23	4	Yes	Yes
9	14.4	1910	132	7	Yes	Yes
10	37.5	819	22	1	Yes	Yes

*   The Binding Site reagent was used for the assay

** Siemens reagent was used for the assay

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/prozone-phenomenon-leads-to-low-igg4-concentrations-in-igg4-related-disease/