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Keywords:

  • High-performance liquid chromatography;
  • pre-eclampsia;
  • pregnancy;
  • proteinuria

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

Please cite this paper as: Baweja S, Kent A, Masterson R, Roberts S, McMahon L. Prediction of pre-eclampsia in early pregnancy by estimating the spot urinary albumin: creatinine ratio using high-performance liquid chromatography. BJOG 2011;118:1126–1132.

Objective  To establish whether a spot urinary albumin: creatinine ratio (ACR) measured before 20 weeks of gestation can predict subsequent pre-eclampsia when urinary albumin is measured by high-performance liquid chromatography (HPLC).

Design  Prospective exploratory study.

Setting  Antenatal clinic in a tertiary teaching hospital, Victoria, Australia.

Population  A cohort of 265 women with a singleton pregnancy, normal renal function, and no evident proteinuria, attending antenatal clinics between 12 and 20 weeks of gestation.

Methods  The ACR was determined from a mid-stream urine (MSU) sample taken between 17 and 20 weeks of gestation. Intact urinary albumin was determined by HPLC; creatinine was measured by modified Jaffe’s method.

Outcome measures  Pre-eclampsia (primary); gestational hypertension, small for gestational age (SGA), gestational diabetes mellitus, gestational age at delivery, and prematurity (secondary).

Results  The median ACR was 28 mg/mmol (IQR 16–46 mg/mmol). Women who subsequently developed pre-eclampsia had a significantly higher ACR (median 50 mg/mmol; IQR 33–90 mg/mmol) compared with women suffering from gestational hypertension (median 27 mg/mmol; IQR 8–35 mg/mmol), and compared with unaffected women (median 28 mg/mmol; IQR 16–46 mg/mmol). Mothers of SGA infants also had a significantly higher median ACR. By ROC analysis, the optimum ACR to predict pre-eclampsia was 35.5 mg/mmol: the relative risk of developing pre-eclampsia in women with a urinary ACR ≥ 35.5 mg/mmol was 7.8 times more than in those with a urinary ACR < 35.5 mg/mmol.

Conclusions  When urinary albumin is measured by HPLC, spot urinary ACR values are higher in early uncomplicated pregnancy compared with previously reported conventional methods. When measured early in the second trimester, an ACR ≥ 35.5 mg/mmol predicted pre-eclampsia well before the onset of clinical manifestations.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

Pre-eclampsia remains a major cause of perinatal and maternal morbidity and mortality, and is one of the most common medical complications of pregnancy.1–3 Although there is no established preventative therapy, there is potential gain in being able to identify the women and fetuses at risk, so that appropriate monitoring can ensue, as well as some evidence to support the prophylactic benefit of the early introduction of aspirin,4,5 calcium (in women at high risk and low baseline calcium intake),6 and/or possibly heparin (in high-risk women with prothrombotic conditions).7–9 Although there is currently no established preventative therapy, early diagnosis is helpful for research studies, and may in the future be beneficial if new preventative therapies are discovered.

The pathophysiological events resulting in pre-eclampsia begin early in gestation, and precede the onset of the clinical features.10 One of the early pathophysiological hallmarks is endothelial cell damage.11,12 Microalbuminuria is a marker of endothelial dysfunction and, in the general population, is associated with hypertension, obesity, diabetes, and covert renal disease, and also with an increased risk for myocardial infarction, stroke, and premature death.13,14 The risk rises with the urinary albumin concentration, even within the so-called normal range.15 Microalbuminuria might be used as an early marker of endothelial dysfunction in pre-eclampsia, before the onset of the overt syndrome, as it is likely that overt proteinuria is preceded by a microalbuminuric phase. Previous studies examining the effectiveness of such a screening procedure have had variable results.16–18 Although the 24-hour collection of urine is the gold standard for quantifying urinary albumin excretion, it is cumbersome, and results in a delay of at least a 24 hours in diagnosis.19 Therefore, the ‘spot’ urinary protein: creatinine ratio, or urinary albumin: creatinine ratio (ACR), has been advocated as an alternative.19,20

Urinary albumin is usually measured by an immunochemical method such as immunonephelometry, immunoturbidimetry, enzyme-linked immunosorbent assays (ELISA), or radioimmunodiffusion. Recently, a number of studies have used high-performance liquid chromatography (HPLC) for urinary albumin measurement in different populations, and demonstrated that the level of albumin detected in the urine by HPLC, when compared with conventional assays, is significantly greater because HPLC is able to measure both immunoreactive and immunounreactive intact albumin.24–27

However, none of the previous studies have used HPLC to measure urinary albumin in pregnant women. In this study we wished to establish prospectively whether a spot urinary ACR measured before 20 weeks of gestation can predict the development of pre-eclampsia when urinary albumin is measured by HPLC.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

This was a prospective exploratory study of women with singleton pregnancies attending an antenatal clinic at a tertiary teaching hospital in Victoria, Australia, from March 2006 to December 2007. All women attending clinics were asked to participate in the study at the time of booking (between 12 and 20 weeks of gestation), and were recruited if they agreed. The study was approved by the regional ethics committee, and written informed consent was obtained from all participating women. Inclusion criteria were women over 18 years of age, singleton pregnancy, ≤20 weeks of gestation at the time of recruitment, and nil proteinuria upon measurement with a dipstick. Women with haematuria, dipstick-positive proteinuria, ongoing urinary tract infection, multiple pregnancy, acute renal failure, chronic kidney disease (CKD), assisted reproduction, or a poor obstetric history were excluded. However, women with a past history of urinary tract infection, renal failure, haematuria, or proteinuria were included if there was no evidence of current disease, if the urine was dipstick-negative for proteinuria, and if the serum creatinine level was within the normal range. CKD was diagnosed if the patient had a personal history of CKD, if there was evidence of impaired renal function, or if the patient was under a nephrologist’s care. Chronic hypertension was diagnosed if the patient was already taking anti-hypertensive medication or displayed hypertension before 20 weeks of gestation. Data regarding demographic profile, blood pressure, body mass index (BMI), and medical and family history (history of chronic hypertension, diabetes mellitus, and/or CKD) were recorded. Obstetric history documented gravidity, parity, past history of pre-eclampsia, prematurity, small for gestational age (SGA), miscarriage, and family history of pre-eclampsia.

Between 17 and 20 weeks of gestation, all women were given a sterile urine container without preservative and, after instruction, a mid-stream urine sample was collected. Routine microscopic and dipstick examination of urine was performed, and within 30–60 minutes of collection the sample was stored at –20°C for ACR analysis at the end of the study. Participants were then followed until delivery. The primary outcome measure was pre-eclampsia, defined according to standard clinical criteria.25 Secondary outcome measures included gestational hypertension (GH), SGA, gestational diabetes mellitus (GDM), gestational age, and prematurity, and a normal range estimate of urinary ACR was established.

The definition of GH was a blood pressure of ≥140/90 mmHg occurring after 20 weeks of gestation in a previously normotensive woman.28 GDM was defined as a positive 50-g oral glucose challenge test [with a venous plasma glucose level 1 hour after glucose challenge of at least 7.8 mmol/l (140 mg/dl)], and a positive 75-g oral glucose-tolerance test at 24–28 weeks of gestation [with venous plasma glucose levels of <7.8 mmol/l after an overnight fast and 7.8–11.0 mmol/l (198 mg/dl) at 2 hours).26 SGA was diagnosed when the birthweight was below the tenth centile for the gestational age,27 and prematurity was defined as birth on or before the end of the last day of the 37th week (259th day) following the onset of the mother’s last menstrual period, or by initial ultrasound estimation.28

Total intact urinary albumin (immunoreactive plus immunounreactive) was determined by analysing urine samples using a Hewlett Packard 1100 HPLC system with UV–visible detector (Hewlett Packard, Waldbronn, Germany). Aliquots of urine (25 μl) were injected onto a Zorbax Bio series preparative GF-250 column. The mobile phase was phosphate-buffered saline run at a flow rate of 0.5 or 2.0 ml/minute in a gradient system. The urinary albumin peak was identified to within ±2% of the elution time of the monomer albumin standard. Urine creatinine was measured by modified kinetic Jaffe reaction without deproteinization using COBAS INTEGRA systems.

Statistical analysis was performed by pasw statistics v18 software. As this is the first exploratory study to use this technique in pregnancy, a meaningful power calculation could not be performed. Comparison of spot urinary ACR and birthweight between unaffected, gestational hypertensive, and pre-eclamptic groups were performed by Kruskal–Wallis test or Mann–Whitney U-test. Comparison of the mean of the continuous data (normally distributed) was performed by analysis of variants (anova), and chi-square analysis was used to compare categorical data between groups. For post hoc analysis of normally-distributed continuous data, Bonferoni’s test was applied. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) at different values of ACR for predicting pre-eclampsia were calculated from the receiver operating curve (ROC).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

After obtaining informed consent, 295 women were enrolled at between 12 and 20 weeks of gestation. Thirty (10.1%) women were excluded subsequently because of missing outcome data (miscarriage, transferral to another facility, withdrawal of consent, or misplaced urine sample during transport); therefore, 265 women were included in the final analysis (Tables 1 and 2).

Table 1.   Baseline clinical parameters and demographic profile
 Unaffected (n = 248)Gestational hypertension (n = 11)Pre-eclampsia (n = 6)SGA (n = 9)P*
  1. ATSI, Australian Torres Strait islanders; CHT, chronic hypertension; DBP, Diastolic blood pressure; FH, family history; MAP, mean arterial pressure; PE, pre-eclampsia; PH, past history; SBP, systolic blood pressure.

  2. Data are means ± SDs, except as indicated.

  3. *Reflects comparison between unaffected, gestational hypertension, and pre-eclampsia groups. (The SGA group was comprised of patients from the unaffected and pre-eclampsia groups).

  4. **Significant difference only between gestational hypertension and unaffected group.

Age (years)29.5 ± 4.731.1 ± 5.826.3 ± 4.927.6 ± 5.30.142
Ethnicity, n (%)
Australian111 (44.8)5 (45.5)3 (50.0)4 (44.4)0.023
European84 (33.9)1 (9.1)04 (44.4)
Asian40 (16.1)3 (27.3)2 (33.3)0
African6 (2.4)1 (9.1)1 (16.7)1 (11.1)
ATSI1 (0.4)1 (9.1)00
Others6 (2.4)000
BMI, kg/m226.8 ± 6.127.8 ± 5.930.3 ± 10.226.4 ± 7.10.334
Primigravida, n (%)82 (33.0)5 (45.4)4 (66.7)4 (44.4)0.98
SBP, mmHg109 ± 10.7120 ± 13116 ± 21108 ± 180.001**
DBP, mmHg65 ± 871 ± 867 ± 1264 ± 100.054**
MAP, mmHg101 ± 10111 ± 12106 ± 18100 ± 160.008**
DM, n (%)15 (6.0)0000.580
CHT, n (%)19 (7.7)2 (18.2)2 (33.3)1 (11.1)0.046
PH renal disease, n (%)9 (3.6)1 (9.1)000.004
PH PE, n (%)5 (2.0)0000.840
FH DM, n (%)101 (40.7)6 (54.5)3 (50.0)4 (44.4)0.612
FH HT, n (%)96 (38.7)7 (63.6)4 (66.7)6 (66.7)0.110
FH CKD, n (%)10 (4.0)1 (9.1)2 (33.3)2 (22.2)0.004
GDM, n (%)20 (8.15)1 (9.1)000.762
Table 2.   Spot urinary ACR, fetal complications, and outcome measures
 Unaffected (n = 248)Gestational hypertension (n = 11)Pre-eclampsia (n = 6)SGA (n = 9)P*
  1. *Reflects the comparison between unaffected, gestational hypertension, and pre-eclampsia groups (as per Table 1).

  2. **Significant difference between pre-eclampsia and other groups.

  3. ***Median and IQR.

ACR (mg/mmol)28 (16–46)27 (8–35)50 (33–90)55 (32–76)0.042
Gestational age at delivery39.2 ± 1.938.4 ± 1.736.7 ± 2.437.4 ± 1.80.003**
Mode of delivery
Normal vaginal delivery179 (72.2)8 (72.7)2 (33.3)5 (55.6)0.115
Caesarean section69 (27.8)3 (27.3)4 (66.7)4 (44.4) 
Birthweight (g)***3420 (3090–3740)3270 (3038–3700)2270 (1378–3420)2250 (1731–2490)0.024
Prematurity, n (%)9 (3.6)1 (9.1)4 (66.7)3 (37.5)<0.001

The mean maternal age was 29.5 ± 4.7 years and the mean BMI was 26.9 ± 6.2 kg/m2. Most women were of Australian (44.5%) or European (33.2%) descent, and 91 (34.3%) were primigravid. Six (2.3%) developed pre-eclampsia, and 11 (4.2%) developed GH. The remainder did not develop hypertensive complications during follow-up. Nine babies (3.4%) were SGA at birth, three of which were born to mothers who had pre-eclampsia. Twenty-one (7.9%) women developed GDM. Fifteen women (5.75%) had pregestational diabetes, 23 women (8.7%) had chronic hypertension, and ten women (3.8%) had a past history of renal or urinary tract disease. Of these, only two of the women with hypertension developed pre-eclampsia. A family history of diabetes, hypertension, and CKD was present in 110 (41.7%), 107 (40.5%), and 13 (4.9%) women, respectively. Four of six women who developed pre-eclampsia were nulliparous; the other two women had no history of pre-eclampsia.

At the time of booking the mean systolic blood pressure (SBP), mean diastolic blood pressure (DBP), and mean arterial pressure (MAP) were significantly higher in women who later developed gestational hypertension than in those who remained unaffected. Although the mean SBP, DBP, and MAP also tended to be higher at study entry in those who developed pre-eclampsia, this difference was not significant.

Overall, the median spot urinary ACR measured between 17 and 20 weeks of gestation was 28 mg/mmol (IQR 16–46 mg/mmol). Women who subsequently developed pre-eclampsia had a significantly higher initial spot urinary ACR (median 50 mg/mmol; IQR 33–90 mg/mmol) in comparison with women suffering gestational hypertension (median 27 mg/mmol; IQR 8–35 mg/mmol; = 0.035) and unaffected women (28 mg/mmol; IQR 16–46 mg/mmol; = 0.036). Mothers of infants with SGA also had a significantly higher median spot urinary ACR between 17 and 20 weeks of gestation than others (median 55 versus 28 mg/mmol; IQR 32–76 versus 16–45 mg/mmol; = 0.008) (Figure 1). However, no significant correlation was found between spot urinary ACR and birthweight (r = 0.087; = 0.160) or gestational age at the time of delivery (coefficient = −0.083; = 0.179). There was no difference in ACR between women with a history of renal or urinary tract disease, chronic hypertension, or diabetes in comparison with those who did not. As expected, babies of mothers with pre-eclampsia were smaller and delivered earlier (see Table 1).

image

Figure 1.  Graph detailing median and IQRs of pre-eclampsia, unaffected, and SGA groups. Individual values for particular subsets are as indicated.

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The sensitivity and specificity of spot urinary ACR were calculated from the ROC (Figures 2 and 3). The optimum spot urinary ACR to predict pre-eclampsia was 35.5 mg/mmol, which had a test sensitivity of 83.3%, specificity of 61.2%, PPV of 63.0%, and NPV of 78.6%. The area under the curve (AUC) was 0.753 (95% CI 0.590–0.916; = 0.034). The relative risk of developing pre-eclampsia in those with a urinary ACR ≥ 35.5 mg/mmol was 7.8 fold higher than in those who had a urinary ACR of <35.5 mg/mmol. Based on ROC, the optimum urinary ACR for predicting SGA was 34.5 mg/mmol, with a test sensitivity of 77.8%, specificity of 59.2%, PPV of 65.6%, and NPV of 72.7%. AUC was 0.762 (95% CI 0.625–0.898; = 0.008).

image

Figure 2.  Receiver operating curve to predict pre-eclampsia.

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image

Figure 3.  Receiver operating curve to predict SGA.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

Pre-eclampsia remains a leading cause of maternal and fetal mortality and morbidity.1 In the last few years some of the molecular mechanisms underlying pre-eclampsia have been clarified. Studies have shown that alterations in the regulation and signalling of angiogenic pathways that contribute to the inadequate cytotrophoblast invasion are seen in pre-eclampsia. High levels of anti-angiogenic factors (soluble fms-like tyrosine kinase-1 and soluble endoglin) and low levels of pro-angiogenic factors (VEGF, PIGF) may subsequently contribute to widespread maternal endothelial dysfunction and the clinical syndrome of pre-eclampsia.10,20,29,30 Endothelial dysfunction has been demonstrated as early as 22 weeks of gestation,12 and the level of anti-angiogenic factors start rising as early as 17 weeks of gestation.10 It could be expected that microalbuminuria, a marker of endothelial dysfunction, might also be apparent by this time, although perhaps at a level undetectable by immunochemical methods. However, HPLC is more sensitive than any of the immunochemical methods, and could be used to estimate a low level of excreted albumin in urine.

To our knowledge, this is the first study that has measured spot urinary ACR in a pregnant population by HPLC. We found that the spot urinary ACR at 17–20 weeks of gestation was significantly higher in women who subsequently developed pre-eclampsia and/or SGA, than in those who did not. Figure 1 shows that although the ACRs for the pre-eclamptic and SGA groups were significantly higher than average, some still fell within the normal range, and therefore this may well limit the usefulness of this test in clinical practice. Comparing the 110 subsequently unaffected patients whose ACR overlapped with those destined to develop pre-eclampsia revealed no marked differences in blood pressure, age, parity, or gestational stage. Other factors that might have been relevant but were not assessed included the woman’s level of activity before the urine was collected and the time of day, either of which could have affected the ACR. Although a 24-hour collection of urine is the gold standard for quantifying urinary albumin, a spot ACR was used in this study because it was more likely to reflect clinical practice for a screening test, and because an attempted 24-hour collection would very likely have been futile and erroneous.19,31 Some but not all past studies have shown an excellent correlation between a spot urinary ACR and albumin excretion in a 24-hour urine sample in normal pregnancy and pre-eclampsia.32–36

Many previous studies have measured microalbuminuria in an attempt to predict pre-eclampsia in early pregnancy, postulating that the stage of gross proteinuria is preceded by the stage of microalbuminuria. Using regression models, Bar et al.16 identified the presence of microalbuminuria at 28–30 weeks of gestation to be predictive of subsequent pre-eclampsia, with an odds ratio of 2.1 (95% CI 1.26–3.53). Shaarawy found that microalbuminuria at 10–12 weeks of gestation had 50% sensitivity, 58% specificity, 50% PPV, and a 91% NPV for the later development of pre-eclampsia.18 Poon found that, although the logarithmic conversion of urinary ACR at 11–13 weeks of gestation helped predict pre-eclampsia, it did not provide additional value above clinical maternal variables.37 It has also been shown that in hypertensive women, the presence of microalbuminuria at 28–30 weeks of gestation has a high specificity and positive predictive value when predicting pre-eclampsia.38 Similarly, its presence even before pregnancy in pregnant women with type-1 diabetes is a strong predictive marker.39 It is likely that the diversity of results from these studies indicates that other risk factors apart from microalbuminuria can predict the establishment of pre-eclampsia, particularly when severe or presenting early in gestation.

A number of studies have used HPLC for urinary albumin measurement in non-pregnant populations, and have demonstrated that the level of albumin detected in the urine by HPLC is significantly greater compared with conventional assays. However, this fact has not been verified in pregnant women. Previous studies from non-pregnant populations have reported urinary albumin excretion to be between two and four times higher when measured by HPLC, than by one of the immunochemical methods.21–23,40,41 The median value of spot urinary ACR (28 mg/mmol; IQR 16–46 mg/mmol) in uncomplicated pregnancy was over 20 times higher in our study compared with other studies using one of the conventional methods.42–44 This suggests that the level of immunounreactive albumin excreted in pregnancy is much greater than in the non-pregnant woman, but the reason for this is unknown. It can be conjectured to be the result of incomplete processing by the lysosomal pathway, as in diabetes (although in our study, women with diabetes or hypertension did not have higher levels than the non-diabetic women). Women with a raised ACR, measured by HPLC rather than by immunochemical measurements, may have a higher cardiovascular mortality risk.45,46

The major limitation of our study was that the number of affected cases was small. The findings should therefore be interpreted with caution. In addition, we were unable to calculate customised birthweight centiles, as the necessary maternal and fetal data were not collected during the study. This prevented us from defining intrauterine growth restriction (IUGR) as an end point in the study. Furthermore, the limited numbers precluded a regression analysis to investigate factors that contributed to changes in urinary ACR and pre-eclampsia. Finally, as the HPLC assay is relatively expensive, our results do not suggest that it is likely to be useful as a screening test, although this can only be established by a much larger study. Additional studies are now required to explore the biochemical nature and underlying molecular mechanisms of the excreted albumin in pregnancy, to confirm the ‘normal’ range of ACR by HPLC, and to corroborate the predictive potential of this technique.

Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

When urinary albumin is measured by HPLC, spot urinary ACR values are higher in uncomplicated pregnancy in comparison with conventional methods. A spot urinary ACR value of ≥35.5 mg/mmol (measured by HPLC between 17 and 20 weeks of gestation) predicted future pre-eclampsia with a sensitivity and specificity of 83.3% and 61.2%, respectively. Additional studies and a cost–benefit analysis are required to confirm these findings before recommending this test for screening purposes. The molecular nature of the excreted albumin fractions and the underlying mechanisms of proteinuria in early pregnancy also need to be explored.

Contribution to authorship

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

SB analysed the data and wrote the article. AK was involved in planning strategies, recruitment, and sample collection. SR was involved in conducting patient visits and in the collection of data. RM was involved in planning strategies and in the enrolment of the patients. LPM was involved in planning strategies, recruitment, analysis of data and writing the article.

Details of ethics approval

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

The study was approved by the Melbourne Health Research and Ethics Committee on 16 June 2004; ref. no. 2004.075.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References

We thank the patients for their participation in the study and the nursing staff of Sunshine Hospital for their co-operation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Disclosure of interests
  9. Contribution to authorship
  10. Details of ethics approval
  11. Funding
  12. Acknowledgements
  13. References
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