Clinica Chimica Acta 502 (2020) 102–110

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/cca

Diagnostic performance of 14-3-3η and anti-carbamylated protein T

antibodies in Rheumatoid Arthritis in Han population of Northern China
⁎
Yuan Zhang, Yongming Liang, Limei Feng, Liyan Cui
Department of Clinical Laboratory, Peking University Third Hospital, Beijing 100191, China

A R T I C LE I N FO A B S T R A C T

Keywords: Objectives: As we already know, Rheumatoid arthritis (RA) cannot be excluded when the rheumatoid factor (RF)
Rheumatoid factor (RF) or anti-cyclic citrullinated peptide antibody (anti-CCP) is negative. Here, we determined the application value of
Anti-cyclic citrullinated peptide antibody (anti- 14-3-3η protein, anti-carbamylated proteins antibodies (anti-CarP), as well as their potential role to diagnose RA
CCP) together with RF or anti-CCP.
14-3-3η
Method: Serum levels of anti-CCP, RF, 14-3-3η and anti-CarP antibodies were detected in 291 RA patients, 223
Anti-carbamylated proteins antibodies (anti-
patients with autoimmune diseases except RA, and 156 healthy subjects recruited from Han population of
CarP)
Rheumatoid arthritis (RA) Northern China. We examined the differences in the levels of these indicators among groups and compared the
correlations between any two of the indicators. At the same time, a total of 12 testing strategies were established
for comparison to maximize the diagnostic value.
Result: The levels of RF, anti-CCP, anti-CarP and 14-3-3η were significantly higher in RA patients (12.5;
[9.36–15.7], 30.7;[25.7–35.6], 1.90;[1.70–2.01], 15.8;[10.8–20.8], respectively) compared with either inter-
ference-control group (1.24;[1.07–1.41], 0.64;[0.42–0.86], 0.51;[0.46–0.57], 0.33;[0.23–0.44], respectively)
(p < 0.0001) or healthy-control group (1.03;[0.99–1.08], 0.49;[0.38–0.59], 0.28;[0.21–0.35], 0.55;
[0.27–0.85], respectively) (p < 0.0001). Among all 12 detection strategies, the YI and κ value of a novel
strategy that either double-positive of any 2 markers or single-positive of anti-CCP can be diagnosed as RA had
the highest diagnostic value.
Conclusion: The results of our study demonstrated that in Han population of Northern China, anti-CarP anti-
bodies and 14-3-3η protein can be treated as valuable indicators of RA, especially when combined with RF and
anti-CCP, the detection value is maximized.

1. Introduction anti-CCP [3]. The formation of immune complexes in RA drives car-

As a long-term autoimmune disorder, Rheumatoid arthritis (RA)
mainly affects joints. When a patient is suspected of suffering from RA,
rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody
(anti-CCP) are two indicators that are frequently detected in serum.
However, RA cannot be excluded when the RF or anti-CCP is negative
[1,2]. Therefore, it is necessary to incorporate some novel RA indicators
into the diagnostic criteria of RA.
1.1. Anti-CarP antibodies
As a non-enzymatic post-translational modification, carbamylation
participated in the pathogenesis of rheumatoid arthritis (RA). In 2011,
Shi J. et al have found that anti-carbamylated proteins antibodies (anti-
CarP) were detected in the serum of patients with RA in addition to
bamylation, thereby disrupting tolerance and inducing the production
of anti-CarP antibodies [4]. They also found that anti-CarP antibodies
were detected in blood donors before the appearance of clinical
symptoms of RA [5]. Actually, Anti-CarP antibodies could exist several
years before the start of RA symptoms [6]. Additionally, it has been
found that in patients not diagnosed with rheumatoid arthritis, anti-
CarP antibodies were mainly present in patients with undifferentiated
arthritis [7] and primary Sjögren's syndrome [8]. A group of re-
searchers from Spain believed that anti-CarP antibodies were different
from anti-CCP risk factors, but were related to radiological damage of
RA, so anti-CarP antibodies could be used as independent RA auto-
antibodies [9]. Although anti-CarP antibodies are increased in RA pa-
tients, increased carbamylation is not sufficient to result in immune
responses against carbamylated proteins. Therefore, after the process of
carbamylation, genetic or environmental factors may still be required to

⁎
Corresponding author.
E-mail addresses: cliyan@163.com, cuiliyan2006@hotmail.com (L. Cui).

https://doi.org/10.1016/j.cca.2019.12.011
Received 12 August 2019; Received in revised form 12 December 2019; Accepted 15 December 2019
Available online 17 December 2019
0009-8981/ © 2019 Elsevier B.V. All rights reserved.
Y. Zhang, et al.
Table 1
Numbers and demographical characteristics of subjects.
Groups
RA group
Number of subjects 291
Age, years 51.6 ± 15.7(17–85)
Female, n (%) 242(83.2%)
RA: Rheumatoid arthritis; IC: Interference-control; HC: Healthy-control.
induce the production of anti-CarP antibodies [10]. Similarly, several
studies have confirmed that anti-CarP antibodies are widely present in
RA patients from US military personnel, indigenous North Americans,
Japanese, French, Swedish and Dutch descent [11–16], as well as more
common in first-degree relatives of RA patients compared to normal
controls [11].
1.2. 14-3-3η protein
14-3-3η proteins are a group of highly conserved protein families,
composed of seven isoforms (β, γ, ε, η, σ, θ, and ζ) [17]. In 2007, Kilani
RT et al. found for the first time that serum 14-3-3η increased in ar-
thritic patients and was significantly associated with two biomarkers of
rheumatoid arthritis [18]. As a novel RA biomarker with a higher
specificity, serum 14-3-3η was significantly elevated in patients with
aggravated RA disease progression as well as involved in the patho-
genesis of RA [17]. Serum 14-3-3η levels are associated to some extent
with RA activity and inflammatory responses [19]. 14-3-3η-positive can
also be detected in arthralgia patients with anti-CCP and/or RF-positive
before the onset of RA and is related to the progression of arthritis [20].
In RA patients treated with Tofacitinib (TOF), 14-3-3η reduction is as-
sociated with remission of disease progression and can therefore be
used as a biomarker for monitoring the effect of TOF [21]. In addition,
RA patients with 14-3-3η-positive may have a higher incidence of os-
teoporosis. 14-3-3η is thought to be involved in the development of
osteoporosis in RA patients, and may be a predictor of osteoporosis in
patients with early RA [22]. Moreover, 14-3-3η can also be used for
differential diagnosis of other type of arthritis and connective tissue
diseases. Detection of serum 14-3-3η levels contributes to early diag-
nosis of poly-arthritis patients [23]. Elevated CRP and 14-3-3η sug-
gested a deteriorating disease process, especially in the elderly popu-
lation [23]. Additionally, Serum 14-3-3η levels were significantly
higher in early RA patients compared with osteoarthritis patients [24].
These studies above have focused on the diagnostic value of 14-3-3η
or Anti-Carp for RA. However, in order to better assist diagnosis and
treatment, the diagnostic value of 14-3-3ηand Anti-Carp combined with
other biomarkers for RA needs to be further investigated. In our present
study, serum levels of anti-CCP, RF, 14-3-3η and anti-CarP antibodies
were detected in RA patients, patients with autoimmune diseases except
RA, and healthy subjects recruited from Han population of Northern
China. We have taken clinical diagnosis as the standard [25,26] to
determine the application value of 14-3-3η protein, anti-CarP anti-
bodies, as well as their potential role to diagnose RA together with RF
or anti-CCP.
2. Methods
2.1. Study population
A total of 670 subjects in Peking University Third Hospital were
enrolled from June1, 2017, to May 31, 2019. They were all from the
Han population in North China. The first group (RA group) was col-
lected from 291 RA patients, aged 17–85 years old, 16.8% were male
and 83.2% were female. We strictly followed the American College of
Rheumatology (ACR) 1987 diagnostic criteria [26] and the 2010 RA
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Clinica Chimica Acta 502 (2020) 102–110
IC group HC group
223 156
41.6 ± 15.5(18–86) 34.1 ± 7.6(23–74)
125(56.1%) 84(53.8%)
classification criteria of the American College of Rheumatology and
European League Against Rheumatism (ACR/EULAR) [25] to determine
RA. The second group (Interference-control group, IC Group) was ob-
tained from 223 patients with autoimmune diseases that excludes the
possibility of RA (non-RA autoimmune patients), which included Sys-
temic lupus erythematosus (SLE), Osteoarthritis (OA), Ulcerative colitis
(UC), Ankylosing spondylitis (AS), Hashimoto's disease, Scleroderma,
Psoriasis, gout, vasculitis, and dermatomyositis. These patients ranged
from 18 to 86 years old, 43.9% were male and 56.1% were female. Each
non-RA autoimmune disease with fewer than 10 patients was combined
into “other” autoimmune disease. The third group (healthy-control
group, HC group) was gathered from 156 healthy individuals, aged 23
to 74 years old, 46.2% were male and 53.8% were female. Both inter-
ference-control group and healthy-control group constituted the Con-
trol group of our study (Table 1). The study was approved by the local
ethical committee and informed consent was provided.
2.2. Measurements of serologic biomarkers
After vacuum blood collection, each serum of the RA group, the
interference group and the healthy control group was separated by
centrifugation at 3500 rpm for 10 min after 1 h at room temperature.
All the sera were stored at –80 °C, and then dissolved and mixed before
measuring.
RF was detected by rate-turbidimetric immunoassay using IMMAGE
® 800 Immunochemistry System (Beckman Coulter, USA), with a cutoff
value of 20 IU/ml according to the manufacturer’s instructions. Anti-
CCP was measured by electro-chemi-luminescence assay (ECLA) using
ROCHE COBAS E601 (Roche Diagnostics GmbH, Germany), with a
cutoff value of 17 U/ml following manufacturer's recommendation.
Both methods are traditionally applied in our laboratory as well as
approved by the Ministry of Health.
The quantity of anti-CarP and 14-3-3η levels in the serum samples
was determined by Light Initiated Chemiluminescent Assay (LiCA)
using LiCA 500 immunoassay system (ChIVD Chemclin Diagnostics
Corp., China). The brief protocol of detection for serological Anti-CarP/
14-3-3η was as follows: Firstly, 50 ul of each sample or calibrator was
added to the sample dilution wells, followed by adding 50 ul of Reagent I
(Tris-buffer, NaCl, sodium lauryl sulfate(SLS), Triton, sodium deox-
ycholate) and then vortexing for 5 min. Secondly, 20 ul of the diluted
sample or calibrator was added to the reaction wells, followed by the
addition of 25 ul of Reagent II(rabbit anti-CarP/14-3-3η antibody coated
luminescent particles, phosphate buffer saline (PBS), and bovine serum
albumin (BSA)) and 25 ul of Reagent III (biotinylated carbaylated-BSA/
14-3-3η antibody and PBS), then incubating at 37 °C for 15 min.
Thirdly, 175 ul of General Buffer(mainly with avidin-coated photo-
sensitive particles) was added to each well and incubated for 10 min at
37 °C. All the reaction wells were then illuminated with laser and the
amount of photons (Relative light units, RLUs) emitted per well was
measured. Finally, a standard curve was obtained based on the cali-
brator results, and the sample concentration was calculated. All re-
agents above were stored at 2–8 °C. To demonstrate the linear range of
the assay kit, the linearity analysis was then performed as previously
described [27,28]. Briefly, dilution linearity was observed in two
samples with different levels, high-level samples (H) and low-level
Y. Zhang, et al. Clinica Chimica Acta 502 (2020) 102–110

samples (L), both for 14-3-3 η and anti-CarP. The levels of the high- Table 2
concentration and low-concentration sample was very close to but ex- Analytical performance of 14-3-3η and anti-CarP.
ceed the upper and lower limit of the linear range provided by the kit 14-3-3 η anti-CarP
instructions. Binary mixtures in proportions of 0L + 1H, 1L + 4H,
2L + 3H, 3L + 2H, 4L + 1H, and 1L + 0H were analyzed in triplicate, Intra-assay precision L 3.98% 2.40%
H 1.16% 1.95%
so that a total of 18 data points, at six different levels, were obtained.
Inter-assay precision L 7.72% 5.54%
Linearity was then assessed by plotting the measured results versus the H 3.57% 4.04%
expected results based on the dilution factor by least-squares fits. Accuracy (recovery rate) 91.3–96.3% 99.1–101.7%
All results were expressed as S/CO ratio (signal-to-cutoff ratio). S/ Minimum detection limit 0.1 ng/mL 10 RU/mL
CO ≤ 1.0 indicated negative results, while S/CO > 1.0 represented Linearity evaluation 0.1–19.6 ng/mL 15–149 RU/mL
R square 0.999 0.999
positive results. All the data were illustrated in accordance with the
Calibrators CAL1 0 ng/mL (362 0 RU/mL (27219
manufacturer's guidelines. RLUs) RLUs)
CAL2 0.2 ng/mL (706 1 RU/mL (22058
2.3. Statistical analysis RLUs) RLUs)
CAL3 0.5 ng/mL (1326 10 RU/mL (16331
RLUs) RLUs)
Statistical analysis was performed using SPSS software (SPSS for CAL4 2 ng/mL (9169 40 RU/mL (9254
Windows, version 22.0; IBM, USA). Quantitative variables were ex- RLUs) RLUs)
pressed as mean ± standard deviation (SD) or 95% confidence interval CAL5 5 ng/mL (29306 100 RU/mL (5172
(CI), while categorical variables were expressed as frequency and per- RLUs) RLUs)
CAL6 20 ng/mL (128517 200 RU/mL (1633
centage. The Mann-Whitney U test was used to compare the median
RLUs) RLUs)
differences between groups. A chi-square test was performed to analyze 4 parameters of logistic A 251.40 27151.00
qualitative data or categorical variable comparisons. Spearman corre- regression (4PL) B 315983.00 −18384.00
lation was used to show the correlation of quantitative results. The C 26.35 115.20
Kappa conformance test and the McNemar test were performed using a D 1.38 0.45
Reference interval ≤0.2 ng/mL ≤25 RU/mL
contingency table. Receiver operating characteristic (ROC) curve ana-
lysis was performed to calculate the diagnostic utility of 14-3-3η protein
and anti-Carp antibodies for the diagnosis of RA. The cutoff point of 14- application potential in anti-CarP and 14-3-3η analysis.
3-3η or anti-Carp antibodies was characterized by the Youden index.
Linear regression analysis (SPSS for Windows, version 22.0; IBM, USA)
3.2. Receiver operating characteristic (ROC) curves of anti-CarP and 14-3-
and a runs test (GraphPad Prism 8 Inc, San Diego, CA, USA) were used
3η
to evaluate the linearity of the assay. To compare more than 2 groups,
one-way analysis of variance (ANOVA) was used to determine if there
We depicted ROC curves to evaluate the performance of anti-CarP
was statistical significance among the groups. The positive rate, sensi-
and 14-3-3η as diagnostic suitable biomarkers for RA. As shown in
tivity, specificity, positive predictive value, negative predictive value,
Fig. 1C, anti-CarP had better discriminatory performance with an area-
and coincidence rate were calculated using 2 × 2 contingency table. A
under-curve (AUC) of 0.83(95%CI = [0.79; 0.86], p < 0.0001) with a
p value < 0.05 was considered statistically significant, and p < 0.01
cut off value of 25 RU/mL (Se = 66.32%, Sp = 88.65%), while the
was considered as a highly statistical significance.
ROC curve for 14-3-3η yielded an AUC value of 0.77(95%CI = [0.74;
0.81], p < 0.0001), with a cut off value of 0.2 ng/mL (Se = 52.23%,
3. Results
Sp = 93.67%) (Fig. 1D).
3.1. Analytical performance of anti-CarP and 14-3-3η assay
3.3. Quantitative performance of indicators
To test the analytical characterization of anti-CarP and 14-3-3η,
precision, accuracy, and linearity were summarized in Table 2 and As we can see in Fig. 2, the levels of RF, anti-CCP, anti-CarP and 14-
depicted in Fig. 1. Precision studies of 14-3-3η and anti-CarP revealed 3-3η were significantly higher in RA patients (12.5;[9.36–15.7], 30.7;
intra-assay CVs of < 5% and inter-assay CVs of < 10%. 14-3-3η and [25.7–35.6], 1.90;[1.70–2.01], 15.8;[10.8–20.8], respectively) com-
anti-CarP could be accurately measured at concentrations as low as pared with either interference-control group (1.24;[1.07–1.41], 0.64;
0.1 ng/mL and 10 RU/mL, respectively. Recovery of 14-3-3η and anti- [0.42–0.86], 0.51;[0.46–0.57], 0.33;[0.23–0.44], respectively)
CarP in mixes of low (L, 0.2 ng/ml and 20 RU/mL, respectively) and (p < 0.0001) or healthy-control group(1.03;[0.99–1.08], 0.49;
high (H, 5.0 ng/ml and 75 RU/mL, respectively) plasma pools was [0.38–0.59], 0.28;[0.21–0.35], 0.55;[0.27–0.85], respectively)
within the pre-defined acceptable limits of 85–115% (Table 2). The (p < 0.0001). Additionally, there was no difference between inter-
assay of anti-CarP and 14-3-3η showed the linearity in the range of ference-control group and healthy-control group in both anti-CCP
15–149 RU/mL (R2 = 0.999) and 0.1–19.6 ng/mL (R2 = 0.999), re- (p = 0.2787) and 14-3-3η (p = 0.1004) (Fig. 2B and C), while a higher
spectively (Fig. S1), included in the linear range given by the kit in- level of either RF (p < 0.05) or anti-CarP (p < 0.05) was observed in
structions. Meanwhile, the slopes showed a value of nearly 1 (1.024 and interference-control group (Fig. 2A and D).
1.082 for anti-CarP and 14-3-3η, respectively), representing the sa-
tisfactory linearity. In order to determine the quantity of anti-CarP and 3.4. Qualitative performance of indicators
14-3-3η, we compared our sample results to those of a set of standards
of known quantities (CAL1–CAL6), which were tested at a range of It is already known that the positivity for RF, anti-CCP, anti-CarP
concentrations that yields results from basically undetectable to max- and 14-3-3η was characterized as values more than 20 IU/ml, 17 U/ml,
imum signal (Table 2). Subsequently, the 4 parameter logistic regres- 25 RU/mL and 0.2 ng/mL, respectively. Fig. 3 shows the prevalence of
sion (4PL) was used to generate a predicted standard curve. The curve RF, anti-CCP, anti-CarP and 14-3-3η in RA group, IC group, and HC
fitted through the 6 points of anti-CarP standards was acceptable group. Similar to the quantitative results described above, the positive
(R2 = 0.998) (Fig. 1A), while the assay of 14-3-3η was reliable rates of RF, anti-CCP, anti-CarP and 14-3-3η in RA group were sig-
(R2 = 0.999) (Fig. 1B) based on the R-square values of the fit. With nificantly higher than that in either IC (p < 0.0001) or HC groups
good precision, accuracy, and reliable range, this method showed good (p < 0.0001). Additionally, the detection rates of RF and Anti-CarP in

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Fig. 1. Four parameters of logistic regression (4PL) Curves and ROC curves of the 14-3-3η and anti-CarP assay. (A) 4PL Curve of anti-CarP (A = 27151.00,
B = −18384.00, C = 115.20, D = 0.45); R square = 0.9988 (B) 4PL Curve of 14-3-3η (A = 251.40, B = 315983.00, C = 26.35, D = 1.38); R square = 0.9988.
ROC curves of anti-CarP (C) and 14-3-3η (D) for RA group versus control group. RLUs: Relative light units. The equation for the model is: F(x) = D + (A − D)/(1 + (x/
C) ^ B). Here, A = the minimum value that can be obtained, D = the maximum value that can be obtained, C = the inflection point, B = the slope of the curve. RA:
Rheumatoid Arthritis; ROC: Receiver Operating Characteristic; AUC: Area Under Curve.

IC group were significantly higher than those in HC group (p < 0.05)
(Fig. 3A and D), while there were small but observable differences
between the IC and HC groups in the positive rates of anti-CCP and 14-
3-3η (Fig. 3B and C). However, these differences were statistically in-
significant.
We performed a qualitative classification of the three groups of
samples. Fig. 4 reports the number of single-positive, double-positive,
triple-positive and quadruple-positive patients in RA group and Control
group (IC and HC groups) according to the Cut-off values of anti-CCP,
RF, anti-CarP and 14-3-3η. As shown in Fig. 4A, the samples with Triple
positive and Quadruple positive result were only found in RA group,
while neither ‘14-3-3η &RF’ nor ‘14-3-3η & anti-CCP’ double positive
case was found in Control group (Fig. 4B). Meanwhile, 23 of all 291
confirmed RA patients were seronegative (approximately 7.9%).
3.5. Mutual associations of RA indicators
As described in Table 3, RF levels were positively correlated with
Anti-CarP (r = 0.505, p < 0.0001), 14-3-3η(r = 0.517, p < 0.0001),
and the highest correlation was found with anti-CCP (r = 0.648,
p < 0.0001) (Fig. S2A, E, F). Anti-CCP were also moderately corre-
lated with 14-3-3η (r = 0.530, p < 0.0001) and Anti-CarP (r = 0.503,
p < 0.0001) (Fig. S2B, C). Meanwhile, weakly positive correlation was
observed between14-3-3η and Anti-CarP (r = 0.378, p < 0.0001) (Fig.
S2D). Collectively, significantly positive associations were discovered
among all these RA biomarkers in our study population.
methodological analysis. Similarly, for the ‘V–VIII’ testing strategies,
RA is determined only if 1 (V), 2 (VI), 3 (VII), or 4 (VIII) of the in-
dicators shows reactive. The goal of these strategies is to maximize
diagnostic value (Yonden index and kappa value) by combining bio-
markers in different ways.
Among the four detection strategies 'I–IV' which were judged by the
results of only one indicator, anti-CCP showed the highest sensitivity
(Se), specificity(Sp), positive predictive value(PPV), negative predictive
value (NPV), Youden index(YI) and kappa value(κ) (Se = 73.20%,
Sp = 97.36%, PPV = 95.52%, NPV = 82.55%, YI = 0.71, κ = 0.73,
respectively). In addition, among the four detection strategies of
'V–VIII', the 'V' had the highest sensitivity (Se = 92.1%), the 'VII' and '
VIII' had the highest specificity (both are Se = 100.00%), while the 'VI'
had the highest comprehensively diagnostic value (YI = 0.72,
κ = 0.74), which were even better than that of anti-CCP (Table 4).
In order to further optimize the diagnostic value, we combined 'VI',
the best one of the first 8 strategies, with RF, anti-CCP, anti-CarP or 14-
3-3ηrespectively, and then four new strategies 'IX–XII ' were derived.
Here, we combined a double-indicator test with a single-indicator test
to maximize the diagnostic value. As shown in Table 4, among the last 4
strategies, ‘X’ has the highest sensitivity, specificity, and positive pre-
dictive value (Se = 79.73%, Sp = 95.78%, PPV = 93.55%), while
accuracy, YI and κ are the highest (accuracy = 88.81%, YI = 0.76,
κ = 0.77) among all the 12 strategies (Fig. 4C).
4. Discussion

3.6. Comparison and selection of testing strategies
Table 4 summarized a total of 12 testing strategies modeled. Briefly,
with the ‘I–IV’ testing strategies, if the test result of RF(I), anti-CCP(II),
14-3-3η(III), or Anti-CarP (IV) is reactive, RA is reported for further
Although ACPA and RF have been generally used in RA diagnosis, it
is proved that RA patients with negative RF and anti-CCP accounted for
approximately 15–25% of the total number of RA patients [1,2]. Thus
we further investigated the diagnostic value of 14-3-3η and Anti-Carp
combined with other biomarkers for RA. So far researches on laboratory

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Fig. 2. Distribution of the biomarker levels in RA, IC and HC groups: (A), of RF; (B), of ACPA; (C), of 14-3-3η; (D), of anti-Carp. *p < 0.05 Compared with HC group;
**p < 0.001 Compared with IC group; ***p < 0.0001 Compared with HC group; S/CO ratio: signal-to-cutoff ratio; RA: Rheumatoid Arthritis; IC: Interference-control; HC:
healthy-control.

diagnosis of RA have focused on single, double, and triple indicators
[7,13,29], while the novelty of our investigation is that single and
multiple biomarkers can be combined together to improve the clinical
diagnostic value of RA. Here, it is worth noting that our study explored
the diagnostic value of the combined analysis of RF, anti-CCP, 14-3-3 η
and anti-CarP, and for the first time found that Strategy ‘X’ (among 4
biomarkers, if any 2 are positive or only anti-CCP is positive, it is
considered to be diagnosed as RA.) had the highest diagnostic value for
assisting the judgment of RA in Han population of Northern China.
Meanwhile, we established the reference intervals of both 14-3-3η
and anti-CarP in serum of our subjects as well as evaluated analytical
performance of both assays. We also found that these four indicators
(RF, anti-CCP, 14-3-3η and anti-CarP) were significantly positively
correlated with each other. Firstly, among the correlation coefficients
between any two indicators, anti-CCP and RF showed the strongest
correlation (r = 0.648). Meanwhile, 14-3-3η had the moderate corre-
lation with either RF (r = 0.517) or anti-CCP (r = 0.530), while anti-
CarP was also moderately related to RF (r = 0.505) and anti-CCP
(r = 0.503). Lastly, the correlation between 14-3-3η and anti-CarP was
demonstrated minimal and mild (r = 0.378). Interestingly, the corre-
lations coefficient between anti-CarP and anti-CCP, RF and anti-CCP,
and14-3-3η and anti-CCP were higher than that in previous researches

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Fig. 3. Distribution of positivity in RA, IC and HC groups: (A), of RF; (B), of ACPA; (C), of 14-3-3η; (D), of anti-Carp. *p < 0.05 Compared with HC group;
**p < 0.001 Compared with IC group; ***p < 0.0001 Compared with HC group; S/CO ratio: signal-to-cutoff ratio; RA: Rheumatoid Arthritis; IC: Interference-control; HC:
healthy-control.
[3,30], whereas the association between 14-3-3η and RF was lower than
other studies [23,30]. It is speculated that the moderate correlation of
these indicators in our study may be due to the fact that these indicators
may have spontaneous increase in RA patients. Particularly, it has been
proved that HLA-DRB1 * 03 was specifically associated with anti-CCP-
negative and anti-CarP-positive RA patients [31], thus the regulatory
mechanisms of anti-CarP antibodies and anti-CCP may be independent
of each other. However, other researchers proposed that affinity-pur-
ified anti-CCP bound to both carbamylated proteins and peptides con-
taining homocitrulline, thus anti-CCP could cross-react with anti-CarP
antibodies [32]. After all, it is not possible to directly derive interac-
tions from correlations, nor can it rule out the effects of racial differ-
ences. Notably, the specificity of anti-CCP or anti-CarP was similar to
the previous study, while the sensitivity was slightly higher [7].
Meanwhile, the specificity of RFor14-3-3η was parallel to the previous
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study, while the sensitivity was slightly lower [20,30]. Differences in
ethnicity, living environment, and test methodology may all be the
cause of this issue. Further investigation is needed to explain the rea-
sons for these dissimilarities.
In RA group, the proportion of patients with Anti-CarP-positive and
anti-CCP-negative accounted for 13.7%, which was similar to several
previous studies [3,15,31]. The levels of RF, anti-CCP, 14-3-3η and anti-
CarP in RA group were significantly higher than those in IC Group and
HC group, suggesting that these indicators are all valuable in RA di-
agnosis. At the beginning, we assumed that there was no difference in
the level of each indicator between IC Group and HC group. Actually, it
is showed that anti-CCP and 14-3-3η met the above assumption, while
RF and anti-CarP were significantly higher in IC group. This may be due
to the low level of expression of RF and anti-CarP in other autoimmune
diseases. Previously, some researchers have found that although anti-
Y. Zhang, et al. Clinica Chimica Acta 502 (2020) 102–110

Fig. 4. Distribution of positive results of each indicator and schematic diagram of RA laboratory diagnostic strategy with the best Youden index (YI). Distribution of
single-positive, double-positive, triple-positive and quadruple-positive patients in RA group (A) and Control group (B). The RA laboratory diagnostic strategy with the
best Youden index (C) is like a parallel circuit controlled by several switches (D). Each of the two parallel circuits has its own switch. When ACPA is positive, switch
(a) is turned on. When any two of the four indicators (RF, ACPA, anti-CarP and 14-3-3η) are positive, switches (b1) and (b2) are turned on. The connection of the
circuit is a metaphor for the diagnosis of RA.

CarP was mainly present in RA patients, it could also be detected in
patients with other autoimmune rheumatic diseases such as systemic
lupus erythematosus and Sjogren's syndrome [33]. It has been reported
that RF was visible in about 10% of healthy people in many other
chronic autoimmune diseases such as Sjögren syndrome (SSc) and
systemic lupus erythematosus (SLE), thus RF is not so specific to RA
[1,2]. Interestingly, after we omitted the subjects of SLE in IC group, the
positive rate of anti-CarP in the IC group was no longer significantly
different from that of NC group (Fig. S3). On the one hand, it may be
due to the low levels of Anti-CarP in SLE [33]. On the other hand, it
may contribute to the possibility that some of the selected SLE subjects
might carry a certain degree of joint damage [34,35]. It also gave us a
hint to further explore the role of anti-CarP in SLE patients with joint
erosion in our living area.
Anti-CCP showed the best performance among the four indicators,
and its sensitivity, specificity, positive predictive value, and negative
predictive value were all optimal. Despite this, a proportion of (about
18.9%) RA patients showed negative in anti-CCP as well as positive in
at least one of other three indicators (Fig. 4A), thus the diagnostic value
of RF, 14-3-3 η and anti-CarP cannot be underestimated. As we ex-
pected, in multi-index criteria, with the increase number of included
biomarkers, the sensitivity gradually decreased, while the specificity
steadily increased. We have found that when RF, anti-CCP, 14-3-3η and
anti-CarP were all positive or triple positive, the specificity for RA was
the highest (100%) while the sensitivity was quite low (30.58–58.76%).
Notably, among the strategies of single, double, triple, and quadruple
indicators, methodological indexes of triple-positive model in the di-
agnosis of RA were similar to those of previous studies [24,29,30],
while double-positive model showed the best comprehensive char-
acteristics. Interestingly, considering the outstanding performance of
anti-CCP in the northern Chinese population, we proposed a novel
strategy that either double-positive of any 2 markers or single-positive
of anti-CCP can be diagnosed as RA (Fig. 4C). The principle is similar to
a parallel circuit (Fig. 4D). As expected, the YI and κ value of this
strategy rose to 0.76 and 0.77, respectively, which led this program
have the highest diagnostic value. Admittedly, it is suspected that the

Table 3
Correlations of serum values among RF, ACPA Anti-CarP, and 14-3-3 η.
ACPA 14-3-3 η Anti-Carp RF

Spearman r 95% CI Spearman r 95% CI Spearman r 95% CI Spearman r 95% CI

ACPA 1.000 1.000–1.000

14-3-3 η 0.530*** 0.471–0.584 1.000 1.000–1.000
Anti-Carp 0.503*** 0.443–0.559 0.378*** 0.310–0.443 1.000 1.000–1.000
RF 0.648*** 0.599–0.691 0.517*** 0.458–0.572 0.505*** 0.444–0.561 1.000 1.000–1.000

Abbreviations: ***p < 0.0001.

95% CI: 95% confidence interval.
Spearman r: spearman’s rank correlation coefficient.

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Y. Zhang, et al. Clinica Chimica Acta 502 (2020) 102–110

Table 4
Comparison of methodological metrics for various criteria.
Sensitivity Specificity Positive Negative Mistake Omission Accuracy Youden’s index Kappa
predictive value predictive value diagnostic rate diagnostic rate value

I RF positive 64.60% 93.40% 88.26% 77.46% 6.60% 35.40% 80.90% 0.58 0.60
II ACPA positive 73.20% 97.36% 95.52% 82.55% 2.64% 26.80% 86.87% 0.71 0.73
III 14-3-3 η positive 52.23% 93.67% 86.36% 71.86% 6.33% 47.77% 75.67% 0.46 0.48
IV Anti-Carp positive 66.32% 88.65% 81.78% 77.42% 11.35% 33.68% 78.96% 0.55 0.56
V Single positive 92.10% 75.99% 74.65% 92.60% 24.01% 7.90% 82.99% 0.68 0.66
VI Double positive 74.91% 97.10% 95.20% 83.45% 2.90% 25.09% 87.46% 0.72 0.74
VII Triple positive 58.76% 100.00% 100.00% 75.95% 0.00% 41.24% 82.09% 0.59 0.62
VIII Quadruple positive 30.58% 100.00% 100.00% 65.23% 0.00% 69.42% 69.85% 0.31 0.33
IX Double positive or RF 77.32% 91.56% 87.55% 84.02% 8.44% 22.68% 85.37% 0.69 0.70
positive
X Double positive or 79.73% 95.78% 93.55% 86.02% 4.22% 20.27% 88.81% 0.76 0.77
ACPA positive
XI Double positive or 14- 75.95% 91.56% 87.35% 83.21% 8.44% 24.05% 84.78% 0.68 0.69
3-3 η positive
XII Double positive or 83.85% 88.39% 84.72% 87.70% 11.61% 16.15% 86.42% 0.72 0.72
Anti-Carp positive

best detection schemes may vary in individuals of other races.
The limitations of this study included that early RA was not ana-
lyzed alone due to the small sample size. Anti-CarP antibodies could be
detected in approximately 25% of seronegative (RF negative and anti-
CCP negative) early-RA patients. At the same time, anti-CarP antibodies
were related to more severe radiological outcomes [15]. Meanwhile,14-
3-3η-positive was detected in arthralgia patients with anti-CCP and/or
RF-positive before the onset of RA [20] and was associated to some
extent with RA activity and inflammatory responses [19]. These find-
ings suggest that Anti-CarP and 14-3-3η have a certain degree of di-
agnostic value for early RA. Therefore, combination of multiple in-
dicator assays may help to discover early RA. In addition, an expanded
sample size is still needed to evaluate the diagnostic and predictive
capacity of 14-3-3η protein and anti-CarP for RA, as well as their cor-
relation with disease activity and therapeutic response.
5. Conclusion
The results of the present study have demonstrated that in Han
population of Northern China, anti-CarP antibodies and 14-3-3η protein
can be treated as valuable indicators of RA, especially when combined
with RF and anti-CCP, the detection value is maximized. In the further
study, we will expand the sample size and recruit subjects from
southern China to confirm this issue, as well as conduct an exploration
of multiple-indicator criteria in diagnosis of early RA.
CRediT authorship contribution statement
Yuan Zhang: Data curation, Writing - original draft, Formal ana-
lysis, Validation, . Yongming Liang: Investigation, Data curation,
Writing - review & editing. Limei Feng: Investigation, Writing - review
& editing. Liyan Cui: Supervision, Project administration, Funding
acquisition, Conceptualization.
Acknowledgments
All authors agreed to publish this work and critically reviewed the
article. Conception and design of this work was discussed with all the
authors.
Funding
This work was supported by grants No. 61771022 from the National
Natural Science Foundation of China.
Declaration of Competing Interest
The authors have no conflicts of interest to disclose.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.cca.2019.12.011.
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