Baseline characteristics of ACLF AKI, ACLF no-AKI and non-liver AKI (diseased control group)
A total of 150 subjects were included in this study in which 91were ACLF (Acute on chronic liver failure) patients, 29 were AKI (non-liver acute kidney injury) patients and 30 were healthy controls (HC). The ACLF group was further stratified based on ICA-AKI criteria (as mentioned in methods), as ACLF no-AKI (n = 35) and ACLF AKI (n = 56). The study design flowchart is depicted in Fig. 1.
The mean age ± S.D. and males n(%) across ACLF no-AKI, ACLF AKI, non-liver AKI and HC, was 40.23 ± 2.12 and 80%, 45.05 ± 1.56 and 80.3%, 30.92 ± 13.42 and 50%, and, 36.90% and 96.67% respectively. Alcohol abuse was the most common chronic (56%) as well as acute (32%) etiology in ACLF patients.
Stratification of ACLF patients based on grades (as per the EASL-CLIF criteria), were- 13 (14.28%) for Grade 1, 32 (35.16%) for Grade 2 and 46 (50.54%) for Grade 3 (Table 1).
Table 1
Baseline characteristics and biomarker measurements of study groups ACLF no-AKI (n = 35), ACLF AKI (n = 56), Non-liver AKI (n = 29) and Healthy controls (n = 30)
Parameters
|
ACLF no-AKI (n = 35)
|
ACLF AKI
(n = 56)
|
Non-liver AKI
(n = 29)
|
Healthy (n = 30)
|
p-value
|
Age, mean ± S.D. (years)
|
40.23 ± 2.12
|
45.05 ± 1.56
|
30.92 (13.419)a,b
|
36.90 (9.10)b
|
0.0009
|
Male, n (%)
|
28 (80.0%)
|
45 (80.3%)
|
15 (50.0%)
|
29 (96.67%)
|
0.006
|
Hemoglobin (g/dL)
|
9.06 ± 0.43
|
8.05 ± 0.27
|
|
|
0.0386
|
TLC (x103 per mm3)
|
12280 (8600–19070)
|
12630 (7950–20650)
|
|
|
0.88
|
Platelet (x103 per mm3)
|
91(68–122)
|
77 (54.5-108.5)
|
|
|
0.236
|
PT (seconds)
|
28(22–38)
|
28(22–36)
|
|
|
0.973
|
INR
|
2.5(1.9–3.6)
|
2.6(1.95–3.25)
|
|
|
0.83
|
Urea (mg/mL)
|
54(36–93)
|
129.5(87-191.5) a
|
110.5 (86–147)a
|
|
< 0.0001
|
Creatinine(mg/dL)
|
1.4 (0.8–1.6)
|
3.15(2.3–4.5) a
|
4.1 (3.2–5.4) a
|
|
< 0.0001
|
Na (mEq/L)
|
138.34 ± 1.40
|
132.43 ± 1.07 a
|
135.63 ± 7.64
|
|
0.0034
|
K (mEq/L)
|
4.25 ± 0.15
|
4.67 ± 0.16
|
4.60 ± 0.93
|
|
0.1784
|
Bilirubin (mg/dL)
|
13.5 (6.8–22.8)
|
9.1 (3.25–21.8)
|
0.4 (0.3–0.6) a, b
|
|
< 0.0001
|
AST (IU/L)
|
96 (69–177)
|
88 (52–144)
|
26 (19–37)a, b
|
|
< 0.0001
|
ALT(IU/L)
|
42 (30–62)
|
33.5 (23-56.5)
|
17.5 (10–30)a, b
|
|
< 0.0001
|
ALP(IU/L)
|
242 (193–301)
|
196 (164–237)
|
250 (182–441)
|
|
0.0243
|
Albumin (g/dL)
|
2.45 (2.1–3.1)
|
2.45 (2.15–2.75)
|
2.85 (2.5–3.5)
|
|
0.336
|
MELD
|
29.38 (25.01–34.65)
|
35.47 (30.02-40)
|
|
|
0.0005
|
CLIF ACLF
|
50.67 (42.15–55.22)
|
52.25 (43.865–60.275)
|
|
|
0.1355
|
CTP
|
12.17 ± 0.32
|
12.64 ± 0.20
|
|
|
0.1914
|
FABP (µg/ml)
|
82.3 (55.7-115.1)
|
113.4 (74.55–180.5) a
|
150.03 (89-178.48)a
|
21.5 (19.32–30.72)a, b, c
|
< 0.0001
|
IL-18 (pg/mL)
|
800.8 (549.9-1056.1)
|
656.85 (428.5-1649.5)
|
372.9 (268.9-725.9)a, b
|
166.95 (124.7-187.5)a, b, c
|
0.003
|
NGAL (ng/mL)
|
60.1 (12.3-120.6)
|
99.75 (29.1-271.6)
|
129 (76.26-183.09)a
|
25.31 (19.96–30.56)a, b,c
|
0.0178
|
Cystatin C (µg/ml)
|
19.6 (13.6–30.2)
|
28 (18.55–45.1)a
|
53.93 (41.09–68.6)a, b
|
10.68 (9.07–13.05)a, b, c
|
0.0001
|
Note: arefers to significant difference between ACLF no AKI v/s other groups (p-value ≤ 0.05) |
brefers to significant difference between ACLF AKI v/s other groups (p-value ≤ 0.05) |
crefers to significant difference between non-liver AKI v/s other groups (p-value ≤ 0.05) |
The 28-day mortality rate for the 91 ACLF patients 72 (79.12%) (Table 1).
Clinical blood parameters measured on the day of admission for all patients revealed that the ACLF no-AKI patients had higher haemoglobin Hb (g/dL) than ACLF AKI patients, with a Mean ± S.D of 9.06 ± 0.43 v/s 8.05 ± 0.27 (p < 0.05) (Table 1). The platelet count (x1000) [Median (Inter-quartile range)] (91 (68–122) v/s 77 (54.5-108.5), prothrombin time PT (seconds) (28 (22–36) v/s 28(22–36) and INR ( 2.5 (1.9–3.6) v/s 2.6 (1.95 vs 3.25)) were not significantly different among the two groups.
The non-liver AKI group had no accompanying liver complications with baseline bilirubin levels (mg/dL) [Median (Inter-quartile range)] of 0.4 (0.3–0.6), AST 26 (19–37) and ALT 17.5 (10–30), which were significantly lower than the ACLF patient groups (p < 0.005) (Table 1).
Among the kidney function parameters, creatinine (mg/dl) [Median (Inter-quartile range)] was similar between non liver AKI (4.1 (3.2–5.4)) and ACLF AKI (3.15 (2.3–4.5)) patients. Blood urea levels (mg/mL) were also comparable (110.5 (86–147) v/s 129.5 (87-191.5) (Table 1).
ACLF patients were stratified into grade 1 (n = 13), grade 2 (n = 32) and grade 3(n = 46) on the day of hospital admission, based on the EASL-CLIF criteria. Baseline characteristics were compared across the three grades and biomarker levels were compared across the different groups (Table 2). The levels of TLC (x103 per mm3) (p ≤ 0.05), bilirubin (mg/dL) (p ≤ 0.001), AST (IU/L) (p ≤ 0.05), ALT (IU/L) (p ≤ 0.05), INR (p ≤ 0.001),were highest in grade 3 ACLF patients intermediate in grade 2 and lowest in grade 1 patients (Table 2). Urea levels (mg/mL) (p ≤ 0.05) was highest in grade 1 patients followed by grade 3 and lowest in grade 2 patients (Table 2). The MELD score which is commonly used to grade severity of ACLF patients and incorporates these baseline parameters were also significantly different across the three grades (p ≤ 0.001) (Table 2).
Table 2
Comparison of baseline characteristics across ACLF grades 1, 2 and 3
Parameters
|
ACLF grade 1 (n = 13)
|
ACLF grade 2 (n = 32)
|
ACLF grade 3 (n = 46)
|
P value
|
Age mean ± S.D. (years)
|
42.0 ± 11.6
|
45.2 ± 14.0
|
42.2 ± 11.0
|
0.545
|
Gender (Male)
|
10 (76.92%)
|
24 (75.0%)
|
39 (84.78%)
|
0.537
|
Hemoglobin (g/dL)
|
8.3 ± 2.46
|
8.15 ± 2.41
|
8.66 ± 2.14
|
0.693
|
TLC(x103 per mm3)
|
8.8 (6.6–12.3)
|
13.0 (10.0–17.6)
|
14.4 (8.0–22.3)
|
0.048
|
Platelet(x103 per mm3)
|
100 (56–122)
|
87 (68–120)
|
66 (40–107)
|
0.096
|
INR
|
1.7 (1.5–2.1)
|
2.4 (1.9–3.1)
|
3.2 (2.5–3.5)
|
< 0.001
|
Na (mEq/L)
|
135.23 ± 6.72
|
136.06 ± 9.52
|
133.64 ± 8.33
|
0.37
|
K (mEq/L)
|
4.41 ± 0.95
|
4.56 ± 1.19
|
4.49 ± 1.09
|
0.668
|
Bilirubin (mg/dL)
|
4.6 (1.9–8.2)
|
11.2 (3.2–20.7)
|
19.3 (12.0–25.3)
|
0.001
|
AST(IU/L)
|
68 (40–102)
|
83 (58–138)
|
108 (79–213)
|
0.009
|
ALT(IU/L)
|
24 (18–56)
|
37 (28–62)
|
46 (32–88)
|
0.029
|
ALP(IU/L)
|
207 (165–232)
|
208 (158–250)
|
198 (164–278)
|
0.88
|
Albumin (gm/dL)
|
2.5 ± 0.6
|
2.6 ± 0.5
|
2.4 ± 0.5
|
0.4
|
Urea (mg/mL)
|
133 (109–196)
|
87 (51–146)
|
110 (63–146)
|
0.049
|
Creatinine(mg/mL)
|
2.2 (1.6–3.7)
|
2.3 (1.6–3.5)
|
2.7 (2.1–3.9)
|
0.484
|
CTP
|
11.3 ± 1.39
|
12.3 ± 1.65
|
12.89 ± 1.62
|
0.773
|
MELD
|
27.4 ± 5.9
|
31.9 ± 6.0
|
36.3 ± 4.3
|
< 0.001
|
FABP(µg/ml)
|
90.8
|
111.3
|
98
|
0.8
|
(60.2–140.9)
|
(70.9–169.6)
|
(64.0–188.1)
|
IL-18 (pg/ml)
|
726.9
|
598
|
913.8
|
0.126
|
(328.9–1043.3)
|
(434.1–1094.4)
|
(580.4–1684.6)
|
NGAL (ng/ml)
|
76
|
78.9
|
116.9
|
0.122
|
(29.5–135.3)
|
(8.1–160.3)
|
(35.4–313.1)
|
Cystatin-C (µg/ml)
|
26.5
|
23.8
|
28.9
|
0.252
|
(17.4–44.5)
|
(13.4–34.5)
|
(19.1–46.3)
|
However, the serum levels of the 4 biomarkers, FABP1 did not differ significantly across the three ACLF grades (Table 2).
Serum levels of FABP1, NGAL, Cystatin C and IL-18 in ACLF, non-liver AKI and Healthy Controls
The serum levels of the four biomarkers i.e., FABP1 (µg/ml), IL-18 (pg/ml), NGAL (ng/ml) and Cystatin C (ng/ml) were estimated on the day of admissionfor all patients and on the day of sample collection for healthy controls (Table 1).The study groups in which these biomarkers were measured were- (i) ACLF no-AKI, (ii) ACLF-AKI, (iii) non-liver AKI and (iv) Healthy Controls (HC). The objective was to evaluate the ability of the biomarkers to discriminate between – (a) ACLF no-AKI and ACLF AKI and, (ii) ACLF-AKI and non-liver AKI. The measured levels were as follows-
1. FABP1 (µg /ml) levels were significantly lower in ACLF no-AKI group (median value 82.3 (IQR 55.7-115.1)), as compared to ACLF-AKI (median value 113.4 (IQR74.55-180.5)) and non-liver AKI (median value 150.03 (IQR 89-178.48)); (p ≤ 0.05). Serum FABP1 did not discriminate between ACLF-AKI and non-liver AKI (p = 0.43) (Table 1; Fig. 2a). Therefore, serum FABP1 levels were not significantly different between either ACLF no-AKI and ACLF-AKI nor, ACLF-AKI and non-liver AKI.
2. IL-18 (pg/ml) levels were comparable among ACLF no-AKI (median value 800.8 (IQR 549.9-1056.1)), and ACLF-AKI (median value 656.85 (IQR428.5-1649.5)). IL-18 levels were significantly lower in the non-liver AKI group (median value 372.9 (IQR 268.9-725.9) as compared to both- ACLF no-AKI (p ≤ 0.005) as well as ACLF AKI (p ≤ 0.05))(Table 1; Fig. 2b). Hence, serum IL-18 was significantly lower in non-liver AKI as compared to both ACLF no-AKI and ACLF-AKI. However, within ACLF, the levels of IL-18 were not significantly different between AKI and no-AKI.
3. NGAL (ng/ml) levels were comparable among ACLF no-AKI (median value 60.1 (IQR12.3-120.6)), and ACLF-AKI (median value 99.75 (IQR29.1-271.6)). The level of NGAL in the non-liver AKI group (median value 129 (IQR76.26-183.09)) was not significantly different as compared the ACLF-AKI group (p = 0.43) (Table 1; Fig. 2c). Hence serum NGAL levels were not significantly different between ACLF-AKI and no-AKI or between ACLF-AKI and non-liver AKI.
4. Cystatin C (CysC) (µg/ml) levels were significantly lower in ACLF no-AKI group (median value 19.6 (IQR 13.6–30.2)), as compared to ACLF-AKI (median value 28 (IQR 18.55–45.1)) and non-liver AKI (median value 53.93 (IQR 41.09–68.6); (p ≤ 0.05 and ≤ 0.0001, respectively) (Table 1, Fig. 2d). Therefore, CysC levels were significantly different between both- (i) ACLF AKI vs no-AKI and (ii) ACLF-AKI and non-liver AKI (p-value < 0.0001).
Utility of FABP1, NGAL, Cystatin C and IL-18 in the Prediction of In-hospital, 28-day and 3 Month Mortality in ACLF patients
Of the total 91 ACLF patients, 72 patients (79.12%) were 28-day non-survivors. These patients had elevated urea levels (mg/mL) (p ≤ 0.05) and decreased albumin levels (g/dL) (p ≤ 0.01). Age (years) was the only confounding factor significantly different between 3-month survivors and non-survivors (p ≤ 0.005) (Table 3).
Table 3
Baseline characteristics and biomarker level comparison between ACLF survivors and non-survivors at 28-day follow-up.
Clinical parameters
|
28-day mortality
|
|
Survivor (n = 19)
|
Non-survivor (n = 72)
|
p-value
|
Age mean ± S.D. (years)
|
38.26 ± 2.39
|
44.5 ± 1.44
|
0.046
|
Gender (Male)
|
13 (%)
|
60(%)
|
0.147
|
Hemoglobin(g/dL)
|
8.45 ± 0.57
|
8.43 ± 0.260
|
0.9703
|
TLC (x103 per mm3)
|
12.1 (7.4- 15.8)
|
12.31 (8.60- 20.65)
|
0.295
|
Platelet (x103 per mm3)
|
107 (56–121)
|
81(54.5–111)
|
0.43
|
INR
|
2.3 (1.7–2.8)
|
2.7 (1.95–3.4)
|
0.138
|
Bilirubin(mg/dL)
|
12.6 (3.5–28)
|
10.78 (4.25–22.6)
|
0.598
|
AST (IU/L)
|
68 (54–208)
|
92 (60–160)
|
0.828
|
ALT(IU/L)
|
33 (22–62)
|
37.5 (28–62)
|
0.755
|
ALP(IU/L)
|
178 (159–228)
|
220 (174.5–272)
|
0.254
|
Albumin(gm/dL)
|
2.85 (2.6–3.3)
|
2.4 (2-2.7)
|
0.008
|
Urea(mg/dL)
|
74 (49–133)
|
107 (62–145)
|
0.05
|
Na (mEq/L)
|
132.73 ± 2.095
|
135.26 ± 0.99
|
0.2548
|
Creatinine (mg/ mL)
|
2.1 (1.4–2.6)
|
2.25 (1.45–3.7)
|
0.714
|
K (mEq/L)
|
4.51 ± 0.28
|
4.50 ±0.12
|
0.99
|
INR
|
2.5 (2.2–2.8)
|
2.5 (1.8–3.3)
|
0.755
|
CTP
|
11.36 ± 0.36
|
12.75 ± 0.18
|
0.001
|
MELD
|
31.17 ± 1.08
|
32.96 ± 0.91
|
0.3461
|
CLIF C-OF
|
42.59 ±1.90
|
52.75 ± 1.07
|
0.0001
|
FABP(µg/ml)
|
90.8 (50.4- 140.3)
|
99 (68.6- 175.7)
|
0.4177
|
IL-18 (pg/ml)
|
584.9 (305.7- 899.1)
|
804.6 (534.2 -1594.05)
|
0.0108
|
NGAL (ng/ml)
|
37.2(8- 137.5)
|
90.4 (27.9- 234.55)
|
0.0375
|
Cystatin-C (µg/ml)
|
21.4 (13 - 28.4)
|
26.5 (17.95- 40.85)
|
0.0458
|
Among the 4 biomarkers, IL-18 (pg/ml), NGAL (ng/ml) and Cystatin C (µg/ml) was higher in non- survivors as compared to survivors (p < 0.01, p ≤ 0.05 and, p ≤ 0.05 respectively). Among the standard clinical scores, CTP and CLIF-OF were significantly higher in non-survivors of 28-day follow up (p ≤ 0.001, and p ≤ 0.001 respectively).
An AUROC analysis was performed for the 4 biomarkers (FABP1, IL-18, NGAL and CysC) as well as clinical scores (MELD, CTP, CLIF) and sensitivity and specificity were calculated for the prediction of 28-day mortality (Table 4).
Table 4
Receiver operating curves for FABP1, IL-18, NGAL, Cystatin C and clinical scores, in predicting 28-day mortality.
Clinical parameters
|
28-day mortality
|
|
AUROC
|
Cut off
|
Sensitivity
|
Specificity
|
FABP (µg/ml)
|
0.56 (0.41–0.71)
|
92
|
52.78
|
52.63
|
IL-18 (pg/ml)
|
0.69 (0.45–0.72)
|
618
|
63.89
|
63.16
|
NGAL (ng/ml)
|
0.65 (0.52–0.79)
|
58.8
|
66.8
|
68.42
|
Cystatin-C (µg/ml)
|
0.65(0.52–0.78)
|
23.8
|
58.33
|
57.89
|
MELD
|
0.59(0.40–0.72)
|
32.27
|
56.52
|
55.56
|
CTP
|
0.74(0.62–0.85)
|
13
|
68.06
|
73.68
|
CLIF-OF
|
0.80(0.68–0.91)
|
47.31
|
73.61
|
73.68
|
None of the biomarkers showed a significant AUROC (≥ 0.7) for discriminating between 28-day survivors and non-survivors (FABP1 (µg/ml) (AUROC 0.56, CI = 0.41–0.71); IL-18 (pg/ml) (AUROC 0.69, CI = 0.45–0.72); NGAL (ng/ml) (AUROC 0.65, CI = 0.52–0.79) and Cystatin C (µg/ml) (AUROC 0.65, CI = 0.52–0.78).
Among the clinical scores, both CTP and CLIF-OF consistently discriminated among the survivors and non-survivors groups (AUROC ≥ 0.70). On the contrary, MELD score which incorporates parameters like bilirubin, INR and creatinine showed a poor performance in predicting mortality (AUC ≤ 0.7).