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Arch Gynecol Obstet (2013) 287:205210

DOI 10.1007/s00404-012-2548-3

MATERNAL-FETAL MEDICINE

Fetal fibronectin (Quick Check fFN test) for detection


of premature rupture of fetal membranes
Ibrahim A. Abdelazim

Received: 17 May 2012 / Accepted: 27 August 2012 / Published online: 12 September 2012
Springer-Verlag 2012

Abstract
Objectives This study was designed to detect the accuracy of the fetal fibronectin (Quick Check fFN test) in
diagnosing premature rupture of fetal membranes.
Study design This comparative prospective study was
carried out over 1 year in Ahmadi Kuwait Oil Company
(KOC) Hospital, Kuwait from March 2011 till March 2012.
Patients and methods Two hundred and twenty (220)
pregnant women [34 and \37 weeks gestation were
included in this study and divided into two groups
according to presence or absence of PROM; 110 patients
with PROM were included in group I, and 110 patients
without PROM were included in group II as controls.
Patients with multiple pregnancies or [37 weeks gestation
or not sure of dates or fetal distress or vaginal bleeding or
preterm labor or chorioamnionitis were excluded from this
study. The diagnosis of PROM was based on patients
history of sudden gush of water, pooling of amniotic fluid,
positive ferning pattern, positive nitrazine test, confirmed
by visualization of fluid passing from the cervical canal
during sterile speculum examination and Trans-abdominal
ultrasound to measure the amniotic fluid index (AFI B
5 cm in PROM). The gestational age was calculate from
the first day of LMP and confirmed by early ultrasound
scan (done before 20 weeks gestation). Patients included in
this study were subjected to standard examination, transabdominal ultrasound and sterile speculum examination to
I. A. Abdelazim
Department of Obstetrics and Gynecology,
Ain Shams University, Abbassia, Cairo, Egypt
I. A. Abdelazim (&)
Ahmadi Hospital, Kuwait Oil Company (KOC),
P.O.Box: 9758, 61008 Ahmadi, Kuwait
e-mail: dr.ibrahimanwar@gmail.com

detect amniotic fluid pooling through the cervical canal and


for collection of samples on admission. Some laboratory
investigations were done to exclude chorioamnionitis
(maternal fever, maternal tachycardia, fetal tachycardia,
maternal leucocytosis, CRP).
Results In this study, the sensitivity and the specificity of
fetal fibronectin in diagnosing PROM were 94.5 and
89.1 %, respectively, as compared with 84.5 % sensitivity
and 78.2 % specificity for Ferning test, respectively, and
87.3 % sensitivity and 80.9 % specificity for Nitrazine test,
respectively. The PPV and NPV of fetal fibronectin were
89.7 and 94.2 %, respectively, as compared with 79.5 %
PPV and 83.5 % NPV for Ferning test, respectively, and
82.1 % PPV and 86.4 % NPV for Nitrazine test, respectively. Fetal fibronectin was more accurate (91.8 %) for
detection of PROM than Ferning (81.4 %) or Nitrazine
(84.1 %) tests.
Conclusion The Quick Check fFN test for detection of
the fetal fibronectin in the vaginal fluid is a simple bedside
test, more sensitive, and specific than Ferning and Nitrazine tests, it can be used as complimentary test to confirm
the clinical diagnosis of premature rupture of fetal
membranes.
Keywords Fetal fibronectin  Quick Check fFN test 
Premature rupture of fetal membranes
Abbreviations
CRP
C-reactive protein
IUGR
Intrauterine growth retardation
KOC
Kuwait Oil Company
NPV
Negative predictive value
PPROM Preterm premature rupture of membranes
PPV
Positive predictive value
PROM
Premature rupture of membranes

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206

TLC

Arch Gynecol Obstet (2013) 287:205210

Total leucocyte count

Introduction
Premature rupture of membranes (PROM) is rupture of the
fetal membranes before the onset of labor, while rupture of
fetal membranes before 37 weeks gestation, is defined as
preterm premature rupture of membranes (PPROM) [1].
PROM is usually associated with significant perinatal and
maternal infectious morbidities [2]. During the management of patients with PROM the clinician weighs the risk
of prolonging gestation against the risks of serious fetal and
maternal consequences [3, 4]. Failure to identify patients
with PROM can result in failure to implement standard
measures and conversely an incorrect diagnosis leads to
inappropriate interventions (such as hospitalization or
induction of labor). Therefore, the diagnosis of PROM is of
critical importance to avoid serious fetal or maternal consequences [4]. Accurate diagnosis of PROM remains a
frequent clinical problem in obstetrics. Unfortunately, a
non-invasive standard diagnostic test is not available at this
time and the currently available tests are inaccurate. The
diagnosis of PROM is usually based on the patients history, identification of gross pooling of amniotic fluids from
the cervical canal during sterile speculum examination,
ferning pattern after microscopic examination and the
nitrazine test [5, 6]. Ferning has been associated with falsepositive results in 530 %; and false-negative results in
512.9 % [6]. Nitrazine evaluation has been associated
with false-positive results in 17.4 % and false negative
results in 12.9 % [6]. The absence of a non-invasive gold
standard for the diagnosis of rupture of fetal membranes
resulted in the appearance of several tests based on alternative biochemical markers. These biochemical markers
include vaginal prolactin, alpha-feto-protein (AFP), fetal
fibronectin and insulin-like growth factor binding protein-1
(IGFBP-1) [79]. However, prolactin and AFP were not
useful markers for PROM because of the overlap in concentrations between women with and without ruptured
membranes [8]. The human chorionic gonadotropin (HCG)
has been evaluated as a marker for PROM, unfortunately,
the quantitative evaluation of HCG as a marker for PROM
is costly and time consuming [1012].
Fetal fibronectin (fFN) ia a complex adhesive glycoprotein (450 KD = kilo Dalton) and a member of extracellular matrix family at the maternal-fetal interface.
During pregnancy, it is expressed in the extracellular
matrix located in the choriodecidual junction between the
maternal decidua and fetal membranes, as well as in the
uterus and placenta [13, 14].

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It is detectable during the first 22 weeks of pregnancy,


where it may reflect normal growth of trophoblasts and
placenta. It is absent between 22 and 34 weeks gestation,
and then is again normally detectable after 34 weeks,
during the final stage of pregnancy. Fetal fibronectin is
thought to be a trophoblast glue that promotes cellular
adhesion at uterine-placental and decidual fetal membrane
interface. It is released into cervicovaginal secretions when
the extracellular matrix of the chorionic/decidual interface
is disrupted [13, 14]. Several authors suggested that the
detection of fetal fibronectin in the vaginal fluid will provide qualitative results that will exceed the current diagnostic methods in sensitivity and specificity [9]. So, this
study was designed to detect the accuracy of fetal fibronectin (Quick Check fFN test) to diagnose premature
rupture of fetal membranes.

Patients and methods


This comparative, prospective study was carried out over
1 year in Ahmadi Kuwait Oil Company (KOC) Hospital,
from March 2011 till March 2012. Two hundred and
twenty (220) pregnant women [34 weeks and \37 weeks
gestation were included in this study after informed consent and approval of the study protocol by the institute
ethical committee and divided into two groups according to
presence or absence of PROM; 110 patients with PROM
were included in group I, and 110 patients without PROM
were included from the antenatal ward, while they were
admitted for control of blood pressure (hypertension) or
blood sugar (diabetic) or assessment of fetal wellbeing
(IUGR) in group II as controls. Patients with multiple
pregnancies or [37 weeks gestation or not sure of dates or
fetal distress or vaginal bleeding or preterm labor or chorioamnionitis were excluded from this study. The diagnosis
of PROM was based on patients history of sudden gush of
water, pooling of amniotic fluid, positive ferning pattern,
positive nitrazine test, confirmed by visualization of fluid
passing from the cervical canal during sterile speculum
examination and Trans-abdominal ultrasound to measure
the amniotic fluid index (AFI B 5 cm in PROM) [6, 9].
The gestational age was calculated from the first day of
LMP and confirmed by early ultrasound scan (done before
20 weeks gestation).
Methods
Patients included in this study were subjected to standard
examination, trans-abdominal ultrasound and sterile speculum examination to detect amniotic fluid pooling through
the cervical canal and for collection of samples on
admission. Some laboratory investigations were done to

Arch Gynecol Obstet (2013) 287:205210

exclude chorioamnionitis (maternal fever, maternal tachycardia, fetal tachycardia, maternal leucocytosis, CRP).
Samples collection
Patients were examined in dorsal Lithotomy position with
good illumination using sterile speculum (without antiseptics). Three sterile swabs were used to collect the
samples from the posterior vaginal fornix after insertion of
the speculum (the swabs should not touch the vaginal wall
during insertion or during removal).
Nitrazine test
The first sterile swab impregnated with nitrazine yellow
dye was inserted in the posterior vaginal fornix for 15 s,
then the color of the swab was interpreted after removal of
the swab from the vagina. The blue color was considered as
positive (PROM) and other colors were considered as
negative (no PROM).
Ferning test
The sample collected from the posterior vaginal fornix
using the second swab was spread on a glass slide, creating
a very thin smear and the smear was examined by low
power microscope, crystallization of amniotic fluid to form
a fern like pattern was considered as positive (PROM).

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immobilized mouse monoclonal anti-fetal fibronectin


antibody, human fibronectin, and goat polyclonal antifibronectin antibody-gold conjugate is placed in the
extraction buffer [1517].
The extraction buffer migrates up the test strip by
wicking action; the polyclonal antibody colloidal gold
conjugate becomes re-suspended and migrates with the
extraction buffer.
If fetal fibronectin is present in the specimen, it will bind
to the anti-human fibronectin colloidal gold conjugate. This
complex migrates by capillary action across a membrane
containing an immobilized monoclonal antibody specific to
fetal fibronectin. The fetal fibronectin-anti-fibronectin-gold
complex then binds to the immobilized anti-fetal fibronectin antibody, producing a visible line. If fetal fibronectin
is absent from the sample, no binding occurs to the
immobilized anti-fetal fibronectin antibody. Residual
unbound anti-human fibronectin polyclonal antibody-gold
migrates further across the membrane and binds to
immobilized plasma fibronectin, providing an assay control. A positive specimen will result in two visible lines and
a negative specimen will result in one visible line [1517].
After delivery the collected data on admission were confirmed, reviewed and statistically analyzed to assess the
sensitivity, specificity, PPV, NPV and accuracy for each
test.

Sample size justification


Quick Check fFN test

Quick Check fFN test is a 10 min, one-step, visual test


consists of a sterile polyester-tipped applicator, fetal
fibronectin test strip, and sample extraction buffer. The
patient should not had sexual intercourse within the last
24 h before the Quick Check fFN test.
According to manufacturers instructions, before collection the patient sample, the tube containing the extraction buffer was removed from the package and its cap was
carefully removed. During a speculum examination, prior
to any vaginal examination, the third polyester tipped
applicator provided with the kit was lightly rotated across
the posterior fornix of the vagina for 10 s, and then inserted
into the tube containing the extraction buffer for 1015 s.
The test strip was removed from the foil pouch and its
lower end (indicated by the arrows) was inserted into the
tube containing the extraction buffer for 10 min [1517].
Principles of the Quick Check fFN test
The Quick Check fFN is a solid-phase immunogold
assay. Specimens obtained from the posterior fornix
are placed into an extractor buffer. A test strip with

Using data of previous studies [6], data from the manufacture, setting the type-1 error (a) at 0.05, the power (1-b)
at 0.8 and assuming a 5 % dropout rate, the number of
participants needed to produce a statistically acceptable
figure was two hundred and twenty (220) women.
Statistical analysis
Data were collected, tabulated then statistically analyzed
using Statistical Package for Social Sciences (SPSS),
computer software version (15). Numerical variables were
presented as mean and standard deviation (SD), while
categorical variables were presented as number and percentage. Chi-square test was used for comparison between
groups as regards to qualitative variables, and a significant
level of p value was set at 0.05.
Sensitivity is the proportional detection of individuals
with the disease of interest in the population. Specificity is
the proportional detection of individuals without the disease of interest in the population. PPV is the proportion of
all individuals with positive tests, who have the disease.
NPV is the proportion of all individuals with negative tests,
who are non-diseased.

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Arch Gynecol Obstet (2013) 287:205210

PROM than ferning (81.4 %) or Nitrazine (84.1 %) tests,


(Table 3).

Results
In this study there was no significant difference between
the two groups studied regarding the mean age, which was
28.2 7.34 in group I (PROM) and 30.6 3.45 in group
II (control group); in addition, there was no significant
difference between the two groups studied regarding the
mean gestational age, which was 34.2 3.38 weeks for
group I, while it was 35.6 6.28 weeks for group II
(Table 1).
In group I, the fetal fibronectin was true positive in 104
patients (94.5 % = sensitivity) and it was false negative in
6 patients (5.5 %), while in group II, the fetal fibronectin
was true negative in 98 patients (89.1 % = specificity) and
it was false positive in 12 patients (10.9 %), (Table 2).
In group I, the Ferning test was true positive in 93
patients (84.5 % = sensitivity) and it was false negative in
17 patients (15.5 %), while in group II, the Ferning test was
true negative in 86 patients (78.2 % = specificity) and it
was false positive in 24 patients (21.8 %), (Table 2).
In group I, the Nitrazine test was true positive in 96
patients (87.3 % = sensitivity) and it was false negative in
14 patients (12.7 %), while in group II, the Nitrazine test
was true negative in 89 patients (80.9 % = specificity) and
it was false positive in 21 patients (19.1 %), (Table 2).
The sensitivity and the specificity of fetal fibronectin in
diagnosing PROM were 94.5 and 89.1 %, respectively, as
compared with 84.5 % sensitivity and 78.2 % specificity
for Ferning test, and 87.3 % sensitivity and 80.9 % specificity for Nitrazine test. The PPV and NPV of fetal fibronectin were 89.7 and 94.2 %, respectively, as compared
with 79.5 % PPV and 83.5 % NPV for Ferning test, and
82.1 % PPV and 86.4 % NPV for Nitrazine test. Fetal
fibronectin was more accurate (91.8 %) for detection of

Discussion
PROM is usually associated with significant perinatal and
maternal infectious morbidities [18]. Ferning has been
associated with false-positive results in 530 % due to
contamination with fingerprints on a slide or contamination
with semen or cervical mucus, and false-negative results in
512.9 % due to dry swabs or contamination with blood [4,
6]. Nitrazine evaluation has been associated with falsepositive results in 17.4 % due to cervicitis, vaginitis,
alkaline urine, blood, semen or antiseptics and false negative results in 12.9 % [4, 6].
Recent studies suggested that the fetal fibronectin in the
vaginal fluid is a good marker for prediction of preterm
labor [18, 19], and few old studies suggested its role in
detection of PROM, so this study was designed to reevaluate the accuracy of fFN (Quick Check fFN test) to
diagnose PROM as compared to the standard diagnostic
methods [20].
Immunoassays specific for fFN can detect fFN in the
cervicovaginal secretions of the pregnant women during
the first 22 weeks of pregnancy until the fetal membranes
completely fuse to the maternal decidua. By 37 weeks
gestation, fFN becomes more heavily glycosylated, loses
its adhesive properties, and can again be detected in cervicovaginal secretions [13, 14], this why pregnant women
[37 weeks gestation were excluded from this study.
In this study, the fetal fibronectin was more sensitive
and specific in diagnosing rupture of fetal membranes than
Ferning or Nitrazine tests were; the sensitivity and the

Table 1 The maternal age and gestational age for the studied population
Variables

Group I (PROM group = 110)

Group II (control group = 110)

p value test used

Significance

Maternal age (years) Mean SD

28.2 7.34

30.6 3.45

p [ 0.05 (0.78)

Non-significant

Gestational age (weeks) Mean SD

34.2 3.38

35.6 6.28

p [ 0.05 (0.88)

Chi-Square test
Non-significant

Chi-Square test

Table 2 The fetal fibronectin, ferning and nitrazine positive and negative cases in the two studied groups
Test

Fetal fibronectin test

Group I (PROM = 110 patients)

Group II (controls = 110 patients)

Positive cases
True Positive
Number (%)

Positive cases
False Positive
Number (%)

Negative cases
True Negative
Number (%)

6 (5.5)

12 (10.9)

98 (89.1)

Ferning test

93 (84.5)

17 (15.5)

24 (21.8)

86 (78.2)

Nitrazine test

96 (87.3)

14 (12.7)

21 (19.1)

89 (80.9)

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104 (94.5)

Negative cases
False Negative
Number (%)

96 ? 89/(96 ? 89 ? 21 ? 14) 9
100 = 84.1 %
104 ? 98/(104 ? 98 ? 12 ? 6) 9
100 = 91.8 %
Accuracy = true positive ? true negative/
(true positive ? true negative ? false
positive ? false negative) 9 100

93 ? 86/(93 ? 86 ? 24 ? 17) 9
100 = 81.4 %

89/(89 ? 14) 9 100 = 86.4 %


98/(98 ? 6) 9 100 = 94.2 %
Negative predictive value (NPV) = true
negative/(true negative ? false negative) 9 100

86/(86 ? 17) 9 100 = 83.5 %

96/(96 ? 21) 9 100 = 82.1 %


93/(93 ? 24) 9 100 = 79.5 %
104/(104 ? 12) 9 100 = 89.7 %
Positive predicative value (PPV) = true
positive/(true positive ? false positive) 9 100

86/(86 ? 24) 9 100 = 78.2 %


98/(98 ? 12) 9 100 = 89.1 %
Specificity = true negative/(true negative ?
false positive) 9 100

93/(93 ? 17) 9 100 = 84.5 %


104/(104 ? 6) 9 100 = 94.5 %
Sensitivity = true positive/(true positive ?
false negative) 9 100

Ferning test
Fetal fibronectin
Variables

Table 3 The sensitivity, specificity, PPV, NPV, accuracy of fetal fibronectin, ferning and nitrazine tests in diagnosis PROM

89/(89 ? 21) 9 100 = 80.9 %

specificity of fetal fibronectin in diagnosing PROM were


94.5 and 89.1 %, respectively, as compared with 84.5 %
sensitivity and 78.2 % specificity for Ferning test, respectively, and 87.3 % sensitivity and 80.9 % specificity for
Nitrazine test, respectively. The PPV and NPV of fetal
fibronectin were 89.7 and 94.2 %, respectively, as compared with 79.5 % PPV and 83.5 % NPV for Ferning test,
respectively, and 82.1 % PPV and 86.4 % NPV for Nitrazine test, respectively.
Similar to this study, Eriksen et al., in a multicenter
clinical trial, pooling, ferning, and nitrazine tests were
compared with fetal fibronectin in 339 women (study
group) with a clinical history of rupture of the membranes
and in 67 women at term receiving routine prenatal care
(controls). Fetal fibronectin in the cervicovaginal secretions
was determined using a qualitative enzyme-linked immunosorbent assay test and it was considered positive at
50 ng/ml. Eriksen et al. [21], concluded that the sensitivity
of fetal fibronectin in the women with ruptured membranes
was 98.2 %.
A. Salfelder and colleagues [20], found that 97.1 %
patients (34 of 35) with clearly visible amniotic fluid in the
vagina had a positive immunoassay test for fetal fibronectin
(study group), whereas 96.5 % of the control group without
any signs of ruptured membranes had negative test results,
and they concluded that a positive test result helps to
confirm the diagnosis of PROM especially in equivocal
cases.
Fetal fibronectin was measured in samples of cervicovaginal secretion from 80 cases with PROM (study group),
and 50 cases of term pregnancy (control group) by Gu et al.
[22], and they found that fetal fibronectin was detected in
95 % of the patients with PROM. Gu et al., concluded that
the fetal fibronectin is a sensitive test that can confirm the
diagnosis of PROM.
Pascal Gaucherand and colleagues, concluded that the
apart from its value in predicting premature labor, fFN
represents a diagnostic test of PROM with good sensitivity
(94 %) and specificity (97 %), also, Aaron and colleagues,
Stated that the detection of the fetal fibronectin in the
amniotic fluid had 9798 % sensitivity, 7097 % specificity, 7493 % PPV and 98100 % NPV for detection of
PROM [9, 23].
In this study, the Quick Check fFN test for detection of
the fetal fibronectin in the vaginal fluid was more accurate
(91.8 %) for detection of PROM than Ferning (81.4 %) or
Nitrazine (84.1 %) tests.
In this study, the Quick Check fFN test was a simple
bedside test for detection of the fetal fibronectin in the
vaginal fluid, also, it was more sensitive and specific than
Ferning and Nitrazine tests, it can be used as complimentary test to confirm the clinical diagnosis of premature
rupture of fetal membranes.

96/(96 ? 14) 9 100 = 87.3 %

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Nitrazine test

Arch Gynecol Obstet (2013) 287:205210

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210

Arch Gynecol Obstet (2013) 287:205210

Acknowledgments I would like to thank Doctor/Hanan H. Makhlouf,


for her continuous advice for publication of the manuscript.
12.
Conflict of interest I declare that no actual or potential conflict of
interest in relation to this article exists.
13.

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