Journal of Automatic Chemistry Vol. 11, No. 2 (March-April 1989), pp.

80-83

An evaluation of osmolality measurement by

freezing point depression using
micro-amounts of sample

G. Koumantakis and L. E. Wyndham Experimental

Biochemistry Department, Royal North Shore Hospital of Sydney, Sydney, 2065,
Australia The Advanced micro-osmometer was supplied by Stan-
sens Scientific (Sydney, Australia) for this study. This
An evaluation of the Advanced micro-osmometer is presented. This instrument uses 20 1 of sample to measure sample
instrument has been shown to have an excellent analytical precision
osmolality by freezing point depression. Supercooling of
(within-run CV 0"59%, between-@ CV 0"58%). It is the sample is initiated by the insertion of a specially
accurate over an analytical range of O-2000 mmol/kg ofosmolality
shown by linearity studies and split sample correlations against designed, disposable sample holder containing the
vapour pressure osmometry, freezing point osmometry and an sample into the instrument’s thermistor probe, which is
in a fixed position. Following a solenoid-induced pulse
external quality assurance programme. Analytical errors due to
and subsequent sample freezing, the liberated heat of
operator technique are almost eliminated because ofgood instrument fusion is related by a microprocessor to the sample’s
design. Preliminary results on whole-blood osmolality are included.
The required sample size of 20 bl permits osmolality measure- freezing point and osmolality is shown on a digital
ments on most clinical samples. It is concluded that the Advanced display. Calibration of the instrument requires no adjust-
ment by the operator. It consists of running 2-6 samples
micro-osmometer satisfies laboratory requirements.
at each of two calibration levels (50 and 850 mmol/kg). If
the repeatability is acceptable, the instrument automatic-
ally performs internal calibration.
Introduction
Samples
Changes in departmental requirements concerning the
measurement of fluid osmolality initiated this study. Our Within-run precision. Five types of sample were used in
laboratory is currently equipped with instrumentation order to evaluate the repeatability of the instrument.
that utilizes the principle of vapour pressure osmometry Aqueous, commercially available solutions of 50, 290 and
but, because of the poor performance of this procedure in 850 mmol/kg (Advanced Instruments, P.N. 3MA005,
our external quality assurance programme, it was Clinitrol 290 and 3MA085, respectively), plasma
decided to investigate the osmolality of solutions by obtained by centrifugation from lithium heparinized
measurement of freezing point depression, as this method whole blood, whole blood, three 24-h urine samples
has been shown to perform well in such programmes. collected with 5 g of thymol and pooled eccrine sweat
collected from volunteers by pilocarpine iontophoresis
Freezing point in osmometry is that temperature (at using the Webster Model 3600 sweat inducer and
atmospheric pressure) at which the solid and liquid microduct kit (Webster Scientific, Sydney, Australia).
phases will co-exist in equilibrium. When a solute is Each sample was analysed ten times (except the sweat
dissolved in a pure solvent, the colligative properties of sample, which was analysed five times) in a single batch
the solvent usually change in direct proportion to the and on the same day. The osmolalities measured were
solute concentration. Measurement of the freezing point selected to span the instrument’s analytical range of
allows concentrations to be determined with greatest 0-2000 mmol/kg.
precision owing to the inherent isolation of the sample
from the environment by the iced blanket generated when Between-@ precision. To avoid sample evaporation and
the sample freezes. subsequent changes in sample osmolality, samples selec-
ted for this part of the study were refrigerated throughout
Apart from the analytical principle requirement, sample and an effort was made to exclude as much air as possible
size was an important consideration. We perform sweat from the sample tube. Prior to each analysis, aliquots
osmolalities for the detection of cystic fibrosis as de- were brought to room temperature and analysed in
scribed by Webster and co-workers [1-3] using a random order amongst a run of other specimens. Samples
collection procedure described by Carter et al. [4] and were selected from patients and quality control material,
receive paediatric samples and body fluids not easily with appropriate osmolality levels, and analysed in
obtained in large amounts. Primary selection of instru- duplicate on nine separate runs over a period of eight
ments was based on impressions gained from commercial days.
advertising and it was decided to assess the performance
of the Model 3MO Advanced micro-osmometer (Advan- Accuracy studies. Ninety-four samples were selected from
ced Instruments, Needham Heights, MA, USA). patients’ plasma and urine specimens analysed by the

80 0142-0453/89 $3.00 (C) 1989 Taylor & Francis Ltd.

G. Koumantakis and L. E. Wyndham: Evaluation of osmolality measurement

Wescor vapour pressure osmometer so that the levels of Table 2. Between-@ precision
osmolality were distributed throughout the analytical
range. Haemolysed, lipaemic and icteric specimens were No. of Mean SD Sample
also included. These samples were analysed in duplicate runs Target (mmol/kg) (mmol/kg) CV (%) type
with the micro-osmometer. In addition, the accuracy of
the micro-osmometer was also investigated by analysing 9 37 36"2 0"45 1"20 Urine
25 urine and plasma samples with both the Advanced 9 290 287"9 1"09 0"38 Plasma
9 569 568" 2" 14 0"38 Urine QC*
micro-osmometer and a Knauer semi-micro osmometer
9 1026 1026 3"53 0"34 Urine Qc-
(Knauer, Berlin, FRG), which also utilizes freezing point
depression thermodynamics. In addition, 11 lyophilized * Lyphocheck Quantitative urine control Normal (I), Lot No.
samples with target values previously assigned and 15300.
linearly related were obtained from the Royal College of J" Lyphocheck Quantitative urine control Abnormal (II), Lot
Pathologists of Australasia Quality Assurance Pro- No. 15400.
gramme (Target Kit No. 13, Samples 1-11), reconsti-
tuted and analysed in duplicate with the Advanced to our laboratory for routine analysis. Acceptable stan-
micro-osmometer. dard deviations (SD) and coefficients of variations (CV)
of 0" 17-1.30% (average 0"59%) were obtained.
Linearity studies. A urine sample with an osmolality of 1918
mmol/kg was selected and 0-,. 0"2-, 0"4-, 0"6-, 0"8- and
l’0-ml aliquots were pipetted into test-tubes in duplicate. Between-day precision
Into each of the aliquots 1.0, 0"8, 0"6, 0"4, 0"2 and 0 ml of
The results are shown in table 2. The four levels of
distilled water were added in sequence, resulting in six
diluted samples with calculated osmolalities of 0, 384, osmolality selected, representing clinically significant
767, 1150, 1534 and 1918 mmol/kg, respectively. Both values, exhibit CVs of 0"34-1"20% (average 0"58%),
which is satisfactory.
sets of aliquots were analysed in duplicate with the
micro-osmometer in two different analytical batches.
A ccuracy
Whole-blood osmolality. Thirty-four heparinized samples
submitted to the laboratory for routine biochemical As shown in figure a, a split sample comparison between
analysis were used to measure whole-blood osmolality. vapour pressure and freezing point measurements indi-
Each sample was analysed for osmolality and after cates good agreement. However, it is important to note
centrifugation the plasma osmolality was measured. that calibration of the Wescor vapour pressure
osmometer is performed with aqueous standards having
Between-operator variation. Because of the small amount of osmolality values established by our laboratory. For this
sample required to perform each analysis on the micro- reason, two additional accuracy studies were performed.
osmometer, we decided to measure the instrument’s First, a split sample comparison was made between the
dependence on operator technique. Seven members ofthe Advanced micro-osmometer and another freezing instru-
laboratory staff were selected from five technical and ment (Knauer). As a result, a regression line ofy 2"5
professional types. Each was instructed on the basic 1.0x was calculated with a correlation coefficient of
operation of the instrument and was then given a sample 0"9999. Second, osmolality values obtained with the
to analyse ten times. Advanced micro-osmometer from samples were com-
pared with values established by an external assessor,
with satisfactory results (figure b).
Results

Within-run precision Linearity

The results are shown in table 1. The samples selected Table 3 indicates that the Advanced micro-osmometer
spanned an analytical range of 37-1917 mmol/kg, gives a linear response up to at least 1917 mmol/kg. It is
sufficient to cover expected results most often submitted also interesting that the diluent used (distilled water, tube
1) has a value of mmol/kg. Distilled water measure-
ments with the vapour pressure osmometer resulted in
Table 1. Within-run precision
osmolalities of 30-50 mmol/kg, indicating a possible
No. of Mean SD deviation from linearity at low levels or contamination of
analyses (mmol/kg) (mmol/kg) CV (%) Sample type the measuring chamber.

10 36"7 0"49 1"30 Urine

10 49"8 0"42 0’85 Aqueous Operator variation
5 124"0 0"92 0-74 Eccrine sweat Previous experience had taught us to investigate this
10 289’3 0"48 0" 17 Aqueous important, but often neglected variable. This becomes
10 293"4 2"98 1"02 Whole blood even more important if micro-amounts of sample are
10 295"6 1"00 0"35 Plasma
10 557"6 0"97 0" 17 Urine required by the analytical principle. Table 4 shows a CV
10 852"4 1"76 0"21 Aqueous range of 0"17-0"81% (average 0"49%) with accuracy
10 1917"6 9"20 0"48 Urine deviations of less than 1% for all of the seven operators
involved.
81
G. Koumantakis and L. E. Wyndham: Evaluation of osmolality measurement

Table 4. Between-operator variation

(a)
Number Target* Mean SD
of (mmol/(mmol/(mmol/ CV Accuracy Operator
analyses kg) kg) kg) (%) (%) type
000-

10 296 295"6 1.00 0"35 -0" 14 Senior

600 scientific
y 1.004x 5.725 10 296 295"2 0"92 0"31 -0"27 Senior
0.999 technical
10 279 277"7 1"77 0’64 -0"47 Senior
0 200 600 1000
Vapor Pressure
14’00 ;00 scientific
10 401 403"6 0"70 0’17 +0’65 Technical
10 285 283"7 2"30 0"81 -0"46 Technical

340] (b)
J
10

10
286

296
288"0

296"2
2’11
1"78
0"73

0"60
+0"70

+0"07
Trainee
scientific
Trainee
technical

* Target values established from previous measurements.

J Y 0.992x
0.996
4.761
Whole-blood osmolality
220 260 300 340
Target Value (mmol/Kg) Although a relatively small number of samples were
analysed (n 34) for whole-blood osmolality, the results
330
shown in figure c indicate a high correlation between
(c) plasma and heparinized whole-blood samples. Note that
310
in table a CV of 1"02% was calculated, which compares
with CVs of 0.17% at 290 mmol/kg and 0"35% at 295
290
mmol/kg for similar plasma measurements.
Discussion
270
1.04gx 10.646
0.919
Our experience over the last 2 years with vapour pressure
measurements of osmolality with all types of samples
270 290
Plasma Osmolallty
310 330 commonly submitted to a clinical laboratory (including
sweat) has shown that this procedure lacks the precision
and accuracy required by our laboratory, and this was
Figure 1. Graphical representation of (a) split sample comparison confirmed by this study. We measured precision of our
of osmolality measurements by the Wescor vapour pressure and vapour pressure osmometer (unpublished results) and
Advanced 3MO osmometers, (b) freezing point osmolality the CV was found to be of the order of 3"3-4"9% for an
measurements of 11 external quality assurance samples and (c) osmolality range of 74-1000 mmol/kg. Similar findings
whole-blood versus plasma osmolality measured by freezing point were reported by Davis [5] and Kaplan [6] in CAP
depression. quality assurance surveys and by the Royal College of
Pathologists of Australasia in quality assurance surveys
in the last 2 years [7,8]. This type of measurement also
tends to produce lower results by an average of about 3%
throughout the analytical range [5-8] which led us to
Table 3. Linearity alter the calibration values of primary standards in order
to compensate for this effect.
Sample Diluent Error
Tube (ml) (ml) Result* Result* Mean (%) The introduction of the Advanced micro-osmometer with
a small sample requirement (20 1) should enable the
0.0 1.0 analyst to perform freezing point measurements with
2 0.2 0.8 381 386 383.5 -0.13 higher precision and accuracy than with earlier models of
3 0.4 0.6 769 762 765.5 -0.20 such instruments with larger sample size requirements.
4 0.6 0-4 1140 1135 1138 1.00
5 0.8 0-2 1420 1530 1525 -0.59
6 1.0 0.0 1904 1918 1911 -0.31 This evaluation of the Advanced micro-osmometer has
shown excellent precision and accuracy throughout the
Regression analysis: analytical range prescribed by the manufacturer. The
Slope 0"9947 CVs for within-run (table 1) and between-day (table 2)
Intercept 0-41 imprecision are well within acceptable limits using five
Correlation coefficient (r) 0"9999 different types of samples. It is linear throughout the
analytical range, especially at low values, which will now
* Mean of duplicate measurements (mmol/kg). enable us to measure sweat osmolality with confidence,
82
G. Koumantakis and L. E. Wyndham: Evaluation of osmolality measurement

whereas this was not possible with earlier models using osmolality measurements by freezing point depression
the freezing point depression principle. [9], we have shown preliminary results in figure lc.
It is recognized that any analytical procedure is more or Our results are in agreement with those ofRocks et al., but
less susceptible to errors introduced by operator tech- we found whole-blood osmolality to be about 4.0 mmol/
niques and expertise. In this instance, we measured this kg higher than plasma osmolality using lithium hep-
important factor and the results shown in table 4 indicate arinized samples. It is believed that this is due to an
a range of CVs between 0"17 and 0.81% with less than artificial elevation caused by anaerobic glycolysis in the
0"7% deviation in analytical accuracy. samples, which is unavoidable unless whole-blood osmo-
lality is measured within h of collection, and we intend
The high degree of precision exhibited by the Advanced to address this problem. Notwithstanding the above
micro-osmometer could be explained by examining the limitation, whole-blood osmolality could prove to be a
attempts made by the manufacturer to minimize factors very useful measurement, especially with subjects where
that contribute to a loss of precision with conventional sample size is of importance.
instruments. For example, bath temperature control,
which is dependent on the fluid used, amount of fluid and In conclusion, we have found the Advanced micro-
volume changes as moisture condenses from the room air, osmometer to be a very precise and accurate instrument
may be important. The supercooling ofthe sample should for the measurement of osmolality with a high degree of
be rapid but at a controlled rate. The Advanced linearity throughout a wide analytical range, and the
instrument uses no fluid in the cooling chamber; instead, instrument design has almost entirely eliminated ana-
temperature is controlled by an electronically adjusted lytical errors introduced by operator techniques.
thermoelectric module that provides steady supercooling
of the sample. The probe senses the temperature in the
sample cell. Because the probe sees only a small portion of Acknowledgements
the sample space, it is important that the sample
temperature be uniform. This is achieved by the stan- The authors thank Stansens Scientific (Australia) for
dardized sample cuvettes which fit perfectly with the supplying the instrument and Miss Sandy Li for typing
sample port. In addition, if the tip of the probe is not the manuscript.
positioned repeatably from sample to sample, the preci-
sion may suffer. The tip of the probe in the Advanced
instrument is in a fixed position and the sample port References
provides for repeatable positioning and centring of the
sample in the probe. In most instruments the probe needs WEBSTER, H. L. and LOCHLIN, H., Medical Journal of
1.
to be wiped in order to minimize carryover from sample Australia, 1 (1977), 923.
to sample, and in laboratories with a large number of staff 2. WEBSTER, H. L., and BARLOW, W. K., Clinical Chemistry,
27 1981 ), 385.
operating the instrument insufficient or incorrect clean- 3. WEBSTER, H. L., CRC Critical Reviews in Clinical Laboratory
ing could be encountered. Cleaning of the probe in the Sciences, 18 (1983), 313.
Advanced instrument is another regulated procedure 4. CARTER, E. P., BARRETT, A. D., HEELEY, A. F., and
which will ensure a clean chamber and probe each time KtZEMKO, J. A., Archives of Disease in Childhood, 59 (1984),
with any operator. 919.
5. DAVIS, J. E., in Clinical Chemistry, Theory, Analysis and
Rocks et al. [9], using a freezing point osmometer, Correlation, Eds Kaplan, L. A., and Pesce, A. J. (C. V.
measured whole-blood osmolality and reported this to be Mosby, St. Louis, 1984), p. 237.
an average of 1"5 mmol/kg higher than that of plasma. 6. KAPLAN, L. A., in Clinical Chemistry theory, analysis and
Certain factors must be considered if whole-blood osmo- correlation, Eds Kaplan, L. A., and Pesce, A. J. (C. V.
Hosby, St. Louis, 1984), p. 1071.
lality is to be measured. These include effects of lithium 7. PENBERTHY, L. A., The Clinical BiochemistmReviews, 6
heparin on whole-blood osmolality, effect of the packed (1986), 146.
cell volume and haemolysis. We did not investigate these 8. PENBERTHY, L. A., The Clinical Biochemist--Reviews, 8
effects in this work, but we are in the process of defining (987), 0.
them in a much larger study. However, as it has been 9. RocKs, B. F., SHERWOOD, R. A., and COOK, J. G. H., Annals
reported that the above factors may have no effect on of Clinical Biochemistry, 23 (1986), 106.

83
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