University of Medical Technology, Mandalay

URINARY CALCIUM CREATININE RATIO

AND ALBUMIN CREATININE RATIO
IN THE DIAGNOSIS OF PRE-ECLAMPSIA

A Protocol Submitted for

Degree of Master of Medical Technology
(Medical Laboratory Technology)
Phyu Hnin May
B. Med. Tech (Medical Laboratory Technology)
2018
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1. TITLE

Urinary Calcium Creatinine Ratio and Albumin Creatinine Ratio in the Diagnosis of
Pre-eclampsia
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2. BACKGROUND

Pre-eclampsia is the development of hypertension and proteinuria

(>300mg urinary protein in 24 hours) after 20th week gestation. Hypertension is
defined as blood pressure greater than 140/90 mm Hg or a rise in blood pressure of
30/15 mm Hg from the baseline confirmed by two measurements 6 hours apart
(Gaurang et al., 2015).
Around the world, an estimated 529,000 women die during pregnancy or
childbirth. Incidence of preeclampsia is around 5-10% of all pregnancies, and in
developing countries around 4-18%. It has been reported that, pre-eclampsia is a
major cause of both maternal and fetal morbidity and mortality (Bringman et al.,
2006). In Myanmar, the number of pre-eclampsia patients is increasing year by year.
According to Central Women’s Hospital (Mandalay) Annual hospital statistics,
approximate 10% of total admission was pre-eclampsia and 11% of operative delivery
were due to pre-eclampsia.
Unfortunately, the pathophysiology of this multisystem disorder, characterized
by abnormal vascular response to placentation, is still unclear. Delivery is the only
curative treatment for pre-eclampsia (Anjali & Meena, 2016). Early diagnosis and
treatment helps to reduce it to a minimum and therefore the importance of identifying
the women at risk. The most important feature in toxemia of pregnancy is
hypertension which is supposed to be due to vasospastic phenomenon in kidney,
uterus, placenta and brain (Gaurang et al., 2015).
Pre-eclampsia can cause changes in virtually all organ systems, most notably
the cardiovascular, renal, hematologic, and immunologic systems. Some of these
changes are present before the clinical diagnosis of pre-eclampsia, as has been clearly
demonstrated by several investigators (Hellen et al., 1988).
The predominant pathology in pre-eclampsia is endothelial dysfunction, which
sets in as early as 8-18 weeks, however, the signs and symptoms appear in the late
mid trimester. In order to arrest the disease process in the initial stages or to prevent
complications especially in women predisposed to pre-eclampsia, various predictors
have been proposed time. Proteinuria and alterations in calcium excretion are
common features of various forms of hypertension and renal disorders. This
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prospective clinical hospital based study is done to find out the validity of urinary
microalbumin and calcium to creatinine ratio, and on the basis of results, to identify
high risk population for the development of pregnancy hypertension. These patients
can be benefited from intense observation and aggressive treatment as primary
preventions (Aherwar & Ahirwar, 2016).
There is hypercalciuria during normal pregnancy, while pre-eclampsia is
associated with hypocalciuria and low urinary calcium to creatinine ratio. This
phenomenon occurs early enough and persists throughout gestation, so it is useful for
early identification of patients at risk. The involvement of the renal system in pre-
eclampsia is in the form of endotheliosis and there is alteration in renal functions. It is
found that urinary calcium excretion can be markedly decreased early in the course of
pre-eclampsia, even before the clinical appearance of signs and symptoms. This is the
basis for using urinary calcium to creatinine ratio as a predictor of pre-eclampsia
(Anjali & Meena, 2016).
Estimation of calcium and creatinine in a spot urine sample is simple test, non-
invasive, and is easy to perform and hence assures good patient compliance. It has a
good predictive value and hence justifies the cost and is suited to be adopted as
screening test for pre-eclampsia (Narendra, Kshama & Manjushree, 2016).
In normal pregnancy, urinary protein excretion increases substantially. The
upper limit of normal protein excretion in pregnancy is <300 mg in 24 hours. Renal
function changes in pre-eclampsia have been documented and several prospective
studies indicate that at least some of these changes are present before the clinical
diagnosis of pre-eclampsia. So, urinary protein excretion increases in pre-eclampsia.
Proteinuria has classically been an important sign in the diagnosis of pre-eclampsia.
However, customary dipstick methods for detecting proteinuria fail to detect minimal
elevations in urinary excretion of albumin that may be present before other clinical
signs and symptoms of pre-eclampsia. With the recent development of the
radioimmunoassay for detection of microalbuminuria, it is now possible to detect
minimal elevations in albumin excretion that would have gone unnoticed in the past
(Hellen et al., 1988). Microalbuminuria refers to sub-clinical elevation of urinary
albumin excretion. It has shown to be preceded the development of chronic renal
failure in patients with insulin dependent diabetes mellitus, and may be evidence of
renal involvement in hypertension. The presence of microalbuminuria should be an
important clinical finding in pregnant women (Salako et al., 2003). The traditional
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method for diagnosing microalbuminuria, collection of 24 hours urine samples, is

time-consuming, cumbersome, and may be associated with collection errors and poor
compliance. Albumin to creatinine ratio in a random urine sample is a more
convenient method of detecting microalbuminuria (Singh et. al., 2013).
So, microalbuminuria using albumin creatinine ratio (ACR) test is a useful
method for predictor of development of subsequent pre-eclampsia as there is a
significant relationship between high ACR and development of pre-eclampsia. The
National Kidney Foundation now recommends the spot albumin creatinine ratio
(instead of 24 hour urine collection) to diagnose proteinuria in most situations,
without specific mention of pregnancy (Anne et al., 2008).
Several researchers have demonstrated the efficacy of decreasing urinary
calcium to creatinine ratio and high albumin creatinine ratio in the prediction of pre-
eclampsia, whereas others have not found it as useful. Hence there is a need to
determine the predictive values of these tests and thereby their feasibility as screening
tools to identify women at risk of developing pre-eclampsia (Sheela, Beena &
Mhaskar, 2011).
Therefore, this study will compare urinary calcium creatinine ratio and albumin
creatinine ratio in 20-34 weeks of gestation of pregnant women and find out whether
urinary calcium creatinine ratio is a better diagnostic marker for pre-eclampsia or not.
By doing so, this study hopes to provide information on predicting the risk of disorder
and start preventive therapy at early stages to prevent maternal and feto-neonatal
morbidity and mortality.
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3. RATIONALE

Pre-eclampsia is a multisystem endothelial disease that leads to glomerulo

endotheliosis, and in severe cases it may lead to renal impairment and failure.
Hypertension is one of the most common complications of pregnancy and a leading
cause of maternal and perinatal morbidity and mortality. It forms deadly triad along
with hemorrhage and infection and contributes greatly to maternal morbidity. An early
diagnosis and treatment helps to reduce it to a minimum, and therefore it is important
to identify women at risk at the earliest. Various methods for screening have been
studied to identify pregnant women at risk of development of pre-eclampsia, but no
ideal screening test has been identified so far.
Measurement of spot urinary calcium creatinine ratio and microalbumin using
albumin creatinine ratio is non-invasive, inexpensive, and easy to carry out. Their use
as early predictors will help to identify pregnant females at high risk of pre-eclampsia
and prompt the initiation of education and prophylactic interventions, thus minimizing
the severity of pregnancy induced hypertension.

In this study, random urinary calcium creatinine ratio and albumin creatinine
ratio are studied. This study would like to compare the sensitivity, specificity, positive
predictive value, negative predictive value and accuracy of urinary calcium creatinine
ratio and albumin creatinine ratio in pre-eclampsia patients to determine which is the
better diagnostic marker in pre-eclampsia.
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4. OBJECTIVES

4.1. General Objective

To compare the diagnostic accuracy of urinary calcium creatinine ratio and

albumin creatinine ratio in the diagnosis of pre-eclampsia among the pregnant
women

4.2. Specific Objectives

1. To determine the proportion of pre-eclampsia among the pregnant women by

using calcium creatinine ratio
2. To determine the proportion of pre-eclampsia among the pregnant women by
using albumin creatinine ratio
3. To determine the proportion of pre-eclampsia among the pregnant women by
senior consultant gynecologist’s clinical diagnosis
4. To compare the diagnostic accuracy of urinary calcium creatinine ratio and
albumin creatinine ratio in the diagnosis of pre-eclampsia among the pregnant
women

5. EXPECTED OUTCOME
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6. METHODOLODY

6.1. Study Design

Laboratory based analytic study

6.2. Study Site

Central Women’s hospital, Mandalay

Department of Clinical Pathology, Mandalay General Hospital
Department of Medical Laboratory Technology, University of Medical
Technology, Mandalay

6.3. Study Period

From April 2018 to March 2019

6.4. Reference Population

All pregnant women

6.5. Study Population

All pregnant women with 20-34 weeks of gestation at OPD or admitted to

CWH.
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6.6. Selection Criteria

6.6.1. Inclusion Criteria

All pregnant women with 20-34 weeks of gestation

6.6.2. Exclusion Criteria

1. Known case of Chronic renal disease

2. History of taking calcium channel blocker ( >7days )
3. History of high dose of calcium supplementation
4. Known case of underlying disorder of hypertension

6.7. Sample Size Determination

6.7.1. Sample Size Calculation

Sample size was calculated by using the following formula with specified
absolute precision for both of sensitivity and specificity;

n = Minimum required sample size

p = Proportion for sensitivity and specificity
d = Absolute precision

Detailed calculations were done in n4studies with the following inputs,

Required Sample Size based on sensitivity of CCR;
Sensitivity = 0.64 (Narendra, Kshama & Manjushree, 2016)
d = 0.1
Minimum required sample size = 89
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Required Sample Size based on specificity of CCR,

Specificity = 0.97 (Narendra, Kshama & Manjushree, 2016)

d = 0.1
Minimum required sample size = 12

Minimum required sample size for both sensitivity and specificity is 101.
So, for the sake of safety, at least 110 pregnant women will be studied in this
research.

6.7.2. Sampling Procedure

From all pregnant women with 20-34 weeks of gestation, all pregnant women
will be selected according to inclusion and exclusion criteria.

6.8. Detailed Procedure

All pregnant women with 20-34 weeks of gestation are included. According to
inclusion and exclusion criteria, all pregnant women admitted to CWH will be
selected. Patients’ past medical history and charts will be explored, and then
explanation about the study will be done and informed consent will be obtained (see
details in appendix III). After that, a sterile urine container will be given to the patient
and the patient will also be instructed thoroughly for urine collection. The spot urine
sample will be collected. Then, the samples will be transported to chemical pathology
department of MGH. The urine sample will be analyzed for calcium, creatinine and
microalbumin level by Pentra 400 auto analyzer (see details in appendix IV, V, VI).
Urinary calcium creatinine ratio and albumin creatinine ratio will be calculated by
formula (see details in appendix VII, VIII). The collected data will be recorded on Pro
forma (see details in appendix I). And then, all pregnant women will be followed up at
the time of delivery about the presence or absence of pre-eclampsia. Data entry will
be done using Microsoft Excel work sheet. The data will be analyzed by SPSS version
20.1 software.
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Materials and Methods

6.8.1. Materials

Materials for urinary calcium determination

1. Disposable sterile urine container to collect urine

2. Adjustable 100-200 µL automated pipette (Human, Wiesbaden Germany)
3. Pipette tips
4. Sample cups specially designed for Pentra-400 clinical chemistry bench-top
automatic analyzer
5. ABX Pentra Autoanalyzer
6. Calcium reagent kit ( HORIBA, ABX , France)
7. Calibrator: ABX Pentra Multical (HORIBA, ABX , France)
8. Control: ABX Pentra control N/P (HORIBA, ABX, France)

Materials for urinary creatinine determination

1. Disposable sterile urine container to collect urine

2. Adjustable 100-200 µL automated pipette (Human, Wiesbaden Germany)
3. Pipette tips
4. Sample cups specially designed for Pentra-400 clinical chemistry bench-top
automatic analyzer
5. ABX Pentra Autoanalyzer
6. Creatinine reagent kit ( HORIBA, ABX , France)
7. Calibrator: ABX Pentra Multical (HORIBA, ABX , France)
8. Control: ABX Pentra control N/P (HORIBA, ABX, France)

Materials for urinary microalbumin determination

1. Disposable sterile urine container to collect urine

2. Adjustable 100-200 µL automated pipette (Human, Wiesbaden Germany)
3. Pipette tips
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4. Sample cups specially designed for Pentra-400 clinical chemistry bench-top

automatic analyzer
5. ABX Pentra autoanalyzer
6. Microalbumin reagent kit ( HORIBA, ABX , France)
7. Calibrator: ABX Pentra Micro ALB Cal (HORIBA, ABX , France)
8. Control: ABX Pentra Micro ALB Control L/H (HORIBA, ABX, France)

6.8.2. Methods

After receiving informed consent form, a sterile urine container will be given
to patient after giving detailed instruction. The urine sample will be collected. The
urine sample will be immediately sent to laboratory. Urinary calcium, creatinine and
microalbumin will be analyzed. Urinary calcium creatinine ratio and albumin
creatinine ratio will be calculated.

6.9. Data Processing and Analysis

All data from each pregnant woman will be recorded in the Pro forma
(Appendix I). Background characteristic of patients including age, gestation period
and gravidity will be collected using pro forma. Then, age, gestation period and
gravidity will be categorized into groups and distribution will be presented as
frequency, percentage table. All data collected will be entered into Microsoft Office
Excel 2010 software. The data will be will be analyzed by SPSS version 20.1
software.
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7. STUDY TIMELINE

2018 2019

Activities
April

April
June

June
May

May
Aug

Nov

Aug

Nov
Mar

July

Mar

July
Dec

Dec
Feb

Sep

Feb

Sep
Oct

Oct
Jan

Jan
1. Preparation
of protocol
2. Protocol
submission
to Board of
Studies
3. Study of the
research
project
4. Writing up
the
dissertation
5. Submission
of the
dissertation
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8. ETHICAL CONSIDERATIONS

Ethical clearance will be obtained from the institutional ethical board of

University of Medical Technology, Mandalay. Written informed consent will be
obtained from patients after thorough explanation about the study. It will be carried
out urinary calcium, creatinine and microalbumin in all pregnant women. The urine
sample will be used for this research only. There will be no intervention to the
management of patients involved and all expenses for this study will be done by the
investigator. The patient does not need to pay for the cost of laboratory testing.
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9. BUDGET ESTIMATION AND BREAKDOWN

No. Item Quantity Estimated Budget

(Kyats)
1 Urine Container 150 15,000
2 Calcium Reagent Kit 1 bot 65,000
3 Calcium Calibrator 1 bot 35,000
4 Calcium Control 1 bot 40,000
5 Creatinine Reagent Kit 2 bots 50,000
6 Creatinine Calibrator 1 bot 35,000
7 Creatinine Control 1 bot 40,000
8 Microalbumin Reagent Kit 1 bot 166,000
9 Microalbumin Calibrator 1 bot 231,000
10 Microalbumin Control 1 bot 507,000
11 Cuvette 5 boxes 150,000
12 Sample Cup 1 pack 70,000
TOTAL 1,404,000 kyats
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10. RESEARCH TEAM

(1) Ma Phyu Hnin May

Principal Investigator
Post-graduate student
Department of Medical Laboratory Technology,
University of Medical Technology, Mandalay

(2) U Win Min Than

Supervisor
Associate Professor
Department of Medical Laboratory Technology,
University of Medical Technology, Mandalay

(3) Professor Dr. Daw Khin Than Nyunt

Professor/Head
Department of Medical Laboratory Technology,
University of Medical Technology, Mandalay.
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11. CURRICULUM VITAE OF RESEARCHER

Name: - Phyu Hnin May

NRC No: - 9/khamasa(Naing)037521
Date of Birth: - 15.7.1992
Sex: - Female
Nationality: - Myanmar
Religion: - Buddhist
Father name - U Win San
E-mail address: - phyuhninmay.111@gmail.com
Contact Phone No: - +95 9 402599210
Education Qualification - B.Med.Tech (Medical Laboratory
Technology)
University of Medical Technology,
Mandalay
Present Education - Post-graduate Student
First year, M.Med.Tech (Medical Laboratory
Technology)
Department of Medical Laboratory
Technology, University of Medical
Technology, Mandalay.
Rank and Work Address: - Medical Technologist, Department of
Pathology, Mandalay General Hospital.
Service Experience - 3 years and 9 months
Research Experience - No
Address: - 484, Tampawadi, Kyae-Thoon Quarter,
Chanmyatharsi Township, Mandalay.

12. AIMED ETHICAL REVIEW BOARD FOR ETHICAL

CLEARANCE PROCEDURE
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(A) Informed consent form

1. Title of research Urinary Calcium Creatinine Ratio and Albumin

Creatinine Ratio in the diagnosis of Pre-eclampsia

2. Researcher Ma Phyu Hnin May, Post graduate student,

Department of Medical Laboratory Technology,
University of Medical Technology, Mandalay

3. Organization University of Medical Technology, Mandalay

4. Introduction This study will be done to compare Urinary

Calcium Creatinine Ratio and Albumin Creatinine
Ratio in the diagnosis of Pre-eclampsia

5. Purpose of study To compare Urinary Calcium Creatinine Ratio and

Albumin Creatinine Ratio in the diagnosis of Pre-
eclampsia

6. Type of research Laboratory based analytic study

7. Subject All pregnant women with 20-34 weeks of gestation

recruitment and admitted to CWH during the study period.
selection of study
8. Procedures All pregnant women will be selected for this study.
The research purpose, procedure and benefit of the
study will be explained clearly to the patients. After
getting informed consent form, a sterile urine
container will be given to the patient and the patient
will also be instructed thoroughly for urine collection.
The samples will be transported to chemical pathology
department of MGH. Urinary calcium, creatinine and
microalbumin will be analyzed and urinary calcium
creatinine ratio and albumin creatinine ratio are
calculated.

9. Duration of study No more extra time will be needed for the participants.
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At the time of delivery, I will connect and question

about the presence or absence of pre-eclampsia.

10. Potential risks of There will be no serious risk or any other

the participants complications.

11. Potential benefits By participating in this study, there will be able to

of the study give additional information about the diagnosis of pre-
eclampsia.

12. Incentives There will be no extra cost and no financial gain for
the patient by participating in this research study. All
expenses for this research will be paid by researcher.

13. The result of the The result can describe the better diagnostic marker
study for the diagnosis of pre-eclampsia.

14. Confidentiality Patient name will never be expressed and coding

system will be used instead. The data from this
research will be kept confidentially and will be used
only for research purpose.

15. Sharing the The information data will be disclosed only in the
results educational discussion and presentation. These data
will not be published in public news journal except
medical journal.

16. Further use of After the research, all the samples will be destroyed
samples according to guidelines for good laboratory practice
and will not be used for other purposes.

17. Right to refuse Participant can decide independently whether to

participate in this research by themselves. Although
she does not participate in this study, it will have no
effect on the treatment. Moreover, the participant can
withdraw from this study at any time.

18. Whom to contact If you have any questions you may ask anytime, even
after the study has started. If you wish to ask questions
later, you may contact me: Ma Phyu Hnin May, 1st
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year M.Med.Sc (Medical Laboratory Technology)

student, Department of Medical Laboratory
Technology, University of Medical Laboratory
Technology, Mandalay, Phone 09-402599210.
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(B) Patient consent form

I have read this consent form. I have already discussed it with the
investigator to my satisfaction. I understand that my permission is voluntary. I
know enough about the purpose, methods, risks and possible benefits of the study. I
know that I can call the investigator if I have any questions. I have a chance to ask
questions. I feel that all of my questions have been answered to my satisfaction.
I was well informed about the research. I understand the procedure of
research well. I consent voluntarily for my participation as a participant in this
research.

Signature of patient or guardian

Name ………………………..
Identification Card ……………………….. Fingerprint of Left thumb
Address ……………………….. (For illiterate patient)

Signature of witness
Name ………………………..
Rank ………………………..
Department ………………………..

Signature of researcher
Name .....................................

(C) Statement by researcher

I have accurately read out the information sheet to the potential participant. In
addition to information in this consent form, I have offered an opportunity for further
explanation of the risks and discomforts, which are or may be associated with this
study. I also answered any further questions relating to it.

Signature ----------------- Hand phone: 09-402599210

Ma Phyu Hnin May Email: phyuhninmay.111@gmail.com