The Journal of Maternal-Fetal and Neonatal Medicine, January 2010; 23(1): 96102

Optimal laboratory panel for predicting preeclampsia

1 & MARIO S 2 TEFANOVIC RATKO DELIC

Department of Obstetrics & Gynecology, General Hospital, Celje, Slovenia, and 2Clinical Institute of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia (Received 27 October 2008; revised 27 June 2009; accepted 1 July 2009)
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Abstract Objective. This study was undertaken to investigate the usefulness of standard biochemical and hematological parameters measurement at third trimester of pregnancy for the individual prediction of preeclampsia. Methods. A retrospective designed study included 113 patients with preeclampsia and a control group of 95 normal, uncomplicated pregnancies. Patients were recruited in the third trimester of pregnancy at the Department of Gynecology and Obstetrics, General Hospital Celje, Slovenia, EU. Erythrocytes, leukocytes, thrombocytes, hemoglobin, hematocrit, aspartate aminotransferase, alanine aminotransferase, g-glutamyl transferase, alkaline phosphatase, total bilirubin, urea, creatinine, uric acid, body mass index, parity, and age were evaluated to predict the occurrence of preeclampsia based on multivariate logistic regression model. Results. When parameters such as uric acid and urea were included into logistic regression model, we correctly classied 79.6% patients. With additional four parameters (thrombocytes, hematocrit, aspartate aminotransferase and leukocytes) we correctly classied 83.8% patients with preeclampsia. Conclusion. Our ndings conrmed that several standard biochemical and hematological parameters, when used as laboratory test panel have signicant prognostic value in the prediction of preeclampsia.

Keywords: Preeclampsia, biochemical markers, hematological markers, 3rd trimester, prediction, laboratory panel, uric acid, urea, thrombocytes, hematocrit, aspartate aminotransferase, leukocytes

Introduction Preeclampsia is a pregnancy specic disorder and refers to the new onset of hypertension (blood pressure 140/90 mmHg) and proteinuria (300 mg in a 24-h urine) after 20 weeks of gestation in a previously normotensive woman [1,2]. This disorder occurs in *35% of pregnancies and is a signicant cause of maternal and fetal morbidity and mortality. The etiology and pathogenesis remain elusive, resulting in a failure to develop specic preventive and treatment options. The clinical spectrum of this disease covers a wide range of presentations, from mild forms of hypertension and proteinuria, to severe forms such as eclampsia, and HELLP (Hemolysis, Elevated Liver enzymes, and Low Platelets count) syndrome [24]. Several screening tests have been proposed for the prediction of preeclampsia, but so far there is no

single screening test that is considered reliable enough [57]. Measurement of angiogenic factors (VEGF vascular endothelial growth factor, sFlT-1soluble fms-like tyrosine kinase 1, PIGF placental growth factor, sEng-soluble endoglin) in blood or urine represent the most promising approach for predicting preeclampsia, however, these tests are investigational and are not available for clinical use at present [711]. Assessment of the risk of preeclampsia is a challenging task because there is no available tool that can separate low to high risk patients. The purpose of this study was to combine standard biochemical and hematological markers at third trimester of pregnancy in assessing the risk of preeclampsia. This includes reevaluation of aspartate aminotransferase (AST), alanine aminotransferase (ALT),

tefanovic , Zorkova , Clinical Institute of Chemistry, Sestre Correspondence: Ratko Delic cka 4, 10000 Zagreb, Croatia. E-mail: rdelic@gmail.com or Mario S milosrdnice University Hospital, Vinogradska 29, 10000 Zagreb, Croatia. E-mail: mstefan6@gmail.com ISSN 1476-7058 print/ISSN 1476-4954 online 2010 Informa UK Ltd. DOI: 10.3109/14767050903156643

Optimal laboratory panel urea, creatinine, uric acid, thrombocytes, erythrocytes, hemoglobin, and hematocrit. We also evaluated several other tests such as leukocytes, alkaline phosphatase (ALP), total bilirubin, g-glutamyl transferase (gGT) that were generally considered less important. For the comparison of clinical usefulness of each test, we performed receiver operating curve (ROC) analysis. For the estimation of best chosen test panel overall effect, we performed logistic regression analysis and presented tests that contribute to diagnosing preeclampsia most signicantly. Materials and methods This retrospective study was carried out on 208 Caucasian subjects: 113 preeclamptic and 95 healthy pregnant women in their third trimester (weeks 32 41) of pregnancy attended the Obstetric Department of the General Hospital Celje, Slovenia. All data were gathered and evaluated for period from January 2002 to December 2007. The study was approved by the Hospital ethical committee. Descriptive data of all the study subjects are presented in Table I. Normal pregnancy was diagnosed on the basis of absence of clinical, biochemical, and ultrasonographical pathological ndings. Diagnosis of preeclampsia was made according to the current guidelines (new onset of hypertension and proteinuria after 20 weeks of gestation in previously normotensive woman) [1,2]. Patients who were diagnosed as eclampsia, or HELLP syndrome according to the current clinical and laboratory criteria [3] were excluded from this study in advance, because we wanted to focus specically on preeclampsia. Patients who did not fulll criteria for preeclampsia, according to guidelines, as well as patients with preeclampsia and concurrent diseases such as diabetes mellitus, systemic lupus erythematosus, or another chronic disease were also excluded from this study. All subjects were evaluated on a daily basis for symptoms of hypertension, proteinuria, weight gain, or other possible complications. As it was not possible to obtain total protein excretion in 24 h from all patients, proteinuria was measured in spot urine and presented as follows: (0) absent, 1 (*0.15 g/l), 2 (0.3g/l), 3 (1 g/l), and 4 (5 g/l) positive proteinuria (Combur10 Test M, Roche Diagnostics, Basel, Switzerland) [1214]. Blood pressure (RRss systolic, RRds diastolic) was measured several times daily by the use of conventional mercury or electronic sphygmomanometer (Riester, Jungingen, Germany) that has been validated for pregnancy. For each subject blood pressure was expressed as median from recorded values [15,16].

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At our Department, 44 out of 112 preeclamptic patients received antihypertensive therapy; however we do not have data of all patients regarding antihypertensive therapy prior their arrival to the department. Subjects were divided into two groups, women with normal, uncomplicated pregnancies (control group), and women with preeclampsia according to current guidelines [1,2]. Because of relatively small number (n 23) of severe preeclamptic pregnancies, we decided to join mild and severe preeclampsias in a single group. The standard biochemical parameters (AST, ALT, gGT, total bilirubin, urea, creatinine, uric acid and ALP) were determined within last week before delivery (3.5 days, mean) by an advanced auto analyzer (Modular, Roche Diagnostics, Basel, Switzerland) using commercially available kits from the same manufacturer. Enzyme catalytic concentrations were originally expressed in mkat/l at 378C, but because of widespread use, we decided to express them as U/l (1 U/l corresponds to 16.67 nkat/l) [17]. The hematological parameters (erythrocytes, leukocytes, thrombocytes, hemoglobin, and hematocrit) were analyzed with Coulter STKS analyzer using reagents from the same manufacturer (ISOTON III, LYSE S III DIFF, and SCATTER PAK) (Fullerton, California). Statistical analyses were performed using MedCalc for Windows, version 9.3.9.0 (MedCalc Software, Mariakerke, Belgium). Variable comparison for signicant difference was performed by independent samples t-test (for normally distributed variables), and by MannWhitney test on independent samples (for variables that were not normally distributed). For the use of uniform presentation, each nominal parameter (both normally and not normally distributed) was described with mean, 95% condence interval (CI), and corresponding standard deviation (Table I). Results The study population consisted of 113 women with preeclampsia in their third trimester of pregnancy. As control group, we investigated the results of 95 healthy pregnant women admitted for labor. Mean systolic and diastolic blood pressures were higher in preeclamptic group as compared with control group values (p 5 0.05). Among laboratory parameters, values of serum uric acid (p 5 0.001), creatinine (p 5 0.001), urea (p 5 0.001), AST (p 5 0.015), and leukocytes (p 5 0.04) were signicantly higher in preeclamptic group (p 5 0.05), compared with values in the control group.

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Table I. Descriptive statistics of tested parameters and their difference between controls and patients. Controls 95% CI N 113 113 112 113 113 97 102 113 102 113 112 109 113 113 109 105 99 108 105 107 28.2 38.1 3.5 91 143 353 72 212 4.1 10.2 35 38 118 4.1 35 14 17.1 27.3 182 6.8 27.25929.166 37.71638.408 2.7184.228 89.37692.482 141.055145.671 338.665366.695 70.21174.084 201.837221.348 3.8124.325 9.66910.704 34.59235.765 26.11250.549 116.082120.237 3.9854.119 18.35551.872 10.80816.542 15.86518.357 26.20028.491 166.151197.723 5.9067.640 26.63728.661 38.14039.081 71.78075.588 119.998124.781 247.488269.024 61.93064.888 237.440261.297 2.8473.185 10.61811.948 35.83637.238 18.82221.627 118.860123.616 4.0734.226 11.70914.147 8.80410.880 14.67317.068 25.33527.832 156.862184.526 5.7487.194 4.9 2.3 9.0 12.0 50.0 7.0 59.0 0.8 3.2 3.0 7.0 12.0 0.4 6.0 5.0 5.6 5.9 67.0 3.5 27.0 39.0 75* 120* 250* 64* 240 2.9 10.8 37* 19 123* 4.2* 11 9 15.0* 25.3 156 5.9 SD Median Mean 95% CI SD 5.1 1.9 4.0 8.0 12.0 70.0 10.0 52.0 1.3 2.8 3.0 64.0 11.0 0.4 88.0 15.0 6.2 6.0 82.0 4.5 Preeclampsia Median 28.0* 38.0 2.0 90* 140 360* 72 206 3.9 9.6 35* 22 117* 4.1* 12 9 17.0 25.5 168 5.4 Difference p value 0.349 0.060* 50.001* 50.001* 50.001* 50.001 50.001 50.001 0.004 0.003* 0.015 0.054* 0.058* 0.108 0.135 0.292 0.334 0.357 0.394

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N 27.6 38.6 74 122 258 63 249 3.0 11.3 37 20 121 4.2 13 10 15.9 26.6 171 6.5

Mean

Age (years) Gestational weeks Timeline RRds (mmHg) RRss (mmHg) Urate (mmol/l) Creatinine (mmol/l) Thrombocyte (103/l) Urea (mmol/l) Leukocyte (109/l) Hct (%) AST (U/l) Hb (g/l) Erythrocyte (1012/l) ALT (U/l) GGT (U) Weight gain (kg) BMI BB (kg/m2) AP (U/l) Bilirubin (mmol/l)

94 95 95 95 86 88 95 88 94 95 93 95 95 93 92 85 89 92 90

N, number of subjects; SD, standard deviation. p level of signicant difference between groups. (* denotes independent samples t-test for parameters distributed normally; otherwise difference is calculated with MannWhitney test on independent samples); italic parameter values are statistically signicant; BMI BB, BMI before birth.

Optimal laboratory panel Thrombocytes (p 5 0.001) and hematocrit (p 5 0.03) were signicantly lower in preeclamptic group in comparison to the control group. Further laboratory values that were evaluated did not show statistically signicant difference (p 4 0.05) were ALT, bilirubin, ALP, gGT, hemoglobin, and erythrocytes. Also, mean age, body mass index (BMI), and parity did not show signicant difference between patients with preeclampsia and control group. For parameters that showed signicant difference between groups (uric acid, thrombocytes, urea, creatinine, AST, hematocrit), we performed ROC analysis (Table II). Comparison of ROC curves for RRss, RRds, urate, urea, and creatinine showed signicant difference between all the values (p 5 0.05) except between urea/creatinine and RRss/RRds which have the values that were too close to each other. In order to assess the probability of occurrence of preeclampsia, the same parameters were employed in logistic regression model (without proteinuria and blood pressure measurements). According to logistic regression model, our optimal laboratory panel correctly classied 83.8% of participants in the study (Table III). To explore the parameters that contributed most to our regression model, we performed additional logistic regression analyses: the model with only hematocrit, uric acid, urea, and leukocytes (without AST and thrombocytes) correctly classied 83.3% of participants, and another model with only uric acid and urea that correctly classied 79.6% of participants (data not shown). Thus, uric acid and urea demonstrate the biggest contribution to our test panel, leaving 54% for other four parameters (AST, hematocrit, leukocytes, and thrombocytes). Discussion A good screening test for preeclampsia should be simple, safe, rapid, inexpensive, reproducible, and

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should provide an opportunity for intervention to prevent development of the disease, or at least result in better outcome. So far, a wide variety of laboratory and imaging tests have been proposed to detect subgroups of women at high risk of developing preeclampsia [7]. Most have not been shown to be sufciently sensitive and specic to be clinically useful as a screening test. As mentioned previously, measurement of angiogenic factors (VEGF, PIGF, sFlt-1, sEng) in blood or urine is the most promising approach for predicting preeclampsia; but these tests are investigational and are not available for clinical use at present. Denitions of preeclampsia based solely on hypertension and proteinuria ignore the wide clinical variability in this syndrome. Women with no proteinuria but who do have hypertension and other features such as severe headache or other symptoms, thrombocytopenia, hyperuricemia, disordered liver function, and fetal compromise are likely to have preeclampsia. Our goal was to offer optimal laboratory test panel available to practically every laboratory at third trimester of pregnancy for the individual prediction of preeclampsia, especially in those patients with non-typical presentations of preeclampsia. Though individually most of the hematological and biochemical parameters are non-specic, combined properly they have signicant prognostic value. The use of certain standard laboratory tests in the prediction of preeclampsia is not a new issue. For example, Martin et al. combined symptoms with certain laboratory tests to predict likelihood of maternal morbidity for the patients with severe preeclampsia [6]. In our retrospective study, the most signicant standard laboratory parameter in preeclamptic patients was elevated concentration of serum uric acid (p 5 0.001) compared with control group [2,7,18]. Hyperuricemia is commonly seen in women with preeclampsia and still produces controversies.

Table II. ROC analysis for tested parameters. Np RRds (mmHg) RRss (mmHg) Urate (mmol/l) Creatinine (mmol/l) Urea (mmol/l) Thrombocyte (103/l) Hct (%) Leukocyte (109/l) AST (U/l) Hb (g/l) 113 113 97 102 102 113 112 113 109 113 Nc 95 95 86 88 88 95 95 94 93 95 AUC 0.909 0.905 0.856 0.785 0.771 0.686 0.627 0.618 0.599 0.591 95% CI 0.8620.945 0.8570.941 0.7960.903 0.7200.841 0.7050.829 0.6190.749 0.5570.693 0.5480.684 0.5280.667 0.5210.658 p (AUC 0.5) 50.001 50.001 50.001 50.001 50.001 50.001 50.001 0.003 0.012 0.022 c/o 480 4130 4288 468 43.2 229 36 10.1 421.6 122

Nc, N (controls); Np, N (patients); AUC, area under the curve; c/o, cut off values.

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Table III. Logistic regression (without RRds). Sample size Controls 85 (47.49%) Patients 94 (52.51%)

Description

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Regression coefcients Variable AST (U/l) Hct (%) Leukocyte (109/l) Thrombocyte (103/l) Urate (mmol/l) Urea (mmol/l) Constant Coefcient 0.094 70.348 70.313 70.011 0.028 0.877 4.773 p 0.003 50.001 0.024 0.028 50.001 0.002

Percentage of cases correctly classied: 0.838 (83.8%).

Interestingly, the rst report of increased uric acid in preeclampsia was published back in 1917, by Slemons and Bogert [19]. Normally, plasma uric acid declines in the second trimester but increases to nonpregnant levels at term [20]. In the year 2006, systematic review of ve studies concluded that there is insufcient evidence to draw rm conclusions about the accuracy of serum uric acid determination in predicting preeclampsia [21]. Another systematic review reached similar conclusion that serum uric acid measurement was not useful for predicting the development of complications in women with preeclampsia [22]. On contrary, Roberts et al. suggest that hyperuricemia is as effective as proteinuria in identifying gestational hypertensive pregnancies at increased risk [23]. On the basis of our results, hyperuricemia strongly correlates with preeclampsia and as such were included in our logistic regression model. Renal function improves during normal, non-complicated pregnancy. Girling in a prospective cross-sectional study presented that creatinine concentration is lower in normal pregnancy than outside pregnancy and that there is a small third trimester rise in creatinine concentration [24]. In our study, serum urea and creatinine concentration showed statistically signicant elevation in preeclamptic group compared with control group, and were included in logistic regression model. Among hematological parameters, thrombocytopenia and lower hematocrit were statistically signicant ndings compared with controls and were also included in logistic regression model. It is known that the frequency and severity of thrombocytopenia and falling hematocrit (formation of microthrombi, microangiopathic hemolysis) increase with the severity of preeclampsia. Regarding liver function tests, serum AST elevation was the most signicant nding in women with preeclampsia

(p 5 0.015). Nevertheless, we have to be aware that some of the AST is not of hepatic origin: AST is widely distributed throughout the body (heart muscle cells, liver cells, skeletal muscle cells and to a lesser degree in the kidneys, pancreas, and red blood cells), whereas ALT is found predominantly in the liver (lesser quantities are found in the kidneys, heart, and skeletal muscle) and is considered quite specic for hepatocellular disease [25,26]. Yet, such result is not surprising as we know that with worsening of preeclampsia (towards severe preeclampsia and HELLP syndrome), and eventual hemolysis additional rise of serum AST is expected [27]. Recently published bivariate meta-analysis about accuracy of BMI in predicting preeclampsia showed that BMI is fairly weak predictor for preeclampsia [28]. Samuels-Kalow et al. also recently published an article; they concluded that elevated prepregnancy BMI is associated with increased risk of hypertensive disorders of pregnancy and with increased long-term maternal mortality rates [29]. In our study BMI as well as parity did not show statistically signicant difference between preeclamptic women compared with controls. By employing two parameters that were statistically most signicant (serum uric acid and urea) we correctly classied nearly 80% of cases. With additional four parameters in logistic regression model (thrombocytes, AST, hematocrit, leukocytes), we increased this percentage to nearly 84% of cases. Blood pressure (140/90 mmHg conrmed by two separate measurements) and proteinuria (0.3 g protein in a 24 h urine specimen or persistent 1 on dipstick) were not included in the calculation because they are diagnostic criteria and must be present to diagnose preeclampsia. There are also few limitations to our study. Our study population included only slightly more than 200 patients, with 113 preeclampsia and 95 controls. Secondly, because of the small number of preeclamptic patients with severe preeclampsia we combined all the readings with preeclampsia in one group. Our readings were obtained from 32nd to 41st week of pregnancy, so selected laboratory panel should be primarily used within those weeks range of gestation. We emphasize on the fact that because of the small number of preeclamptic patients, we could not further divide the patients into subgroups according to their gestational age; this aspect should be considered in future investigations on a larger patient samples. Conclusion The goal of our study was to assess prognostic potential of standard laboratory tests for the

Optimal laboratory panel individual prediction of preeclampsia during third trimester of pregnancy. On the basis of current criteria which dene preeclampsia, by using standard laboratory tests (without blood pressure measurements and proteinuria), evaluated through the logistic regression model, we presented an optimal laboratory panel that explained almost 84% of the cases. Even with two most signicant parameters (serum uric acid and urea) we correctly classied nearly 80% of the cases. We showed that selected laboratory tests (serum uric acid, urea, thrombocytes, AST, hematocrit, leukocytes), when taken as a test panel, have signicant prognostic value. Our optimal laboratory panel does not have the intention to enter the everyday use as a calculated equation, but rather as a combination of optimal tests that should be considered when dealing with the preeclamptic patients. Acknowledgement We would like to thank primarius Vladimir Weber, MD, (Head of the Department of Obstetrics and Gynecology, General Hospital, Celje, Slovenia) for giving opportunity this study to realize. We would tefka Krivec, MSc., (Head of the also like to thank S Department of the laboratory medicine of the same hospital), for providing additional information regarding the laboratory results. Declaration of interest: The authors report no conicts of interest. The authors alone are responsible for the content and writing of the paper.

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