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Student Original Article |
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Karthik N Rao, Krishnamurthy U, Vasudha KC
Department of Biochemistry, M S Ramaiah Medical College, Bangalore 560054, India
Corresponding Author: Dr. Krishnamurthy U, Department of Biochemistry, M S Ramaiah Medical College, Bangalore 560054. India.
Email: kmurthyu@yahoo.co.in
Abstract
Back ground and Objectives: Urine albumin measurements are used in patient care, epidemiological studies,
and in clinical trials for which the samples are often stored at different temperatures and for a variable
duration of time. Proper storage technique is the need of the hour to preserve the albumin concentration as
measurement errors can have serious implications on the study results. Therefore, this study intended to
observe the effect of storage temperatures and duration of storage on low concentrations of urinary albumin
(microalbuminuria) in patients of diabetes mellitus.
Methods: Single random urine sample which is dipstick negative for protein was collected from 44 patients
with diabetes mellitus. The samples were analysed immediately and then stored at -20ºC and 4ºC for 1week
and 4weeks and reanalysed. The urinary albumins in the above samples were re-estimated at the end of 1
week and again at the end of 4 weeks. The difference in albumin levels between the fresh samples and after
storage was noted and statistically analysed.
Results: Urinary albumin concentration decreased with the increase in duration of storage. Storage at -20°C
showed a larger decrease in urinary albumin concentration as compared to those stored at 4°C.
Conclusion: Estimation of microalbumin should be performed in a fresh sample of urine and if storage is
imperative, the sample may be stored at 4°C and the estimation carried out in less than a week.
Key words: Albuminuria; preservation;diabetes mellitus.
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Introduction
Urine albumin measurements are used in patient
care, epidemiological studies, and in clinical trials
for which the samples are often stored at lower
temperatures and for a variable duration of time.
Practically, most of the samples are stored at -20ºC
or 4ºC. Studies have shown that freezing the urine
samples for storage can result in measurement
errors, especially in samples with low
concentrations [1,2,3]. Also, there are conflicting
reports on choosing the temperature for urine
storage [4,5]. Importantly, this change in urinary
albumin concentrations (UACs) induced by freezing
can result in a significant decrease in predictive
properties of urine albumin for mortality. The
variations in concentrations can also occur because
of the different methods of estimations where the
properties of albumin play a key role [6]. Therefore,
it is necessary for every laboratory to derive its own
protocol to store its urine samples depending on
their method of estimation. Urine matrix
components exert potentially significant effects on
assays for urine albumin which varies with the
population and their dietary habits [7,8]. Since
there are no such studies in the Indian population,
there is a need for the same. This study reports here
the determinations of low albumin concentration by
immunoturbidimetric method, of fresh samples,
frozen aliquots (stored at -20 ºC for one and four
weeks), and refrigerated samples (stored at 4 ºC for
one and four weeks). Albumin was measured in 220
samples of urine obtained from 44 subjects with
diabetes mellitus.
Methods
This study was under taken in the department of
Biochemistry, M S Ramaiah Medical College,
Bangalore after the approval of the research and
ethical committee. This study was conducted during
the months of June & July, 2010. Type 2 diabetes
mellitus patients attending the OPD of M S Ramaiah
medical college teaching hospital were tested for
urinary albumin by albustix method. Patients with
gross proteinura were excluded and albustix
negative diabetic patients (n=44) were recruited
into the study. Turbid urine samples were also
excluded from the study. Single random urine
samples were collected and aliquoted into 5
polyurethane bottles. One sample was analysed
afresh, 2 were frozen at -20ºC and 2 were
refrigerated at 4ºC. Later the samples were
reanalysed after 1 week and 4 weeks of duration of
storage. The difference in albumin levels between
the fresh samples and the stored samples was noted
and statistically analysed. Urinary albumin was
measured by immunoturbidimetric method, using
fully automated analyser (ROCHE, Cobas 6000).
Quantitative data was summarised to test the
difference in mean values obtained for different
temperatures and period of storage. A statistical
analysis was done by using SPSS 14.0. Analysis of
variation (ANOVA) was employed to understand the
variables. Data is presented as mean (SD) & mean
(SD) percentage changes in albumin. Multiple
comparisons were perfored with log-transformation of variables and significance was
taken at 0.05 level.
Results
For analysis, results were categorised into 9 groups
which are as follows:
Group 1; included the fresh sample values which
acted as the controls.
Group 2; were the samples stored at 4ºC for 1 week
Group 3; samples stored at -20ºC for 1 week
Group 4; samples stored and 4ºC for 4 weeks
Group 5; samples stored at -20ºC for 4 weeks
The urinary albumin concentrations of the 5 groups
were tabulated. The difference between the values
in fresh samples (Group 1) and the values in stored
samples i.e. Group 2, 3, 4 & 5 were tabulated as
Group 6, 7, 8 & 9 respectively. Mean ± SD values of
the groups are shown in the tables 1 & 2.
Table 1 shows that there is fall in urinary albumin
values with storage at both 4 °C and -20°C. From
table 2 it is found that storage for 1 and 4 weeks at
-20°C resulted in mean (±SD) urine albumin changes
of 40.78 (± 28.82) % & 62.18 (± 21.39) % respectively.
Storage at 4 °C resulted in 32.14 (± 30.74) % changes
after 1 week and 52.17 (± 24.77) % changes after 4
weeks. Table 3 shows the multiple comparisons of
the changes after storage. It shows Group 6 is
significantly different from groups 7, 8 & 9.
Table 1- Mean ± SD values of fresh and stored
samples
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Group |
Mean ± SD (mg/L) |
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Group 1 |
17.02 ± 21.04 |
Group 2 |
11.55 ± 17.3 |
Group 3 |
10.08 ± 16.82 |
Group 4 |
8.14 ± 13.8 |
Group 5 |
6.43 ± 12.00 |
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Table 2- The Mean± SD of amount of fall in
concentration after storage
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Group |
Mean ± SD (mg/L) |
In percent |
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Group 6 |
17.02 ± 21.04 |
32.14 ± 30.74 |
Group 7 |
11.55 ± 17.3 |
40.78 ± 28.82 |
Group 8 |
10.08 ± 16.82 |
52.17 ± 24.77 |
Group 9 |
8.14 ± 13.8 |
62.18 ± 21.39 |
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Table 3- Multiple comparisons of the changes
after storage by Log transformation
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Group |
Mean of the changes |
Significance |
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Group 6 Vs Group 7 |
-0.0928 |
0.050 |
Group 6 Vs Group 8 |
-0.1604 |
0.001 |
Group 6 Vs Group 9 |
-0.2320 |
0.000 |
Group 7 Vs Group 8 |
-0.0675 |
0.169 |
Group 7 Vs Group 9 |
-0.1391 |
0.005 |
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Discussion
The measurement of albumin in urine is important in
evaluating kidney disease in people with diabetes
mellitus, hypertension, or possible adverse health effects from exposure to nephrotoxins [9].
Microalbuminuria is persistent proteinuria below
level of detection by routine dipstick testing but
above normal (30–300 mg/day in a 24 hour urine
sample or 20-200 mg/L in a spot sample). It is an
early predictor of diabetic nephropathy and also a
marker of cardiovascular morbidity and mortality
[10,11]. Therefore there is a great interest in
accurate measurement of very low levels of urinary
albumin. Epidemiological studies require that
several samples be collected, stored for various
periods and eventually transported to the
laboratory before the assay. Hence, the
temperature for storage should be appropriate so as
to preserve the concentration of albumin at almost
the same as that of fresh sample [12]. In this study
we tried to determine the effect of storage
temperatures of 4°C and -20°C when stored for 1
week and 4 weeks. It was observed that urinary
albumin concentration decreased with increase in
duration of storage and storage at -20°C showed
larger decrease in urinary albumin concentration as
compared to samples stored at 4°C, for the storage
duration of both 1 week & 4 weeks. On comparing
the amount of decline in albumin concentration,
group 6 had the least fall (32%) and group 9 (62%)
had the maximum fall in albumin concentration.
Therefore, it is suggested that urine samples should
be refrigerated rather than frozen and analysed at
the earliest. It is thought that freezing urine
specimens may cause conformational change in
urinary proteins, resulting in a partial precipitation
yielding low values. Precipitation of albumin at
-20°C may be responsible for decreased values in
this study. Also, it is found by comparing the various
techniques of albumin estimation that antigen
based techniques yielded low values as freezing
affected the antigenic properties of albumin [13].
This study used immunoturbidimetric method which
is based on the antigen–antibody reaction.
Therefore, altered antigenic properties may be
responsible for decrease in the albumin values at
both the temperatures in this study. This false
decrease could lead to classifying the albumin
content as normal rather than “microalbuminuria”,
a stage which can be reversed with tight glycemic
control, and thus missing an opportunity for
intervention. An exploration for better technique
for storage is still at large. This study is limited by
the fact that it did not investigate the matrix of the
urine to reveal the probable factors that can cause
the decline in albumin concentration.
It can be concluded that urine specimens for
microalbuminuria measurements should either be
analysed as fresh specimens or stored at 4ºC and
assayed as soon as possible preferably within a week.
Acknowledgements
The authors wish to acknowledge the STS 2010
Programme of Indian Council of Medical Research
under which this study was executed and to M S
Ramaiah Medical College Student Research
Committee for funding this project.
Key Points
- Detecting low albumin concentrations in urine
samples (microalbuminuria) is important for clinical management.
- Storage of urine samples for microalbuminuria
determination results in reduced estimations.
- There is a greater reduction of concentration if
the urine samples are stored at -20°C as
compared to 4°C.
- The least decline in urine albumin
concentrations is seen if the samples are
estimated within a week and stored at 4°C.
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