321ORIGINAL
The value of Neutrophil gelatinase-associated lipocalin and
Neutrophil / Lymphocyte Ratio in the diagnosis ofpreeclampsia and its severity
Eham Amer Ali1, Ban Hadi Hameed2, and Alea Farhan Salman3Ass. Prof. Department of Chemistry and Biochemistry, College of Medicine/Mustansiriyah University, Baghdad, Iraq, 2Ass. Prof. Department
of Gynaecology And Obstetrics, Mustansiriyah university \ College of medicine, Baghdad, Iraq, 3Lecturer Alea Farhan Salman, The National
Centre of Haematology, Mustansiriyah University, Baghdad, Iraq
1

Abstract : Many hypotheses underlie the pathogenesis of preeclampsia. This study aims to evaluate Neutrophil
gelatinase-associated lipocalin (a marker of immune hypothesis) and Neutrophil / Lymphocyte ratio (a marker of
inflammation) in the diagnosis of preeclampsia and its severity and to determine the correlation between them.
Study design : This randomized case-control study involved 132 pregnant women ; 88 were diagnosed with PE(divided into non-severe and severe groups), and 44 healthy pregnant women as a control group. Results : Themean serum level of NGL was significantly higher in PE (535.37 ± 158.61 ng / ml for severe PE, 522.5 ± 106.3 ng / mlfor non-severe PE, and 161.96 ± 17.48 ng / ml for the control group). The ROC Curve NGL criteria of more than
204.4 ng / ml showed 100% sensitivity and specificity in both severe and non-severe cases versus control. The N / L
ratio showed a significant difference (5.81 ± 5.24 for severe PE, 4.1 ± 3.41 for non-severe PE, and 3.89 ± 1.79 for thecontrol group), but the ROC curve criterion was not significant. Both showed a non-significant positive correlation. Conclusion : NGL is an excellent diagnostic factor, whereas N / L might have lower diagnostic performancecompared with NGL. Both are related independently to the pathophysiology of PE. J. Med. Invest. 68 : 321-325,
August, 2021
Keywords : Neutrophil gelatinase-associated lipocalin, Preeclampsia, Neutrophil/lymphocyte ratio

INTRODUCTION
Preeclampsia (PE) is a common significant health issue affecting pregnant women for which the placenta has been accusedas the cause of its systemic effects on the mother and fetus (1).
After twenty weeks of gestation, it is characterized by new-onset
hypertension and an increased level of protein secretion in urine.
It is one of the major causes for restricted fetal growth, multi-organ maternal affection, and even mortality of both the motherand the fetus in severe cases (2). Approximately five to ten
percent of pregnancies worldwide are affected by preeclampsia(3). The exact pathogenesis remains controversial ; however, theimpaired coagulation-fibrinolysis systems, abnormal immune
responses, and inflammation are often mentioned in addition to
the abnormal gestational trophoblast cell invasion and endothelial cell injuries, which are also widely studied (4, 5).
In general, possible mechanisms presented to elucidate thepathophysiology behind preeclampsia included : abnormal remodeling of the spiral arteries, abnormal trophoblast differentiation, and abnormal placentation with the resulting hypoxia,
placental hypoperfusion, and ischemia (6). Some factors have
key roles in the pathogenesis of PE, for example, genetic factors,
immunological factors, inflammation, and systemic endothelial
damage (7).
The wide diversity of mechanisms that underlie the pathophysiology of PE directed the researchers to investigate theusefulness of combined biomarkers analysis in the enhancement
Received for publication May 12, 2021 ; accepted July 1, 2021.
Address correspondence and reprint requests to Dr. Ban Hadi
Hameed, Mustansiriyah university, P O Box : 14022.

of their diagnostic performance (8). Neutrophil gelatinase-associated lipocalin (NGL) is a 25-KDa protein of the lipocalin family
and is considered a biomarker for ischemic injury. It was used asa biomarker for diagnosing acute kidney injury and was linked
to the immune pathway of PE (8, 9). Very few studies have been
conducted on serum NGL in human pregnancy. However, thecharacteristics of this molecule, which increases in hypertensive
pregnant women and correlates with angiogenic factors, imply
that NGL might be related to the pathogenesis of preeclampsia
and eclampsia. The increase in NGL level in blood during thesecond trimester of pregnancy is a sensitive indicator for theoccurrence of preeclampsia (10, 11). The kidney injury, proteinuria, and endothelial inflammatory reactions in preeclampsia
represent the possible role of NGL in PE pathogenesis.
This study evaluated the Neutrophil / lymphocyte ratio (N / L),
one of the markers of systemic inflammation and a hot research
point in recent years, as it is involved in developing various diseases related to the prognosis of neoplasms (12). The complete
blood count (CBC) parameter of inflammation and / or ischemia
is the ratio of absolute neutrophils count to the absolute count oflymphocytes. It is a sign of the body’s immune response to offending factors. It is also a quick, cost-effective, and straightforward
parameter that indicates systemic inflammation and stress (13).
The relation of this ratio with PE was evaluated in many studies
with contradicting results due to the study’s different design andsampling (14). The combination of two important mechanisms
in the pathogenesis of PE that are abnormal immune response
and inflammation is the cornerstone behind evaluating thesetwo variables in the current study which aims to assess the diagnostic performance of NGL and N / L in PE and to investigate
their prognostic utility to determine disease severity on the same
sample of pregnant women and to determine the correlation ofthese two variables in the pathophysiology of PE.

The Journal of Medical Investigation Vol. 68 2021

E. Amer Ali, et al. NGL and N / L in the diagnosis of PE

322
PATIENTS AND METHOD

A case-control study had been conducted at Al Yarmouk
teaching hospital / Baghdad \ Iraq, for six months from August2020 till February 2021. The study enrolled 132 women who
were allocated into three groups : 44 cases with severe PE, 44
cases with non-severe PE, and 44 pregnant normotensive womenas a control group, the maternal age and gestational age werematched to minimize the effect of these variables on the study.
The patients were newly diagnosed cases selected from the
Obstetric ward and outpatient clinic who fill the diagnostic
criteria of PE : blood pressure (BP) equal or more than 140 / 90
mmHg after 20 weeks in previously normotensive women withproteinuria of 300 mg / 24h or more. The following features specify the patients in the severe PE group : systolic BP equal or morethan 160 mmHg, diastolic BP equal or more than 110 mmHg,
headache, visual disturbances, upper abdominal pain, oliguria,
convulsion, elevated serum creatinine, thrombocytopenia <100,000 / μL, elevated serum transaminase, fetal-growth restriction, and pulmonary edema. Whereas the absence of severefeatures categorizes the patients for non-severe PE (15).
The study’s protocol was approved by the medical of Mustansiriyah University (MOG 112 on 22 July 2020). Informed consent had been obtained from all participants before enrolment
in the study.
Thorough Obstetric history and complete physical examination had been performed to document the demographic andclinical criteria. Body mass index (BMI) was calculated asfollows : Weight (kg) / Height (m2), blood pressure (BP) record insitting or semi-sitting position using a mercurial sphygmomanometer to document both systolic BP (SBP) and diastolic BP(DBP). A blood sample was taken by venepuncture, centrifuged,
and kept in the freezer till analysis for all participants. Complete
blood count, renal function, liver function, and urine analysis
were performed, and NGL assay by ELISA.

Statistical Analysis
The data were analyzed using a statistical package for social
science (SPSS 24). Parametric data were analyzed using One-Way
ANOVA and posthoc Scheffe tests whereas the non-parametricdata were analyzed using the Kruskal-Wallis test. The inter-assay coefficient of variability for NGL was 10.7% whereas theintra-assay coefficient of variability was 8.5% with a sensitivityof 92%. Spearman’s correlation was used to determine the correlation coefficient between NGL and N / L ratio. A P-value lessthan 0.05 was considered significant.

RESULTS
The study groups’ demographic, clinical, and biochemicalvariables are clarified in table 1, showing a significantly higher
BMI, SBP, DBP, liver enzymes, and renal function parametersin PE groups compared to the control group as p values were lessthan 0.05.
Regarding NGL, the study showed a highly significant risein this biomarker in PE cases when compared with the control
group (535.37 ± 158.61) ng / ml for severe PE, (522.5 ± 106.3)
ng / ml for non-severe PE, and (161.96 ± 17.48) ng / ml for thecontrol group) as the p-value was less than 0.001. The neutrophil / lymphocyte ratio showed a significant difference among thestudy groups (5.81 ± 5.24) for severe PE, (4.1 ± 3.41) for non-severe PE, and (3.89 ± 1.79) for the control group, and the p-value
was 0.027 as shown in table 2.
The ROC curve calculated the NGL criterion value for diagnosing PE for severe and non-severe cases versus control,
clarified in figures 1 and 2. For both severe and non-severe PE
cases at a more than 204.4 ng / ml criterion, the sensitivity andspecificity were 100%, suggesting an excellent diagnostic marker
as the area under the curve (AUC) was 1.00, 95% confidence
interval (0.92-1.00), P-value < 0.001.
Despite the significant difference in N / L ratio among thestudy groups, the ROC curve showed a non-significant criterion
for severe and non-severe cases as P-values were more than 0.05,
AUC 0.56, and 95% confidence interval (0.4-0.7), P-value 0.46 as
shown in figures 3 and 4.
Spearman’s correlation revealed a weak positive non-significant correlation between NGL and N / L ratio with a correlation
coefficient of 0.107 and P-value 0.39 as shown in table 3.

Table 1. The demographic, clinical, and biochemical variables of the study groups
Non-severe
PE Number
44

Variable(Mean ± SD)

Severe PE
Number 44

Age (years)

28.59 ± 7.84

26.63 ± 6.85

27.36 ± 5.4

0.096

Gestational age (weeks)

35.81 ± 1.78

36.31 ± 1.93

35.13 ± 2.56

0.644

1.59 ± 2.06

1.6 ± 1.86

1.5 ± 1.18

0.084

ParityControl
Number 44

P- Values
ANOVA

Pre-pregnancy BMI (Kg / m )24.56 ± 1.67

25.75 ± 2.43

28.54 ± 2.54

0.72

Weight in pregnancy (Kg)

92.43 ± 7.62

85.95 ± 12.05

87.58 ± 9.23

0.307

Weight gain during pregnancy (Kg)

12.34 ± 4.24

11.87 ± 4.65

9.54 ± 5.46

0.14

2

SBP (mmHg)

163 ± 7.85

144 ± 5.09

115 ± 5.9

< 0.001

DBP (mmHg)

105 ± 9.59

96.36 ± 7.26

80 ± 6.17

0.005
0.032

SGOT (IU / L)35.75 ± 38.22

20.53 ± 7.78

19.02 ± 5.9

SGPT (IU / L)

25.89 ± 18.6

17.87 ± 8.6

18.51 ± 5.6

0.03

B. urea (mg / dl)

24.38 ± 8.46

19.39 ± 7.44

19.32 ± 6.76

0.047

S. creatinine (mg / dl)

0.78 ± 0.13

0.65 ± 0.12

0.69 ± 0.1

0.003

Abbreviations : BMI : Body mass index, SBP : Systolic blood pressure, DBP : Diastolic blood pressure,
SGOT : Serum glutamic-oxaloacetic transaminase, SGPT : Serum glutamic-pyruvic transaminase, B.
urea : Blood urea, S. creatinine : Serum creatinine.

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The Journal of Medical Investigation Vol. 68 August 2021
Table 2. Comparison of NGL and N / L ratio among the study groups
StatisticNGL(ng / ml)

N/L
ratio

GroupsNumber

Range

Mean

SD

controls

44

142.13-180.54

161.97

17.49

non-severePE44410.5-633.23

522.5

106.3

severe PE

44383.18-693.3

535.37158.62

controls

44

2.09-5.87

3.90

1.80

non-severePE440.89-7.6

4.10

3.41

severe PE

44

0.68-11.04

5.81

5.25

P- Values
ANOVA< .0010.027

Abbreviations : SD : Standard deviation.

Figure 1. ROC Curve of NGL in severe PE cases versus control

Figure 2. ROC Curve for NGL in non-severe PE versus control

Figure 3. ROC Curve for N / L ratio in severe PE versus control

Figure 4. ROC curve for N / L ratio in non-severe PE versus control

324

E. Amer Ali, et al. NGL and N / L in the diagnosis of PE

Table 3. Spearman’s Correlation between NGL and N / L ratio.
Spearman’s correlation
N/L

NGL
Correlation Coefficient

0.107

Sig. (2-tailed)

0.390

DISCUSSION
The estimation of Neutrophil gelatinase-associated lipocalinhas been investigated by many researchers, as it is closely related to acute inflammatory conditions. The role of inflammationin the pathogenesis of PE is well established so that researchers
had estimated NGL levels in PE (16).
Our study showed significantly higher serum NGL concentrations in preeclampsia cases as compared to normotensive
pregnant women. These findings are consistent with Yalman
et al. and Yolli et al., (17, 18). Karmapas et al. reported that NGLlevels showed higher levels in pregnant women. However, inconcordance with the results of our study, the increase in NGL
in cases with PE was significantly higher than in normotensive
pregnant women. Moreover, serum NGL levels were also raisedin the first and second trimesters of pregnant women who later
developed PE (19).
Interpretation of those findings in preeclampsia is that ; vascular endothelial dysfunction leads to oxidative stress, ischemic
placenta, systemic inflammation during pregnancy, and renal
dysfunction. It is observed that NGL production occurs at thematernal-fetal interface (20). Consequently, serum NGL levels
may increase in preeclampsia cases and can be utilized to assess
PE progression and assess kidney functions. In contrast, Ahmad
et al. (21) found a non-significant difference between the PE andcontrol groups, but the sample size in this study was small. The
same results were shown by Dhivya (22) study where nothing
was mentioned about the severity of the disease.
Many studies linked the severity of inflammatory response
in PE to the development of clinical symptoms. For example,
activation of inflammatory cells and immune responses releases reactive oxygen species (ROS), inflammatory cytokines by
the neutrophils, and lymphocytes, leading to endothelial dysfunction. Many papers addressed the association of N / L withpregnancy complications such as preeclampsia (23). Recently,
neutrophil extracellular traps (NETs), sent out by neutrophils,
was suggested to affect the development of the fetus in humans.
NETs could take the space inside trophoblast villi causing
fetal hypoxia by reducing blood flow. Another mechanism that
includes the facilitation of NETs production is by activating endothelial cells, which damages the endothelium and may cause
preeclampsia (24). The morbidity associated with severe disease
necessitates predictive markers for preeclampsia severity, sill
little of them proved significant.
In the present study, we aimed to assess the relation of N / L
with the severity of preeclampsia. The N / L showed a highly
significant rise in PE cases than the control group, in line withearlier studies (25, 26). Our findings support that N / L may serve
as a promising diagnostic tool due to its significant elevation inPE, especially in severe cases but the ROC curve criterion was
not significant. At the same time, another study, in contrast to
our study, showed a non-significant difference and ROC curve
criterion between PE and control groups (14).
The value of N / L in pregnancy was discussed in terms ofpredicting pregnancy outcomes in the first trimester. A study by
Biyik I et al. showed a higher ratio in cases with missed miscarriage comparing to normal pregnancy (27). High ratio suggested
an imbalance in the inflammatory reaction and it is assigned asa marker of severity in sepsis and inflammatory diseases (28).
As the severity of PE is the most important determinate ofcomplications occurrence and in clinical work the discriminationof severe from non-severe cases is crucial, the present study summarizes the relationship between non-severe and severe casesof preeclampsia with the normotensive group by using the ROC
curve to calculate the cut off value associated with the highest
sensitivity and specificity for NGL and N / L ratio. The NGL
was proved to be an excellent diagnostic biomarker with a 100%
sensitivity and specificity in both severe and non-severe caseswith no discrimination regarding the severity of the disease. The
N / L showed a better performance for non-severe PE cases thansevere PE cases as higher sensitivity was detected in non-severe cases. Whereas the results suggested that N / L might be auseful laboratory tool for clinical evaluation of disease severity
due to high specificity 86.4% in severe cases. There was a weak
positive non-significant correlation between the studied two parameters suggesting that they are related independently in thepathophysiology of PE contradicting another study that showed asignificant correlation between them in cases with renal failureand heart disease (29).

CONCLUSIONNGL is an excellent diagnostic factor, whereas N / L might
have lower diagnostic performance compared with NGL. The
two variables are related independently in the pathophysiologyof PE.

ACKNOWLEDGMENT
Our great appreciation for the valuable support of medical
staff members at Al Mustansiriyah University and Al Yarmouk
hospital.

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