Laboratory Medicine

Won, Dong, II

doi: 10.1093/labmed/lmz087pmid: 31872218

Abstract Background Automated systems for antinuclear antibody (ANA) testing provide endpoint titers that are predicted based on the fluorescence intensity (FI) value at a screening dilution (single-well titration [SWT]) showing frequent titration errors (more than plus or minus 1 dilution). Methods Line slope titration (LST) was based on the trend of FI values on dilutions. Three dilutions per specimen were prepared considering a patient’s previous titer or FI at the screening dilution. On the XY plot, with the reciprocal of dilution as the X-axis and FI value as the Y-axis, a fitted line was drawn to obtain the endpoint titers. Results The titration error rate (no. of errors/total no.) of LST using a regression line was lower than that of SWT (31/710 [4.4%] and 152/674 [22.6%], respectively; P < .000000001), with serial dilution as a reference. When comparing a regression line using 3 dilution points with a line using 2 dilution points, the error rate of the former was not significantly different from that of the latter (31/710 [4.4%] and 31/746 [4.2%], respectively; P = .842). Conclusions This LST method is useful as an accurate, cost-effective, and rapid approach to measure endpoint titers in routine ANA testing. anti-nuclear antibody testing, endpoint titer, fluorescence intensity, serial dilution, automated system, systemic autoimmune rheumatic disease Typically, the first laboratory testing to be performed on a patient presenting with clinical manifestations of systemic autoimmune rheumatic disease (SARD) is anti-nuclear antibody (ANA) testing by indirect immunofluorescence (IIF) using the HEp-2 cells of the human epithelioma of the larynx. This testing is the preferred screening method for SARD due to its high sensitivity.1 Different possible patterns, intensities, and endpoint titers on a consecutive dilution series have to be carefully examined with this testing method.2 As there are limited data suggesting a correlation between ANA titer and disease activity in patients with systemic lupus erythematosus (SLE), the ANA titer is of little value in monitoring SLE disease activity.3 However, the ANA titer, as well as the ANA pattern, strongly enhances our ability to discriminate between ANA-positive healthy individuals and patients with SARD, and unusually elevated ANA titers are important in the diagnosis of SARD.2,4 Thus, the determination of endpoint titers is offered by a majority of the routine autoimmune laboratories.5 Automated systems perform the acquisition of digital images of stained HEp-2 cells, measurements of fluorescence intensity (FI), assignment of preliminary categorization of the test specimen as positive or negative, and interpretation of the staining pattern. In addition, they enable the prediction of endpoint titers based on the measured FI value avoiding serial dilution.5–8 In the NOVA View system (INOVA Diagnostics, San Diego, CA), this function is called single-well titration, which considers both the FI value and ANA pattern at a single well of the screening dilution (1:80) based on the built-in standard curves for the 8 individual ANA patterns (homogeneous, speckled, centromere, nucleolar, nuclear dots, fine speckled, coarse speckled, and dense fine speckled). However, when comparing the titers by single-well titration with those by serial dilution (the method of reference), the agreement (within plus or minus 1 dilution) is known to be poor regardless of the automated system used, as evidenced from the following agreement data (no. of agreement/total no.): 25/37 (67.6%) by the NOVA View system,7 110/176 (62.5%) by the PolyTiter system (Polymedco, Inc., Cortlandt Manor, NY),9 3,239/3,610 (89.7%) by the AKLIDES system,5 and 34/41 (82.9%) by the EUROPattern Suite system (Euroimmun AG, Lübeck, Germany).10 Additionally, the agreement rate was 139/176 (79.0%) using the 3-well dilution protocol by the EUROPattern Suite system,9 and the agreement (different by zero dilution) was 41/94 (43.7%) by the NOVA View system.11 As noted above, the usual error rate in predicting endpoint titers in a single well is ~20%. Moreover, when the ANA pattern is mixed, cytoplasmic, or undetermined, no prediction can be made. In this case, the only available method is serial dilution up to the endpoint, which is uneconomical and time consuming. The author hypothesizes that endpoint titers can be predicted based on the decreasing trend of FI grades on the minimum number of dilutions prepared considering a patient’s previous titer or FI at the screening dilution even before reaching or passing through the negative conversion of FI. In this study, the author has investigated how the decreasing trend line is fitted onto the plot of FI as a function of dilution (ie, dilution vs FI plot) and whether the calculated endpoint titers based on the slope of this line are more accurate than are single-well titers. Materials and Methods Patient Sera Patient blood was sampled for diagnostic purposes only and was submitted for ANA testing at the Clinical Immunology Laboratory at the Kyungpook National University Hospital (Daegu, South Korea). A total of 759 patient serum specimens were characterized as ANA positive and grouped according to their ANA patterns (Table 1). For adherence to the tenets of the Declaration of Helsinki (1964) ethical guidelines, specimens were blinded for research to maintain confidentiality. The study protocol was determined to be exempt by the Institutional Review Board of the Kyungpook National University Hospital. Table 1. ANA Pattern and Endpoint Titer of Positive Patient Specimens Enrolled in this Study Endpoint Titer . ANA Pattern, no. of Specimens . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Total . 80 50 37 15 1 17 120 160 48 30 12 6 10 106 320 26 39 14 13 12 104 640 36 33 9 35 17 130 1280 31 35 3 59 18 146 2560 18 19 2 35 2 76 5120 4 17 1 13 2 37 10,240 6 11 0 5 0 22 20,480 0 12 1 2 0 15 40,960 0 3 0 0 0 3 Total 219 236 57 169 78 759 Endpoint Titer . ANA Pattern, no. of Specimens . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Total . 80 50 37 15 1 17 120 160 48 30 12 6 10 106 320 26 39 14 13 12 104 640 36 33 9 35 17 130 1280 31 35 3 59 18 146 2560 18 19 2 35 2 76 5120 4 17 1 13 2 37 10,240 6 11 0 5 0 22 20,480 0 12 1 2 0 15 40,960 0 3 0 0 0 3 Total 219 236 57 169 78 759 ANA, anti-nuclear antibody. Open in new tab Table 1. ANA Pattern and Endpoint Titer of Positive Patient Specimens Enrolled in this Study Endpoint Titer . ANA Pattern, no. of Specimens . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Total . 80 50 37 15 1 17 120 160 48 30 12 6 10 106 320 26 39 14 13 12 104 640 36 33 9 35 17 130 1280 31 35 3 59 18 146 2560 18 19 2 35 2 76 5120 4 17 1 13 2 37 10,240 6 11 0 5 0 22 20,480 0 12 1 2 0 15 40,960 0 3 0 0 0 3 Total 219 236 57 169 78 759 Endpoint Titer . ANA Pattern, no. of Specimens . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Total . 80 50 37 15 1 17 120 160 48 30 12 6 10 106 320 26 39 14 13 12 104 640 36 33 9 35 17 130 1280 31 35 3 59 18 146 2560 18 19 2 35 2 76 5120 4 17 1 13 2 37 10,240 6 11 0 5 0 22 20,480 0 12 1 2 0 15 40,960 0 3 0 0 0 3 Total 219 236 57 169 78 759 ANA, anti-nuclear antibody. Open in new tab ANA Testing Conventional ANA Testing ANA slide processing, from specimen dilution to the final wash steps, was completely performed using the NOVA Lite HEp-2 IgG ANA with a 4′,6-diamidino-2-phenylindole kit and QUANTA-Lyser instrument. Digital images of stained HEp-2 cells were acquired using the NOVA View instrument with software version 2.0. The cutoff value to discriminate between a positive and negative FI value was 48 light intensity units (LIU). Two independent experts reviewed these images on liquid crystal display monitors with high resolution and high contrast ratio using the QUANTA Link (all from INOVA Diagnostics). The FI values at a screening dilution are summarized according to their ANA patterns and endpoint titers by serial dilution in Figure 1. Figure 1 Open in new tabDownload slide Endpoint titer vs FI value plot for all enrolled specimens according to their assigned ANA patterns (n = 759). The endpoint titers determined by serial dilution and digital images were assigned on the X-axis. The fluorescence intensity (FI) values measured at a screening dilution (1:80) were assigned on the Y-axis. The lowest measured titer (80) is marked with interrupted vertical blue lines for each ANA pattern. An interval between ticks on the X-axis scale corresponds to a doubling dilution. Figure 1 Open in new tabDownload slide Endpoint titer vs FI value plot for all enrolled specimens according to their assigned ANA patterns (n = 759). The endpoint titers determined by serial dilution and digital images were assigned on the X-axis. The fluorescence intensity (FI) values measured at a screening dilution (1:80) were assigned on the Y-axis. The lowest measured titer (80) is marked with interrupted vertical blue lines for each ANA pattern. An interval between ticks on the X-axis scale corresponds to a doubling dilution. For ANA screening, serum was diluted 1:80. Even when the FI value was less than 48, any specimen showing HEp-2 cells with distinct fluorescence and a discernible ANA pattern on digital images or fluorescence microscopy was also considered positive. Cytoplasmic staining alone was also determined to be ANA positive. For positive specimen, the experts assigned their reaction patterns and scored their FI using a 1 to 4 grading scale (grade 4+ for FI is comparable to a positive control). To obtain endpoint titers, positive serum specimens were diluted serially from 1:80 to 1:2560 in phosphate-buffered saline. When required, further dilutions were prepared for the next batch of testing. On a dilution series, the endpoint titer was determined as the reciprocal of the dilution immediately before the dilution of negative conversion. Single-Well Titration To perform single-well titration at a screening dilution, positive specimens were categorized into the following 5 patterns depending on their HEp-2 cell staining: homogeneous, speckled, nucleolar, centromere, and cytoplasmic. When a specimen showed a mixed pattern, the pattern characterization was based on based on the brighter fluorescence to perform single-well titration. Single-well titration by the NOVA View system could not be applied to the following important ANA patterns: (1) mixed and (2) cytoplasmic. Line Slope-Based Titration To prepare the minimum number of dilutions for line slope titration (LST), a patient’s result of the previous ANA testing was considered. Only 2 more dilutions per specimen were prepared in addition to a screening dilution (D1, 1:80), submaximal dilution (D2) and maximal dilution (D3). When a patient’s previous titer was known, D3 was the corresponding dilution to that titer. Otherwise, D3 was prepared based on an FI grade at the screening dilution, as follows: 1:160 for grade 1+, 1:320 for grade 2+, 1:640 for grade 3+, and 1:1280 for grade 4+. D2 was always D3 minus a dilution. For example, when D3 was 1:320, D2 was 1:160, and a doubling dilution series of 1:80–1:160–1:320 was prepared. When the previous titer of a patient was 1280, a dilution series of 1:80–1:640–1:1280 was prepared. The dilution series prepared for LST (n = 759) are summarized in Table 2. Table 2. Types of Dilution Series Prepared for Line Slope Titration Dilution Series . No. . 1:80–1:160–1:320 411 1:80–1:320–1:640 128 1:80–1:640–1:1280 118 1:80–1:1280–1:2560 64 1:80–1:2560–1:5120 38 Total 759 Dilution Series . No. . 1:80–1:160–1:320 411 1:80–1:320–1:640 128 1:80–1:640–1:1280 118 1:80–1:1280–1:2560 64 1:80–1:2560–1:5120 38 Total 759 Open in new tab Table 2. Types of Dilution Series Prepared for Line Slope Titration Dilution Series . No. . 1:80–1:160–1:320 411 1:80–1:320–1:640 128 1:80–1:640–1:1280 118 1:80–1:1280–1:2560 64 1:80–1:2560–1:5120 38 Total 759 Dilution Series . No. . 1:80–1:160–1:320 411 1:80–1:320–1:640 128 1:80–1:640–1:1280 118 1:80–1:1280–1:2560 64 1:80–1:2560–1:5120 38 Total 759 Open in new tab The calculation principle of LST is schematized in Figure 2. In the XY plot with reciprocals of dilutions as the X-axis and FI values as the Y-axis, 3 points (coordinates) were named P1, P2, and P3, which corresponded to the 3 prepared dilutions, D1, D2, and D3, respectively. With these 3 points, the number of lines to be drawn was 4, as follows: regression (trend) line for all 3 points, the P1–P2 line, the P2–P3 line, and the P1–P3 line. The latter 3 lines linked a pair of points among the 3 points. The X-coordinate value on this line was calculated at a Y-coordinate value of 48 LIU (cutoff FI value, NOVA View system). Finally, endpoint titers were determined as the reciprocal of the maximal doubling dilution, which was less than or equal to the calculated X-coordinate value. Figure 2 Open in new tabDownload slide The calculation principle of line slope titration using a regression line for 3 points on the XY plot with dilution on the X-axis and fluorescence intensity on the Y-axis. A line was fitted on the linear–log plot, where the natural logarithm with a base of e was used. For visual clarity in this figure, the X-axis is scaled as logarithmic with base 2 and the Y-axis as logarithmic with base 10, which are different from those of the plot that was actually used. Therefore, the fitted line is curved in this figure instead of being straight. Figure 2 Open in new tabDownload slide The calculation principle of line slope titration using a regression line for 3 points on the XY plot with dilution on the X-axis and fluorescence intensity on the Y-axis. A line was fitted on the linear–log plot, where the natural logarithm with a base of e was used. For visual clarity in this figure, the X-axis is scaled as logarithmic with base 2 and the Y-axis as logarithmic with base 10, which are different from those of the plot that was actually used. Therefore, the fitted line is curved in this figure instead of being straight. Statistical Analysis The current United States federal regulations for proficiency testing allow deviations from the target value within plus or minus 2 doubling dilutions.12 In routine ANA testing, a titer difference of a dilution is not considered significant.13 In this study, the difference within plus or minus a dilution was considered to represent agreement. A titration error was declared when a given titration method yielded a value that was 2 or more dilutions deviant from that obtained from digital images and serial dilution as a reference. Statistical analyses were performed using SPSS version 23.0 (IBM Corp., Armonk, NY). The linearity for 3 dilution points on a dilution series per specimen on the XY plot was evaluated with an R2 value by simple regression analysis. Line slope values were compared among different ANA patterns by one-way analysis of variance (ANOVA). Titration error rates were compared between different titration methods by a χ 2 test or Fisher’s exact test, with P <.05 being considered significant. Data are expressed as the mean ± standard deviation. Results Decreasing Trend of FI Values among 3 Dilutions Prepared for LST In principle, a lower FI value should be obtained at a higher dilution for a specimen. To verify this principle, the FI values of 3 dilutions (D1, D2, and D3) were compared within each specimen (Figure 3). Most cases (710/754, 93.9%) showed a decreasing trend of D1, D2, and then D3. However, reverse (ie, increasing) trends of D1 less than D2, D2 less than D3, and D1 less than D3 were often observed as follows: 29/756 (3.8%), 14/755 (1.9%), and 9/755 (1.2%), respectively. At the screening dilution (D1), the specimens with D1 less than D2 showed higher FI values than did the other specimens with D1 greater than D2 (1955 ± 880 LIU [n = 29] and 1069 ± 1037 LIU [n = 727], respectively; P <.00001). This finding suggests that prozone effects (antibody excess) should occur at the screening dilution of the reversed specimens. Figure 3 Open in new tabDownload slide Decreasing trend of FI values among 3 dilutions prepared for line slope titration. Three points were named P1, P2, and P3 for the 3 dilutions D1, D2, and D3, respectively. This plot is presented in a log–log model for visual clarity. Only partial data (n = 250) of total enrolled specimens (n = 759) are presented for the same reason. This figure shows some cases with reversed trend of FI values on a dilution series. The proportion of specimens of (D1 < D2) suspected of prozone effects was 29/756 (3.8%). Abbreviations: D1, dilution 1; P1, point 1; and FI, fluorescence intensity. Figure 3 Open in new tabDownload slide Decreasing trend of FI values among 3 dilutions prepared for line slope titration. Three points were named P1, P2, and P3 for the 3 dilutions D1, D2, and D3, respectively. This plot is presented in a log–log model for visual clarity. Only partial data (n = 250) of total enrolled specimens (n = 759) are presented for the same reason. This figure shows some cases with reversed trend of FI values on a dilution series. The proportion of specimens of (D1 < D2) suspected of prozone effects was 29/756 (3.8%). Abbreviations: D1, dilution 1; P1, point 1; and FI, fluorescence intensity. Linearity of 3 Dilution Points to Compare Line-Fitting Effectiveness among Different Models for the XY Plot In the first experiment, the best model for line fitting (ie, standard curve fitting) was selected for plotting in the XY plot. In this plot, reciprocals of dilutions were assigned the X-axis and FI values the Y-axis. Two types of scales, linear and logarithmic, were tried for each axis. Thus, the total number of candidate models to be applied was 4, linear–linear, log–linear, linear–log, and log–log. The logarithmic base was always the mathematical constant e (ie, natural logarithm [ln]). On such a plot, the linearity for 3 dilution points on a dilution series per specimen was evaluated by simple regression analysis. Two statistical parameters for a regression line (R2 value [coefficient of determination] and P), which reflect the line-fitting effectiveness, were compared among these 4 scale models. In a total of 759 cases of enrolled specimens, the cases showing a reversed trend (D1 < D2, D2 < D3, or D1 < D3, n = 49) were excluded from this experiment because these cases were suspected to have prozone effects. Among the remaining cases showing a decreasing trend (n = 710), the proportion of P value of <0.05 and the proportion of R2 value of >0.96 are summarized in Table 3 according to the scale model. The log–log model showed a relatively low proportion of P values of <0.05 despite the highest proportion of R2 value of >0.96 (27.9% and 64.6%, respectively). The linear–log model appeared to be the most adequate in both rates (100% and 60.6%, respectively). Furthermore, this model showed a titration error rate of 4.2%, which was the lowest among the 4 models using P1–P3 line slope. Therefore, this linear–log model was adopted for application to the XY plot throughout this study. Table 3. Regression Line Analysis for 3 Dilution Points on a Dilution Series per Specimen in 4 Modes for the XY Plota Scale Model (X–Y) . Regression Line for 3 Dilutions, Mean ± Standard Deviation, Coefficient of Variance (%) . . Titration Error Rate by P1–P3 Line Slope, no. of Errors/Total no. (%) . . R2 > 0.96 . P < .05 . . Linear–linear scale 88/710 (12.4) 710/710 (100) 84/747 (11.2) Log–linear scale 398/710 (56.1) 198/710 (27.9) 53/747 (7.1) Linear–log scale 430/710 (60.6) 710/710 (100) 31/746 (4.2) Log–log scale 459/710 (64.6) 198/710 (27.9) 226/746 (30.3) Scale Model (X–Y) . Regression Line for 3 Dilutions, Mean ± Standard Deviation, Coefficient of Variance (%) . . Titration Error Rate by P1–P3 Line Slope, no. of Errors/Total no. (%) . . R2 > 0.96 . P < .05 . . Linear–linear scale 88/710 (12.4) 710/710 (100) 84/747 (11.2) Log–linear scale 398/710 (56.1) 198/710 (27.9) 53/747 (7.1) Linear–log scale 430/710 (60.6) 710/710 (100) 31/746 (4.2) Log–log scale 459/710 (64.6) 198/710 (27.9) 226/746 (30.3) aReciprocals of dilutions were assigned to abscissa (X-axis), and fluorescence intensity (FI) values were assigned to ordinate (Y-axis). Open in new tab Table 3. Regression Line Analysis for 3 Dilution Points on a Dilution Series per Specimen in 4 Modes for the XY Plota Scale Model (X–Y) . Regression Line for 3 Dilutions, Mean ± Standard Deviation, Coefficient of Variance (%) . . Titration Error Rate by P1–P3 Line Slope, no. of Errors/Total no. (%) . . R2 > 0.96 . P < .05 . . Linear–linear scale 88/710 (12.4) 710/710 (100) 84/747 (11.2) Log–linear scale 398/710 (56.1) 198/710 (27.9) 53/747 (7.1) Linear–log scale 430/710 (60.6) 710/710 (100) 31/746 (4.2) Log–log scale 459/710 (64.6) 198/710 (27.9) 226/746 (30.3) Scale Model (X–Y) . Regression Line for 3 Dilutions, Mean ± Standard Deviation, Coefficient of Variance (%) . . Titration Error Rate by P1–P3 Line Slope, no. of Errors/Total no. (%) . . R2 > 0.96 . P < .05 . . Linear–linear scale 88/710 (12.4) 710/710 (100) 84/747 (11.2) Log–linear scale 398/710 (56.1) 198/710 (27.9) 53/747 (7.1) Linear–log scale 430/710 (60.6) 710/710 (100) 31/746 (4.2) Log–log scale 459/710 (64.6) 198/710 (27.9) 226/746 (30.3) aReciprocals of dilutions were assigned to abscissa (X-axis), and fluorescence intensity (FI) values were assigned to ordinate (Y-axis). Open in new tab When considering only the cases with a decreasing trend of FI values on a dilution series (excluding the reversed cases suspected of having prozone effects), R2 values were not the same among the different ANA patterns (ANOVA, P < .000005) as shown by the following values: 0.919 ± 0.159 of total cases, 0.943 ± 0.137 in the homogeneous pattern (highest), 0.904 ± 0.175 in the centromere pattern (the lowest of nuclear patterns), 0.909 ± 0.175 in the speckled pattern, 0.926 ± 0.084 in the nucleolar pattern, and 0.904 ± 0.166 in the cytoplasmic pattern. Slope Values of the Regression Line According to Individual ANA Patterns When considering only the cases showing a decreasing trend of FI values of 3 points on a dilution series, the slope value of the regression line was not the same among different ANA patterns (P < .0005, ANOVA; Table 4). The difference in the slope values between 2 different ANA patterns was also significant (P < .05, post-hoc test) in 6 out of 10 pairs of ANA patterns (Table 5). This finding suggests that the line slope of a serum should be suggestive of its ANA pattern, and thus, its endpoint titer can be predicted based on the calculated value of this line slope. Table 4. Line Equation [slope (a) and Y-intercept (b)] on the XY Plota Using Linear–Log Model [ln(y) = ax + b] According to Individual ANA Patterns ANA Pattern . Regression Line for 3 Dilution Points, Mean ± Standard Deviation, Coefficient of Variance (%) . . . P1–P3 Line, Mean ± Standard Deviation, Coefficient of Variance (%) . . . . Slope . Y-Intercept . No. . Slope . Y-Intercept . No. . Homogeneous −0.0034 ± 0.0061, 182 7.36 ± 13.47, 183 210 −0.0015 ± 0.0107, 690 3.59 ± 6.48, 181 218 Speckled −0.0018 ± 0.0036, 200 7.69 ± 15.74, 205 215 −0.0010 ± 0.0033, 324 5.51 ± 13.07, 237 230 Nucleolar −0.0048 ± 0.0084, 175 6.72 ± 13.00, 193 57 −0.0045 ± 0.0080, 177 6.33 ± 12.25, 193 57 Centromere −0.0022 ± 0.0022, 104 7.37 ± 8.36, 113 158 −0.0020 ± 0.0020, 104 6.67 ± 7.49, 112 164 Cytoplasmic −0.0041 ± 0.0054, 132 6.05 ± 8.70, 144 70 −0.0030 ± 0.0042, 138 5.11 ± 7.14, 140 77 Total −0.0025 ± 0.0046, 185 7.40 ± 14.44, 195 710 −0.0015 ± 0.0070, 459 5.16 ± 12.11, 235 746 ANA Pattern . Regression Line for 3 Dilution Points, Mean ± Standard Deviation, Coefficient of Variance (%) . . . P1–P3 Line, Mean ± Standard Deviation, Coefficient of Variance (%) . . . . Slope . Y-Intercept . No. . Slope . Y-Intercept . No. . Homogeneous −0.0034 ± 0.0061, 182 7.36 ± 13.47, 183 210 −0.0015 ± 0.0107, 690 3.59 ± 6.48, 181 218 Speckled −0.0018 ± 0.0036, 200 7.69 ± 15.74, 205 215 −0.0010 ± 0.0033, 324 5.51 ± 13.07, 237 230 Nucleolar −0.0048 ± 0.0084, 175 6.72 ± 13.00, 193 57 −0.0045 ± 0.0080, 177 6.33 ± 12.25, 193 57 Centromere −0.0022 ± 0.0022, 104 7.37 ± 8.36, 113 158 −0.0020 ± 0.0020, 104 6.67 ± 7.49, 112 164 Cytoplasmic −0.0041 ± 0.0054, 132 6.05 ± 8.70, 144 70 −0.0030 ± 0.0042, 138 5.11 ± 7.14, 140 77 Total −0.0025 ± 0.0046, 185 7.40 ± 14.44, 195 710 −0.0015 ± 0.0070, 459 5.16 ± 12.11, 235 746 ANA, anti-nuclear antibody. aReciprocals of dilution are on the X-axis; ln (fluorescence intensity value) is on the Y-axis. See Table 5 for multiple comparison of the slope values between 2 different ANA patterns. Open in new tab Table 4. Line Equation [slope (a) and Y-intercept (b)] on the XY Plota Using Linear–Log Model [ln(y) = ax + b] According to Individual ANA Patterns ANA Pattern . Regression Line for 3 Dilution Points, Mean ± Standard Deviation, Coefficient of Variance (%) . . . P1–P3 Line, Mean ± Standard Deviation, Coefficient of Variance (%) . . . . Slope . Y-Intercept . No. . Slope . Y-Intercept . No. . Homogeneous −0.0034 ± 0.0061, 182 7.36 ± 13.47, 183 210 −0.0015 ± 0.0107, 690 3.59 ± 6.48, 181 218 Speckled −0.0018 ± 0.0036, 200 7.69 ± 15.74, 205 215 −0.0010 ± 0.0033, 324 5.51 ± 13.07, 237 230 Nucleolar −0.0048 ± 0.0084, 175 6.72 ± 13.00, 193 57 −0.0045 ± 0.0080, 177 6.33 ± 12.25, 193 57 Centromere −0.0022 ± 0.0022, 104 7.37 ± 8.36, 113 158 −0.0020 ± 0.0020, 104 6.67 ± 7.49, 112 164 Cytoplasmic −0.0041 ± 0.0054, 132 6.05 ± 8.70, 144 70 −0.0030 ± 0.0042, 138 5.11 ± 7.14, 140 77 Total −0.0025 ± 0.0046, 185 7.40 ± 14.44, 195 710 −0.0015 ± 0.0070, 459 5.16 ± 12.11, 235 746 ANA Pattern . Regression Line for 3 Dilution Points, Mean ± Standard Deviation, Coefficient of Variance (%) . . . P1–P3 Line, Mean ± Standard Deviation, Coefficient of Variance (%) . . . . Slope . Y-Intercept . No. . Slope . Y-Intercept . No. . Homogeneous −0.0034 ± 0.0061, 182 7.36 ± 13.47, 183 210 −0.0015 ± 0.0107, 690 3.59 ± 6.48, 181 218 Speckled −0.0018 ± 0.0036, 200 7.69 ± 15.74, 205 215 −0.0010 ± 0.0033, 324 5.51 ± 13.07, 237 230 Nucleolar −0.0048 ± 0.0084, 175 6.72 ± 13.00, 193 57 −0.0045 ± 0.0080, 177 6.33 ± 12.25, 193 57 Centromere −0.0022 ± 0.0022, 104 7.37 ± 8.36, 113 158 −0.0020 ± 0.0020, 104 6.67 ± 7.49, 112 164 Cytoplasmic −0.0041 ± 0.0054, 132 6.05 ± 8.70, 144 70 −0.0030 ± 0.0042, 138 5.11 ± 7.14, 140 77 Total −0.0025 ± 0.0046, 185 7.40 ± 14.44, 195 710 −0.0015 ± 0.0070, 459 5.16 ± 12.11, 235 746 ANA, anti-nuclear antibody. aReciprocals of dilution are on the X-axis; ln (fluorescence intensity value) is on the Y-axis. See Table 5 for multiple comparison of the slope values between 2 different ANA patterns. Open in new tab Table 5. Multiple Comparison (post-hoc test, Tamhane) of Slope Values of Regression Lines between 2 Different ANA Patternsa ANA Pattern . n . P Value . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Homogeneous 210 <0.05 0.792 <0.05 0.986 Speckled 215 <0.005 0.955 <0.005 Nucleolar 57 <0.001 1.000 Centromere 158 <0.001 Cytoplasmic 70 ANA Pattern . n . P Value . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Homogeneous 210 <0.05 0.792 <0.05 0.986 Speckled 215 <0.005 0.955 <0.005 Nucleolar 57 <0.001 1.000 Centromere 158 <0.001 Cytoplasmic 70 ANA, anti-nuclear antibody. an = 710. The average slope value of the regression line was not the same among different ANA patterns (P < .0005, ANOVA). See Table 4 for the line equations for each ANA pattern. Open in new tab Table 5. Multiple Comparison (post-hoc test, Tamhane) of Slope Values of Regression Lines between 2 Different ANA Patternsa ANA Pattern . n . P Value . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Homogeneous 210 <0.05 0.792 <0.05 0.986 Speckled 215 <0.005 0.955 <0.005 Nucleolar 57 <0.001 1.000 Centromere 158 <0.001 Cytoplasmic 70 ANA Pattern . n . P Value . . . . . . . Homogeneous . Speckled . Nucleolar . Centromere . Cytoplasmic . Homogeneous 210 <0.05 0.792 <0.05 0.986 Speckled 215 <0.005 0.955 <0.005 Nucleolar 57 <0.001 1.000 Centromere 158 <0.001 Cytoplasmic 70 ANA, anti-nuclear antibody. an = 710. The average slope value of the regression line was not the same among different ANA patterns (P < .0005, ANOVA). See Table 4 for the line equations for each ANA pattern. Open in new tab Titration Error Rates by Various Methods Titration error rates according to individual prediction methods are summarized in Table 6 (n = 759). Considering all specimens irrespective of their ANA patterns, the error rate (no. of errors/total no.) of single-well titration was 152/674 (22.6%), which is similar to the previously reported values.5,7,9–11 Among the methods of LST, the regression line or P1–P3 line scored the lowest error rates (31/710 [4.4%] and 31/746 [4.2%], respectively], both of which were significantly lower than the error rates of single-well titration (both P < .000000001). Table 6. Error Rates of the Predicted Endpoint Titer by Various Methods with Serial Dilution as a Reference in ANA Testinga ANA Pattern Groupb . Error Typec . Error Rate, no. of Errors/Total no. (%) . . . . . . . Single-Well Titration . Line Slope Titrationd . . . . . . . 2-Point Lines . . . 3-Point Regression Line . . . . P1–P2 . P1–P3 . P2–P3 . . Enrolled cases 674/759 (88.8) 727/759 (95.8) 746/759 (98.3) 740/759 (97.5) 710/759 (93.5) Homogeneous Higher 25/217 (11.5) 4/213 (1.9) 2/218 (0.9) 0/215 (0.0) 1/210 (0.5) Lower 4/217 (1.8) 4/213 (1.9) 1/218 (0.5) 118/215 (54.9) 1/210 (0.5) Hi/Lo 29/217 (13.4) 8/213 (3.8)d 3/218 (1.4) 118/215 (54.9) 2/210 (1.0) Speckled Higher 11/233 (4.7) 3/219 (1.4) 1/230 (0.4) 0/232 (0.0) 0/215 (0.0) Lower 32/233 (13.7) 7/219 (3.2) 2/230 (0.9) 163/232 (70.3) 2/215 (0.9) Hi/Lo 43/233 (18.5) 10/219 (4.6) 3/230 (1.3) 163/232 (70.3) 2/215 (0.9) Nucleolar Higher 2/56 (3.6) 2/57 (3.5) 0/57 (0.0) 0/57 (0.0) 0/57 (0.0) Lower 2/56 (3.6) 3/57 (5.3) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Hi/Lo 4/56 (7.1) 5/57 (8.8) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Centromere Higher 76/168 (45.2) 0/162 (0.0) 1/164 (0.6) 0/164 (0.0) 0/158 (0.0) Lower 0/168 (0.0) 35/162 (21.6) 15/164 (9.1) 157/164 (95.7) 18/158 (11.4) Hi/Lo 76/168 (45.2) 35/162 (21.6) 16/164 (9.8) 157/164 (95.7) 18/158 (11.4) Cytoplasmic Higher N/A 1/76 (1.3) 0/77 (0.0) 0/72 (0.0) 0/70 (0.0) Lower N/A 11/76 (14.5) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Hi/Lo N/A 12/76 (15.8) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Total Higher 114/674 (16.9) 10/727 (1.4) 4/746 (0.5) 0/740 (0.0) 1/710 (0.1) Lower 38/674 (5.6) 60/727 (8.3) 27/746 (3.6) 514/740 (69.5) 30/710 (4.2) Hi/Lo 152/674 (22.6) 70/727 (9.6) 31/746 (4.2) 514/740 (69.5) 31/710 (4.4) ANA Pattern Groupb . Error Typec . Error Rate, no. of Errors/Total no. (%) . . . . . . . Single-Well Titration . Line Slope Titrationd . . . . . . . 2-Point Lines . . . 3-Point Regression Line . . . . P1–P2 . P1–P3 . P2–P3 . . Enrolled cases 674/759 (88.8) 727/759 (95.8) 746/759 (98.3) 740/759 (97.5) 710/759 (93.5) Homogeneous Higher 25/217 (11.5) 4/213 (1.9) 2/218 (0.9) 0/215 (0.0) 1/210 (0.5) Lower 4/217 (1.8) 4/213 (1.9) 1/218 (0.5) 118/215 (54.9) 1/210 (0.5) Hi/Lo 29/217 (13.4) 8/213 (3.8)d 3/218 (1.4) 118/215 (54.9) 2/210 (1.0) Speckled Higher 11/233 (4.7) 3/219 (1.4) 1/230 (0.4) 0/232 (0.0) 0/215 (0.0) Lower 32/233 (13.7) 7/219 (3.2) 2/230 (0.9) 163/232 (70.3) 2/215 (0.9) Hi/Lo 43/233 (18.5) 10/219 (4.6) 3/230 (1.3) 163/232 (70.3) 2/215 (0.9) Nucleolar Higher 2/56 (3.6) 2/57 (3.5) 0/57 (0.0) 0/57 (0.0) 0/57 (0.0) Lower 2/56 (3.6) 3/57 (5.3) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Hi/Lo 4/56 (7.1) 5/57 (8.8) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Centromere Higher 76/168 (45.2) 0/162 (0.0) 1/164 (0.6) 0/164 (0.0) 0/158 (0.0) Lower 0/168 (0.0) 35/162 (21.6) 15/164 (9.1) 157/164 (95.7) 18/158 (11.4) Hi/Lo 76/168 (45.2) 35/162 (21.6) 16/164 (9.8) 157/164 (95.7) 18/158 (11.4) Cytoplasmic Higher N/A 1/76 (1.3) 0/77 (0.0) 0/72 (0.0) 0/70 (0.0) Lower N/A 11/76 (14.5) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Hi/Lo N/A 12/76 (15.8) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Total Higher 114/674 (16.9) 10/727 (1.4) 4/746 (0.5) 0/740 (0.0) 1/710 (0.1) Lower 38/674 (5.6) 60/727 (8.3) 27/746 (3.6) 514/740 (69.5) 30/710 (4.2) Hi/Lo 152/674 (22.6) 70/727 (9.6) 31/746 (4.2) 514/740 (69.5) 31/710 (4.4) ANA, anti-nuclear antibody; P1, point 1; P2, point 2; P3, point 3; Hi/Lo, higher or lower; N/A, not available by the NOVA View system. an = 759. bOnly cases with a decreasing trend of FI values were included in the evaluation of LST. cTitration error was defined as more than plus or minus a dilution compared to serial dilution (digital images). dWhen the error rate obtained using a given method of LST was significantly lower than that obtained using single-well titration, the corresponding cell data are in bold. Open in new tab Table 6. Error Rates of the Predicted Endpoint Titer by Various Methods with Serial Dilution as a Reference in ANA Testinga ANA Pattern Groupb . Error Typec . Error Rate, no. of Errors/Total no. (%) . . . . . . . Single-Well Titration . Line Slope Titrationd . . . . . . . 2-Point Lines . . . 3-Point Regression Line . . . . P1–P2 . P1–P3 . P2–P3 . . Enrolled cases 674/759 (88.8) 727/759 (95.8) 746/759 (98.3) 740/759 (97.5) 710/759 (93.5) Homogeneous Higher 25/217 (11.5) 4/213 (1.9) 2/218 (0.9) 0/215 (0.0) 1/210 (0.5) Lower 4/217 (1.8) 4/213 (1.9) 1/218 (0.5) 118/215 (54.9) 1/210 (0.5) Hi/Lo 29/217 (13.4) 8/213 (3.8)d 3/218 (1.4) 118/215 (54.9) 2/210 (1.0) Speckled Higher 11/233 (4.7) 3/219 (1.4) 1/230 (0.4) 0/232 (0.0) 0/215 (0.0) Lower 32/233 (13.7) 7/219 (3.2) 2/230 (0.9) 163/232 (70.3) 2/215 (0.9) Hi/Lo 43/233 (18.5) 10/219 (4.6) 3/230 (1.3) 163/232 (70.3) 2/215 (0.9) Nucleolar Higher 2/56 (3.6) 2/57 (3.5) 0/57 (0.0) 0/57 (0.0) 0/57 (0.0) Lower 2/56 (3.6) 3/57 (5.3) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Hi/Lo 4/56 (7.1) 5/57 (8.8) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Centromere Higher 76/168 (45.2) 0/162 (0.0) 1/164 (0.6) 0/164 (0.0) 0/158 (0.0) Lower 0/168 (0.0) 35/162 (21.6) 15/164 (9.1) 157/164 (95.7) 18/158 (11.4) Hi/Lo 76/168 (45.2) 35/162 (21.6) 16/164 (9.8) 157/164 (95.7) 18/158 (11.4) Cytoplasmic Higher N/A 1/76 (1.3) 0/77 (0.0) 0/72 (0.0) 0/70 (0.0) Lower N/A 11/76 (14.5) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Hi/Lo N/A 12/76 (15.8) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Total Higher 114/674 (16.9) 10/727 (1.4) 4/746 (0.5) 0/740 (0.0) 1/710 (0.1) Lower 38/674 (5.6) 60/727 (8.3) 27/746 (3.6) 514/740 (69.5) 30/710 (4.2) Hi/Lo 152/674 (22.6) 70/727 (9.6) 31/746 (4.2) 514/740 (69.5) 31/710 (4.4) ANA Pattern Groupb . Error Typec . Error Rate, no. of Errors/Total no. (%) . . . . . . . Single-Well Titration . Line Slope Titrationd . . . . . . . 2-Point Lines . . . 3-Point Regression Line . . . . P1–P2 . P1–P3 . P2–P3 . . Enrolled cases 674/759 (88.8) 727/759 (95.8) 746/759 (98.3) 740/759 (97.5) 710/759 (93.5) Homogeneous Higher 25/217 (11.5) 4/213 (1.9) 2/218 (0.9) 0/215 (0.0) 1/210 (0.5) Lower 4/217 (1.8) 4/213 (1.9) 1/218 (0.5) 118/215 (54.9) 1/210 (0.5) Hi/Lo 29/217 (13.4) 8/213 (3.8)d 3/218 (1.4) 118/215 (54.9) 2/210 (1.0) Speckled Higher 11/233 (4.7) 3/219 (1.4) 1/230 (0.4) 0/232 (0.0) 0/215 (0.0) Lower 32/233 (13.7) 7/219 (3.2) 2/230 (0.9) 163/232 (70.3) 2/215 (0.9) Hi/Lo 43/233 (18.5) 10/219 (4.6) 3/230 (1.3) 163/232 (70.3) 2/215 (0.9) Nucleolar Higher 2/56 (3.6) 2/57 (3.5) 0/57 (0.0) 0/57 (0.0) 0/57 (0.0) Lower 2/56 (3.6) 3/57 (5.3) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Hi/Lo 4/56 (7.1) 5/57 (8.8) 2/57 (3.5) 30/57 (52.6) 2/57 (3.5) Centromere Higher 76/168 (45.2) 0/162 (0.0) 1/164 (0.6) 0/164 (0.0) 0/158 (0.0) Lower 0/168 (0.0) 35/162 (21.6) 15/164 (9.1) 157/164 (95.7) 18/158 (11.4) Hi/Lo 76/168 (45.2) 35/162 (21.6) 16/164 (9.8) 157/164 (95.7) 18/158 (11.4) Cytoplasmic Higher N/A 1/76 (1.3) 0/77 (0.0) 0/72 (0.0) 0/70 (0.0) Lower N/A 11/76 (14.5) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Hi/Lo N/A 12/76 (15.8) 7/77 (9.1) 46/72 (63.9) 7/70 (10.0) Total Higher 114/674 (16.9) 10/727 (1.4) 4/746 (0.5) 0/740 (0.0) 1/710 (0.1) Lower 38/674 (5.6) 60/727 (8.3) 27/746 (3.6) 514/740 (69.5) 30/710 (4.2) Hi/Lo 152/674 (22.6) 70/727 (9.6) 31/746 (4.2) 514/740 (69.5) 31/710 (4.4) ANA, anti-nuclear antibody; P1, point 1; P2, point 2; P3, point 3; Hi/Lo, higher or lower; N/A, not available by the NOVA View system. an = 759. bOnly cases with a decreasing trend of FI values were included in the evaluation of LST. cTitration error was defined as more than plus or minus a dilution compared to serial dilution (digital images). dWhen the error rate obtained using a given method of LST was significantly lower than that obtained using single-well titration, the corresponding cell data are in bold. Open in new tab The statistics according to the individual ANA patterns (Table 6) show that the titration error rates of LST (regression line or P1–P3 line) were always significantly lower than those of single-well titration for any ANA pattern, except the nucleolar pattern. In the nucleolar pattern, the difference in error rates between LST and single-well titration did not reach statistical significance (2/57 and 4/56, respectively; P = .438). This may be because specimens with high titers were relatively infrequent in this pattern. To investigate the effects of the number of dilutions to be prepared (Table 6), the titration error rate was compared between the 2 methods of LST, regression line using 3 dilution points vs P1–P3 line using 2 dilution points. Interestingly, the error rate of the former was not significantly different from that of the latter (31/710 [4.4%] and 31/746 [4.2%], respectively; P = .842). In a comparison of the methods of LST using 2-point lines (Table 6), the P1–P3 line scored the lowest error rate (31/746, 4.2%) of the 3 lines. The titration error rate obtained using a distant point (ie, P1–P3 line) was significantly lower than that obtained using a nearby point (ie, P1–P2 line) (31/746 [4.2%] and 70/727 [9.6%], respectively; P < .00005). Therefore, when choosing only one more dilution point to be linked to the screening dilution (1:80) point in a given doubling dilution series, a point distant from the screening dilution point may be better than a nearby point in predicting an accurate titer. Discussion The line slope-based titration developed in this study was useful for measuring endpoint titers using the minimum number of dilutions instead of complete concentration series by serial dilution. In order to prepare an optimal dilution series for LST, a patient’s previous titer or FI at the screening dilution was considered. This LST method is more rapid and cost-effective than is titration using serial dilution and is more flexible and accurate than is single-well titration. The calculated slope value of a fitted line was proven to reflect the ANA pattern of the specimen, and thus, it is not necessary to determine its ANA pattern or to use the built-in standard curves for its ANA pattern. For the XY plot with dilution as the X-axis and FI as the Y-axis, the linear–log model was adopted after comparing the line-fitting effectiveness of the 4 scale models. This adopted model proved to be adequate in regression analysis statistics, with the proportions of R2 value >0.96 of 100% and the proportion of P <.05 of 60.6% (Table 3). This model also showed the best accuracy in titration. In contrast, Peng et al8 had used the log–linear model to present the linearity between dilutions and FI values. In this LST method, a fitted line was drawn for 3 points on a dilution series per specimen. The average R2 value for the fitted lines was not the same among the different ANA nuclear patterns, and this value was the lowest for the centromere pattern (0.904 ± 0.175). Fortunately, even in the centromere pattern, the titration error rate obtained using P1–P3 LST was lower than that obtained using single-well titration (16/164 [9.8%] and 76/168 [45.2%], respectively; P < .000000000001). This acceptable linearity for dilution points in any ANA pattern supports the principle of using a line slope value to predict the endpoint titer, where the fitted line is extended to obtain the X-coordinate value (reciprocal of dilution) at a Y-coordinate cutoff FI value of 48 LIU (NOVA View system). Theoretically, a higher number of dilution points leads to a better-fitted line. However, the titration error rate was significantly lower with only 2 dilutions than with single-well titration and was not significantly different from that using 3 dilutions. When choosing only one more dilution point to be linked to the screening dilution (1:80) point in a given doubling dilution series (eg, 1:80–1:160–1:320), a distant point (eg, 1:320) was better than a nearby point at accurately predicting the titer (eg, 1:160), as summarized in Table 6. In principle, FI should be measured as a smaller value in a higher dilution. However, in this study, the incidences of the reversed cases were 29/756 (3.8%) for D1 less than D2, 14/755 (1.9%) for D2 less than D3, and 9/755 (1.2%) for D1 less than D3 (Figure 3), occurring more frequently in lower dilutions (between D1 and D2) than in higher dilutions (between D2 and D3; P < .05). The FI values at the screening dilution D1 in the reversed cases of D1 less than D2 were higher than those in the cases of D1 greater than D2 (1955 ± 880 LIU and 1069 ± 1037 LIU, respectively; P < .00001). Taken together, the prozone effects may be an important cause for most reversed cases of D1 less than D2, and these prozone effects may induce frequent titration errors in single-well titration, with a higher error rate in the reversed cases than in the cases of D1 greater than D2 (17/27 [63.0%] and 135/645 [20.9%], respectively; P < .0000005). SLE is characterized by the presence of a broad spectrum of autoantibodies (including antibodies to native DNA, chromatin, Smith antigen, U1 nuclear ribonucleoprotein, anti-Sjögren’s-syndrome-related antigen A, anti-Sjögren’s-syndrome-related type antigen B, complement component 1q, ribonucleoprotein, and several other nonhistone proteins, or nonhistone protein–ribonucleic acid [RNA] complexes).14,15 As SLE frequently shows a mixed ANA pattern, the prediction of endpoint titers has to be possible irrespective of ANA patterns. LST possible in any mixed pattern is a solution to this requirement. The recognition of cytoplasmic ANA patterns can be a hint as to some specific autoantibodies associated with idiopathic inflammatory myopathies (IIM), a heterogeneous group of SARD. Autoantibodies directed against the aminoacyl transfer RNA (tRNA) synthetases (anti-synthetase autoantibodies) are found in IIM patients, for example, autoantibodies to histidyl (Jo-1) and threonyl (PL-7) tRNA synthetases.16 For this cytoplasmic ANA pattern, LST could predict endpoint titers with an error rate of 7/77 (9.1%), whereas single-well titration by the NOVA View system could not predict the endpoint titer at all. Single-well titration has many shortcomings, as follows: (1) this method showed a relatively high titration error rate (~20%); (2) checking prozone effects is not possible with only a single screening dilution; (3) this method cannot be applied to mixed ANA patterns; and (4) in the NOVA View system, titration is possible only at a screening dilution. This limitation has to be overcome; titration has to be possible at any last dilution with positive HEp-2 cell staining to avoid the screening well affected by the prozone phenomenon. LST also has a few shortcomings, as follows: (1) obtaining decreasing FI values on 2 or more dilutions is a prerequisite to obtaining an accurate line slope value (when no such FI values are obtained on prepared dilutions, the specimen should be further diluted to escape from potential prozone effects), (2) either a patient’s previous titer or FI at the screening dilution is necessary to prepare an optimal dilution series, and (3) LST using at least 2 wells is more costly than is SWT using a single well. If the current automated systems are used without a major redesign, an interfacing software to calculate the endpoint titer using LST should be implemented between middleware software (QUANTA Link in the NOVA View system) and the laboratory information system (LIS). Otherwise, the middleware software should be upgraded such that it can receive information about patients’ previous titers from the LIS, which will minimize operator intervention (eg, manual input of dilutions to be performed) and save the cost and turnaround time for ANA testing. Conclusively, line slope titration on the minimum number of dilutions prepared considering a patient’s previous titer or FI at the screening dilution (1) is available in any ANA pattern, including mixed, cytoplasmic, or undetermined patterns; (2) enables prozone effects to be evaluated with a higher dilution; (3) enhances titration accuracy; and (4) is useful even with only 2 dilutions. Therefore, this LST method is useful as an accurate, cost-effective, and rapid approach to obtain an endpoint titer in routine ANA testing. LM Abbreviations Abbreviations ANA antinuclear antibody FI fluorescence intensity SWT single-well titration LST line slope titration SARD systemic autoimmune rheumatic disease IIF indirect immunofluorescence SLE systemic lupus erythematosus LIU light intensity units ANOVA analysis of variance ln natural logarithm RNA ribonucleic acid IIM inflammatory myopathies tRNA transfer RNA LIS laboratory information system References 1. Briolay J , Gioud M , Monier JC , et al. Antinuclear antibodies detected by indirect immunofluorescence on HEp2 cells and by immunoblotting in patients with systemic sclerosis . Autoimmunity. 1989 ; 2 ( 2 ): 165 – 176 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Soto ME , Hernández-Becerril N , Perez-Chiney AC , et al. Predictive value of antinuclear antibodies in autoimmune diseases classified by clinical criteria: analytical study in a specialized health institute, one year follow-up . Results Immunol. 2015 ; 5 : 13 – 22 . 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