High Resolution Gel Electrophoresis vs. Combined Light Chain Immunofixation (CLIF) for the detection of Monoclonal Gammopathies

Joel Smith,1 Geoffrey Raines,1 Hans G Schneider1 1Clinical Biochemistry, Alfred Pathology Service, Melbourne, Australia.

INTRODUCTION RESULTS (Continued) Monoclonal gammopathies are characterised by the production of a monoclonal HR SPEP was abnormal in 62 of 64 (97%) of patients with a known paraprotein, immunoglobulin or free light chains by an abnormal plasma cell or B-cell clone and whereas CLIF was abnormal in 57 of 64 (89%) cases. There was good inter- adjudicator agreement for both HR SPEP (κFleiss = 0.78) and CLIF (κFleiss = 0.82) may indicate malignancy or a precursor (MGUS). There is currently no consensus interpretations. HR SPEP was deemed abnormal in 76 cases (49%) and CLIF was on the initial test or combination of tests to be performed in suspected monoclonal deemed abnormal in 60 cases (38%). gammopathies. In our laboratory, serum protein electrophoresis (SPEP) and urine protein electrophoresis (UPEP) are commonly performed as initial investigations. If Sensitivity and specificity for the detection of paraprotein by HR SPEP was 97% abnormal, immunofixation electrophoresis (IFE) is then performed to confirm the and 85%, respectively. For CLIF, sensitivity and specificity was 89% and 97%, presence of paraprotein and to determine its heavy and light chain type. Recently respectively. (Table 2). some groups have developed simplified “screening” IFE methods for use in parallel to SPEP for the detection monoclonal gammopathies. Table 2: Detection of Paraprotein by both HR SPEP and CLIF in 156 patients (64 with known paraprotein) Jenner et al., found that the systematic implementation of a screening IFE known as combined κ and λ light chain immunofixation (CLIF) led to an increase in True False True False Sensitivity Specificity monoclonal bands missed by SPEP alone and improved turn-around times and Positives positives Negatives Negatives reduced the utilisation of laboratory consumables (1). This was in comparison to a HR SPEP 62 14 78 2 97% 85% low resolution SPEP system (Sebia Hydragel β1 β2 (SE4146)). We evaluated the diagnostic performance of a combined κ and λ light chain CLIF 57 3 89 7 89% 97% immunofixation (CLIF) as described by Jenner et al.(1), for the detection of monoclonal immunoglobulin in serum in comparison to our current laboratory There were six cases of monoclonal gammopathy missed by CLIF but detected by method of serum protein electrophoresis on a high resolution (Sebia Hydragel 15 HR SPEP, including one trace level IgA(λ), one IgA(λ) at 5 g/L, one trace level HR) agarose gel system. IgD(λ), one trace level IgG(λ) and two patients with monoclonal free light chains (one λ and one κ). The one case missed by HR SPEP but detected by CLIF was a trace level IgA(κ). There was only one case missed by both techniques. This was shown by full immunofixation to be a trace (<1g/L) IgG(λ) paraprotein. Examples of MATERIALS AND METHODS CLIF (Figure 1A, 1B) and HR SPEP (Figure 1C) are shown below. This project was approved by The Alfred Ethics Committee (project no. 584/15). 156 serum samples with requests for SPEP received consecutively over the A B periods 21/8/15 - 27/8/15 and 13/10/15 - 20/10/15 were used in the comparison. These periods were representative of the labs normal routine operation. All electrophoresis was performed on a Sebia Hydrasys 2-scan instrument.

High resolution Serum Protein Electrophoresis High resolution protein electrophoresis was performed according to the manufacturers’ protocol on Sebia Hydragel 15 HR gels (SE4122). Gels were stained with Acid Violet.

Combined Light Chain Immunofixation

Combined κ and λ light chain immunofixation was performed as described by

Jenner et al. Initial protein electrophoresis was performed on Sebia Hydragel β1 β2 C gels (SE4146) on a Hydrasys 2-Scan instrument. Gels were stained with Amido- black. CLIF was then performed on Sebia Hydragel 9 IF (SE4809, a total of 54 lanes) gels in the same sequence as applied to the Sebia Hydragel β1 β2 gels.

Samples for CLIF were diluted with Sebia diluent according to the total globulin result (> 30 g/L 1:11, 20-30 g/L 1:6, <20 g/L 1:3 g/L) Immunofixation was then performed by applying 20μl of a 1:1 preparation of Anti-κ (SE4606) and Anti-λ

(SE4607) anti-sera to each lane via 54 lane standard mask (SE1265). CLIF gels were stained with acid violet.

Interpretation Each Sebia hydrasys 15 HR gel and the 54 lane Hydragel β1 β2 gels and CLIF gels were interpreted by 3 staff members (JS, GIR, HGS) separately. An abnormal Sebia Hydragel 15 HR SPEP was defined as any one of: a quantifiable band, Figure 1: (A) 54 lane Sebia Hydragel β1 β2 gel and corresponding CLIF (B). HR SPEP is shown in (C). Samples in black box in A and B appear in C in the same hypogammaglobulinaemia, or an increased β-region. An abnormal CLIF was order for comparison. Sample in the red box in each is an IgA(λ) at 5 g/L that was defined as a discrete band visualised on CLIF, hypogammaglobulinaemia or an missed by CLIF but detected on HR SPEP increased β-region. Where discrepancies existed between interpretations, the majority interpretation The 14 false positives detected by HR SPEP included one case with acute (2/3 adjudicators) was taken as the final interpretation. Inter-adjudicator agreement inflammation, two patients with transferrin variants, two patients with mild hypogammaglobulinaemia and eight patients with trace oligoclonal bands. With was assessed using the method of Fleiss (2) regard to the 3 false positives detected by CLIF, two had trace oligoclonal bands A final reference diagnosis was based on a combination of clinical history and and one had a polyclonal increase in IgA. standard immunofixation results.

CONCLUSIONS RESULTS This study found that high resolution gel serum protein electrophoresis using a A total of 156 patient samples were analysed. Samples were from 83 male and 73 Sebia Hydrasys 15 HR system was more sensitive than CLIF for the detection of female patients with a median age of 62 years (range 18-90 years). No samples paraprotein in patient serum in this patient cohort. The drawback of the greater were excluded from the analysis. sensitivity of HR SPEP was the higher false positive rate requiring an increased utilisation of follow up immunofixation electrophoresis. Of the 156 patients, a total of 64 (41%) had a paraprotein detectable by standard immunofixation. The final diagnoses for these patients is shown in table 1.

Table 1: Diagnoses of patients with paraprotein in serum detectable by immunofixation References 1. Jenner W, Klingberg S, Tate JR, Wilgen U, Ungerer JP, Pretorius CJ. Combined Diagnosis N (%) light chain immunofixation to detect monoclonal gammopathy: a comparison to standard electrophoresis in serum and urine. Clin Chem Lab Med. 2014;52(7):981- Total 64 7. 2. Fleiss, J. L.. Measuring nominal scale agreement among many raters. Psychol. Multiple Myeloma 43 (67%) Bull., 1971 76, 378382. MGUS 15 (23%) Waldenströms 1 (2%) Macroglobulinaemia Primary Amyloidosis 2 (3%) Lymphoma 3 (5%)