Andre Mattman (Presenter)
University of British Columbia
Bio: I am a medical biochemist who works in the clinical chemistry laboratory at St Paul's Hospital in Vancouver BC. I have an interest in the laboratory diagnosis of disorders of plasma proteins including in particular alpha-1-antitrypsin deficiency and disorders of IgG subclasses.
Authorship: A Mattman (1), M DeMarco (1), DT Holmes (1), M Carruthers (2), L Chen (3), JG van der Gugten (1)
(1) Clinical Chemistry Laboratory, Dept of Pathology and Laboratory Medicine St Paul’s Hospital, (2) Division of Rheumatology, Dept of Medicine, (3) Division of Hematology, Dept of Medicine
| Short Abstract IgG subclasses are measured clinically to diagnose IgG4 related disease. In some patient samples, the sum of the immunonephelometric quantification of the individual subclasses (sum (IgGs)) is much greater than the immunonephelometric total IgG test. 84 samples with a range of IgG4 values were retrieved. In the IG4RD cohort, the sum (IgGs), when measured by immunonephelometry, was greater than total IgG as measured by any of three methods (immunonephelometry, electrophoresis or LC-MS/MS). This bias between sum (IgGs) and total IgG was was predicted by IgG4 levels and was absent when all measurements were by LC-MS/MS. |
Long Abstract
Introduction:
The test: Serum IgG subclasses test includes the measurement of the following subclasses which are normally present in the g/L range with a total IgG level of 5 – 15 g/L and relative proportions of IgG1 (60%), IgG2 (30%), IgG3 (5%) and IgG4 (<5%).
Indications to order the test: Identification of a) individuals at high risk for the IgG4 related disease syndrome, and b) individuals with low levels of select IgG subclasses in a pattern suggestive of an inherited immune deficiency disorder
IgG4 Related Disease: Clinicopathologic features of this relatively new disease syndrome include: a) Multi-organ disease involvement and b) Organ involvement showing tumefactions with characteristic histopathologic findings: i) lymphoplasmacytic infiltrate, stortiform fibrosis and obliterative phlebitis, ii) Plasma cell infiltrate characterized by a high proportion of cells that stain positive for an IgG4 immunoperoxidase stain and iii) Abnormally high serum IgG4 levels (> 1.25 g/L) in approximately 90% of patients as measured by immunonephelometry.(1)
IgG4 is a sensitive biomarker of unknown pathophysiologic significance. There is also elevation in the IgG2 subclass in a subset of patients(2) with IgG4RD that is of unknown pathophysiologic significance
Analytic issues: Immunonephelometry errors in IgG subclass measurement include the error of antigen excess leading to low or normal serum IgG4 measurement(3). To avoid the error of antigen excess, manufacturer recommends comparison of the total IgG test to the sum of the individual subclasses. There is frequent discordance between the sum of the individual subclass concentrations and the total IgG test(4). The discordance is more frequently seen in patients with high IgG4 levels. This latter error suggests an analytic error of interference, calibration or test sensitivity.
Hypothesis: In patients with high IgG4 levels characteristic of IgG4RD: a) There is an overestimation of one or more of the IgG subclasses when the subclass measurements are performed by immunonephelometry in IgG4RD patients, OR b) There is an underestimation of the total IgG test when the total IgG is measured by immunonephelometry
Objective: To compare individual and sum (IgGs) and total IgG immunonephelometric measurements with those as measured by tryptic digest LC-MS/MS and by protein electrophoresis (PEP).
Methods: Patient serum separator tube samples were sequestered at the request of local specialists in hematology and rheumatology where IgG subclasses had been ordered. Samples stored at -20C for up to 6 months.
Analytic Methods and Definitions:
Total IgG (neph): Total IgG as measured with Siemens BNII nephelometer and Siemens reagents (g/L)
Total IgG (PEP): Total IgG as estimated by agarose gel electrophoresis with coomassie blue stain (g/L)
Total IgG (MS): Total IgG as measured by tryptic digest with LC-MS/MS as per Ladwig(5) method and Binding site calibrator (g/L)
IgGx (neph): IgGx subclass as measured with Siemens BNII nephelometer and Binding Site reagents (g/L)
IgGx (MS): IgGx subclass as measured with LC-MS/MS as per Ladwig method and Binding site calibrator (g/L)
Results: 38 samples with immunonephelometry results were retrieved for LC-MS/MS testing. Results from 46 samples with both immunonephelometry and PEP results on file were reviewed. The major findings were: 1) The Difference between Sum (IgGs, neph) and Total IgG is correlated with IgG4 concentration when total IgG is measured by nephelometry, LC-MS/MS or electrophoresis, 2) The Difference between Sum (IgGs) and Total IgG is markedly lower when both parameters are measured by mass spectrometry, and 3) In a cohort of 12 patients with IgG4 related disease, the four patients with the highest IgG4 concentrations (3.7, 6.9, 7.1 and 10. 2 g/L) also had high IgG2 concentrations by immunonephelometry (5.1, 15.7, 17.7, and 14.6 g/L) but not by LC-MS/MS (2.4, 5.4, 2.3, and 4.6 g/L). There was strong agreement in IgG(x) measurements for IgG1, IgG3 and IgG4.
Discussion: IgG(x) and total IgG correlate well with measurements by immunonephelometry. Some patients with IgG4 related disease have elevations in IgG2 that are detected by immunonephelometry but not by LC-MS/MS suggesting analytic error in the IgG2(neph) method as the cause of the IgG2 elevations. Further study will include clarification of the mechanism of analytic interference and verification of its potential specificity with regard to the IgG4D syndrome.
References & Acknowledgements:
1- Carruthers MN Ann Rheum Dis 2015 PMID 24651618
2- Mavragani CP Arthritis Care Res 2014. PMID 25100215
3- Khosroshahi A Arhritis Rheumatol 2014. PMID 24431286
4- Mattman A Clin Biochem 2014 PMID 24717727
5- Ladwig PM Clin Chem 2014 PMID 24799527
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |