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Routine Quantification of Rheumatoid Factor by Rate Nephelometry

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Routine Quantification of Rheumatoid Factor by Rate Nephelometry Ann Rheum Dis: first published as 10.1136/ard.44.6.379 on 1 June 1985. Downloaded from Annals of the Rheumatic Diseases, 1985, 44, 379-383 Routine quantification of rheumatoid factor by rate nephelometry P J ROBERTS-THOMSON, R McEVOY, T LANGHANS AND J BRADLEY From the Department of Clinical Immunology, Flinders Medical Centre, South Australia 5042 SUMMARY In a cross-sectional study of over 3000 consecutive serum specimens the levels of rheumatoid factor (RF) measured by rate nephelometry (Beckman ICS II) were compared with values obtained by the more traditional methods of sheep cell agglutination (Rose-Waaler) and latex agglutination. Similar values for sensitivity and specificity were found for all three methods for rheumatoid arthritis, with nephelometry giving slightly higher levels of sensitivity for other rheumatic disorders. A significant correlation (r=0-46, p<001) was found between the nephelometric and Rose-Waaler method for 147 consecutive seropositive specimens. Of interest, however, several disparate results were observed, and explanations for these were sought. Longitudinal studies of RF were performed in 49 seropositive patients over a two-year period. The nephelometric method was considered superior compared with the other techniques because of its ability to detect changes in absolute levels at earlier stages and its low interassay coefficient of variance (11%). copyright. We conlcude that the nephelometric technique appears suitable for routine diagnostic use, offers several advantages compared with more traditional methods, and is no more expensive per test specimen than the Rose-Waaler technique. http://ard.bmj.com/ Traditionally, rheumatoid factor (RF) has been interaction of RF with heat aggregated human IgG. measured in the routine diagnostic laboratory by the There are two principal forms of nephelometric latex agglutination test and by methods utilising analysis: (1) end point nephelometry, which sheep cell agglutination (e.g. Rose-Waaler). These measures the final light scatter after a fixed period of tests are semiquantitative in that they require a reaction time, e.g., 60 min, and is dependent on subjective interpretation of an end point and have background scatter;45 and (2) rate nephelometry, poor reproducibility (e.g. coefficient of variance which measures the initial rate of scatter and hence on September 26, 2021 by guest. Protected approximately 20%'). In addition the absolute level is relatively independent of background scatter and of RF has to be changed by over 50% before there is results can be obtained within seconds.3 7 There are a change in titre if a doubling dilution technique is several advantages in using nephelometric tech- used. Nevertheless these agglutination tests have niques, particularly the latter form, in measuring become established as the methods most commonly levels of RF; it is fast, truly quantitative, has a used at the present time to measure RF in the desirably low coefficient of variance, and has been diagnostic laboratory. reported to have a low false positive rate in the Over the last five years nephelometric methods elderly non-rheumatoid population.' 3Of the seven have been described to measure RF. l-7 Nephe- published reports concerning the nephelometric RF lometry is a simple and sensitive technique for assay all have concluded that this assay measures RF measuring the degree of light scatter caused by the accurately, and two1 2 have specifically recom- mended it in preference to the established agglutina- tion techniques. Accepted for publication 17 December 1984. In the present study we have undertaken a large Correspondence to Dr P J Roberts-Thomson, Department of Clinical Immunology, Flinders Medical Centre, Bedford Park, comparative and analytical study to determine the South Australia 5042. usefulness of the nephelometric technique in 379 Ann Rheum Dis: first published as 10.1136/ard.44.6.379 on 1 June 1985. Downloaded from 380 Roberts-Thomson, McEvoy, Langhans, Bradley measuring RF in the routine diagnostic laboratory. Results Cross-sectional and sequential studies of RF levels in over 3000 consecutive specimens submitted to our Over a two-year period RF was measured in 3000 laboratory were performed, and the results con- consecutive specimens received by our laboratory. firmed our initial favourable opinions of the nephe- For all specimens RF was measured by three lometric method.7 methods, and negative and positive RF controls were included in each assay batch. For the nephe- Materials and methods lometric assay the coefficient of variance of the positive control was 11.0% over the two-year period and for a 6/12 month period it was 6*1%. In 29 SERA specimens (i.e. <1% of total) it was not possible to RF was measured in over 3000 consecutive serum record a RF level accurately because of excess samples received by our laboratory over a two-year background scatter due to gross lipaemia. period in which a request for RF estimation had The nephelometric RF level measured in 50 blood been made. The sera were assigned to various donors is seen in Fig. 1. Four individuals had levels diagnostic categories, the diagnosis being assumed > 80 IU/ml, and this level was arbitrarily chosen as to be that clearly identified by the referring source, the upper limit of normal in our non-hospital and in approximately 80% of instances this referral 'healthy' population. The RF levels found in various was from the rheumatology and immunology clinics. disease categories are shown in Fig. 1. As predicted In many mistances RF was quantified in the same the greatest prevalence and highest values were patient on multiple occasions, and 49 patients with found in RA, and 80% of our rheumatoid patients rheumatoid arthritis (RA) were identified in whom were seropositive by the nephelometric assay. RF had been measured on five or more occasions. The sensitivity and specificity of the three assays Sera were also obtained from 50 blood donors. are given in Table 1. In general the methods gave Serum were stored at and RF samples -20°C, similar results; the nephelometric assay was slightlycopyright. estimations were performed twice weekly. more sensitive than the agglutination assays but a little less specific (than the Rose-Waaler assay). The RF ASSAYS cost of each assay (cost of reagents, technicians time RF was measured in all sera by three different etc.) is also shown in this table. A comparison of the assays-namely, the slide agglutination test RF levels measured by the nephelometric and (Behring Rapi-Tex), sheep cell agglutination (Rose- Rose-Waaler assays for 359 random consecutive Waaler), and rate nephelometry (Beckman ICS II). specimens is shown in Fig. 2. Excluding thehttp://ard.bmj.com/ The latex agglutination test was performed accord- ing to the manufacturer's instructions at a final serum dilution of 1:10 and 1:50 in diluting buffer, a10ooo- and agglutination was detected visually after gentle mechanical rocking at room temperature for two 5000- minutes. The Rose-Waaler sheep cell agglutination test was performed in accordance with published methods, and a positive titre was defined as 1:32 or on September 26, 2021 by guest. Protected greater. The rate nephelometric test was performed 1000 . on a Beckman ICS II with Beckman reagents exactly as described by the manufacturer's technical 500- brochure. The rate and total scatter were also A It. recorded graphically on a Rikadenki dual channel :-..*.- chart recorder connected to the Beckman ICS II .I. two 100- j. instrument via outputs (labelled 'rate' and I -1. on the back of the instrument. RF ------- 'scatter') panel *b6 1 -"'-- - was also measured in some sera and column ..... fL 0: AM.Ltm fractions with a Behring laser nephelometer as previously described.4 SLE PSS SS RA SPOF PSOR 1 E0 Fig. 1 RF levels (lUlml) measured by rate nephelometry in patients with rheumatoid arthritis (RA), systemic lupus COLUMN CHROMATOGRAPHY erythematosus (SLE), progressive systemic sclerosis (PSS), Selective sera were fractionated on a 90x2-5 cm Sjogren's syndrome (SS), spondylarthritis (SPOND), Sepharose 6B (Pharmacia) column, and RF was psoriatic arthritis (PSOR), or osteoarthritis (OA) and measured in the eluate by laser nephelometry. in 50 blood donors (BD). Ann Rheum Dis: first published as 10.1136/ard.44.6.379 on 1 June 1985. Downloaded from Routine quantification of rheumatoid factor by rate nephelometry 381 Table 1 Sensitivity and specificity of RF assayed by three methods BD* RA SLE PSS SS SPOND PSOR PMR OA Cost ($) No. 50 100 33 12 7 14 23 19 14 - Neph. >80 IU/mI 4 80 13 4 5 1 4 1 1 1-60 Latex (1:10 dil) 2 83 8 3 4 1 2 2 0 0-8 Rose-Waaler >1:32 0 75 6 2 4 1 1 1 0 1-60 *BD=blood donors; RA=rheumatoid arthritis; SLE=systemic lupus erythematosus; PSS=progressive systemic sclerosis; SS=Sjogren's syndrome; SPOND=spondylarthritis; PSOR=psoriatic arthritis; PMR=polymyalgia rheumatica; OA=osteoarthritis. 10000 E 5000 I 4- a a)0 A "A E0. 1000 Fig. 2 Comparison ofRFlevels z0 measured by the Rose-Waaler copyright. .0ca agglutination assay and expressed 500 as reciprocal oftitre (x abscissa) with RF levels measured by rate 0co a~~~~~~~~~~~~~~~~~~~~ nephelometry (y ordinate). * - cu .-0 E 100 http://ard.bmj.com/ .-C 460 St 4 '16 64 256 1024 >4096 t~~~~~~~~~~~~~~~Reciprocal of Rose-Waaler Titre on September 26, 2021 by guest. Protected seronegative specimens a significant correlation Table 2 High RF by nephelometry with low Rose-Waaler (r=046, p<0-01) was found between the two titre assays. More importantly, however, a number of disparate results were obtained, and possible Patient Diagnosis Rose-Waaler titre Nephelometric reasons for these were examined. In a review of the RF (lUlml) comparative results for all sera studied over two 1 HP* 1:16 5340 years six patients were observed who gave a 2 HP 1:128 3560 persistently high or very high nephelometric reading 3 SS 1:256 2140 and negative or low positive Rose-Waaler titre 4 SS 1:128 2090 2240 (Table 2).
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