Clin. exp. Immunol. (1979) 38, 181-185.
A simple, rapid micro-latex fixation test J. C. CICCIARELLI, P. I. TERASAKI, D. CHIA, E. V. BARNETT, M. R. MICKEY, S. PRINCE, Y. IWAKI & SUSAN SHIRAHAMA Departments of Surgery and Medicine, UCLA School of Medicine, University of California, Los Angeles, California 90024, USA (Accepted for publication 2 April 1979)
SUMMARY
A micro-latex fixation test (LFT) for the determination of rheumatoid factor (RF) is presented. Its advantages compared to similar tests are greater precision, simplicity, increased sensitivity, lower cost, reproducibility and adaptibility to large-scale testing. Micro-LFT titres are presented from a wide range of sample populations. The majority of normal samples show measureable titres whereas rheumatoid patients show high titres. A large sampling of pre- and post-transplant sera from kidney patients was studied and the micro-LFT titres were in the range of normal persons. The transplant sera were tested for lymphocytotoxic antibodies and no correlation was observed with the micro-LFT titres. INTRODUCTION The latex fixation test (LFT) introduced in 1956 is the most widely used method for determination of rheumatoid factor (RF) (Singer & Plotz, 1956). Some modifications of the test have been proposed, such as the use of aggregated IgG (Singer et al., 1960) and more recently, the nephelometric measurement of the agglutination titre (Virella, Waller & Fudenberg, 1978). Nephelometric measurement eliminates the subjective aspects in the determination of endpoint, although the useful range and sensitivity seem to be slightly less than in the LFT. Although the LFT is a relatively simple test, we describe here modifications that make the test easier to perform, easier to read and give it greater sensitivity. MATERIALS AND METHODS The test procedure was as follows. One pil of heat-inactivated sera in two-fold serial dilutions from 1:20 to 1: 10,240 were added to microtest trays (Terasaki) containing 5 pil of mineral oil per well. Latex beads coated with pooled aggregated IgG (Behring Diagnostics) were diluted 1:50 in glycine buffer (0-1 M glycine, 0-17 M NaCl pH 8-2) and 1 pl of this dilution was added to each well. Following incubation for 90 min at room temperature, the trays were read using inverted darkfield microscopy with a 6X objective. The endpoint titre was determined by the absence of agglutination and the absence of Brownian movement in more than 70% of the latex particles. The endpoint can be determined by absence of agglutination alone but use of the absence of Brownian movement generally yields a sharper endpoint and one which can be agreed upon by a number of independent observers. The equipment used was basically that developed for the microlymphocyte cytotoxicity test (Terasaki et al., 1978): the 60-96-well microtest tray, the Hamilton microsyringe or the Terasaki multichannel pipette (Flow Laboratories, McLean, Virginia) with a repeating dispenser for the delivery of 0-001 ml, and the inverted phase contrast microscope with an attached electric typewriter device. The standard latex fixation tube test was performed for comparison (Singer, 1973). Anti-IgG antibodies were prepared by passing rheumatoid arthritis (RA) serum through a column containing IgG covalently bound to Sepharose 4B (Pharmacia, Rahway, New Jersey) (Cuatrecasas, 1970). Anti-IgG antibodies were eluted off the column with 0.1 M glycine buffer pH 3-5. The concentration of IgM immunoglobulins was measured by radial immunodiffusion. The titre of these antibodies was assessed in the micro-LFT to determine the sensitivity of this method. Correspondence Dr J. C. Cicciarelli, 1000 Veteran Avenue, Los Angeles, CA 90024, USA. 0099-9104/79/1000-0181$02.00 (D 1979 Blackwell Scientific Publications
181
J. C. Cicciarelli et al.
182
Factors of variability affecting the micro-LFT results were studied by arranging the 'blind' testing of sera from thirty-six subjects into eighteen trays such that each serum was titrated on each of three different trays; each tray contained six sera with the ten dilutions for a given serum arranged in order within one row. Data in the form of log2 titres were analysed by regression analysis using the computer programme BMDP1R (Dixon & Brown, 1977). Results were expressed as estimates of the standard deviations corresponding to components of the variability. The geometric mean titre for the triplicate titres from the micro-LFT was compared to LFT tube titres for the same subjects by regression analysis. Micro-LFT titres for the various populations of sera were summarized by the geometric mean titre and the percentage relative standard error. The percentage relative error of the geometric mean was calculated as: relative error = 100 (2'- 1), s = standard error of log2 (geometric mean). The calculations were done by expressing the titres as logarithms to the base 2, by computing the mean, standard deviation and standard error of the mean for the 1og2 titres and by transforming the summary statistics to the geometric mean (i.e. anti-log2 of mean log2 titre) and percentage relative standard error. B and T lymphocytes were isolated and tested by the lymphocyte microcytotoxicity test (Terasaki et al., 1978). B-cold, cytotoxic antibody was tested according to Iwaki et al. (1978). HLA-A, -B, -C, and -DR cytotoxic antibody was tested using the method of Terasaki et al. (1978). Each serum was tested on a random panel of thirty B and T lymphocytes. Linear regression analysis was performed by comparing the percentage cytotoxicity of post-transplant sera with log2 micro-LFT titres.
RESULTS Fig. 1 shows the linear regression analysis of the micro-LFT method as compared to the LFT tube method. There was a strong correlation (r=0.95) between the two tests and the slope of the line demonstrates an increase in sensitivity utilizing the micro-LFT. Furthermore, the sensitivity of the micro-LFT had the ability to detect 0-5 ng of anti-IgG antibody, as shown by agglutination of the latex particles with purified IgM anti-IgG. The precision of the micro-LFT was assessed by a double-blind experiment in which thirty-six sera were titred in triplicate (Table 1). Factors of variability associated between trays were of an approximately equal effect as the factors controlling variability of titrations within a tray. Estimates were approximately 0 5 for the standard deviation of log2 titre within a tray and 0-6 for the standard deviation corresponding to the tray effect. The two combined (1(0.5)2 + (0.6)2) to give an estimate of 0-8 for the standard deviation of log2 titre for the uncertainty of a single titration. The estimated precision of the average of triplicate log2 titres was 0 7 for titrations within a tray and 0 5 for titrations in different trays, thus indicating that for titrations of large numbers of sera the precision was not markedly affected by the positioning of the sera. The geometric mean of triplicate titres was accordingly quite likely to be within 1-1.5 dilutions of the 'true' value. The variation among the sera tested was estimated by the value of 3.3 for the standard deviation. The sera were thus well differentiated by the micro-LFT. 10 _ 9
,'/
, -1 6
*
0
5
X _/~~~~~~ 4
0
2
3 4
5
6
7
8
9
10
Mijcro- LFT test 092 (dilution x 10) -) FIG. 1. Linear regression analysis of the LFT tube method compared to the micro-LFT method. -) represents the observed correlation (r = 0 95) and the slope Represents an exact correlation (r = 1); ( (0.85) shows an increased sensitivity of the micro-LFT method.
Micro-LFT
183
TABLE 1. Comparison of triplicate micro-LFT titres
Sample No.
1
2
3
4
5
6
Cordsera
A simple, rapid micro-latex fixation test J. C. CICCIARELLI, P. I. TERASAKI, D. CHIA, E. V. BARNETT, M. R. MICKEY, S. PRINCE, Y. IWAKI & SUSAN SHIRAHAMA Departments of Surgery and Medicine, UCLA School of Medicine, University of California, Los Angeles, California 90024, USA (Accepted for publication 2 April 1979)
SUMMARY
A micro-latex fixation test (LFT) for the determination of rheumatoid factor (RF) is presented. Its advantages compared to similar tests are greater precision, simplicity, increased sensitivity, lower cost, reproducibility and adaptibility to large-scale testing. Micro-LFT titres are presented from a wide range of sample populations. The majority of normal samples show measureable titres whereas rheumatoid patients show high titres. A large sampling of pre- and post-transplant sera from kidney patients was studied and the micro-LFT titres were in the range of normal persons. The transplant sera were tested for lymphocytotoxic antibodies and no correlation was observed with the micro-LFT titres. INTRODUCTION The latex fixation test (LFT) introduced in 1956 is the most widely used method for determination of rheumatoid factor (RF) (Singer & Plotz, 1956). Some modifications of the test have been proposed, such as the use of aggregated IgG (Singer et al., 1960) and more recently, the nephelometric measurement of the agglutination titre (Virella, Waller & Fudenberg, 1978). Nephelometric measurement eliminates the subjective aspects in the determination of endpoint, although the useful range and sensitivity seem to be slightly less than in the LFT. Although the LFT is a relatively simple test, we describe here modifications that make the test easier to perform, easier to read and give it greater sensitivity. MATERIALS AND METHODS The test procedure was as follows. One pil of heat-inactivated sera in two-fold serial dilutions from 1:20 to 1: 10,240 were added to microtest trays (Terasaki) containing 5 pil of mineral oil per well. Latex beads coated with pooled aggregated IgG (Behring Diagnostics) were diluted 1:50 in glycine buffer (0-1 M glycine, 0-17 M NaCl pH 8-2) and 1 pl of this dilution was added to each well. Following incubation for 90 min at room temperature, the trays were read using inverted darkfield microscopy with a 6X objective. The endpoint titre was determined by the absence of agglutination and the absence of Brownian movement in more than 70% of the latex particles. The endpoint can be determined by absence of agglutination alone but use of the absence of Brownian movement generally yields a sharper endpoint and one which can be agreed upon by a number of independent observers. The equipment used was basically that developed for the microlymphocyte cytotoxicity test (Terasaki et al., 1978): the 60-96-well microtest tray, the Hamilton microsyringe or the Terasaki multichannel pipette (Flow Laboratories, McLean, Virginia) with a repeating dispenser for the delivery of 0-001 ml, and the inverted phase contrast microscope with an attached electric typewriter device. The standard latex fixation tube test was performed for comparison (Singer, 1973). Anti-IgG antibodies were prepared by passing rheumatoid arthritis (RA) serum through a column containing IgG covalently bound to Sepharose 4B (Pharmacia, Rahway, New Jersey) (Cuatrecasas, 1970). Anti-IgG antibodies were eluted off the column with 0.1 M glycine buffer pH 3-5. The concentration of IgM immunoglobulins was measured by radial immunodiffusion. The titre of these antibodies was assessed in the micro-LFT to determine the sensitivity of this method. Correspondence Dr J. C. Cicciarelli, 1000 Veteran Avenue, Los Angeles, CA 90024, USA. 0099-9104/79/1000-0181$02.00 (D 1979 Blackwell Scientific Publications
181
J. C. Cicciarelli et al.
182
Factors of variability affecting the micro-LFT results were studied by arranging the 'blind' testing of sera from thirty-six subjects into eighteen trays such that each serum was titrated on each of three different trays; each tray contained six sera with the ten dilutions for a given serum arranged in order within one row. Data in the form of log2 titres were analysed by regression analysis using the computer programme BMDP1R (Dixon & Brown, 1977). Results were expressed as estimates of the standard deviations corresponding to components of the variability. The geometric mean titre for the triplicate titres from the micro-LFT was compared to LFT tube titres for the same subjects by regression analysis. Micro-LFT titres for the various populations of sera were summarized by the geometric mean titre and the percentage relative standard error. The percentage relative error of the geometric mean was calculated as: relative error = 100 (2'- 1), s = standard error of log2 (geometric mean). The calculations were done by expressing the titres as logarithms to the base 2, by computing the mean, standard deviation and standard error of the mean for the 1og2 titres and by transforming the summary statistics to the geometric mean (i.e. anti-log2 of mean log2 titre) and percentage relative standard error. B and T lymphocytes were isolated and tested by the lymphocyte microcytotoxicity test (Terasaki et al., 1978). B-cold, cytotoxic antibody was tested according to Iwaki et al. (1978). HLA-A, -B, -C, and -DR cytotoxic antibody was tested using the method of Terasaki et al. (1978). Each serum was tested on a random panel of thirty B and T lymphocytes. Linear regression analysis was performed by comparing the percentage cytotoxicity of post-transplant sera with log2 micro-LFT titres.
RESULTS Fig. 1 shows the linear regression analysis of the micro-LFT method as compared to the LFT tube method. There was a strong correlation (r=0.95) between the two tests and the slope of the line demonstrates an increase in sensitivity utilizing the micro-LFT. Furthermore, the sensitivity of the micro-LFT had the ability to detect 0-5 ng of anti-IgG antibody, as shown by agglutination of the latex particles with purified IgM anti-IgG. The precision of the micro-LFT was assessed by a double-blind experiment in which thirty-six sera were titred in triplicate (Table 1). Factors of variability associated between trays were of an approximately equal effect as the factors controlling variability of titrations within a tray. Estimates were approximately 0 5 for the standard deviation of log2 titre within a tray and 0-6 for the standard deviation corresponding to the tray effect. The two combined (1(0.5)2 + (0.6)2) to give an estimate of 0-8 for the standard deviation of log2 titre for the uncertainty of a single titration. The estimated precision of the average of triplicate log2 titres was 0 7 for titrations within a tray and 0 5 for titrations in different trays, thus indicating that for titrations of large numbers of sera the precision was not markedly affected by the positioning of the sera. The geometric mean of triplicate titres was accordingly quite likely to be within 1-1.5 dilutions of the 'true' value. The variation among the sera tested was estimated by the value of 3.3 for the standard deviation. The sera were thus well differentiated by the micro-LFT. 10 _ 9
,'/
, -1 6
*
0
5
X _/~~~~~~ 4
0
2
3 4
5
6
7
8
9
10
Mijcro- LFT test 092 (dilution x 10) -) FIG. 1. Linear regression analysis of the LFT tube method compared to the micro-LFT method. -) represents the observed correlation (r = 0 95) and the slope Represents an exact correlation (r = 1); ( (0.85) shows an increased sensitivity of the micro-LFT method.
Micro-LFT
183
TABLE 1. Comparison of triplicate micro-LFT titres
Sample No.
1
2
3
4
5
6
Cordsera
