JIM-11776; No of Pages 7 Journal of Immunological Methods xxx (2013) xxx–xxx

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Research paper

Novel method for ANA quantitation using IIF imaging system Xiaodong Peng ⁎, Jiangtao Tang 1, Yongkang Wu, Bin Yang, Jing Hu Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu 610041, China

a r t i c l e

i n f o

Article history: Received 17 August 2013 Received in revised form 18 November 2013 Accepted 16 December 2013 Available online xxxx Keywords: Antinuclear antibodies (ANAs) HEp-2 cells IIF image analysis system Quantitative measurement

a b s t r a c t A variety of antinuclear antibodies (ANAs) are found in the serum of patients with autoimmune diseases. The detection of abnormal ANA titers is a critical criterion for diagnosis of systemic lupus erythematosus (SLE) and other connective tissue diseases. Indirect immunofluorescence assay (IIF) on HEp-2 cells is the gold standard method to determine the presence of ANA and therefore provides information about the localization of autoantigens that are useful for diagnosis. However, its utility was limited in prognosing and monitoring of disease activity due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. On the other hand, ELISA for the detection of ANA can quantitate ANA but could not provide further information about the localization of the autoantigens. It would be ideal to integrate both of the quantitative and qualitative methods. To address this issue, this study was conducted to quantitatively detect ANAs by using IIF imaging analysis system. Serum samples from patients with ANA positive (including speckled, homogeneous, nuclear mixture and cytoplasmic mixture patterns) and negative were detected for ANA titers by the classical IIF and analyzed by an image system, the image of each sample was acquired by the digital imaging system and the green fluorescence intensity was quantified by the Image-Pro plus software. A good correlation was found in between two methods and the correlation coefficients (R2) of various ANA patterns were 0.942 (speckled), 0.942 (homogeneous), 0.923 (nuclear mixture) and 0.760 (cytoplasmic mixture), respectively. The fluorescence density was linearly correlated with the log of ANA titers in various ANA patterns (R2 N 0.95). Moreover, the novel ANA quantitation method showed good reproducibility (F = 0.091, p N 0.05) with mean ± SD and CV% of positive, and negative quality controls were equal to 126.4 ± 9.6 and 7.6%, 10.4 ± 1.25 and 12.0%, respectively. In conclusion, our novel ANA quantitation method can provide both of the fluorescence density, which could precisely reflect the fluctuate of ANAs level in patient's serum and the useful information about the localization of the autoantigens for clinician in diagnosing and monitoring diseases. © 2013 Elsevier B.V. All rights reserved.

1. Introduction A variety of antinuclear antibodies (ANAs) are found in patients with a number of autoimmune diseases. The detection and quantitation of ANAs is pivotal to the diagnosis of many autoimmune disease (Solomon et al., 2002; Agmon-Levin et al., 2010). Among others, the frequency of ANA is much higher in patients with systemic lupus erythematosus (SLE), mixed ⁎ Corresponding author. Tel.: +86 28 85422752. E-mail address: [email protected] (X. Peng). 1 Co-first author.

connective tissue disease, Sjögren's syndrome, scleroderma and other connective tissue diseases (Kavanaugh et al., 2000; Sinico et al., 2002). The degree of ANA positivity is diagnostically important in SLE since the positive predictive value increases with high titers (Pham et al., 2005). Hence, it was internationally recommended that the detection of ANA is the first level for laboratory diagnosis of systemic autoimmune-rheumatic diseases (SARD) (Agmon-Levin et al., 2013). Indirect immunofluorescence assay (IIF) on HEp-2 cells remains the golden standard method for screening of ANAs and commonly used in most laboratories in the world to date (Tozzoli et al., 2002; Invernizzi et al., 2007; Meroni and

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Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

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Schur, 2010). IIF can determine the presence of ANAs and provide information about the localization of the autoantigens. The staining patterns of ANAs, which include homogeneous, speckled, centromere, nucleolar, cytoplasmic and other mixed patterns, etc., could provide useful information for clinicians to diagnose diseases because of some patterns are associated with certain autoantigen and/or autoimmune diseases (Tan, 1989; Agmon-Levin et al., 2013; Mariz et al., 2011). However, the utility of ANA titer detected by IIF in diagnosing, prognosing and monitoring of disease activity has been limited due to the lack of standardization in performing the technique, subjectivity in interpreting the results and the fact that it is only semi-quantitative. Numerous enzyme immunoassays have been developed as alternative methods for ANA screening in an attempt to make the method more objective and less labor intensive. For example, enzyme-linked immunosorbent assay (ELISA) is a quantitative method for ANAs detection and is more convenient for large number of samples than IIF method. But it may vary in the specificity and/or purity of antigenic epitopes, concentration and binding capacity of antigens, and the secondary antibodies prepared. It could not provide further information about the localization of the autoantigens aid in the diagnosis of disease. It is only able to detect a limited number of ANAs and may result in false negative (Anon, 2009). Therefore, to date, none of enzyme immunoassays has been widely accepted as an alternative to IIF for detection and titration of ANAs (Hiepe et al., 2000; Nifli et al., 2006). It would be optimal to integrate the advantage of the qualitative and quantitative methods mentioned above. Recently, some researchers have attempted to make the classical IIF method more objective and less labor intensive. The use of digital images of HEp-2 cell testing was found to be reliable and helpful in determining ANA titers. It is more objective and could reduce the variation among different laboratories and even among different observers in the same laboratory (Peterson et al., 2009; Peng et al., 2002). However, the amount of ANAs is still reported as endpoint titers which are only semi-quantitative measurement. In this study, we established a really quantitative method for measurement of ANA levels in patient sera by using a combination of IIF method (the classic standard method), a digital imaging system and an image analysis system. 2. Materials and methods 2.1. Serum samples and reagents Serum samples were obtained from in- and out-patients who submitted for ANA-IIF testing at the Clinical Immunology Laboratory of West China Hospital, Sichuan University, China. 51 ANA-IIF negative and 1699 ANA-IIF positive samples with various fluorescence patterns (412 speckled, 300 homogeneous, 542 nuclear mixture and 445 cytoplasmic mixture) were analyzed as shown in Table 1. Positive and negative ANA controls provided by ANA test kits (EUROIMMUN Inc., Germany) were tested in parallel with patient sera. The instruments used in this study include fluorescent microscope (Nikon EC600, Japan), the digital Imaging System (SPOT32, American) and the image analysis system (Image-Pro plus software ipwin32, American).

Table 1 Serum samples from patients with various pattern and titer of ANAs (n). Pattern

Total Number of serial ANA titer number 1:100 1:320 1:1000 1:3200 1:10000

Speckled Homogeneous Nuclear mixturea Cytoplasmic mixtureb Negative

412 300 542 445

a b

50 50 50 52

98 66 132 81

139 75 234 205

69 55 71 54

56 54 55 53

51

Mixture of speckled/homogeneous and other patterns. Mixture of cytoplasmic and/or centromere/nucleolar/dots.

2.2. Detection of ANA titer by indirect immunofluorescence assay (IIF) Serum samples and quality controls were detected for ANAs titer by IIF with ANA test kit according to the manufacturer's instruction (EUROIMMUN Inc., Germany). In brief, serum samples were performed a dilution series (1:100, 1:320, 1:1000, 1:3200 and 1:10000) in phosphate-buffered saline (PBS). Diluted serum samples and quality controls were applied to HEp-2 cells on slides and incubated for 30 min at room temperature. Then the slides were flushed and immersed in PBS for about five minutes. After another incubation with secondary FITC-conjugated antibody for 30 min at room temperature, the slides were washed again and covered by glycerol. Each slide was observed under fluorescence microscope (amplification, 400×) and the highest dilution of serum to stain HEp-2 cells is reported as the endpoint titer of ANA. 2.3. Quantitation of ANA by the Image analysis system For quantitative determination of ANAs, the IIF images of each slide were acquired by a digital Imaging System (SPOT32, American), and the green fluorescence density of each image was analyzed by an image analysis system (Image-Pro plus software, ipwin32, American). Briefly, the serum samples from patients were performed by IIF as described above. Sera dilution at 1:100 was chosen for screening ANAs based on the recommend screening dilution from the manufacturer's instruction (EUROIMMUN Inc., Germany) as well as reports of a decreased percentage of false-positive results in healthy individuals (Rigon et al., 2007; Ghosh et al., 2007; Nifli et al., 2006; Dahle et al., 2004). The 4-s exposure time was chosen for taking each image, and the green fluorescence density of each image was measured in certain parameters (green density, area N 1000) (Fig. 1). Each sample was imaged and measured triplicate, and the average of three densities was reported as quantitative result of each sample. The quality controls provided in the kit were tested in parallel with sera samples. 2.4. Quality control of ANA by the Image analysis system Quality control is very important and necessary in performing a quantitation assay. To control the quality of our quantitation method for ANA detection, 5 positive and 5 negative standards with the same Lot number provided in ANA

Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

X. Peng et al. / Journal of Immunological Methods xxx (2013) xxx–xxx

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Fig. 1. The image analysis system. The image was analyzed by an image analysis system (Image-Pro plus software, ipwin32, American). The fluorescence density of each image was measured in certain parameters (green density, area N 1000), and the value of mean (e.g., 137) is reported as the quantitative result of each sample .

test kit (Euroimmue, Germany) were tested in parallel with patient sera.

respectively (Fig. 2). The results showed significant correlation (significance = 0.000, α = 0.01) and suggested that the fluorescence density was better correlated with ANA titer.

2.5. Statistical methods 3.2. Linearity of ANA density followed the change of ANA level Spearman's equation test was used to assess the correlation between ANA titer and ANA density. The linearity of ANA density was drawn curves and calculated the correlation coefficients (R2). To evaluate the reproducibility of our quantitative measurement, three groups of fluorescence density were statistically analyzed by a one-way ANOVA (p b 0.05 was considered significant difference). The precision of our method was assessed by standard deviation (SD) and coefficient of variation (CV%) of controls. 3. Results 3.1. Correlation between ANA titer and ANA quantitation IIF on HEp-2 cells is commonly used to assess ANA titers in sera of patients with autoimmune disease. To examine the usefulness of the ANA quantitation by the IIF imaging analysis system, 1254 serum samples from patients with IIF-ANA positive and 51 sera from healthy controls with IIF-ANA negative were detected by both the classical IIF method and our novel IIF quantitation method. The fluorescence density of ANAs measured by the novel IIF Image analysis system was found closely correlated with sera dilution titer (Table 2) and showed good correlation between density and titer in various ANA patterns with the correlation coefficients (R2) of 0.942 (speckled), 0.942 (homogeneous), 0.923 (nuclear mixture) and 0.760 (cytoplasmic mixture),

To evaluate whether the fluorescence density measured by our novel quantitative method can reflect the fluctuation of ANA in patients or not, 20 sera (5 speckled, 5 nuclear mixture, 5 cytoplasmic and 5 homogeneous) from patients with high ANA titer of ≥ 1:10000 were diluted at 1:100, 1:320, 1:1000, 1:3200 and 1:10000, respectively, for the measurement of fluorescence density using the IIF imaging analysis system. It was shown that the fluorescence density of each sample exhibited good linearity, and the correlation coefficients (R2) were all more than 0.95 in various ANA patterns (Fig. 2). These results indicated that, for individual patient, the fluorescence density can better reflect the change of ANA level in patient sera. 3.3. Reproducibility of ANA quantitation by the IIF imaging analysis system To determine the reproducibility of the IIF fluorescence density for ANA quantitation, 135 samples (32 speckled, 26 homogeneous, 52 nuclear mixture and 25 cytoplasmic mixture) were detected for ANA in triplicate, and three groups of data were obtained. The fluorescence density showed comparable repeatability in all ANA patterns with various dilutions. The correlation coefficients (R2) of every two groups were 0.974 (group 1 and group 2), 0.979 (group 1 and group 3) and 0.982 (group 2 and group 3), respectively. There was no

Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

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X. Peng et al. / Journal of Immunological Methods xxx (2013) xxx–xxx

Table 2 Correlation between ANA titer and ANA density (median ± SD, percentiles). Pattern

Total number

Speckled

412

ANA titer 1:100

Homogeneous Nuclear mixture

300 a

Cytoplasmic mixture Negative a b

542 b

445 51

1:320

48.5 ± 7.2 71.5 ± 9.4 (44.0–54.2) (63.0–78.2) 48.0 ± 7.3 74.0 ± 9.1 (41.5–52.0) (69.0–80.0) 49.5 ± 9.5 74.0 ± 8.9 (42.0–55.2) (67.0–79.0) 46.0 ± 5.5 60.0 ± 8.8 (42.0–49.0) (54.5–64.5) 36.0 ± 7.6 (32.0–39.0)

1:1000

1:3200

1:10000

117.0 ± 24.3 (99–139) 118.0 ± 20.8 (105.0–137.0) 111.0 ± 21.4 (101.0–130.0) 83.0 ± 20.7 (70.0–97.0)

179.0 ± 20.4 (162.5–188.0) 180.0 ± 18.7 (169.0–193.0) 174.0 ± 22.0 (161.0–186.0) 106.0 ± 24.6 (89.0–117.8)

198.0 ± 20.7 (180.2–211.8) 197.5 ± 22.5 (171.8–203.0) 197.0 ± 27.0 (180.0–213.0) 112.0 ± 25.4 (91.0–126.0)

Mixture of speckled/homogeneous and other patterns. Mixture of cytoplasmic and/or centromere/nucleolar/dots.

significant difference among three groups (one-way ANOVA; F = 0.091, p = 0.913). 3.4. Quality control for the ANA quantitative assay To perform quality control of our ANA quantitation assay using this IIF Image analysis system, 6 positive and 6 negative standards with the same Lot number were tested in parallel with patient sera. The quality control chart was mapped by using Excel software. The results showed good repeatability of the QC fluorescent density with mean values equal to 126.4 and 10.4, SD 9.6 and 1.25, and coefficient of variation (CV%) 7.6% and 12.0%, respectively (Fig. 3). 4. Discussion The level of autoantibody present in patient serum is determined using classical immunofluorescence assay (IIF) by performing a series of dilution to stain HEp-2 cells. The highest dilution of serum to stain HEp-2 cells is the endpoint titer. This is currently recommended the standard method for ANA screening (Agmon-Levin et al., 2013); however, it is labor intensive and requires a significant amount of training to become proficient at interpreting the results. There exits much variation among different laboratories and different technicians mainly due to its semi-quantitative and objective, despite being calibrated using an international reference preparation or equivalent standard (2006 College of American Pathologists Survey; Pham et al., 2005). These issues limit the reproducibility and therefore the validity of the results, making it difficult for physicians to adjust therapeutic strategy in time only based on an IIF ANA titer. To overcome some drawbacks of IIF, researchers have developed several ELISAs and multiplexing technologies to reduce the subjectivity in ANA titer determination, the different interpretation of results among individuals and laboratories, the high skills requirement for operators, etc. (Ghirardello et al., 2009; Liu et al., 2010; Somers et al., 2009). However, these methods are only able to detect a limited number of autoantibodies. The task force reported that many laboratories are not using IIF has resulted in a significant increase in false-negative tests. A recent case report from the Massachusetts General Hospital is well illuminated this issue, where a diagnosis of SLE was significantly delayed because of a false-negative ANA test detected using a non-IFA method.

(Anon, 2009). Therefore, IIF on HEp-2 cells still remains the standard method in the current screening of ANAs and cannot be entirely replaced by other methods (Bossuyt et al., 2008; Meroni and Schur, 2010). Base on method of IIF on HEp-2 cells, some digital imaging systems for ANA detection have been developed, such as near-infrared imaging (NII) system (Peterson et al., 2009), IIF-fluorescence imaging and analysis system (Peng et al., 2002), automated IIF-HEp-2 reader and interpretation systems (Egerer et al., 2010; Kivity et al., 2012; Nakabayashi et al., 2001; Bossuyt et al., 2013), etc. Digital images of stained HEp-2 cells were captured and analyzed for the determination of ANA titer, and it showed good correlation with the gold manual IIF method in ANA titer measurement. Similar titers were determined when compared with the gold manual IIF method, indicating that ANA detection by these methods can be used to screen for and/or determine the titer of antibodies against ANA within the acceptable range (±2 dilutions) for proficiency/competency defined by current federal regulations (Kavanaugh et al., 2000). By using these methods, the advantages are time saving and cost-effective, subjectivity minimized. However, these methods are still qualitative, and the ability to distinguish between ANA positive and negative samples was limited in some methods (e.g., NII system) (Peterson et al., 2009). In the practical work, it was noticed that the fluorescent density of patients ANA had already changed even when it maintained at the same titer (our unpublished data). This encouraged us to develop a novel method for quantitative ANA measurement in patient serum, and the quantitation of ANAs would more precisely reflect the fluctuation of antibodies and provide accurate results for clinicians. In this study, we have established a novel method for ANA quantitation combining the classical IIF method, a digital imaging system and an image analysis system. 1699 ANA-IIF positive and 51 ANA-IIF negative samples were detected by both of the classical IIF and our novel method. It showed good correlation between density and titer in various ANA patterns (significance = 0.000, α = 0.01) (Fig. 2), suggesting that fluorescence density could reflect the change of ANAs in patient sera as well as its titer. Good reproducibility is a prerequisite of quality assurance for quantitative experiment. By analyzing 135 samples detected in triplicate using our novel method, it showed high correlation (R2 N 0.97) and no significant difference among

Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

(b)

(c)

250.00

(d)

(e)

250.00 180.00

250.00

48

341

13 31

353

50.00

342

200.00

291

200.00

150.00 338

density

density

dentisty

density

303

40.00

150.00

28 45

20.00

135 148 327 223 141

213

150.00 426 444 111

100.00 100.00

120.00

90.00

113 77 60 97

65 53

100.00

30.00

150.00

density

200.00

50.00

60.00

0.00

30.00

102

50.00

117

50.00

50 10

100

320

1000

titer

3200

10000

100.00

320.00

1000.00 3200.00 10000.00

titer

100.00

320.00

1000.00 3200.00 10000.00

titer

83

100.00

320.00

1000.00 3200.00 10000.00

titer

Fig. 2. Correlation between ANA titer and ANA quantitation. The fluorescence densities of ANA measured by the novel ANA quantitation method were positive correlated with the ANA titers determined by the classical IIF method in all ANA patterns. The correlation coefficients (R2) were 0.942 of speckled (b), 0.942 of homogeneous (c), 0.923 of nuclear mixture (d) and 0.760 of cytoplasmic mixture (e), respectively.

X. Peng et al. / Journal of Immunological Methods xxx (2013) xxx–xxx

Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

(a)

5

6

X. Peng et al. / Journal of Immunological Methods xxx (2013) xxx–xxx

a) Speckled 250

b) Nuclear-mixture 250

2

R1 = 0.9881 2 R2 = 0.9877 2 R3 = 0.996 2 R4 = 0.9947 2 R5 = 0.9753

200

140

2

R1 = 0.9708 2 R2 = 0.9902 2 R3 = 0.9554

200

c) Cytoplasmic

2

R4 = 0.9617 2 R5 = 0.9907

250

2

R1 = 0.9889 2 R2 = 0.9923 2 R3 = 0.9842 2 R4 = 0.9915

120 100

d) Homogeneous 2

200

R5 = 0.9895

150

150

80

100

100

60

100

40

50

50

50

0

0 2

4

6

20

0

0 0

2

4

6

2

R3 = 0.9632 2 R4 = 0.9642 2 R5 = 0.9811

2

150

0

R1 = 0.9975 2 R2 = 0.9616

0 0

2

4

6

2

4

6

-50

Fig. 3. Linearity of ANA density and ANA titer. The serum samples were diluted at 1:100, 1:320, 1:1000, 1:3200 and 1:10000, respectively, for the quantitative measurements of the fluorescence density using the IIF imaging analysis system. Curves were drawn with log ANA titer on the X-axis and fluorescence density on the Y-axis. The correlation coefficients (R2) were all more than 0.95 in various ANA patterns. (a) Speckled, (b) nuclear mixture, (c) cytoplasmic and (d) homogeneous.

three groups (one-way ANOVA; F = 0.091, p = 0.913). These data indicated that our method had good reproducibility and could ensure the precision of the results. Good accuracy is another prerequisite of quality assurance for quantitative experiment. It is usually conducted by using controls. In our study, the positive and negative controls were performed in parallel with patient samples for 48 times. The mean ± SD of the QC fluorescence density were 126.4 ± 9.6 and 10.4 ± 1.25, respectively, and the CV% were 7.6% and 12.0%, respectively (Fig. 4). The results indicated that this method demonstrates good reproducibility and good accuracy in the measurement of ANAs, and it appears to provide clinicians reliable results in diagnosing and treating patients. The patterns of ANA may be various and complex in an individual patient. There may be variety combinations of different patterns in different patients. For example, one patient has mixture pattern of speckled, dots and cytoplasmic, while another one has mixture pattern of homogeneous and centromere. It is difficult to create a standard curve of the correlation between ANA density and ANA level for these two patients because each patient may have its own special curve. Whether the fluorescence density of ANAs changed along with its level in patient serum remains unknown. To address to this issue, 25 samples with ANAs titer at 1:10000 from each ANA pattern (speckled, nuclear mixture, cytoplasmic and homogeneous) were analyzed by a series of dilution

(1:100, 1:320, 1:1000, 1:3200 and 1:10000, respectively). With the increase of ANAs dilution, the fluorescence density decreased and showed good linear relationship with titer. The correlation coefficients (R2) were all more than 0.95 in various ANA patterns and every sample (Fig. 3). These results indicated that fluorescence density could reflect the change of ANA level and provide us a possibility to precisely monitor the fluctuation of ANA level by quantitative measurement of ANAs. Our study also showed that ANA density was different and may range from 11 to 55 U when ANA titers remain the same (Table 2). In other words, ANA levels in patient sera have already changed, and it could not be observed when ANAs were tested and reported as end point titers. So if we detect density instead of titer, it could more precisely reflect the fluctuation of antibodies in patient sera and provide accurate information for clinicians in treating patients. Although the correlation of ANA with disease activity is controversial, some specific antibodies of the ANA family may present years before the appearance of overt disease and may fluctuate over time followed disease activity (Arbuckle et al., 2003; Eriksson et al., 2011; Arora-Singh et al., 2010). By using this quantitative measurement of ANAs, it may be aid to reevaluate the correlation between ANA level and disease activity. Furthermore, since only one slide (dilution at 1:100) per sample was required in our novel method while 1 to 3 slides

Positive Negative

160 140 120 100 80 60 40 20 0 1

3

5

7

9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47

Fig. 4. Quality control chart of ANA controls. Positive and negative quality controls were detected in parallel with patient sera for 48 times. The mean ± SD and CV% of positive and negative controls were 126.4 ± 9.6, 7.6%, and 10.4 ± 1.25, 12.0%, respectively.

Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

X. Peng et al. / Journal of Immunological Methods xxx (2013) xxx–xxx

per sample were needed in the gold manual IIF method, our novel method could partly reduce labor intensity and be more simple and economical than the gold manual IIF method. In conclusion, our novel quantitative method is as specific as the widely used ANA manual method, and the information of ANA patterns can also be provided for clinicians in diagnosing patients. The measurement of fluorescence density showed good reproducibility, linearity and correlation with ANA titration by ANA manual IIF. Once a normal reference value is established in different laboratories, it may lead to objective results with minimum differences among individuals and laboratories. The establishment of the novel quantitative method may provide a possibility for the standardization of both ANA testing and ANA quality control system, and it may play an important role in improving ANA testing accuracy, which results in comparability among laboratories and clinical application in disease therapy.

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Please cite this article as: Peng, X., et al., Novel method for ANA quantitation using IIF imaging system, J. Immunol. Methods (2013), http://dx.doi.org/10.1016/j.jim.2013.12.004

Novel method for ANA quantitation using IIF imaging system.

A variety of antinuclear antibodies (ANAs) are found in the serum of patients with autoimmune diseases. The detection of abnormal ANA titers is a crit...
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