Urinary Protein Assessment in Preeclampsia: Which Sample is More Suitable?
T. Galini Moghadam
Mild or severe preeclampsia is responsible for about
70% of hypertensive disorders observed during pregnancy and 24 h urine
collection is a gold standard for diagnosis of preeclampsia. This study
was performed to determine whether the gold standard of 24 h urine protein
value in pre-eclampsia can be substituted with 8 or 12 h urine protein
values and to evaluate the effect of ambulation and immobilization on
amount of protein excretion. A cross sectional study was conducted on
40 women with pre-eclampsia (BP≥140/90 mmHg), who referred to the Department
of Obstetric and Gynecology, Imam Khomeiny hospital in Sari, Iran from
April 2005 to September 2005. Positive urinary strip for protein of at
least 1+ samples were collected over 24 h in subsequent periods: the first
8 h and the next 4 h and remaining 12 h urine, in separate containers.
The correlation between groups was determined by Pearson`s correlation.
A total of 40 women were recurited in this study of which 36 had completed
urine collection. A total of 21 had mild proteinuria, 5 had severe proteinuria
and 10 had no proteinuria. There was significant correlation between the
8 or 12 h (day) and 12 h (night) with 24 h urine protein. Total protein
values of 8 and 12 h (day) and 12 h (night) samples, positively correlated
with values of 24 h samples in pre-eclampsia and could be substituted
for assessment of proteinuria instead of 24 h urine collection in women
with pre-eclampsia, as a simpler, faster and cheaper method for diagnosis
of pre-eclampsia. And ambulation and immobilization in preeclamptic patients
has not any effect on protein excretion.
Preeclampsia is a pregnancy-specific multisystem disorder of unknown
etiology. The disorder affects approximately 5 to 7% of pregnancies and
a significant cause of maternal and fetal morbidity and mortality. Preeclampsia
is defined by the new onset of elevated blood pressure and proteinuria
after 20 weeks of gestation (Baumwell and Karumach, 2004; Wagner, 2004).
The minimum criteria for the diagnosis of preeclampsia is hypertension
plus minimal proteinuria, which evident after 20 weeks gestation (Huppertz,
2008). The combination of proteinuria and hypertension during pregnancy
markedly increases the risk of perinatal mortality and morbidity (Ferrazzani
et al., 1990; Chan et al., 2005).
Therefore, repeated urine analysis to screen proteinuria are part of
standard antenatal care. These urine analysis are performed on random
spot urine specimens using a test strip assay. However, if a test strip
is positive for protein 2+ or more in the absence of bacteriuria, the
next step is usually a 24 h urine collection for quantification of proteinuria.
The 24 h urine collection is a time consuming procedure and may result
in a delay in diagnosis and treatment or possibly prolonged hospital stay
and inaccurate due to incomplete collection. Shortening the period for
diagnosis of preeclampsia would be valuable for management, as well as
decreasing hospital stay and patient inconvenience. However, for pregnant
women, particularly if in hospital, the circadian variation in protein
excretion is smaller or absent and it may therefore be possible to use
shorter collection period (Kieler et al., 2003; Tribl et al.,
2005). The aim of this study was to evaluate, whether a 24 h urine collection
for measuring urinary protein in preeclamptic women could be substituted
by a 8 or 12 h collection. In addition we wanted to know if we could collect
urine in a short time, which period of time is most suitable, collecting
urine in day or night. In other word, we wanted to evaluate the effect
of ambulation and immobilization on the amount of proteinuria.
MATERIALS AND METHODS
Forty women with preeclampsia admitted to the Ward at the Department
of Obstetric and Gynecology, Imam Khomeiny hospital Sari, Iran from April
2005 to September 2005 were included in the study. The criteria for inclusion
were a positive urinary test for protein of at least 2+ and a planned
24 h urine collection for quantitative protein measurement. Women with
upper urinary tract infections, defined as a positive urine culture and
fever, were not included. Informed consent was obtained from all women.
The patients were not confined to bed rest from 8 am to 8 pm and were
allowed to move around in the antenatal ward. The patients were asked
to have complete bed rest from 8 pm to 8 am regardless of admitting time.
Urine collection started on the first morning after admission to the hospital
and all samples were collected within a period of 24 h. Prior to the urine
collection, all women were carefully instructed regarding the procedure.
At 8 am, the urine was collected to determine RBC, WBC, protein, urine
culture and creatinine. In order to increase the accuracy of the test,
patients were assisted by a nursing staff for urine collection. The urine
was collected in three, separate, clearly marked containers. The first
container held the first 8 h of urine, the second container held the next
4 h of urine, while the third container held the remaining 12 h urine
sample. Each container was marked with the patient`s name, number of the
container, collection time and file number (Fig. 1).
The containers were sent to the laboratory of the hospital, where the
urine volume of each container was measured with a graduated cylinder
and recorded separately. The total 24 h specimen volume was calculated
from the summation of all three containers. The urine was stirred to ensure
homogeneity and a 6 mL aliquot of urine was taken from the first 8 h collection
(sample 1). The remaining urine from the first container was added to
the second container and stirred then 6 mL aliquot was obtained as described
for the first container (sample NO2 or day sample). Also 6
mL aliquot of urine was taken from the third container (sample NO3
or night sample). The remaining urine of the first 12 h (day sample) was
added to the second 12 h (night sample), which then represented the entire
24 h urine collection. The urine was stirred and a 6 mL aliquot sample
was obtained (sample 4). Then analysis for protein in each of the four
aliquot was performed by using modified Fujita method (Sigma Aldrich,
2000, 2001) the absorption that occurs when the Pyrogallol Red-Molybdate
reacts with protein to form a bluish purple complex that absorbs at 600
nm proteinuria from the 24 h urine result (sample 4) was categorized as
follow: no proteinuria <300 mg, mild proteinuria >300 to <2000
mg and severe proteinuria >2000 mg (Cunningham et al., 2005;
Tara et al., 2008). Data were analyzed using SPSS version 11. Statistical
analysis was determined by calculating a correlation coefficient (r) with
the p-value less than 0.05 was considered statistically significant. Also
prognostic test were analyzed by the use of sensitivity, specificity and
positive and negative predictive values.
There were a total of 40 patients with over 20 weeks of gestation and
BP = 140/90 mmHg. Two patients were excluded because of positive urine
culture and two for delivery prior to collection of urine sample and the
data were analyzed statistically.
Patients were classified in three groups based on protein level of 24
h urine: no proteinuria <300 mg (10 cases), mild proteinuria ≥300
to <2000 mg (21 cases) and severe proteinuria ≥2000 mg (5 cases).
The 8 and 12 h (day) and 12 h (night) of urine protein results correlated
with 24 h results for patients with mild and severe proteinuria (p-value
= 0.000 and r = 0.97, r = 0.99, r = 0.99) (Table 1).
|| Flow chart for collection of urinary samples
|| Calculation of Cut of values, Sensitivity, Specificity,
PPV, NPV, Pearson correlation coefficient and p-value of different
urine samples in mild and sever preeclampsia
The cut off value of protein level for 24 h urine as compared to the
protein results of 8 and 12 h (day) and 12 h (night) urine was obtained.
In this study, the mode was used for determination of cut off value of
mild and severe proteinuria. Cut off value for diagnosis of mild preeclampsia
was determined to be 105 mg for an 8 h sample, with sensitivity of 87%,
specificity and positive predictive value (PPV)of 100% and negative predictive
value (NPV) of 76% (r = 0.98, p<0.001). Cut off value for diagnosis
of mild preeclamsia was determined to be 148 mg for a 12 h sample (day),
with sensitivity of 94%, specificity of 80% and (PPV) of 90% and (NPV)
of 88% (r = 0.99, p<0.001). Cut off value for diagnosis of mild preeclamsia
was determined to be 151 mg for a12 h sample (night), with sensitivity
of 91% , specificity of 100%, (PPV) of 100% and (NPV) of 66% (r = 0.96,
p<0.001). Cut off value for diagnosis of severe preeclampsia was determined
to be 730 mg for a 8 h sample with sensitivity of 80%, specificity and
PPV of 100% and NPV of 96% . Cut off value for diagnosis of severe preeclampsia
was determined to be >1100 mg for a 12 h sample (day), with sensitivity,
specificity, (PPV) and (NPV) of 100%. Cut off value for diagnosis of severe
preeclampsia was determined to be >1100 mg for the 12 h sample
(night), with sensitivity of 83%, specificity of 100%, PPVof 80% and NPV
of 100%. There was no statistically significant correlation between maternal
age and gestational age in patients, with severity of the disease.
The measurement of proteinuria has its own problems. The urinary protein
excretion in preeclampsia arises due to glomerular endoteliosis. However,
it also indicates a generalized increase in capillary permeability in
other organ systems of the body. Presence of significant proteinuria acts
as a marker for the severity of preeclampsia and patients who are suffering
from hypertension and proteinuria are at an increased risk of small for
gestational age fetuses and prenatal mortality as well as maternal morbidity
(Ferrazzani et al., 1990). The most common screening method for
detection of proteinuria in preeclampsia is dipstick testing of a random
urine sample. The dipstick is inexpensive, easy to use and provides a
rapid result, but has been shown to have low sensitivity and specificity
for urinary protein excretion over 24 h (Brown and Buddle, 1995; Price
et al., 2005; Myers et al., 2006; Phelan et al., 2004;
Waugh et al., 2003). However, all dipstick testing needs to be
confirmed with a 24 h urine collection which is currently accepted as
the gold standard for quantification of urinary protein loss. The 24 h
urine collection, which has its difficulty, is inconvenient for the patients,
costly and may be inaccurate due to incomplete collection. Several investigators
explored other means of quantifying proteinuria in a short period of time.
The spot urine protein/creatinin ratio is a new method for quantifying
protein loss and correlate well with the 24 h urine protein. This technique
improves detection of proteinuria and allows for the concentration of
the urine in quantifying protein loss. The results are evidently quicker
than the 24 h urine collection, which is inconvenient and difficult practically
for some of patients (Taherian et al., 2006). However, the protein/creatinine
ratio of a single urine sample of pregnant woman has been shown to correlate
significantly with a 24 h collection of patients with protein values of
lesser than 1 g in 24 h. Above this level, the variation between the samples
increases (Jaschevatzky et al., 1990). Boler et al. (1987)
studied protein/creatinine ratio in pregnant women with preeclampsia and
revealed that the degree of correlation to the 24 h sample was lower in
patients with values of greater than 2 g in 24 h.
Therefore, the protein/creatinine ratio is not sensitive enough to determine
mild versus severe disease for patients with significant proteinuria and
is not suitable to replace the 24 h collection as the standard recommended
methodology. Côté et al. (2008) on the basis of their
systematic review on earlier studies say that we do not advocate use of
the spot protein:creatinine ratio or spot albumin:creatinine ratio for
monitoring or quantifying proteinuria in pregnancy. The results of this
study revealed that the protein values for the 8 and 12 h day and 12 h
night are correlated with the entire 24 h sample with high diagnostic
value and can predict or diagnose the preeclampsia in the patients with
mild and severe proteinuria. In this study, we calculated cut off values
for 8 and 12 (day) and 12 (night) of proteinuria. Total urine protein
value of >105 mg in the 8 h sample or 148 mg in the first 12 h sample
(day) or >151 mg in the second 12 h sample (night) were predictive
of mild preeclampsia, while, 8 h protein values of >730 mg and 12 h
values >1100 mg (day and night) were predictive of severe proteinuria.
The important point in this study is the low number of patients with severe
preeclampsia. Today, due to improvement of prenatal care, there is significant
reduction in the incidence of severer preeclampsia and often for the patients
who are admitted for severe preeclampsia there is not any sufficient time
for the 24 h urinary collection. Several studies have been done for evaluation
of proteinuria in a shorter duration of time (2, 4, 6, 8 or 12 h) and
all of them revealed that it is possible to determine proteinuria and
its severity in a shorter timed urine collections (Adelberg et al.,
2001; Evans et al., 2000; Rinehart et al., 1999; Somanathan
et al., 2003; Wongkitisophon et al., 2003) but more studies
are needed to generate an exact and reliable cut off values for predicting
mild and specially severe preeclampsia. The results of this study in sensitivity
and cutoff values of mild preeclampsia were similar to those of Adelberg
et al. (2001). However, there was a significant difference in cutoff
values of 8 and 12 h urine samples for the diagnosis of severe preeclampsia.
This difference might be due to this policy in defining severe proteinuria.
We categorized present results in severe proteinuria when there was ≤2000
mg protein in 24 h urine (Cunningham et al., 2005) versus Adelberg
et al. (2004), who used 5000 mg as a cut off point.
It is suggested that protein excretion varies. Throughout the day and
tends to increase with ambulation and up right position, which produces
renal vasoconstriction and alters permeability of the glomerular barrier
(Higby et al., 1994). These physiologic factors affect protein
excretion and produce a circadian rhythm that makes a 24 h collection
necessary (Koopman et al., 1985; Douma et al., 1995). In
this study, high correlation between the rate of proteinuria in samples
that were collected from 8 am to 8 pm (ambulation period) and from 8 pm
to 8 am (immobilization period) revealed that collection of urine can
be done at any time (day or night) and daily activity has no effect on
proteinuria. This finding is in agreement with McCaw et al. (1985),
who concluded that proteinuria is not influenced by collection period
and bed rest. According to the results of this study, we recommend the
8 h urine collection for the measurement of the peroteinuria in the patient
with suspected preeclampsia because of it is faster and more convenience
for the patients.
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