Quantification of Citrullinated Histone H3 as a Marker for Neutrophil Extracellular Traps Correlated to Clinical Characteristics of Patients with Systemic Lupus Erythematosus

Maciej Dubaj; Iwona Homa-Mlak; Aleksandra Majdan; Radosław Mlak; Maria Majdan; Teresa Małecka-Massalska

doi:10.2478/aite-2025-0002

Figures & Tables

Box–whisker plots presenting the comparison of the occurrence of arthralgia (A) and reduced C4 levels (B) depending on H3Cit levels [ng/μL]. The graphs show a statistically significant difference in H3Cit levels along with their median and IQR in patients with (1) and without (0) arthralgia (median [IQR], respectively: 1.68 ng/μL [1.67–1.69] vs. 1.67 ng/μL [1.62–1.68]) and reduced C4 levels (median [IQR], respectively: 1.68 ng/μL [1.67–1.70] vs. 1.68 ng/μL [1.67–1.69]). C4, complement component 4; H3Cit, citrullinated histone H3; IQR, interquartile range.

ROC curves presenting the diagnostic utility of H3Cit concentration [ng/μL] in detection of arthralgia (A), seizures (B), hepatomegaly (C) and reduced C4 level (D) in the course of SLE. They present the specificity and sensitivity of H3Cit as a differentiation tool for patients with: arthralgia (spec: 55%, sens.: 88.2%, cut-off point: >1.6705 ng/μL), seizures (spec: 73. 1%, sens.: 100%, cut-off point: >1.685 ng/μL), hepatomegaly (spec: 66.7%, sens.: 80%, cut-off point: 1.6831 ng/μL) and reduced C4 level (spec: 72.2%, sens.: 60.6%, cut-off point: >1.6777 ng/μL). C4, complement component 4; H3Cit, citrullinated histone H3; ROC, receiver operating characteristic; SLE, systemic lupus erythematosus.

Scatter diagram showing the correlation between WBC count [G/L] and H3Cit [ng/μL] (A), lymphocyte count [G/L] and H3Cit concentration [ng/μL] (B), dsDNA level [IU/mL] and H3Cit concentration [ng/μL] (C) and C3 level [mg/dL] and H3Cit concentration [ng/μL] (D). They show that in the study group, as the value of the H3Cit level increases, the level of dsDNA increases significantly, and the count of WBC and lymphocytes and the level of C3 decreases. C3, complement component 3; dsDNA: double stranded DNA; H3Cit, citrullinated histone H3; LYM, lymphocytes; WBC, white blood cells.

Correlation between selected clinical and laboratory factors and H3Cit level

Variables	rho	p
Age (years)	−0.0768	0.5307
Duration of the disease (years)	−0.1070	0.3890
RBC [T/L]	−0.1942	0.1099
HGB [g/dL]	−0.1864	0.1251
WBC [G/L]	−0.2602	0.0309^*
NEUTR [G/L]	−0.2159	0.0748
LYM [G/L]	−0.2788	0.0204^*
PLT [G/L]	−0.0448	0.7148
OB [mm/h]	−0.0260	0.8322
CRP [mg/L]	0.1302	0.2861
Total protein [g/dL]	−0.0373	0.7612
Creatinine [mg/dL]	−0.1178	0.3350
GFR [mL/min/1.73 m²]	−0.0042	0.9729
Glomerulonephritis class	0.3770	0.4615
ANA [IU/mL]	−0.0475	0.6983
dsDNA [IU/mL]	0.3794	0.0036^*
C3 [mg/dL]	−0.2442	0.0447^*
IgG [mg/dL]	0.0939	0.4430
IgM [mg/dL]	0.0686	0.5755

Results of blood and urine laboratory tests of patients with SLE

Variables	N (%)

Protein in daily urine collection [mg/day]
0	57 (71.3)
<150	4 (5)
150–500	11 (13.7)
>500	8 (10)

RBC [T/L]
<3	1 (1.3)
3–3.99	12 (15)
4–5	61 (76.3)
>5	6 (7.5)

HGB [g/L]
8–9.99	4 (5)
10–11.99	15 (18.7)
12–14.99	55 (68.7)
>15	6 (7.5)

WBC [G/L]
<4	17 (21.3)
4–10	56 (70)
>10	7 (8.7)

NEUTR [G/L]
<1.5	4 (5)
1.5–6	62 (77.5)
>6	14 (17.5)

LYM [G/L]
<1.5	45 (56.3)
1.5–4	35 (43.7)
>4	0 (0)

PLT [G/L]
<150	14 (17.5)
150–400	63 (78.7)
>400	3 (3.7)

ESR [mm/h]
<3	14 (17.5)
3–5.99	10 (12.5)
6–11.99	16 (20)
12–19.99	13 (16.3)
20–30	14 (17.5)
>30	13 (16.3)

CRP [mg/L]
<5	61 (76.3)
≥5	19 (23.7)

Total protein [g/L]
<60	33 (41.3)
60–80	46 (57.5)
>80	1 (1.3)

Creatinine [mg/dL]
<0.6	7 (8.7)
0.6–1.3	66 (82.5)
>1.3	7 (8.7)

GFR [mL/min/1.73 m²]
<30	3 (3.7)
30–59	6 (7.5)
60–89	21 (26.3)
≥90	50 (62.5)

Differences in H3Cit levels depending on clinical factors in the study group

Variables	N (%)	Median (IQR) [ng/μL]	p

Photosensitivity
Yes	54 (67.5)	1.68 (1.67–1.69)	0.3238
No	26 (32.5)	1.68 (1.66–1.69)

Skin lesions
Yes	54 (67.5)	1.68 (1.67–1.69)	0.3238
No	26 (32.5)	1.68 (1.66–1.69)

Mucosal erosions
Yes	56 (70)	1.68 (1.67–1.69)	0.7761
No	24 (30)	1.68 (1.67–1.69)

Fatigue
Yes	61 (76.3)	1.68 (1.67–1.69)	0.1070
No	19 (23.7)	1.67 (1.62–1.68)

Fever
Yes	19 (23.7)	1.68 (1.67–1.68)	0.1478
No	61 (76.3)	1.68 (1.67–1.69)

Raynaud's syndrome
Yes	7 (8.8)	1.68 (1.68–1.70)	0.3654
No	73 (71.2)	1.68 (1.67–1.69)

Hair loss
Yes	33 (41.3)	1.68 (1.67–1.69)	0.7760
No	47 (58.7)	1.68 (1.67–1.69)

Myopathy
Yes	5 (6.2)	1.68 (1.68–1.69)	0.5548
No	75 (93.7)	1.68 (1.67–1.69)

Vasculitis
Yes	15 (18.8)	1.68 (1.67–1.69)	0.4118
No	65 (81.2)	1.68 (1.67–1.69)

Arthritis
Yes	43 (53.7)	1.68 (1.67–1.69)	0.4140
No	37 (46.3)	1.68 (1.67–1.69)

Arthralgia
Yes	59 (73.7)	1.68 (1.67–1.69)	0.0150^*
No	21 (26.3)	1.67 (1.62–1.68)

Pleuritis
Yes	7 (8.8)	1.68 (1.68–1.70)	0.1695
No	71 (91.2)	1.68 (1.67–1.69)

Pericarditis
Yes	6 (7.5)	1.69 (1.66–1.73)	0.5550
No	74 (92.5)	1.68 (1.67–1.69)

Epilepsy
Yes	6 (7.5)	1.68 (1.62–1.69)	0.6779
No	74 (92.5)	1.68 (1.67–1.69)

Seizures
Yes	2 (2.5)	1.70 (1.69–1.70)	0.1377
No	78 (97.5)	1.68 (1.67–1.69)

Psychosis
Yes	2 (2.5)	1.68 (1.68–1.68)	0.6879
No	78 (97.5)	1.68 (1.67–1.69)

Other neurological symptoms
Yes	24 (30)	1.68 (1.67–1.68)	0.6434
No	56 (70)	1.68 (1.67–1.69)

Proteinuria
Yes	47 (58.7)	1.68 (1.67–1.69)	0.8916
No	33 (41.3)	1.68 (1.67–1.68)

Hematuria
Yes	17 (21.3)	1.68 (1.67–1.70)	0.6015
No	63 (78.7)	1.68 (1.67–1.69)

Chronic kidney disease
Yes	9 (11.3)	1.67 (1.61–1.69)	0.3404
No	71 (88.7)	1.68 (1.67–1.69)

Sterile leukocyturia
Yes	8 (10)	1.68 (1.67–1.75)	0.4880
No	72 (90)	1.68 (1.67–1.69)

Hemolytic anemia
Yes	4 (5)	1.69 (1.64–1.74)	0.7776
No	76 (95)	1.68 (1.67–1.69)

Hepatomegaly
Yes	5 (6.3)	1.69 (1.68–1.72)	0.0728
No	75 (93.7)	1.68 (1.67–1.69)

Splenomegaly
Yes	8 (10)	1.68 (1.67–1.76)	0.3252
No	72 (90)	1.68 (1.67–1.67)

Lymphadenopathy
Yes	4 (5)	1.68 (1.67–1.68)	0.9180
No	76 (95)	1.68 (1.67–1.69)

RF-IgM
Yes	17 (21.3)	1.68 (1.67–1.70)	0.1784
No	63 (78.7)	1.68 (1.67–1.69)

SSA
Yes	34 (42.5)	1.68 (1.68–1.70)	0.0924
No	46 (57.5)	1.68 (1.67–1.69)

SSB
Yes	14 (17.5)	1.69 (1.67–1.70)	0.1292
No	66 (82.5)	1.68 (1.67–1.69)

Scl-70
Yes	2 (2.5)	1.65 (1.60–1.70)	0.8440
No	78 (97.5)	1.68 (1.67–1.69)

Rib-P
Yes	13 (16.3)	1.68 (1.67–1.68)	0.3734
No	67 (83.7)	1.68 (1.67–1.69)

Anti-dsDNA
Yes	60 (75)	1.68 (1.67–1.69)	0.8589
No	20 (25)	1.68 (1.67–1.69)

pANCA
Yes	3 (3.7)	1.67 (1.67–1.67)	0.4525
No	77 (96.3)	1.68 (1.67–1.69)

Anti-Sm
Yes	18 (22.5)	1.68 (1.67–1.70)	0.7655
No	62 (77.5)	1.68 (1.67–1.69)

Anti-RNP
Yes	22 (27.5)	1.68 (1.67–1.70)	0.8519
No	58 (72.5)	1.68 (1.67–1.69)

Anti-Jo-1
Yes	1 (1.3)	1.61 (1.61–1.61)	0.1320
No	79 (98.7)	1.68 (1.67–1.69)

Anti-beta-2-GPI-2 IgM
Yes	5 (6.3)	1.68 (1.65–1.70)	0.9354
No	75 (93.7)	1.68 (1.67–1.69)

Anti-beta-2-GPI-2 IgG
Yes	6 (7.5)	1.68 (1.67–1.70)	0.6187
No	74 (92.5)	1.68 (1.67–1.69)

Anti-CCP
Yes	1 (1.3)	1.70 (1.70–1.70)	0.2481
No	79 (98.7)	1.68 (1.67–1.69)

Anti-Cl IgM
Yes	25 (31.3)	1.68 (1.67–1.71)	0.0528
No	55 (78.7)	1.68 (1.67–1.69)

Anti-Cl-IgG
Yes	21 (26.3)	1.68 (1.67–1.70)	0.0808
No	59 (73.7)	1.68 (1.67–1.69)

Reduced level of C3
Yes	52 (65)	1.68 (1.67–1.69)	0.1491
No	28 (35)	1.68 (1.67–1.68)

Reduced level of C4
Yes	37 (46.3)	1.68 (1.67–1.70)	0.0297^*
No	43 (53.7)	1.68 (1.67–1.69)

LAC
Yes	17 (21.3)	1.68 (1.67–1.69)	0.6171
No	63 (78.7)	1.68 (1.67–1.69)

ASMA
Yes	1 (1.3)	1.70 (1.67–1.70)	0.2692
No	79 (98.7)	1.68 (1.67–1.69)

aNuA
Yes	11 (13.7)	1.68 (1.67–1.68)	0.3707
No	69 (86.3)	1.68 (1.67–1.69)

Anti-histone
Yes	9 (11.3)	1.68 (1.67–1.70)	0.5807
No	71 (88.7)	1.68 (1.67–1.69)

IgG
Yes	28 (35)	1.68 (1.67–1.70)	0.5690
No	52 (65)	1.68 (1.67–1.69)
Median (range)	0 (0–2781)

IgM
Yes	28 (35)	1.68 (1.67–1.70)	0.5690
No	52 (65)	1.68 (1.67–1.69)
Median (range)	0 (0–312)

IgA
Yes	28 (35)	1.68 (1.67–1.70)	0.5690
No	52 (65)	1.68 (1.67–1.69)
Median (range)	0 (0–551)

Autoimmune comorbidities
Yes	56 (53.7)	1.68 (1.67–1.69)	0.2908
No	24 (46.3)	1.68 (1.67–1.68)

Other comorbidities
Yes	43 (68.7)	1.68 (1.67–1.69)	0.5356
No	37 (32.3)	1.68 (1.67–1.70)

Solu-Medrol i.v. during hospitalization
Yes	22 (27.5)	1.68 (1.67–1.71)	0.0761
No	58 (72.5)	1.68 (1.67–1.69)

Glucocorticoids p.o.
Yes	67 (83.7)	1.68 (1.67–1.69)	0.1169
No	13 (16.3)	1.66 (1.65–1.69)

Evaluation of diagnostic usefulness of H3Cit histones in the detection of SLE and specific clinical conditions by means of ROC curve analysis

Variables	AUC [95% CI]	Sensitivity (%)	Specificity (%)	Cut-off point	p
Form of SLE articular vs. renal and hematological and cutaneous and neuropsychological	0.505 [0.381–0.628]	0.00	82.69	≤1.6482	0.9502
Form of SLE renal vs. articular and hematological and cutaneous and neuropsychological	0.609 [0.483–0.725]	28.00	97.67	≤1.6432	0.1445
Form of SLE hematological vs. articular and renal and cutaneous and neuropsychological	0.605 [0.479–0.722]	66.67	72.31	≤1.6473	0.5391
Form of SLE cutaneous vs. articular and renal and hematological and neuropsychological	0.621 [0.495–0.736]	41.18	88.24	>1.6941	0.1495
Form of SLE neuropsychological vs. cutaneous and articular and renal and hematological	0.585 [0.460–0.704]	85.71	50.82	>1.6765	0.4126
Photosensitivity	0.573 [0.448–0.692]	95.65	21.74	>1.6213	0.3230
Skin lesions	0.573 [0.448–0.692]	95.65	21.74	>1.6213	0.3286
Mucosal erosions	0.522 [0.398–0.644]	95.52	28.57	>1.6705	0.7637
Raynaud's syndrome	0.618 [0.495–0.732]	83.33	51.56	>1.6765	0.2509
Myopathy	0.595 [0.471–0.710]	100.00	50.00	>1.6762	0.3022
Vasculitis	0.579 [0.455–0.696]	100.00	25.00	>1.6632	0.3253
Arthritis	0.570 [0.446–0.688]	97.22	20.59	>1.6904	0.3144
Arthralgia	0.709 [0.589–0.812]	88.24	50.00	>1.6705	0.0115^*
Pleuritis	0.676 [0.553–0.783]	83.33	62.50	>1.6786	0.0633
Pericarditis	0.586 [0.462–0.703]	40.00	92.31	>1.7043	0.6493
Epilepsy	0.543 [0.420–0.663]	33.33	92.19	≤1.6202	0.7663
Seizures	0.813 [0.701–0.896]	100.00	73.13	>1.6485	0.0005^*
Other neurological symptoms	0.524 [0.401–0.625]	76.19	38.78	≤1.6831	0.7434
Proteinuria	0.521 [0.398–0.642]	25.00	92.31	≤1.685	0.7640
Hematuria	0.551 [0.427–0.670]	29.41	90.57	>1.7012	0.5546
Chronic kidney disease	0.638 [0.515–0.750]	40.00	96.67	≤1.6122	0.2516
Sterile leukocyturia	0.583 [0.459–0.699]	37.50	95.16	>1.7116	0.5099
Hemolytic anemia	0.549 [0.426–0.669]	50.00	84.85	>1.6961	0.8207
Hepatomegaly	0.746 [0.628–0.843]	80.00	68.75	>1.6831	0.0111^*
Splenomegaly	0.620 [0.496–0.734]	42.86	88.89	>1.7012	0.3965
Lymphadenopathy	0.510 [0.388 to −0.631]	100.00	34.33	≤1.6839	0.9522
RF IgM	0.621 [0.497–0.735]	53.33	70.91	>1.6831	0.1407
SSA	0.628 [0.505–0.741]	83.33	45.00	>1.6733	0.0571
SSB	0.657 [0.534–0.766]	70.00	68.33	>1.6812	0.1579
Scl-70	0.533 [0.410–0.653]	50.00	95.59	≤1.6032	0.9377
Rib-P	0.570 [0.446–0.688]	76.92	54.39	≤1.6767	0.4425
Anti-dsDNA	0.505 [0.383–0.627]	24.07	93.75	>1.6929	0.9489
pANCA	0.647 [0.524–0.758]	100.00	54.41	≤1.6762	0.2012
Anti-Sm	0.533 [0.410–0.653]	33.33	87.27	>1.6961	0.7340
Anti-RNP	0.504 [0.382–0.626]	50.00	64.58	≤1.6746	0.9620
Anti-beta-2-GPI-2 IgM	0.505 [0.383–0.627]	40.00	83.08	≤1.6602	0.9792
Anti-beta-2-GPI-2 IgG	0.574 [0.450–0.691]	80.00	43.08	>1.6749	0.5843
Anti-Cl IgG	0.642 [0.518–0.753]	55.00	72.00	>1.6815	0.0658
Anti-Cl IgM	0.654 [0.531–0.764]	66.67	67.35	>1.6777	0.0519
Reduced level of C3	0.593 [0.469–0.709]	52.17	75.00	>1.6777	0.1896
Reduced level of C4	0.662 [0.539–0.771]	60.61	72.22	>1.6777	0.0224^*
LAC	0.534 [0.411–0.654]	100.00	12.50	≤1.6733	0.6995
aNuA	0.579 [0.455–0.696]	100.00	33.33	≤1.6855	0.3135
Anti-histone	0.548 [0.425–0.668]	77.78	52.46	≤1.6767	0.6798
Autoimmune comorbidities	0.586 [0.462–0.703]	62.86	60.00	>1.6777	0.2186
Other comorbidities	0.556 [0.433–0.675]	95.92	23.81	≤1.7043	0.4718
Solu-Medrol i.v. during hospitalization	0.642 [0.519–0.753]	42.86	93.88	>1.6961	0.0642
Glucocorticoids p.o.	0.652 [0.529–0.762]	81.97	66.67	>1.6717	0.2019

General characteristic of the study group

Variables	N (%) or Median (IQR), [min–max]

Sex
Male	9 (11.3%)
Female	71 (88.7%)

Age (years)	36.5 (31–48), [19–72]

Age at first symptoms (years)	27 (21–36), [7–69]

Age at diagnosis (years)	29 (23–38), [14–69]

Duration of the disease (years)	6 (2–13), [0–37]

Form of the disease
Renal	28 (35%)
Hematological	7 (8.7%)
Cutaneous	20 (25%)
Neurological	3 (3.75%)
Neuropsychiatric	8 (10%)
Arthritic	17 (21.3%)

Glomerulonephritis class
1B	1 (1.3%)
II	1 (1.3%)
III	1 (1.3%)
IV	2 (2.5%)
V	1 (1.3%)
No	74 (92.5%)

ANA	1 (1–1), [0–1]

H3 level [ng/mL]	1.7 (1.6–1.8), [1.6–1.8]

dsDNA level [IU/mL]	44.8 (14–107), [6–900]

C3 level [mg/dL]	90.9 (71–105), [25–177]

Quantification of Citrullinated Histone H3 as a Marker for Neutrophil Extracellular Traps Correlated to Clinical Characteristics of Patients with Systemic Lupus Erythematosus

Figures & Tables

Fig 1.

Fig 2.

Fig 3.

Correlation between selected clinical and laboratory factors and H3Cit level

Results of blood and urine laboratory tests of patients with SLE

Differences in H3Cit levels depending on clinical factors in the study group

Evaluation of diagnostic usefulness of H3Cit histones in the detection of SLE and specific clinical conditions by means of ROC curve analysis

General characteristic of the study group

Paradigm

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