Received: 3 December 2017  | Revised: 23 March 2018  | Accepted: 22 May 2018  | First published online: 13 June 2018

DOI: 10.1002/ijgo.12536

CLINICALARTICLE
Obstetrics

Accuracy of 12-hour urine collection in the

diagnosis of pre-eclampsia
Rita M. Silva* | Sara R. Pereira | Susana Rego | Nuno Clode

Department of Obstetrics, Gynecology and

Reproductive Medicine, North Lisbon
Hospital Centre, Santa Maria University
Hospital, Lisbon, Portugal
*Correspondence
Rita M. Silva, Department of Obstetrics,
Gynecology and Reproductive Medicine,
North Lisbon Hospital Centre, Santa
Maria University Hospital, Lisbon,
Portugal. Email: ritaimsilva@gmail.com
Abstract
Objective: To evaluate the accuracy of a 12-hour urine collection to diagnose pre-
eclampsia and to determine whether time of collection influences the performance of
this test.
Methods: A prospective observational study was conducted in a tertiary obstetric center in
Portugal between November 1, 2015, and November 30, 2016. Pregnant women (≥20
weeks) admitted for observation with suspected pre-eclampsia were eli-gible for inclusion.
Two consecutive 12-hour urine samples were collected (07:00– 19:00 hours vs 19:00–
07:00 hours). Protein levels were measured in each 12-hour sample, as well as in a
pooled sample (07:00–07:00 hours). The diagnostic cut-off val-ues for pre-eclampsia were
150 mg (12-hour samples) and 300 mg (24-hour sample). Results: The study included 99
patients and diagnoses of pre-eclampsia were con-firmed among 42 (42%) patients. In all,
456 12-hour urine samples were analyzed (equivalent to 228 24-hour samples).
Qualitative analysis (pre-eclampsia vs no pre- eclampsia) indicated substantial agreement
between the 12- and 24-hour samples (Cohen κ 0.779). The sensitivity was 85.9% (95%
confidence interval [CI] 81%–90%) and the specificity was 91.7% (95% CI 88%–95%). No
statistically significant differ-ence was found between the two 12-hour collections.
Conclusion: The 12-hour test showed acceptable accuracy for detecting pre--
eclampsia, regardless of the time of collection.

KEYWORDS
24-hour urine collection; Hypertensive disorders of pregnancy; Non-inferiority;
Pre-eclampsia; Proteinuria

1 | INTRODUCTION equivalent by many obstetric societies.

Pre-eclampsia and other hypertensive disorders of pregnancy account
1 given the wide range of sensitivity (65%–96%), specificity (49%–
for 10.3%–22.1% of all maternal deaths worldwide. The current diag-
nostic criteria indicate that proteinuria should no longer be considered an
2,3
obligatory component of pre-eclampsia ; however, quantification of
urinary protein levels is still routine practice.
A 24-hour urine collection, with a 300 mg cut-off level for protein-uria,
remains the gold standard to diagnose the presence of substan-tial
proteinuria. Nonetheless, alternative methods—including the urine spot
protein-to-creatinine ratio with a cut-off of 0.3—are considered
2,3
The protein-to-creatinine
ratio test is undoubtedly more practical and convenient to perform
than the 24-hour urine collection test but some controversy remains
4–6
100%), and clinical heterogeneity reported.
7
A systematic review and meta-analysis by Stout et al. found that
12-hour urine collection, with a 150 mg cut-off level for proteinuria,
performed well among women with hypertension during pregnancy.
However, the main limitations were the small sample sizes of the stud-ies
included in the analysis, the heterogeneous methods used for urine
collection, and the lack of information on time of collection.

Int J Gynecol Obstet 2018; 142: 277–282 wileyonlinelibrary.com/journal/ijgo © 2018 International Federation of  |  277
Gynecology and Obstetrics
278  |
The primary aim of the present study was to test the hypothesis
that a 12-hour urine collection was non-inferior to a 24-hour urine
col-lection for the diagnosis of pre-eclampsia. The secondary goal
was to determine whether time of collection (diurnal vs nocturnal)
influenced the performance of the 12-hour test.
2 | MATERIALS AND METHODS
The present prospective observational study was conducted in the
Department of Obstetrics, Gynecology and Reproductive Medicine, North
Lisbon Hospital Centre, Santa Maria University Hospital, Lisbon,
Portugal, between November 1, 2015, and November 30, 2016. The
present sample comprised consecutive pregnant women (≥20 weeks)
who were admitted to the obstetrics ward of Santa Maria University
Hospital for maternal–fetal surveillance owing to hypertension (>140/90
mm Hg) and suspicion of pre-eclampsia. The exclusion cri-teria were
known nephropathy and vaginal discharge or bleeding, as these
conditions could interfere with urinary protein excretion (UPE). Urine
cultures were requested whenever symptoms of urinary tract infections
developed; women with positive test results were excluded from the
present analysis. The protocol was approved by the local eth-ics
committee and informed consent was obtained from all participants.
The daily routine of the participants included at least three blood
pressure measurements and reports on the presence of relevant
Diurnal Nocturnal
urine urine
07:00– 19:00–
19:00 h 07:00 h
Daily
urine
07:00–
07:00 h
FIGURE 1 Schematic of study protocol.
S ilva ET AL.
symptoms (e.g. dyspnea, epigastric pain, and cerebral or visual
distur-bances). Physical exercise was limited as the participants
spent the majority of their time either sitting or lying on their beds or
walking within the ward. As per local protocols, women with
suspected pre- eclampsia underwent blood tests (i.e. complete blood
cell count plus measurement of liver enzymes and creatinine) and
urinalysis (24- hour collection) three times per week, under the close
supervision of nursing staff.
In the present study, the 24-hour urine collection was replaced by
two consecutive 12-hour collections (Fig. 1). Collection vessels were
distributed to participants at 07:00 hours, following the first morning void
(which was not included in the collection). The first 12-hour col-lection
took place from 07:00 hours until 19:00 hours (diurnal sample). Women
then received a new vessel for the second 12-hour collection, which took
place from 19:00 hours until 07:00 hours (nocturnal sam-ple). This
second sample included the first morning void.
The diurnal and nocturnal samples were first independently ana-
lyzed for the presence of substantial proteinuria (≥150 mg). These
two samples were then pooled to create a 24-hour or “daily” sample
(07:00–07:00 hours) that was also analyzed for the presence of sub-
stantial proteinuria (≥300 mg).
Each patient underwent a varying number of urine collections,
depending on the length of hospital stay; however, only the 24- hour
values were considered in the clinical-decision making process.
Demographic and clinical data were prospectively recorded.
Urine collection, two consecutive 12-h periods
Quantification of total proteinuria in each 12-h
collection
Mixing and homogenization of both 12-h
samples
Quantification of 24-h proteinuria in the total urine
volume
Silva ET AL.      |  279
The primary end point was a binary outcome of either having or
3 | RESULTS
not having pre-eclampsia. This outcome was based on the presence
of positive proteinuria using the cut-off values outlined above for the
A total of 99 women were included in the present analysis. The
12- hour and 24-hour samples. The secondary end point was testing
mean number of urine collections during hospitalization was 2.3
the equivalence of diurnal and nocturnal proteinuria values.
(range 1–11). In all, there were 456 12-hour urine collections
As each 24-hour measurement comprised two matching 12-hour
(equivalent to 228 24-hour urine measurements).
measurements, the present analysis included three comparisons (Fig. 2).
The demographic and clinical characteristics of the participants
Comparison 1 considered all 12-hour samples and all 24-hour measure-ments
were summarized (Table 1). Most of the women were white, the
(2:1 sample ratio) as an indicator of the global performance of the 12- hour
median age was 34 years (range 18–50 years), and most patients
test. Comparisons 2 and 3 considered the individual perfor-mance of the
diurnal and nocturnal samples, respectively. The qualita-tive analysis
a,b
assessed reliability and validity of the 12-hour samples for diagnosis of
TABLE 1 Characteristics of the participants (n=99).

substantial proteinuria. The quantitative analysis assessed equivalence of the Characteristic Distribution
diurnal versus nocturnal values for proteinuria. Ethnicity

The data were analyzed using SPSS version 24 (IBM, Armonk, NY, White 79(79)
USA). The Kolmogorov-Smirnov test was used to assess normal African 17(17)
distribution of the variables. Systematic effects were assessed using the Other 3(3)
McNemar test (categorical) and the Wilcoxon signed-rank test
Age, y 34(18–50)
(numerical). Reliability of the 12-hour method versus the 24-hour method
Parity
was assessed using the Cohen κ test, where a κ coefficient of 1 indicates
Nulliparous 57(57)
perfect agreement and a κ coefficient of 0 indicates agree-ment similar to
chance. The validity of this concordance was evaluated by determination Multiparous 41(42)

of sensitivity, specificity, positive predictive value (PPV), negative Conception

8 Spontaneous 83(83)
predictive value (NPV), and accuracy. The Pearson coefficient and
IVF or ICSI 16(16)
intraclass correlation coefficient were used to deter-mine agreement
among the diurnal and nocturnal samples (pairwise, quantitative) based No. of fetuses

on a single-measure, absolute agreement, two- way random-effect 1 87(87)

9 2 12(12)
model. An α value of 5% was considered to be sta-tistically significant;
all tests were two-sided. The incidence of positive proteinuria (24-hour BMI

urinary protein level ≥300 mg) from a series of 100 collections among the Before pregnancy 27.4(17.5–49.9)
population from the present study setting (42%) was used to calculate the During admission 32.7(20.0–56.4)
appropriate sample size. A sample size of 372 measurements would Gestational age of the fetus, wk 35(20–39)
allow testing of the non-inferiority of the 12-hour method, with a 10% Blood pressure during hospital stay, mm Hg
margin and 80% power at a 5% signifi-cance level
Normal (≤140/90 mm Hg) 14(14)
(powerandsamplesize.com). Multiple measurements were obtained from
Raised (>140/90 mm Hg) 80(85)
the participants, which allowed a reduction in the total number of patients
Diagnosis
10
recruited.
Pre-eclampsia 42(42)
Gestational hypertension 54(54)

24-h Chronic hypertension 3(3)

24-hour urine collection

Comparison No. of samples 228

“12–24h” Protein excretion, mg/24 hours 611.4 [48.6–6106.5]
12-hour urine collection

12-h Diurnal 12-h Nocturnal No. of samples (diurnal + nocturnal) 456

Daily protein excretion, mg/24 hours 278.5 [16.9–3398.9]
Nocturnal protein excretion, mg/24 hours 312.1 [18.6–2975.4]
Comparison Comparison
“Diurnal and 24-h” “Nocturnal and 24-h”
Abbreviations: BMI, body mass index (calculated as weight in kilograms
divided by the square of height in meters); ICSI, intracytoplasmic sperm
24-h injection; IVF, in vitro fertilization.
a
Values are given as number (percentage), median (range), of mean
FIGURE 2 Summary of main comparisons made in the b
[range]. Percentages refer to valid data (missing data excluded). For
present study. each category, the missing data were inferior to 5%.
280  | S ilva ET AL.

were nulliparous. A diagnosis of pre-eclampsia was made among 42

(95% CI), % Accuracy (95% CI), %

89. (86.2–92.4)3

486. (81.5–91.3)

192. (88.8–95.4)
(42%) women, after confirmation of positive proteinuria in the 24--
hour samples.
The McNemar test found no evidence of a systematic effect of
the data in the three comparisons (P>0.100; data not shown). The
reli-ability and validity of the 12-hour test versus the 24-hour test for
a diagnosis of pre-eclampsia is shown in Table 2. Comparison 1 and

(86.4–93.6)

(81.2–92.5)

(88.9–97.4)
2 gave a substantial level of agreement (as assessed by the Cohen
κ test) and comparison 3 an almost perfect agreement.
To validate these results, the sensitivity, specificity, PPV, NPV,
and accuracy for detection of pre-eclampsia were assessed in each

90
(95% CI), % NPV

(78.5–92.9) 986.

(84.8–96.5) 293.
of the three comparisons (Table 2). When compared with the 24--

(83.6–92.9)
hour sample, the two 12-hour samples showed 85.9% sensitivity,
91.7% specificity, and 89.3% accuracy. The diurnal sample slightly
underesti-mated the proportion of patient with proteinuria when
compared with the 24-hour sample (39% vs 42%). The nocturnal

91. (88.3–95.0)7 88.2

290. (85.0–95.2) 785.

293. (88.9–97.5) 690.

(95% CI), % PPV
sample detected the same proportion of proteinuria as did the 24--
hour sample (42%). The accuracy for this comparison was 92.1%

PPV,pos iti vepredictivevalue.Values aregiv enasm ean±SDornumber(perc entage),unles sindicatedotherwise.Thethreecom paris ons areoutl inedsc hem atically in Figure2.Defin edas aprote inlevelof≥150mg/12hours or≥ 300mg/24hours .χA12- hours am ple (07:0 0–19:0 0hours ).A24-hours am ple (07:0 0–07:0 0hours ).A12-hours am ple (19:0 0–07:0 0hours).
Specificity
thediagnosisofpre-eclampsia.
(nine false positives and nine false negatives).
Although nocturnal proteinuria appeared to be more accurate
than diurnal proteinuria for detecting pre-eclampsia, this difference
was not statistically significant (P=0.151; related-samples Wilcoxon
signed- rank test, data not normally distributed). The equivalence

71930.<0.00181.(73.4–89.1)

83860.<0.00190.(84.8–96.5)
0.<0.00185.(81.0–90.9)7799

g
b

f
Sensitivity

between both measurements was also tested the using Pearson and
intraclass coefficient correlation (Fig. 3). Both measures showed
good (but not excellent) correlation (<0.9).
(95%CI),%
a

Some discrepancy was observed between the 12-hour and 24- hour
absolute values. Theoretically, the difference between the 24- hour
measure and the sum of two 12-hour measures should be close to zero.
d

However, the mean difference was 5.2 mg/dL (95% confidence interval –
Cohen κ valueP

58 to 101), which was attributed to both measurement vari-ability and

measurement error. Therefore, all of the statistical tests were repeated
after excluding the automatically generated outlier val-ues (n=10). This
approach did not affect the results reported above.
c

Abbreviations:CI,confidenceinterval;NPV,negativepredictivevalue;abcdefg
TABLE2 Reliabilityandvalidityof12-hoururinecollection for

Comparison2
Proteinuria,mgComparison proteinuria

91 (39)
96 (42)
Comparison3
96 (42)
96 (42)
Positive

Comparison15All12- hoursamples(n=456)278.±437.9187 (41)24-hoursamples(n=228)611.±918.696(42)3

4 | DISCUSSION

The present study found that a 12-hour urine collection provided

4278. ± 437.8Diurnal(n=288)
611. ± 918.63Daily(n=288)

Nocturnal(n=288)

3611. ± 918.6Daily(n=288)

acceptable accuracy for the detection of proteinuria when compared

with the current gold standard (24-hour urine collection). These find-
1312. ± 490.0

ings suggested that the 12-hour test could offer a reasonable

alterna-tive to the 24-hour test in the diagnosis of pre-eclampsia.
Pre-eclampsia is generally characterized by raised blood pressure in
test.

association with increased UPE as a marker of proteinuria. However, in

the absence of proteinuria, a diagnosis of pre-eclampsia can still be
made on the basis of new-onset hypertension with signs of end-organ
lesion, including thrombocytopenia, renal insufficiency, impaired liver
2,11
function, pulmonary edema, and cerebral or visual symptoms.
Elevated levels of UPE are detected among most women with pre-
eclampsia owing to increased vascular permeability and altered tubu-lar
12,13
charge selectivity of the glomerular filtration barrier. Adverse obstetric
2

outcomes were thought to be related to elevated UPE;

Silva ET AL.
19:00–07:00h (mg/12h) 4000
3000
proteinuria
2000 r (Pearson): 0.852
(P<0.001)
1000 ICC: 0.845
95%CI 0.803–0.879
Nocturnal
0 Wilcoxon:
0 1000 2000 3000
Diurnal proteinuria 07:00–19:00 h (mg/12h)
FIGURE 3 Agreement between diurnal and nocturnal 12-hour
values; data were not normally distributed. Abbreviation: ICC,
intraclass correlation coefficient.
however, studies have failed to demonstrate a strong causality.
Given that the absence of proteinuria could delay prompt intervention,
substantial proteinuria is no longer considered to be either an indica-tor
of disease severity or an obligatory component for the diagnosis of pre--
eclampsia in the presence of another sign of end-organ dysfunc-tion.
Nevertheless, proteinuria undoubtedly remains an important clinical
feature of pre-eclampsia and is fundamental to the diagnosis for many
patients. Quantification of proteinuria in a 24-hour urine collection is the
current gold-standard test because it is representa-tive of circadian
variability in UPE. Collection of urine samples in an inpatient setting is
probably more reliable than collection in an outpa-tient setting. Indeed,
many women with suspected pre-eclampsia are hospitalized for optimum
clinical management. By contrast, the cost of collection in an inpatient
setting will be higher than that incurred in an outpatient setting.
The present study design followed the suggestions of Stout et
7 acquisition of data and to revising the manuscript. NC contributed to the
al. by obtaining a large sample size, ensuring that the collection of
urine was homogeneous, and stratifying the samples by diurnal ver-
sus nocturnal. An effort was made to implement only minor changes
in the established routines of the obstetric ward so as to minimize
procedure errors.
In the qualitative analysis, the 12-hour test detected a similar
pro-portion of patients with proteinuria as the 24-hour test, with a
dis-crepancy of less than 10% (as was proposed to test with the
sample size). However, global reliability was not as satisfactory as
expected, with Cohen k values of less than 0.8.
Nocturnal proteinuria seemed to perform better than diurnal pro-
teinuria for the diagnosis of pre-eclampsia, with high sensitivity, speci-
ficity, PPV, NPV, and accuracy. Nevertheless, the quantitative analysis
(comparing absolute values of proteinuria) found no statistically sig-
nificant difference between the two collection times. Therefore, it was not
possible to make firm conclusions about the performance of nocturnal
collection versus diurnal collection. Levels of UPE are known to be
raised by physical exercise, high blood pressure, urinary tract infection,
massive obesity, cardiac failure, reduced water intake, hematuria, uterine
bleeding, vaginal discharge, and some severe stressful situations.
the present study, several of these confound-ers were controlled by the
exclusion criteria but also by the fact that the participants undertook only
limited physical exercise, even during
     |  281
the day. Regarding blood pressure, the available records only allowed
the identification of women with either normal values or raised values
during their whole hospital stay. A detailed record of blood pressure
values could aid understanding of the importance of hypertension for
UPE variance between the diurnal and nocturnal periods.
An optimum method to both quickly and accurately diagnose pre--
eclampsia is still lacking; however, some promising work is being
developed using angiogenesis-related biomarkers. Consistent evi-dence
P=0.151
indicates that the levels of placental growth factor decrease, whereas the
4000
levels of soluble fms-like tyrosine kinase 1 (also known as sFLT-1)
increase, among pre-eclamptic women before the clinical onset of this
17
condition. Prospective data have already suggested a potential role for
the ratio of these two growth factors as a screening method for pre--
eclampsia that offers high NPV (99.3%–99.9%) and a 78.6% detection
17
14,15 rate of the disease within 1 week. These biomarkers provide rapid
18
results (approximately 30 minutes) and seem to be cost- saving in
identifying patients who would benefit from hospitaliza-tion. However,
recommendations on their use have yet to be approved.
2 In conclusion, the present study demonstrated that a 12-hour
urine collection could offer an alternative to the gold-standard
method of 24-hour urine collection when a fast result is required or
when it is the only financially viable solution. Further studies—
including those designed to assess cost-effectiveness—are now
required to investi-gate this possibility.
AUTHOR CONTRIBUTIONS
RMS contributed to the conception and design of the study, the
acquisition, analysis, and interpretation of data, literature review, and
writing and revising the manuscript. SRP and SR contributed to the
conception and design of the study, the acquisition, analysis, and
interpretation of data, and revising the manuscript.
CONFLICTS OF INTEREST
The authors have no conflicts of interest.
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