Vol. 10, No. 5
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p. 712-718 0095-1137/79/1 1-0712/07$02.00/0
Comparison of a Semiautomated Fluorescent Immunoassay System and Indirect Immunofluorescence for Detection of Antinuclear Antibodies in Human Serum CONRAD H. CASAVANT,* ANN C. HART, AND DANIEL P. STITES Clinical Immunology Laboratory, Department of Laboratory Medicine, University of California at San Francisco, San Francisco, California 94143
Received for publication 31 August 1979
A semiautomated fluorescent immunoassay system (FIAX) for detecting antinuclear antibodies (ANAs) in human serum was compared with conventional indirect immunofluorescence using microscope slides (Meloy Laboratories) coated with mouse fibroblasts. The FIAX system uses quantitative indirect immunofluorescence to measure the specific binding of ANA to a sampler coated with human epithelial cells. A total of 101 serum samples were examined for the presence of ANAs by employing both methods. At an initial 1:10 screening dilution, 23 samples were negative for ANAs by the slide method, whereas 21 samples were negative with the FIAX system. Using 2+, 3+, and 4+ subjective brightness, 68 (100%) samples were positive by the slide method, whereas 67 (98.5%) were positive with the FIAX system. ANA-positive samples were diluted twofold from 1:10 to 1:640, and positive titers were determined by both methods. Sixty (77%) of 78 positive samples titrated by FIAX came within ±1 dilution of the titers determined by the slide method, and 75 (96%) of the samples fell within +2 dilutions. Results indicate good correlation between the FIAX system and indirect immunofluorescence for the detection of ANAs in human serum. The FIAX system has the advantage of speed, reproducibility, and the elimination of subjective microscopic assessment of ANA titers.
Autoantibodies to a variety of nuclear and cytoplasmic antigens are characteristically present in the sera of patients with many rheumatic diseases. Currently, the standard method for detecting antinuclear antibodies (ANAs) is indirect immunofluorescence as initially described by Holman and Kunkel (4) and Friou et al. (G. Friou, S. Finch, and K. Detre, Fed. Proc. 16:413, 1957). A variety of indirect immunofluorescent protocols for detecting ANAs have been described; these methods are technically similar except for the use of different tissues or cell lines as substrates. Recently a semiautomated quantitative fluorescent immunoassay system termed
rescence by using the microscope slide technique for detecting and partially quantitating ANAs in a variety of human sera. MATERIALS AND METHODS Sera. A total of 101 serum samples with routine ANA requests received in the Clinical Immunology Laboratory were used in this study. The serum samples were fresh and were examined for the presence of ANAs by both methods before freezing. FIAX ANA determinations. The components of the FIAX system include a fluorometer with a sample stage into which samplers coated with human epithelial cells are inserted and a microcomputer for calculating ANA indices. The fluorometer digitally displays fluorescence as FSU. Human epithelial cells were grown in Eagle minimal essential medium supplemented with 10% fetal calf serum, harvested, and fixed to the sampler surface to give a uniform cell monolayer. The FIAX protocol is similar to that for indirect immunofluorescence, except that cell-coated samplers are immersed in reagent tubes, whereas, for indirect immunofluorescence, serum dilutions and reagents are subsequently added to slides with fixed substrates. All FIAX reagents are marketed in a kit with 50 deter-
FIAX (International Diagnostics Technology, Santa Clara, Calif.) has been developed for measuring ANAs in serum. This method employs a cellular monolayer fixed to a plastic surface which is serially incubated in sera and fluorescent anti-immunoglobulin conjugate. Fluorescence bound to the cellular substrate is measured in a fluorometer and converted to an arbitary system of fluorescent signal units (FSU) by a computer. In the present study we compared the FIAX system with conventional indirect immunofluo-
minations.
Briefly, the FIAX method employs a 1:10 screening dilution of patient sera and negative, low-positive, and 712
VOL. 10, 1979
high-positive control sera prepared in pH 7.1 phosphate-buffered saline supplied by International Diagnostics Technology. Samplers are immersed in the diluted sera and shaken for 30 min on a shaker available from International Diagnostics Technology. ANAs present in patient sera or positive controls bind to the cell-coated samplers. Unreacted serum components are removed by transferring samplers to tubes containing phosphate-buffered saline, which are shaken for 5 min. The samplers are then transferred to tubes containing fluorescein isothiocyanate-conjugated goat anti-human immunoglobulins and are shaken for 20 min. Samplers are then transferred to a final phosphate-buffered saline wash and remain in buffer until read in the fluorometer. Determination of FLAX ANA index. Results of FIAX determinations are expressed as ANA indices. The index represents the relationship between the fluorescence obtained with an unknown sample and the fluorescence obtained with the high-positive and negative ANA control sera, expressed in the following equation: ANA index = 1 + 4 [(F8 - F8)/(Fh - F.)], where F. is fluorescence signal of sample; F. is fluorescence signal of negative control; Fh is fluorescence signal of high-positive control; and 1 + 4 is the constant for the fluorometer. The more intense the fluorescent signal, the greater the binding of ANAs to cell-coated samplers and the higher the FSU displayed by the fluorometer. High-positive control serum prepared from human serum known to contain ANAs is assigned an ANA index of 5 by the manufacturer. The computer automatically assigns readings of 100 ± 5 FSU upon insertion of the high-positive control sampler. The negative control serum, prepared from human serum from ANA-negative individuals, is assigned a reference ANA index of 1.0. The FSU values obtained from the negative and high-positive control sera are the reference points for construction of the ANA calibration curve. The low-positive and unknown samplers are inserted into the fluorometer with the subsequent calculation of the ANA index by the computer. For manual calculation of ANA indices, the fluorometer is adjusted to zero without a sampler in the sampler stage. The high-positive control sampler is inserted, and the fluorometer is manually adjusted to read 100 ± 5 FSU. The low-positive, negative, and unknown patient samplers are then inserted into the fluorometer, and the FSU value of each sampler is recorded. Using linear graph paper, the FSU value is plotted on the y axis and the ANA index is plotted on the x axis. Points for high-positive and negative controls are plotted using measured FSU values and preassigned ANA indices (see above). The two points are connected, constructing the ANA calibration curve and illustrating the linear relationship between the FSU values and ANA indices. The low-positive control and the unknown patient indices can then be determined from the curve. Slide ANA determinations. Slide ANA testing was carried out using Meloy reagents (Meloy Laboratories, Inc., Springfield, Va.) throughout this study. The Meloy system employs mouse fibroblasts fixed to microscope slides as a substrate. ANA screening and titering were carried out according to the manufac-
713
ANTINUCLEAR ANTIBODIES
turer's instructions. All specimens were screened at a 1:10 dilution, and fluorescence was subjectively graded as negative, ±, 1+, 2+, 3+, or 4+. Slides were examined with a Zeiss fluorescent microscope equipped with a fluorescein isothiocyanate exciter filter at a magnification of x25 or x40. Samples positive at screening were titrated by diluting twofold from 1:10 to 1:640 in phosphate-buffered saline. Types of ANA patterns of positive specimens were recorded. FIAX screening ANA index. ANA indices from a series of known ANA-negative individuals were used to determine a negative cutoff index for ANA. A 1.25 ANA index, representing 1 standard deviation from the mean index derived from 23 ANA-negative individuals, was used as the cutoff index throughout this study. FIAX ANA titers. Serum samples with an index greater than 1.25 were serially diluted twofold from 1: 10 through 1:640 in phosphate-buffered saline and carried through the same protocol as for screening. That dilution which gave an ANA index of 1.0 or slightly greater, when compared to the negative control index of 1.0, was considered the endpoint for that serum specimen. Precision. Between-run coefficients of variation were calculated using negative and low-positive control sera provided by International Diagnostics Technology. Coefficient of variation values of FSU and ANA indices were calculated for 16 separate runs. Freezing and heat inactivation. To determine the effect of freezing on ANA-positive samples, 12 samples positive in the FLAX screening assay were subjected to one cycle of freezing at -20°C and thawing on 3 consecutive days. Aliquots of original samples were heated at 56°C for 30 min. Treated samples were then tested for significant changes in ANA indices or titers by using the FIAX system.
RESULTS Calibration curve. Figure 1 illustrates a typical ANA calibration curve automatically computed by the microcomputer based on the FSU values of the high-positive and negative control ,
120 r
.
.
.
2
3
4
)
I
6
7
S.-
z
100 80
z D.-
60
w
u
40
0 20 0 1
5
FIAX ANA INDEX
FIG. 1. FIAX ANA calibration curve.
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J. CLIN. MICROBIOL.
CASAVANT, HART, AND STITES
The microcomputer calculates the ANA index for all serum samples and the low-positive control. Alternatively, the same curve can be manually constructed by plotting the FSU values of the high-positive and negative controls against their respective preassigned ANA indices on graph paper. Calculation of FLAX ANA titers. Table 1 illustrates examples of FIAX titer calculations. Sample 30 gave a titer of 40, sample 25 gave a titer of 320, and sample 37 yielded a titer of greater than 640, since the ANA index remained above the negative control index of 1.0. Screening correlation. For preliminary comparisons, fluorescent microscopic assessment of 101 serum samples was compared with screening FIAX ANA indices. Microscopic fluorescence was graded as negative, +, 1+, 2+, 3+, or 4+, as described in Materials and Methods (Fig. 2). Two samples graded ± by microscopic examination were negative by FIAX. In the 1+ fluorescent range, 7 samples (38%) were negative by FIAX, whereas 11 samples (61%) were positive. Of more significance was the finding that only 1 out of 30 samples graded 2+ microscopically gave a FIAX index in the negative range, and none of the 38 samples graded 3+ or 4+ gave a negative FIAX ANA index. Thus, only 1 of 68 (1.4%) definitely positive samples could be considered potentially false-negative by using the FIAX system. Titer correlation. A total of 78 serum samples positive by FIAX or microscopic screening were serially diluted twofold from 1:10 through 1:640. A total of 61 (78%) of the FIAX titers were identical to or within ±1 dilution of the slide titers (Fig. 3), whereas 75 (96%) of the FIAX titers fell within ±2 dilutions of the slide titers, indicating that FIAX titers correlated well with those determined microscopically. Three discordant samples which fell out of the ±2 dilution range were retested with both methods and gave similar results. Precision. The precision data for the FIAX low-positive and negative controls are given in Table 2. Between-run coefficients of variation for the FSU values of the low-positive and negative controls were 6.8% and 10.9%, respectively, sera.
and 8.8% for the low-positive control ANA index. Coefficients of variation fell within acceptable limits. Effect of freezing and heat inactivation. Table 3 illustrates that repeated freezing of 12 ANA-positive samples did not markedly alter the ANA index (R = 0.990). Heat inactivation also had no significant effect on the ANA indices (R = 0.993). ANA titers also remained virtually unchanged after repeated freezing and heat inactivation, varying at most by one dilution. DISCUSSION Since the earliest description of indirect immunofluorescence for detecting ANAs, this procedure has become the most widely employed screening test for ANAs in clinical laboratories. The procedure is highly sensitive; a negative test almost excludes a diagnosis of systemic lupus erythematosus unless a patient is on corticosteroids or immunosuppressive therapy (5). ANA determinations are procedures of potentially large volume due to their documented clinical value. Any modification of existing indirect immunofluorescence which contributes to more efficient and objective measurement of the presence of ANAs would be a significant advance in a clinical immunology laboratory. Results of this study indicated good correlation between the semiautomated FIAX ANA system and conventional indirect immunofluorescence. A cutoff index was established to quickly screen the ANA-positive from negative samples. Subjective brightness correlated well with the screening FIAX ANA index values. FIAX titers were easily determined and correlated well with those determined microscopically using commercial slides. Agreement of ANA titers between FIAX and slide indirect immunofluorescence was similar to the agreement reported by Meloy Laboratories (Meloy ANA kit insert) when their commercial slides were compared with mouse liver as substrates. Eighty percent of the ANA titers with Meloy commercial slides were identical or within one dilution of the reference method. Variation between FIAX and slide titers could
TABLE 1. Calculation of FIAX ANA titers ANA index at dilution:
FIAX ti-
Sample no.
30 25 37
'ND, Not done.
ter
1:10
1:20
1:40
1:80
1:160
1:320
1:640
ter
2.30 3.70 2.90
1.30 2.65 3.05
1.15 2.05 2.90
0.70 1.55 2.80
0.50 1.25 2.30
ND" 1.05 1.90
ND 0.90 1.55
40 320 640
VOL. 10, 1979
ANTINUCLEAR ANTIBODIES
0O
5.5
0 0
5.0 00 0
0O
4.5
00
0
4.0 0 0
x
S
0
3.5
2
0
0 0 0 0
x
3.0
0
S
0
0
0
2.5
0* 00 00
0 0 0 0 0
0
0
2.0
0 0
0@ 0
0 0 000
0
0
*0-
00
00
1.5
0
I
1.0 .7
5
0
qp '__ * N.
-0 2. SLIDE FLUORESCENCE
3.
40
FIG. 2. Comparison of FIAX ANA indices with microscopic fluorescence of mouse fibroblasts.
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J. CLIN. MICROBIOL.
CASAVANT, HART, AND STITES
SLIDE TITER
FIG. 3. Comparison of FIAX ANA titers with slide titers. (I) FIAX titer fourfold above slide titer; (II) FIAX titer twofold above slide titer; (III) FIAX titer twofold below slide titer; and (IV) FIAX titer fourfold below slide titer.
be due to differences in the nature as well as the concentration of nuclear antigens present in substrates employed in this study. At least 13 subroups of ANAs with immunological specificity for deoxyribonucleic acid, deoxyribonucleic acidhistone, non-histone proteins, or nucleolar ribonucleic acid have been described (6). Since ANAs present in sera from different individuals rarely have the same specificity for nuclear antigens, the variety of substrates currently employed in ANA testing (1) could account for discrepancies in detection and titration of ANAs as well as differences in fluorescent patterns. Hahon et al. (3) evaluated the sensitivity of 10 human and animal cell lines as well as tissue sections as ANA substrates. These authors rated commercial rat kidney and liver tissue the least sensitive, whereas baby hamster kidney and human lung cell monolayers were the most sensitive substrates for detecting ANAs. Although
both the FIAX and Meloy slide substrates consisted of continuous cell lines, the difference in species source might account, in part, for the variation in titer agreement between the two methods. ANAs are only one species of antibodies present in serum from patients with systemic lupus erythematosus. Antibodies directed against cytoplasmic antigens can also be detected (2, 7-9). The number of antibodies against cytoplasmic determinants and the significance of these antibodies in systemic lupus erythematosus remain unclear. In our experience, serum samples without detectable ANAs, but producing cytoplasmic fluorescence with mouse fibroblasts and human epithelial cells, are occasionally encountered. Cytoplasmic fluorescence might produce falsepositive ANA results with the FIAX system if samples are screened without microscopic examination of fluorescent staining patterns. How-
VOL. 10, 1979
ANTINUCLEAR ANTIBODIES
ever, screening and titering of samples containing ANAs with the FIAX system, followed by examination of the fluorescent pattern using a substrate slide, allows cytoplasmic and nuclear fluorescence to be differentiated. TABLE 2. Between-run coefficients of variation of FIAX negative and low-positive control sera Run no.
FSU . . Low-posiNegative tive control control
2 4 5A 6A 6B 7A 7B 8A 8B 8C 8D 9 10 11 12 13
69 69 73 71 73 76 66 69 70 68 72 72 67 55 65 67
41 28 35 36 38
N X SDa
16 68.88 4.72 6.8%
16 35.59 3.9 10.9%
CVb
39 36 35 41 36 32 32 36 30 41 33
ANA index,
low-positive cotl control
2.85 3.28 3.30 3.17 3.25 3.40 2.85 3.20 2.98 3.00 3.30 3.35 2.95 2.40 2.70 3.10 16 3.07 0.27 8.8%
aSD, Standard deviation. CV, Coefficient of variation.
b
717
In addition to acceptable correlation of ANA results, the relative cost of reagents and technologist time was considered when comparing the FLAX system with the slide indirect immunofluorescence method for performing ANA testing. Reagent costs for the two methods were comparable, based on reagents being marketed in 50-determination kits. Less technologist time is required with the FIAX method than with the slide procedure. The FIAX protocol requires approximately 1 h of incubation time from sample preparation to final readout of ANA titers. However, slide indirect immunofluorescence required approximately 1 h 45 min from sample preparation to preparedness for reading. At that point, a technologist must begin visually screening slides for the presence or absence of ANAs. The FIAX system also offers the advantage of freeing technologist time, since the only activity required during the assay is transferring samplers from rows of reagent tubes, requiring only a few seconds. In contrast, with conventional indirect immnunofluorescence a technologist must manually wash and blot slides at each step in the protocol. Results of FIAX precision studies suggest that objective instrumental reading of fluorescence may eliminate technologist variation in determining endpoints when screening and titrating ANA-positive sera. The FIAX system potentially offers a more efficient, objective methodology for determining the presence of ANAs in human serum. The assay time savings is considerable when large numbers of serum samples are screened.
TABLE 3. Effect offreezing, thawing, and heat inactivation on FIAX ANA indices and titers FLAX ANA index FIAX ANA titer Sample no.
1 2 3 4 5 6 7 8 9 10 11 12
Untreated' 2.1 1.4
1.6 4.6 1.3 7.6 1.4 1.9 2.4 1.1 2.1 1.2
-200C
560C
Untreated
-200C
2.0 1.7 1.9 4.2 1.4 6.5 1.1 1.7 2.5 1.2 1.7 1.0
2.4 1.4 1.4 4.3 1.5 7.5 1.6 2.1 2.8
160 40 40 640 80 640 80 160 320
160 80 40 320 80 640 40 160 320
1.4 2.1 1.0
40 160 40
80 160 40
0.990 0.993 R 0.95 0.83 Slope 0.175 0.252 Intercept 'Serum samples were screened at a 1:20 dilution in phosphate-buffered saline.
560C 160 40
40 320 80 640 80 80 320 80 160 40
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J. CLIN. MICROBIOL.
CASAVANT, HART, AND STITES LITERATURE CITED
1. Barnett, E. V. 1970. Substrates for antinuclear factors, p. 75-84. In E. J. Holborrow (ed.), Standardization in 2.
3. 4.
5.
immunofluorescence. Blackwell Scientific Publications, Oxford. Deicher, H. R., H. R. Holman, and H. G. Kunkel. 1960. Anticytoplasmic factors in the sera of patients with systemic lupus erythematosus and certain other diseases. Arthr. Rheum. 3:1-15. Hahon, N., H. L. Eckert, and J. Stewart. 1975. Evaluation of cellular substrates for antinuclear antibody determinations. J. Clin. Microbiol. 2:42-45. Holman, H. R., and H. G. Kunkel. 1957. Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science 125:162-163. Nakamura, R. M. 1974. Immunopathology, clinical lab-
6.
7.
8.
9.
oratory concepts and methods, p. 247-288. Little, Brown and Co., Boston. Nakamura, R. M., and C. A. Greenwald. 1978. Current status of laboratory test for autoantibodies to nuclear antigens (ANA) in systemic rheumatic diseases. Lab. Res. Meth. Biol. Med. 3:318-338. Schur, P. R., L. E. Morox, and H. G. Kunkel. 1967. Precipitating antibodies to ribosomes in the serum of patients with systemic lupus erythematosus. Immunochemistry 4:447-453. Sturgill, B. C., and R. R. Carpenter. 1965. Antibody to ribosomes in systemic lupus erythematosus. Arthr. Rheum. 8:213-218. Wiedermann, G., and P. A. Miescher. 1965. Cytoplasmic antibodies in patients with systemic lupus erythematosus. Ann. N.Y. Acad. Sci. 124:807-815.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1979, p. 712-718 0095-1137/79/1 1-0712/07$02.00/0
Comparison of a Semiautomated Fluorescent Immunoassay System and Indirect Immunofluorescence for Detection of Antinuclear Antibodies in Human Serum CONRAD H. CASAVANT,* ANN C. HART, AND DANIEL P. STITES Clinical Immunology Laboratory, Department of Laboratory Medicine, University of California at San Francisco, San Francisco, California 94143
Received for publication 31 August 1979
A semiautomated fluorescent immunoassay system (FIAX) for detecting antinuclear antibodies (ANAs) in human serum was compared with conventional indirect immunofluorescence using microscope slides (Meloy Laboratories) coated with mouse fibroblasts. The FIAX system uses quantitative indirect immunofluorescence to measure the specific binding of ANA to a sampler coated with human epithelial cells. A total of 101 serum samples were examined for the presence of ANAs by employing both methods. At an initial 1:10 screening dilution, 23 samples were negative for ANAs by the slide method, whereas 21 samples were negative with the FIAX system. Using 2+, 3+, and 4+ subjective brightness, 68 (100%) samples were positive by the slide method, whereas 67 (98.5%) were positive with the FIAX system. ANA-positive samples were diluted twofold from 1:10 to 1:640, and positive titers were determined by both methods. Sixty (77%) of 78 positive samples titrated by FIAX came within ±1 dilution of the titers determined by the slide method, and 75 (96%) of the samples fell within +2 dilutions. Results indicate good correlation between the FIAX system and indirect immunofluorescence for the detection of ANAs in human serum. The FIAX system has the advantage of speed, reproducibility, and the elimination of subjective microscopic assessment of ANA titers.
Autoantibodies to a variety of nuclear and cytoplasmic antigens are characteristically present in the sera of patients with many rheumatic diseases. Currently, the standard method for detecting antinuclear antibodies (ANAs) is indirect immunofluorescence as initially described by Holman and Kunkel (4) and Friou et al. (G. Friou, S. Finch, and K. Detre, Fed. Proc. 16:413, 1957). A variety of indirect immunofluorescent protocols for detecting ANAs have been described; these methods are technically similar except for the use of different tissues or cell lines as substrates. Recently a semiautomated quantitative fluorescent immunoassay system termed
rescence by using the microscope slide technique for detecting and partially quantitating ANAs in a variety of human sera. MATERIALS AND METHODS Sera. A total of 101 serum samples with routine ANA requests received in the Clinical Immunology Laboratory were used in this study. The serum samples were fresh and were examined for the presence of ANAs by both methods before freezing. FIAX ANA determinations. The components of the FIAX system include a fluorometer with a sample stage into which samplers coated with human epithelial cells are inserted and a microcomputer for calculating ANA indices. The fluorometer digitally displays fluorescence as FSU. Human epithelial cells were grown in Eagle minimal essential medium supplemented with 10% fetal calf serum, harvested, and fixed to the sampler surface to give a uniform cell monolayer. The FIAX protocol is similar to that for indirect immunofluorescence, except that cell-coated samplers are immersed in reagent tubes, whereas, for indirect immunofluorescence, serum dilutions and reagents are subsequently added to slides with fixed substrates. All FIAX reagents are marketed in a kit with 50 deter-
FIAX (International Diagnostics Technology, Santa Clara, Calif.) has been developed for measuring ANAs in serum. This method employs a cellular monolayer fixed to a plastic surface which is serially incubated in sera and fluorescent anti-immunoglobulin conjugate. Fluorescence bound to the cellular substrate is measured in a fluorometer and converted to an arbitary system of fluorescent signal units (FSU) by a computer. In the present study we compared the FIAX system with conventional indirect immunofluo-
minations.
Briefly, the FIAX method employs a 1:10 screening dilution of patient sera and negative, low-positive, and 712
VOL. 10, 1979
high-positive control sera prepared in pH 7.1 phosphate-buffered saline supplied by International Diagnostics Technology. Samplers are immersed in the diluted sera and shaken for 30 min on a shaker available from International Diagnostics Technology. ANAs present in patient sera or positive controls bind to the cell-coated samplers. Unreacted serum components are removed by transferring samplers to tubes containing phosphate-buffered saline, which are shaken for 5 min. The samplers are then transferred to tubes containing fluorescein isothiocyanate-conjugated goat anti-human immunoglobulins and are shaken for 20 min. Samplers are then transferred to a final phosphate-buffered saline wash and remain in buffer until read in the fluorometer. Determination of FLAX ANA index. Results of FIAX determinations are expressed as ANA indices. The index represents the relationship between the fluorescence obtained with an unknown sample and the fluorescence obtained with the high-positive and negative ANA control sera, expressed in the following equation: ANA index = 1 + 4 [(F8 - F8)/(Fh - F.)], where F. is fluorescence signal of sample; F. is fluorescence signal of negative control; Fh is fluorescence signal of high-positive control; and 1 + 4 is the constant for the fluorometer. The more intense the fluorescent signal, the greater the binding of ANAs to cell-coated samplers and the higher the FSU displayed by the fluorometer. High-positive control serum prepared from human serum known to contain ANAs is assigned an ANA index of 5 by the manufacturer. The computer automatically assigns readings of 100 ± 5 FSU upon insertion of the high-positive control sampler. The negative control serum, prepared from human serum from ANA-negative individuals, is assigned a reference ANA index of 1.0. The FSU values obtained from the negative and high-positive control sera are the reference points for construction of the ANA calibration curve. The low-positive and unknown samplers are inserted into the fluorometer with the subsequent calculation of the ANA index by the computer. For manual calculation of ANA indices, the fluorometer is adjusted to zero without a sampler in the sampler stage. The high-positive control sampler is inserted, and the fluorometer is manually adjusted to read 100 ± 5 FSU. The low-positive, negative, and unknown patient samplers are then inserted into the fluorometer, and the FSU value of each sampler is recorded. Using linear graph paper, the FSU value is plotted on the y axis and the ANA index is plotted on the x axis. Points for high-positive and negative controls are plotted using measured FSU values and preassigned ANA indices (see above). The two points are connected, constructing the ANA calibration curve and illustrating the linear relationship between the FSU values and ANA indices. The low-positive control and the unknown patient indices can then be determined from the curve. Slide ANA determinations. Slide ANA testing was carried out using Meloy reagents (Meloy Laboratories, Inc., Springfield, Va.) throughout this study. The Meloy system employs mouse fibroblasts fixed to microscope slides as a substrate. ANA screening and titering were carried out according to the manufac-
713
ANTINUCLEAR ANTIBODIES
turer's instructions. All specimens were screened at a 1:10 dilution, and fluorescence was subjectively graded as negative, ±, 1+, 2+, 3+, or 4+. Slides were examined with a Zeiss fluorescent microscope equipped with a fluorescein isothiocyanate exciter filter at a magnification of x25 or x40. Samples positive at screening were titrated by diluting twofold from 1:10 to 1:640 in phosphate-buffered saline. Types of ANA patterns of positive specimens were recorded. FIAX screening ANA index. ANA indices from a series of known ANA-negative individuals were used to determine a negative cutoff index for ANA. A 1.25 ANA index, representing 1 standard deviation from the mean index derived from 23 ANA-negative individuals, was used as the cutoff index throughout this study. FIAX ANA titers. Serum samples with an index greater than 1.25 were serially diluted twofold from 1: 10 through 1:640 in phosphate-buffered saline and carried through the same protocol as for screening. That dilution which gave an ANA index of 1.0 or slightly greater, when compared to the negative control index of 1.0, was considered the endpoint for that serum specimen. Precision. Between-run coefficients of variation were calculated using negative and low-positive control sera provided by International Diagnostics Technology. Coefficient of variation values of FSU and ANA indices were calculated for 16 separate runs. Freezing and heat inactivation. To determine the effect of freezing on ANA-positive samples, 12 samples positive in the FLAX screening assay were subjected to one cycle of freezing at -20°C and thawing on 3 consecutive days. Aliquots of original samples were heated at 56°C for 30 min. Treated samples were then tested for significant changes in ANA indices or titers by using the FIAX system.
RESULTS Calibration curve. Figure 1 illustrates a typical ANA calibration curve automatically computed by the microcomputer based on the FSU values of the high-positive and negative control ,
120 r
.
.
.
2
3
4
)
I
6
7
S.-
z
100 80
z D.-
60
w
u
40
0 20 0 1
5
FIAX ANA INDEX
FIG. 1. FIAX ANA calibration curve.
714
J. CLIN. MICROBIOL.
CASAVANT, HART, AND STITES
The microcomputer calculates the ANA index for all serum samples and the low-positive control. Alternatively, the same curve can be manually constructed by plotting the FSU values of the high-positive and negative controls against their respective preassigned ANA indices on graph paper. Calculation of FLAX ANA titers. Table 1 illustrates examples of FIAX titer calculations. Sample 30 gave a titer of 40, sample 25 gave a titer of 320, and sample 37 yielded a titer of greater than 640, since the ANA index remained above the negative control index of 1.0. Screening correlation. For preliminary comparisons, fluorescent microscopic assessment of 101 serum samples was compared with screening FIAX ANA indices. Microscopic fluorescence was graded as negative, +, 1+, 2+, 3+, or 4+, as described in Materials and Methods (Fig. 2). Two samples graded ± by microscopic examination were negative by FIAX. In the 1+ fluorescent range, 7 samples (38%) were negative by FIAX, whereas 11 samples (61%) were positive. Of more significance was the finding that only 1 out of 30 samples graded 2+ microscopically gave a FIAX index in the negative range, and none of the 38 samples graded 3+ or 4+ gave a negative FIAX ANA index. Thus, only 1 of 68 (1.4%) definitely positive samples could be considered potentially false-negative by using the FIAX system. Titer correlation. A total of 78 serum samples positive by FIAX or microscopic screening were serially diluted twofold from 1:10 through 1:640. A total of 61 (78%) of the FIAX titers were identical to or within ±1 dilution of the slide titers (Fig. 3), whereas 75 (96%) of the FIAX titers fell within ±2 dilutions of the slide titers, indicating that FIAX titers correlated well with those determined microscopically. Three discordant samples which fell out of the ±2 dilution range were retested with both methods and gave similar results. Precision. The precision data for the FIAX low-positive and negative controls are given in Table 2. Between-run coefficients of variation for the FSU values of the low-positive and negative controls were 6.8% and 10.9%, respectively, sera.
and 8.8% for the low-positive control ANA index. Coefficients of variation fell within acceptable limits. Effect of freezing and heat inactivation. Table 3 illustrates that repeated freezing of 12 ANA-positive samples did not markedly alter the ANA index (R = 0.990). Heat inactivation also had no significant effect on the ANA indices (R = 0.993). ANA titers also remained virtually unchanged after repeated freezing and heat inactivation, varying at most by one dilution. DISCUSSION Since the earliest description of indirect immunofluorescence for detecting ANAs, this procedure has become the most widely employed screening test for ANAs in clinical laboratories. The procedure is highly sensitive; a negative test almost excludes a diagnosis of systemic lupus erythematosus unless a patient is on corticosteroids or immunosuppressive therapy (5). ANA determinations are procedures of potentially large volume due to their documented clinical value. Any modification of existing indirect immunofluorescence which contributes to more efficient and objective measurement of the presence of ANAs would be a significant advance in a clinical immunology laboratory. Results of this study indicated good correlation between the semiautomated FIAX ANA system and conventional indirect immunofluorescence. A cutoff index was established to quickly screen the ANA-positive from negative samples. Subjective brightness correlated well with the screening FIAX ANA index values. FIAX titers were easily determined and correlated well with those determined microscopically using commercial slides. Agreement of ANA titers between FIAX and slide indirect immunofluorescence was similar to the agreement reported by Meloy Laboratories (Meloy ANA kit insert) when their commercial slides were compared with mouse liver as substrates. Eighty percent of the ANA titers with Meloy commercial slides were identical or within one dilution of the reference method. Variation between FIAX and slide titers could
TABLE 1. Calculation of FIAX ANA titers ANA index at dilution:
FIAX ti-
Sample no.
30 25 37
'ND, Not done.
ter
1:10
1:20
1:40
1:80
1:160
1:320
1:640
ter
2.30 3.70 2.90
1.30 2.65 3.05
1.15 2.05 2.90
0.70 1.55 2.80
0.50 1.25 2.30
ND" 1.05 1.90
ND 0.90 1.55
40 320 640
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ANTINUCLEAR ANTIBODIES
0O
5.5
0 0
5.0 00 0
0O
4.5
00
0
4.0 0 0
x
S
0
3.5
2
0
0 0 0 0
x
3.0
0
S
0
0
0
2.5
0* 00 00
0 0 0 0 0
0
0
2.0
0 0
0@ 0
0 0 000
0
0
*0-
00
00
1.5
0
I
1.0 .7
5
0
qp '__ * N.
-0 2. SLIDE FLUORESCENCE
3.
40
FIG. 2. Comparison of FIAX ANA indices with microscopic fluorescence of mouse fibroblasts.
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J. CLIN. MICROBIOL.
CASAVANT, HART, AND STITES
SLIDE TITER
FIG. 3. Comparison of FIAX ANA titers with slide titers. (I) FIAX titer fourfold above slide titer; (II) FIAX titer twofold above slide titer; (III) FIAX titer twofold below slide titer; and (IV) FIAX titer fourfold below slide titer.
be due to differences in the nature as well as the concentration of nuclear antigens present in substrates employed in this study. At least 13 subroups of ANAs with immunological specificity for deoxyribonucleic acid, deoxyribonucleic acidhistone, non-histone proteins, or nucleolar ribonucleic acid have been described (6). Since ANAs present in sera from different individuals rarely have the same specificity for nuclear antigens, the variety of substrates currently employed in ANA testing (1) could account for discrepancies in detection and titration of ANAs as well as differences in fluorescent patterns. Hahon et al. (3) evaluated the sensitivity of 10 human and animal cell lines as well as tissue sections as ANA substrates. These authors rated commercial rat kidney and liver tissue the least sensitive, whereas baby hamster kidney and human lung cell monolayers were the most sensitive substrates for detecting ANAs. Although
both the FIAX and Meloy slide substrates consisted of continuous cell lines, the difference in species source might account, in part, for the variation in titer agreement between the two methods. ANAs are only one species of antibodies present in serum from patients with systemic lupus erythematosus. Antibodies directed against cytoplasmic antigens can also be detected (2, 7-9). The number of antibodies against cytoplasmic determinants and the significance of these antibodies in systemic lupus erythematosus remain unclear. In our experience, serum samples without detectable ANAs, but producing cytoplasmic fluorescence with mouse fibroblasts and human epithelial cells, are occasionally encountered. Cytoplasmic fluorescence might produce falsepositive ANA results with the FIAX system if samples are screened without microscopic examination of fluorescent staining patterns. How-
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ANTINUCLEAR ANTIBODIES
ever, screening and titering of samples containing ANAs with the FIAX system, followed by examination of the fluorescent pattern using a substrate slide, allows cytoplasmic and nuclear fluorescence to be differentiated. TABLE 2. Between-run coefficients of variation of FIAX negative and low-positive control sera Run no.
FSU . . Low-posiNegative tive control control
2 4 5A 6A 6B 7A 7B 8A 8B 8C 8D 9 10 11 12 13
69 69 73 71 73 76 66 69 70 68 72 72 67 55 65 67
41 28 35 36 38
N X SDa
16 68.88 4.72 6.8%
16 35.59 3.9 10.9%
CVb
39 36 35 41 36 32 32 36 30 41 33
ANA index,
low-positive cotl control
2.85 3.28 3.30 3.17 3.25 3.40 2.85 3.20 2.98 3.00 3.30 3.35 2.95 2.40 2.70 3.10 16 3.07 0.27 8.8%
aSD, Standard deviation. CV, Coefficient of variation.
b
717
In addition to acceptable correlation of ANA results, the relative cost of reagents and technologist time was considered when comparing the FLAX system with the slide indirect immunofluorescence method for performing ANA testing. Reagent costs for the two methods were comparable, based on reagents being marketed in 50-determination kits. Less technologist time is required with the FIAX method than with the slide procedure. The FIAX protocol requires approximately 1 h of incubation time from sample preparation to final readout of ANA titers. However, slide indirect immunofluorescence required approximately 1 h 45 min from sample preparation to preparedness for reading. At that point, a technologist must begin visually screening slides for the presence or absence of ANAs. The FIAX system also offers the advantage of freeing technologist time, since the only activity required during the assay is transferring samplers from rows of reagent tubes, requiring only a few seconds. In contrast, with conventional indirect immnunofluorescence a technologist must manually wash and blot slides at each step in the protocol. Results of FIAX precision studies suggest that objective instrumental reading of fluorescence may eliminate technologist variation in determining endpoints when screening and titrating ANA-positive sera. The FIAX system potentially offers a more efficient, objective methodology for determining the presence of ANAs in human serum. The assay time savings is considerable when large numbers of serum samples are screened.
TABLE 3. Effect offreezing, thawing, and heat inactivation on FIAX ANA indices and titers FLAX ANA index FIAX ANA titer Sample no.
1 2 3 4 5 6 7 8 9 10 11 12
Untreated' 2.1 1.4
1.6 4.6 1.3 7.6 1.4 1.9 2.4 1.1 2.1 1.2
-200C
560C
Untreated
-200C
2.0 1.7 1.9 4.2 1.4 6.5 1.1 1.7 2.5 1.2 1.7 1.0
2.4 1.4 1.4 4.3 1.5 7.5 1.6 2.1 2.8
160 40 40 640 80 640 80 160 320
160 80 40 320 80 640 40 160 320
1.4 2.1 1.0
40 160 40
80 160 40
0.990 0.993 R 0.95 0.83 Slope 0.175 0.252 Intercept 'Serum samples were screened at a 1:20 dilution in phosphate-buffered saline.
560C 160 40
40 320 80 640 80 80 320 80 160 40
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CASAVANT, HART, AND STITES LITERATURE CITED
1. Barnett, E. V. 1970. Substrates for antinuclear factors, p. 75-84. In E. J. Holborrow (ed.), Standardization in 2.
3. 4.
5.
immunofluorescence. Blackwell Scientific Publications, Oxford. Deicher, H. R., H. R. Holman, and H. G. Kunkel. 1960. Anticytoplasmic factors in the sera of patients with systemic lupus erythematosus and certain other diseases. Arthr. Rheum. 3:1-15. Hahon, N., H. L. Eckert, and J. Stewart. 1975. Evaluation of cellular substrates for antinuclear antibody determinations. J. Clin. Microbiol. 2:42-45. Holman, H. R., and H. G. Kunkel. 1957. Affinity between the lupus erythematosus serum factor and cell nuclei and nucleoprotein. Science 125:162-163. Nakamura, R. M. 1974. Immunopathology, clinical lab-
6.
7.
8.
9.
oratory concepts and methods, p. 247-288. Little, Brown and Co., Boston. Nakamura, R. M., and C. A. Greenwald. 1978. Current status of laboratory test for autoantibodies to nuclear antigens (ANA) in systemic rheumatic diseases. Lab. Res. Meth. Biol. Med. 3:318-338. Schur, P. R., L. E. Morox, and H. G. Kunkel. 1967. Precipitating antibodies to ribosomes in the serum of patients with systemic lupus erythematosus. Immunochemistry 4:447-453. Sturgill, B. C., and R. R. Carpenter. 1965. Antibody to ribosomes in systemic lupus erythematosus. Arthr. Rheum. 8:213-218. Wiedermann, G., and P. A. Miescher. 1965. Cytoplasmic antibodies in patients with systemic lupus erythematosus. Ann. N.Y. Acad. Sci. 124:807-815.
