Objective
The purpose of this study was to evaluate the performance of the 12-hour urine protein >165 mg and protein:creatinine ratio >0.15 for the prediction of 24-hour urine protein of ≥300 mg in patients with suspected preeclampsia.
Study Design
We performed a prospective observational study of 90 women who had been admitted with suspected preeclampsia. Protein:creatinine ratio and 12- and 24-hour urine specimens were collected for each patient. Test characteristics for the identification of 24-hour urine protein ≥300 mg were calculated.
Results
A 12-hour urine protein >165 mg and protein:creatinine ratio of >0.15 correlated significantly with 24-hour urine protein ≥300 mg (r = 0.99; P < .001; and r = 0.54; P < .001, respectively). A 12-hour urine protein >165 mg performed better than protein:creatinine ratio as a predictor of a 24-hour urine protein ≥300 mg (sensitivity, 96% and 89%; specificity, 100% and 49%; positive predictive value, 100% and 32%; negative predictive value, 98% and 91%, respectively).
Conclusion
The high correlation of a 12-hour urine protein >165 mg with a 24-hour urine protein ≥300 mg (with the benefit of a shorter evaluation time) and the high negative predictive value of protein:creatinine ratio suggest that the use of both these tests have a role in the evaluation and treatment of women with suspected preeclampsia.
Hypertensive disease occurs in approximately 5-10% of pregnancies and is responsible for approximately 15% of maternal deaths in the developed countries. The exact incidence of preeclampsia in the United States is not known; however, it is estimated to range from 6–8% of all pregnancies and remains a leading cause of maternal and neonatal mortality and morbidity worldwide. It is a pregnancy-specific syndrome of reduced organ perfusion that is related to vasospasm and activation of the coagulation cascade.
Multiple causes have been hypothesized for preeclampsia, including abnormal trophoblast invasion of uterine blood vessels, immunologic intolerance between fetoplacental and maternal tissues, maladaptation to the cardiovascular changes or inflammatory changes of pregnancy, dietary deficiencies, and genetic abnormalities. Risk factors that are associated with preeclampsia include nulliparity, multifetal gestation, obesity, maternal age >35 years, African American ethnicity, family history of preeclampsia-eclampsia, preeclampsia in previous pregnancy, abnormal Doppler studies at 18 and 24 weeks, pregestational diabetes mellitus, presence of thrombophilias, hypertension, and renal disease.
Preeclampsia is associated with increased risk of maternal and fetal morbidity and mortality. These depend on the gestational age at onset of preeclampsia, timing of delivery, the severity of disease process, presence of multifetal gestation, and the presence of preexisting medical conditions such as pregestational diabetes mellitus, renal disease, or thrombophilias. In women with mild preeclampsia, the perinatal death rate and rates of preterm delivery, small-for-gestational-age infants, and abruptio placentae are similar to those of normotensive pregnancies.
The standard threshold value for proteinuria in the setting of hypertension for the diagnosis of preeclampsia is a 24-hour urine protein ≥300 mg. Urine dipstick, protein:creatinine ratio, and 12-hour urine protein collection have been compared with the 24-hour urine protein as methods of quantitating proteinuria in pregnancy in the hope of finding a test that is more readily available, easy to perform, inexpensive, and that yields a quick result. However, evaluation of a timed collection as opposed to a random specimen has been classically recommended because protein excretion is variable in the setting of preeclampsia. Preliminary studies have suggested that 12-hour urine protein collection (as opposed to a 24-hour urine protein collection) and/or protein:creatinine ratio may be adequate for the evaluation of preeclampsia with the advantage of an earlier diagnosis and treatment of preeclampsia as well as the potential for earlier hospital discharge and increased compliance with specimen collection.
Adelberg et al, in a prospective observational study of 65 patients, used receiver operating characteristic (ROC) curves to determine the optimal cutoff for proteinuria (>165 mg protein) in the 12-hour sample to diagnose preeclampsia accurately (with 78% sensitivity, 100% specificity, 100% positive predictive value, and 71% negative predictive value; P < .001). Similarly, Schubert and Abernathy, in a small observational study of 15 patients, identified an optimal protein:creatinine cutoff of >0.15 (with 100% sensitivity, 50% specificity, 75% positive predictive value, 100% negative predictive value) for the diagnosis of preeclampsia. However, these cutoffs have not been tested prospectively. The purpose of our study was to determine prospectively the performance of the 12-hour urine protein >165 mg and protein:creatinine ratio >0.15 for the prediction of 24-hour urine protein ≥300 mg in patients undergoing evaluation for preeclampsia.
Materials and Methods
This was a prospective observational study of pregnant women aged 18-55 years and >20 weeks’ gestation who were admitted to the Lehigh Valley Health Network antepartum unit who were undergoing a 24-hour urine collection for the diagnosis and/or management of preeclampsia from July 1, 2010 to December 31, 2011. Women were excluded if they had known prepregnancy renal disease (defined as baseline 24-hour urine protein ≥300 mg), had a clinical indication for delivery at the time of admission, were outside the maternal or gestational age parameters as defined earlier, did not speak English, did not give informed consent for any reason, or had been enrolled previously in the study.
Spot urine for protein:creatinine ratio, 12-hour urine collection, and 24-hour urine collection were obtained for each patient. The 24-hour urine collection was started at the time of admission, regardless of the time of day, in a standard fashion. For study purposes, the 24-hour urine specimen was collected in 2 consecutive 12-hour urine collections. Each container was marked with the patient’s name, medical record number, number of the container, and collection time. The protein:creatinine ratio was sent on the initial urine specimen (which was otherwise discarded, consistent with standard timed urine collection protocol). We chose to send the protein:creatinine ratio on this specimen, as opposed to the timed collection, to simulate how the protein:creatinine ratio would be used in clinical practice (at the time of presentation). In a small number of subjects, the protein:creatinine ratio was erroneously not collected on the initial urine specimen and was therefore collected either from the timed specimen itself or immediately after the timed collection was completed. The containers were sent to the Lehigh Valley Hospital Health Network Laboratories for analysis. The urine volume, total protein, and creatinine level were measured to determine the protein:creatinine ratio, 12-hour urine protein, and 24-hour urine protein. Only the 24-hour urine result was used for clinical management; providers were blinded to the results of the protein:creatinine ratio and 12-hour urine protein. Antepartum management was otherwise routine and at the discretion of the patient’s provider and institutional clinical protocol, which included modified bed rest, laboratory evaluation for HELLP (hemolysis elevated liver enzymes low platelets) syndrome, and serial assessment of maternal blood pressure.
Analysis for protein in the first 12-hour urine sample was performed by Lehigh Valley Hospital Health Network Laboratories with the use of an urine assay (ADVIA Total Protein [urine] assay; Siemens Healthcare Diagnostics Inc., Tarrytown, NY), which is a modified Fujita method. This was the routine commercial assay that was used by the Health Network Laboratories for calculating total urine protein. The assay consisted of aspirating 30 μL of the urine sample and making 1:5 dilution with 120 μL of on-system isotonic saline solution to make a “working dilution,” then 13.3 μL of the “working dilution” was dispensed into a cuvette that contained 80 μL of UPRO_2 R1 reagent (ADVIA Total Protein [urine] assay) and was incubated in the oil bath at 37°C for 10 minutes. The resulting blue-colored complex was read at 596/694 nm to determine the protein concentration in milligrams per deciliter. Total protein for the 12-hour urine was calculated by multiplying the total urine volume (dL) by the concentration of protein in the test sample (mg/dL) and was considered diagnostic for preeclampsia if the result was >165 mg. Total protein for the 24-hour urine protein was calculated by combining both 12-hour urine specimens and running the Health Network Laboratories ADVIA Total Protein assay as described previously. It was considered diagnostic for preeclampsia if the result was ≥300 mg. Creatinine clearance was calculated on the 24-hour urine sample by standard methods as a routine part of the preeclampsia workup. Spot urine for protein:creatinine ratio was calculated by random urine protein (mg/dL)/random urine creatinine (mg/dL) and was considered diagnostic for preeclampsia if the result was >0.15.
The primary outcome was test characteristics of protein:creatinine ratio >0.15 and 12-hour urine protein >165 mg to predict a 24-hour urine protein ≥300 mg. Baseline maternal characteristics (which included age, ethnicity, parity, insurance type, gestational age at admission, indication for admission, comorbidities, and blood pressure during admission) and delivery outcomes (which included gestational age at delivery, induction indication, mode of delivery, preeclampsia complications, and neonatal outcomes) were collected, and the data for patients with and without a 24-hour urine protein ≥300 mg were compared. For a few subjects, pregnancy outcomes were not available because they delivered at an outside hospital. The sensitivity, specificity, and positive and negative predictive values were calculated for the 12-hour urine protein >165 mg and protein:creatinine ratio >0.15; a 24-hour urine protein ≥300 mg served as the reference. Correlation coefficient and ROC curves were generated for the 12-hour urine protein and protein:creatinine ratio vs the 24-hour urine protein.
Our hypothesis was that at least the 12-hour urine protein would perform very well as a predictor of 24-hour urine protein ≥300 mg. We based our initial sample size estimate on the ability of the 12-hour protein level of >165 mg to detect the abnormal 24-hour level. We assumed that approximately 40% of subjects would have the abnormal 24-hour protein level of ≥300 mg. We used an alpha level of .05 and beta level of .2 for our calculation. Based on this assessment, we estimated that, to be able to have a sensitivity of 90% for the 12-hour sample to identify the abnormal 24-hour sample, we would need a total of 150 subjects to be enrolled (75 per group) with up to a 10% attrition rate. An interim analysis was performed after approximately two-thirds of the sample size was achieved to determine what additional resources would be needed. That analysis showed a higher sensitivity than expected, so enrollment was stopped early after 102 patients were enrolled. Data were analyzed with Stata statistical software (version 9.0; StataCorp, College Station, TX). The Student t test, χ 2 test, Mann Whitney U test, and Fisher’s exact test were used to compare characteristics of women with and without 24-hour urine protein ≥300 mg. A probability value of < .05 was considered statistically significant. Institutional review board approval was obtained.
Results
One hundred two patients were enrolled in the study ( Figure 1 ). Twelve subjects were subsequently excluded: 11 women did not complete the 24-hour urine protein collection because of a clinical indication for delivery and one woman’s sample was processed incorrectly in the laboratory; the final cohort comprised 90 subjects. In addition, 4 spot urine samples for protein:creatinine ratio were inadvertently not collected, and four 24-hour urine collections were not separated into two 12-hour jugs for a total of 86 subjects with protein:creatinine ratio and 86 subjects with a 12-hour urine sample ( Figure 1 ). Twenty-eight subjects (31%) had a 24-hour urine protein ≥300 mg. Baseline maternal characteristics by 24-hour urine category are summarized in Table 1 . Women with a 24-hour urine protein ≥300 mg were more likely to be multiparous (71% vs 47%; P = .03) and have pregestational diabetes mellitus (14% vs 2%; P = .015). They were also admitted at an earlier median gestational age (32.8 weeks [range, 24.0–35.4 weeks] vs 34.3 weeks [range, 25.9–39.0 weeks]; P = .007) and were more likely to have proteinuria or abnormal laboratory values as part of their criteria for admission (68% vs 26% [ P < .001] and 39% vs 11% [ P = .002]). Baseline characteristics, including median systolic and diastolic blood pressures at time of admission and during collection period, were otherwise similar.
| Variable | 24-hr protein <300 mg (n = 62) | 24-hr protein ≥300 mg (n = 28) | P value |
|---|---|---|---|
| Maternal age, y a | 29 (19-42) | 30 (19-38) | .76 |
| Race/ethnicity, n (%) | .41 | ||
| White | 49 (79) | 22 (79) | |
| Black | 2 (3) | 3 (11) | |
| Asian | 3 (5) | 0 | |
| Hispanic | 1 (2) | 1 (4) | |
| Private insurance, n (%) | 45 (73) | 20 (71) | .91 |
| Multiparous, n (%) | 29 (47) | 20 (71) | .03 |
| Multiple gestation, n (%) | 8 (13) | 3 (11) | .77 |
| Body mass index, kg/m 2 a | 33.1 (19.5–69.9) | 36.4 (25.4–54.9) | .13 |
| Gestational age, wk a | 34.3 (25.9–39.0) | 32.8 (24.0–35.4) | .007 |
| Smoking, n (%) | 13 (21) | 4 (14) | .45 |
| Any comorbidity, n (%) b | 57 (91) | 26 (93) | .88 |
| Chronic hypertension | 12 (19) | 8 (29) | .33 |
| Gestational hypertension or preeclampsia | 15 (24) | 7 (25) | .93 |
| Pregestational diabetes mellitus | 1 (2) | 4 (14) | .015 |
| Gestational diabetes mellitus | 8 (13) | 6 (21) | .30 |
| Indication for admission, n (%) b | |||
| Elevated blood pressure | 51 (82) | 26 (93) | .19 |
| Proteinuria | 16 (26) | 19 (68) | < .001 |
| Symptoms c | 28 (45) | 14 (50) | .67 |
| Laboratory abnormalities | 7 (11) | 11 (39) | .002 |
| Fetal growth restriction | 10 (16) | 3 (14) | .50 |
| Other d | 9 (15) | 6 (21) | .42 |
| Previous 24-hr urine protein done, n (%) | 31 (50) | 19 (68) | .11 |
| Previous 24-hr urine protein, mg a | 155 (50–440) | 210 (64–2240) | .14 |
| Median systolic blood pressure on admission, mm Hg a | 137 (105–168) | 140 (117–158) | .51 |
| Median diastolic blood pressure on admission, mm Hg a | 83 (55–103) | 82 (64–112) | .85 |
| Median systolic blood pressure during collection, mm Hg a | 131 (99–165) | 136 (105–152) | .11 |
| Median diastolic blood pressure during collection, mm Hg a | 76 (53–98) | 78 (55–99) | .41 |
c Includes headache, scotomata, abdominal pain, and significant weight gain that was associated with edema;
d Includes shortness of breath, seizure of uncertain origin, oligohydramnios, visual changes other than scotomata.
Pregnancy outcomes were available for 85 women and are summarized in Table 2 by 24-hour urine protein category. Women with a 24-hour urine protein ≥300 mg delivered at an earlier median gestational age (34.3 weeks [range, 24.9–38.1 weeks] vs 37.0 weeks [range, 27.3–40.6 weeks]; P < .001), had a lower median neonatal birthweight (2100 g [range, 425–3815 g] vs 2733 g [range, 600–4025 g]; P = .004), and were more likely to be delivered during the index admission (67% vs 33%; P = .003). They were also more likely to experience an intrauterine fetal demise or intrapartum/neonatal demise. There was no difference in overall rates of preeclampsia morbidity, induction rates, cesarean delivery rates, or rates of neonatal intensive care unit admission.
| Variable | 24-hr protein <300 mg (n = 58) | 24-hr protein ≥300 mg (n = 27) | P value |
|---|---|---|---|
| Delivery during study admission, n (%) b | 19 (33) | 18 (67) | .003 |
| Gestational age at delivery, wk c | 37.0 (27.3–40.6) | 34.3 (24.9–38.1) | < .001 |
| Induction, n (%) | 30 (52) | 16 (59) | .47 |
| Indication for induction, n (%) d | |||
| Hypertension/preeclampsia | 22 (38) | 13 (48) | .55 |
| Growth restriction | 4 (7) | 0 (0) | .13 |
| Oligohydramnios | 3 (5) | 1 (4) | .68 |
| Fetal death | 0 | 2 (7) | .048 |
| Preterm premature rupture of membranes | 1 (2) | 1 (4) | .64 |
| Maternal medical condition | 1 (2) | 0 | .46 |
| Other | 2 (3) | 0 | .29 |
| Cesarean delivery, n (%) | 33 (57) | 17 (63) | .66 |
| Maternal preeclampsia morbidity, n (%) d | 18 (31) | 12 (44) | .23 |
| Eclampsia | 1 (2) | 0 | .49 |
| Pulmonary edema | 0 | 1 (4) | .14 |
| HELLP syndrome (hemolysis elevated liver enzymes low platelets) | 1 (2) | 1 (4) | .58 |
| Abruption | 2 (3) | 0 | .33 |
| Fetal death | 0 | 2 (7) | .04 |
| Growth restriction | 13 (22) | 8 (30) | .49 |
| Transfusion | 3 (5) | 1 (4) | .77 |
| Other e | 2 (40) | 3 (11) | .20 |
| Birthweight, g c | 2733 (600–4025) | 2100 (425–3815) | .004 |
| Male sex, n (%) | 29 (50) | 13 (48) | .83 |
| 5-minute Apgar score <7, n (%) | 0 | 1 (4) | .28 |
| Arterial cord pH c | 7.27 (7.06–7.38) | 7.27 (7.01–7.36) | .78 |
| Neonatal intensive care unit admission, n (%) | 30 (52) | 18 (67) | .17 |
| Intrapartum/Neonatal demise, n (%) | 0 (0) | 3 (11) | .006 |
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