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Short report

Verification of serum reference intervals for free light chains in a local South African population Annalise E Zemlin,1 Megan A Rensburg,1 Hayley Ipp,2 Jurie J Germishuys,1 Rajiv T Erasmus1 l

Division of Chemical Pathology, Faculty of Health Sciences, National Health Laboratory Service (NHLS) and University of Stellenbosch, Tygerberg Hospital, Cape Town, South Africa 2 Division of Haematology, Faculty of Health Sciences, National Health Laboratory Service (NHLS) and University of Stellenbosch, Tygerberg Hospital, Cape Town, South Africa Correspondence to Dr Annalise E Zemlin, Division of Chemical Pathology, Faculty of Health Sciences, National Health Laboratory Service (NHLS) and University of Stellenbosch, Tygerberg Hospital, Cape Town 7505, South Africa; [email protected] Received 6 February 2013 Revised 30 April 2013 Accepted 3 June 2013 Published Online First 25 June 2013

ABSTRACT Monoclonal serum free light chain measurements are used to follow up and manage patients with monoclonal gammopathies, and abnormal serum free light chain ratios are associated with risk of progression in certain diseases. We aimed to validate the reference intervals in our population. Reference intervals for κ and λ free light chains were established on 120 healthy adults. Creatinine levels were measured to exclude renal dysfunction and serum protein electrophoresis was performed. All creatinine values were within normal limits. After exclusion of subjects with abnormal serum protein electrophoreses, 113 subjects were available for analysis. The 95% reference interval was 6.3– 20.6 mg/L for κ free light chains, 8.7–25.9 mg/L for λ free light chains and 0.46–1.23 for free light chain ratio. Most of the values fell within the manufacturer’s recommended limits and therefore could be used for our population.

INTRODUCTION Serum free light chains (FLC) may be of either κ or λ type and abnormal values are associated with imbalances in heavy and light chain immunoglobulin production. Automated assays are available for their determination and they are used in the management of multiple myeloma and other disorders associated with monoclonal gammopathies.1 Abnormal κ/λ ratios have been associated with risk of progression in B-cell abnormalities.2 FLC measurements may eliminate the need for urine investigations when assessing a patient for multiple myeloma.3 4 Immunoturbidimetric and immunonephelometric assays (Freelite, The Binding Site, UK) are available on routine chemistry analyzers.2 Polyclonal antihuman-FLC antisera are used and require acceptable imprecision, specificity, accuracy and reproducibility between reagent batches. Katzmann et al5 established FLC reference intervals on 282 healthy donors in 2002 and these ranges are used worldwide. However, these were established on an elderly Caucasian population, and therefore, it was considered important to verify these in our local population of younger and mainly Coloured (mixed race) and Black individuals.

Table 1

To cite: Zemlin AE, Rensburg MA, Ipp H, et al. J Clin Pathol 2013;66: 992–995. 992

Previous studies have shown that there is a need to verify local FLC reference intervals.6 7 Racial differences in immunoglobulin and B-cell abnormalities have been described 8–10 and our laboratory uses a higher reference interval for total immunoglobulin G (IgG) in Black individuals.

MATERIALS AND METHODS Participants Blood was obtained from 120 healthy subjects: 78 HIV negative blood donors from a local donation clinic and 42 volunteers participating as controls in a voluntary HIV prevention and testing clinic (HPT) study. All signed an informed consent approved by our Ethics committee, demographics were anonymised and confidentiality was maintained at all times. All subjects were older than 21 years of age. Those attending the HPT were clinically well, with no symptoms of tuberculosis and not on any medications.

Analytical methods The following blood tests were performed: total protein, IgG, creatinine, serum protein electrophoresis (SPEP) and κ and λ FLC. Serum total protein, IgG and creatinine were determined on the ADVIA 1800 (Siemens) chemistry analyser using the biuret, polyethylene immunoturbidimetric and the standardised Jaffe methods, respectively. SPEP was performed on the semi-automated Sebia Hydrasys system. κ and λ FLC were determined nephelometrically using Freelite (The Binding Site, UK) on the Beckman Coulter IMMAGE. The serum sample is added to a solution containing the antibody in a cuvette. A light beam passes through the cuvette and as the antigen–antibody reaction proceeds, the light is scattered as insoluble immune complexes are formed. The scattered light is monitored by measuring the decrease in intensity of the light beam. As the antibody is in excess, the amount of immune complex is proportional to the antigen concentration.

Mean values serum creatinine, protein and immunoglobulin G (IgG)

Parameter

Laboratory reference interval

Range for study population

Creatinine(μmol/L)

Women: 49–90 Men: 64–104 60–85 10–23 (Black adults)*

44–116

73 (71)

61–92 6.1–25.6

73 (72) 14.1 (13.6)

Protein(g/L) IgG(g/L)

Mean (Median)

*No reference interval determined for coloured or mixed ancestry population.

Zemlin AE, et al. J Clin Pathol 2013;66:992–995. doi:10.1136/jclinpath-2013-201511

Short report Table 2 Reference intervals for free light chains obtained in our local population (n=112)

κ FLC (mg/L) λ FLC (mg/L) FLC Ratio

95% reference limit (manufacturer)

95% reference limit (study–entire group)

Range

Median (SD)

3.3–19.4 5.7–26.3 0.26–1.65 (100% reference interval)

6.3–20.6 8.7–25.9 0.46–1.23

6.4–26.5 8.1–32.3 0.44–1.79

11.6 (0.3) 14.7 (0.3) 0.77 (0.25)

FLC, free light chains.

Statistical analysis Reference intervals were established using the Analyse-it software programme V.2.26. The parameters followed a nonparametric distribution (reported as range and median), and due to the low sample numbers, log-transformed parametric statistics were used to determine the reference intervals. Reference intervals obtained were deemed acceptable if 20 g/L) and one with hypogammaglobulinaemia). No significant interobserver variation was detected.

Table 3

DISCUSSION Reference intervals for FLC, as recommended by the manufacturer, were originally determined on an older Caucasian population.5 Therefore we sought to verify these levels in our local population. Previous studies have shown racial differences in reference intervals of IgG.8–10 Therefore, we hypothesised that free light chain levels may also vary in different races. Additionally, studies have highlighted the need to verify FLC reference intervals in different population groups.6 7 Local studies have described elevated IgG levels in our Black population.11 However, these studies were performed on

Reference intervals for free light chains for Coloured and Black subjects separately

κ FLC (mg/L) λ FLC (mg/L) FLC Ratio

95% reference limit (manufacturer)

95% reference limit (study–Coloured)

95% reference limit (study–Black)

3.3–19.4 5.7–26.3 0.26–1.65 (100% reference interval)

5.9–20.4 7.7–26.1 0.49–1.22

7.3–21.1 9.8–26.7 0.45–1.23

FLC, free light chains.

Figure 1 Histogram with reference interval for κ free light chains.


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Short report Figure 2 Histogram with reference interval for λ free light chains.

subjects of unknown HIV status. In our current study, all participants were HIV-negative. Multiple myeloma is the most common haematological malignancy in the Black population and, the incidence of monoclonal gammopathy of unknown source also has a twofold increased incidence in this population group8 However, Waxman et al9 showed that the Black population have a more indolent course of multiple myeloma and hypothesised that different susceptibility genes may play a role. In addition to a large local Black population, we have a mixed (Coloured) population that is unique to South Africa and largely populates the Western Cape (our hospital’s drainage area). FLC reference intervals have not been established previously for either Black or mixed-race populations; therefore, the verification of recommended reference intervals are particularly important in our setting. We concluded that our whole population’s 95% reference intervals were within acceptable limits and accepted the manufacturer’s recommended intervals. Interestingly, when we studied the Coloured and Black populations separately, we found the reference intervals to be acceptable as well. At the time of this study, there were no defined ‘allowable errors’ for FLC and the bias reported in our method validation was small in proportion to disease state levels and thus not clinically significant. In

consultation with the Binding Site, it was determined that we could accept these reference ranges. Interestingly, the lower limit of the reference intervals in our population did not reach the lower limits claimed by the manufacturers. Although there was a significant difference observed between the medians of the λ only (Mann-Whitney U test), it was still within acceptable limits. Our study had the following strengths: All subjects were HIV-negative. HIV is known to cause polyclonal increases in immunoglobulins, which may affect FLC levels. Creatinine levels were determined to exclude renal impairment, which may affect FLC renal excretion. SPEP was performed on all subjects and those with abnormal patterns were excluded. However, our study had several limitations: It was a small study population and therefore we could not establish new reference intervals. Partitioned according to race, we had even smaller numbers. Ideally, this study should be performed on a larger cohort. Creatinine alone is not an ideal marker of renal dysfunction and may underestimate renal impairment. Lastly, total allowable errors for the FLC assay and knowledge of their biological variation were not available at the time this study was performed. Katzmann et al12 have since published a study showing a biological variation of 27.8% for FLC. We used a value of 20%, which is therefore still acceptable.

Figure 3 Histogram with reference interval for free light chain ratio.

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Short report CONCLUSION Based on our findings we can accept the current manufacturer’s recommended cut-offs in our population, although a larger reference interval study with more subjects per racial group would be ideal.

Ethics approval The study was approved by the University of Stellenbosch Ethics committee and performed according to the Declaration of Helsinki. Provenance and peer review Not commissioned; externally peer reviewed.

REFERENCES 1

Take-home messages 2

▸ Abnormal serum free light chains are associated with imbalances in heavy and light chain immunoglobulin production ▸ Free light chain reference intervals were established on an elderly Caucasian population ▸ Free light chain reference intervals need to be verified ▸ Racial differences in IgG and B-cell abnormalities have been described ▸ Reference intervals were verified in a local population.

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Acknowledgements We wish to thank the Western Province Blood Transfusion Services for allowing us to obtain blood from their donors and the Emavundleni clinic site in Crossroads, Cape Town for their HIV-negative controls. Contributors The authors declare that they all contributed as follows to the article: (1). They contributed to the conception and design of the study (AEZ, HI and RTE), the acquisition of data (AEZ, JJG and HI,) and the analysis and interpretation (AEZ, MAR and JJG) of the data. (2). They contributed to the drafting of the article and the revision of the intellectual content (All). (3). These data formed part of JJG’s Master’s thesis. (4). They all gave their approval of the final version to be submitted for publication. AEZ is guarantor. Funding This research was supported by a research grant (number 94230) from National Health Laboratory Services South Africa and all the kits for free light chain determination were supplied by the Binding Site. The above-mentioned funding sources played no role in this publication besides funding the project and the review of this manuscript by their scientific advisor. Competing interests None. Patient consent Obtained.


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