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Medical Engineering & Physics 29 (2007) 449458

A refractometry-based glucose analysis of body fluids


Kai Zirk a, , Harald Poetzschke b
a D-58313 Herdecke, Germany
b D-65191 Wiesbaden, Germany

Received 27 December 2005; received in revised form 13 February 2006; accepted 22 February 2006

Abstract

In principle, refractometry appears to be a suitable method for the measurement of glucose concentrations in body fluids (such as blood
and the intercellular fluid), even though the refractive index of the measured samples, as an additive property, is not specific. But, if certain
conditions are fulfilled, the glucose content can be calculated using the refractive index in combination with values from a further measurement.
This study describes the determination of the glucose content using refractometry in human blood serum derivates, which were selected due
to their ready availability to be used as a model for interstitial fluid.
Refractometry of body fluids requires the elimination of disturbing components from the measurement sample. First of all, a homogenous
fluid (i.e. consisting of one phase) is required, so that all cells and components in suspension need to be separated out. Furthermore, certain
dissolved macromolecular components which are known to disturb the measurement process must also be removed. In human serum samples
which had been ultrafiltrated with a range of ultrafilters of different pore sizes, a comparative evaluation showed that only ultrafiltration
through a filter with a separation limit of between 3 and 30 kDa resulted in maximal reduction of the refractive index (compared to native
serum), whereas ultrafilters with greater separation limits did not.
The total content of osmotically active solutes (the tonicity) also exerts a clear influence. However, exemplary measurements in blood plasma
fluid from one volunteer showed that the electrical conductivity is (without an additive component) directly proportional to the osmolality:
physiological changes in the state of body hydration (hyperhydration and dehydration) do not lead to any considerable changes in the relation
between ionised and uncharged solute particles, but instead result in a sufficiently clear dilution or concentration of the blood fluids low
molecular components. This finding allows the use of the technically easy to measure electrical conductivity as a measure for the tonicity
of the measurement samples.
Using measurements of these two parameters refractive index and electrical conductivity in blood serum obtained from a healthy
volunteer, a two-dimensional calibration function (calibration matrix) for the assessment of the glucose content of ultrafiltrated human blood
serum was constructed, and the measurement of blood glucose levels in non-diabetic (four females and four males) volunteers in comparison
to a reference method was evaluated showing (as a proof of concept) a linear association.
Assessment of the inaccuracy of these measurements made with the described measuring devices and methods showed a deviation from
the reference values of less than 10%. An estimation of the maximum possible error showed relative deviations (maximum measurement
uncertainties) of up to 20%.
2006 IPEM. Published by Elsevier Ltd. All rights reserved.

Keywords: Glucose; Analysis; Body fluid; Refractometry; Resistometry

1. Introduction and aim

The symptomatic therapy of diabetes mellitus requires the


This work contains parts of the doctoral thesis of K. Zirk.
Correspondence to: Kai Zirk, c/o SES-Entwicklung GmbH, Konrad-
reliable assessment of blood glucose levels (glucose con-
Adenauer-Allee 11, D-44263 Dortmund, Germany. Tel.: +49 231 47730420;
tents) at sufficiently frequent intervals. Established methods
fax: +49 231 47730430. require freshly obtained blood. Due to the pain associated
E-mail address: zirk@ses-entwicklung.de (K. Zirk). with obtaining such a blood sample (for example, by pierc-

1350-4533/$ see front matter 2006 IPEM. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.medengphy.2006.02.011
450 K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458

ing a fingertip), patients often carry out such measurements cally by the use of a two-dimensional calibration function
too infrequently. which includes the measurement (or determination) and
A possible improvement in the treatment of these patients consideration of this influencing factor.
could be achieved by the development of probes which could (4) Further possible substantial influencing factors must
be inserted (insertion probes), presumably in the subcuta- either be identified or their presence experimentally
neous fatty tissue, or surgically implantable glucose probes excluded.
(implantation probes) capable of delivering measurement val-
ues over long periods of time; for example, over months The aim of this study was to assess the possibility of
or even years in the case of implanted probes. Such mea- determining, with sufficient accuracy (initially at a constant
surements could exploit the fact that the glucose content temperature), the blood glucose content in blood fluids from
of the interstitial fluid which could be measured by such various volunteers (to begin with, only blood samples from
probes provides a good measure of the blood glucose healthy subjects were used) using refractometry together with
content which is therapeutically interesting in this context a suitable method for the assessment of the degree of dilu-
[1]. tion (the tonicity), whereby blood fluids served as a model
If such a probe is to be used to allow a (quasi-)continuous for all body fluids [3]. In this way, the fundamental question
detection of the glucose content of the intercellular fluid in of whether refractometry can be used to determine glucose
the subcutaneous connective tissue, the sensor needs to be content could be addressed. Furthermore, the manner of the
based on a purely physical (rather than biochemical) mea- dependency of the temperature coefficients of the measured
surement principle, since only such a method would be able values in the samples assessed was to be characterised in
to provide the necessary signal stability, while at the same order to evaluate the extent of the technical efforts which
time overcoming the need for a complex tailoring of the sen- would be necessary to enable a compensation of the temper-
sor to each individual patient [2]. For in vitro assessment, a ature effects.
purely physical measurement method could also prove to be
advantageous, for example, with respect to the stability of the
measuring systems signal. 2. Fundamental considerations
A suitable and from a design point of view potentially
advantageous physical parameter for the detection of the glu- The initial goal of this study was to confirm the hypothe-
cose content in body fluids is the optical refractive index. The sis that the refractive index measured in ultrafiltrates of body
principal use of refractometry for an estimation of glucose (d- fluids (which still only contain their low molecular weight
(+)-glucose) content in cell-free body fluids has already been components) is predominantly with the exception of the
documented [3]. glucose content a function of the state of hydration of
A refractometrical determination of the glucose content the human body; in other words, that the inter-individual
of pure aqueous solutions is technically simple and pos- differences in the composition of the glucose-free ultra-
sible using direct measurements. A measurement of body filtrate are small enough so as not to confound the described
fluids (with additional components of low and macromolec- purpose.
ular weights) does however require certain additional steps The influence of the total content of dissolved low molec-
during the sample preparation together with additional mea- ular weight components on the accuracy of the estimation
surements [3]: of glucose content of body fluids is substantial [3]. In order
to compensate for this influence in refractometry measure-
(1) The sample to be assessed must be a homogenous (single ments, the total solute content needs to be known. However,
phase) body fluid, which has been freed of cells (such as the direct measurement of a colligative property (e.g. the
erythrocytes), cell fragments (thrombocytes) and other osmotic pressure) for the osmolarity or osmolality is not
suspended or emulsified components (such as lipopro- possible, at least with an inserted or implanted probe. An
teins). alternative approach therefore needs to be found.
(2) Dissolved macromolecular components which could dis- In a protein-free serum-ultrafiltrate such as the one under
turb the measurement must also be eliminated. This can, consideration here, the low molecular weight solutes are
in principle, be carried out using ultrafiltration as has electrolytes (EL, i.e. ions) or uncharged molecules (UM,
been demonstrated previously. The choice of the ultrafil- e.g. glucose, urea and non-dissociated lactic acid). The com-
ter to be used however still needs to be substantiated. plete range of osmotically active solutes in extracellular fluid
(3) The influence of the osmotically active solute content belongs to one of these two groups of substances.
of the sample to be measured (in healthy subjects, this, The majority of electrolytes in serum and subsequently
in particular, expresses the variable state of hydration) also in its ultrafiltrates are small inorganic ions (91% of
needs to be mathematically compensated for during the cations are sodium, potassium, calcium or magnesium
the evaluation of the primary measurement results. The ions and 84% of the anions are chloride, phosphate, sulphate
determination of the glucose content in blood serum and or hydrogen carbonate ions [4]), most of which are strong
other body fluids can only be carried out refractometri- electrolytes which dissociate to a pronounced extent in blood
K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458 451

serum. Their total concentration can be determined using con- 3. Experimental methodology
ductance measurements.
If the relative proportion of the substance quantities for the 3.1. Materials and preparative methods
two groups is constant, then the measurement of the specific
electrical conductance which only applies to the ions can The water used was highly purified and of pharmaceutical
provide reliable information concerning the total quantities grade, so-called Water for Injection (WFI).
of the solutes. Glucose is d-(+)-glucose, crystalline -d-(+)-glucose
In an earlier investigation [3], the consumption of a con- (dextrose) was obtained from Roth (Karlsruhe, D, product
siderable amount of low-salt water or a restriction of water number: M 198,17, purity: 99.5%, Ph.Eur., DAB).
intake as examples of physiological changes in the bodys For the preparation of standard solutions (e.g. glucose,
state of hydration (hyperhydration and dehydration) showed amino acids, urea), the solute was weighed, then water (WFI)
that in vivo (at least in the case of an acute hypotonic hyper- added to obtain the required volume, and the solution mixed
volaemia or a moderate hypertonic hypovolaemia), the spe- by stirring. In the case of glucose, the known temperature-
cific electrical conductance of intravascular and interstitial dependent kinetics for the attainment of a balance of mutaro-
body fluids (i.e. blood, interstitial fluids and lymph) is directly tation between the - and -glucose forms were taken into
proportional to the osmolality of these fluid without further consideration [3]. In the same manner glucose was added to
additional terms [3]. These investigations showed (within the ultrafiltrates of blood serum and mixed for a correspondingly
limits of measurement errors) solely a dilution or concentra- adequate period of time.
tion of the body fluids. Blood samples were taken from healthy (non-diabetic)
By means of inductive reasoning, the general conclusion men and women of between 23 and 42 years of age
can be reached that the ratio of ions to uncharged solute par- (probands), who participated voluntarily. These test per-
ticles in blood fluids is not considerably influenced by the sons were not fasting, and blood-withdrawals were not done
hydration state of the human body. Therefore, the specific immediately post-prandial. Native venous blood samples
electrical conductivity (the measurement of which is fur- were obtained by aspiration puncture of a slightly congested
thermore uncomplicated in technical terms) appeared, prin- cubital vein by a physician. To obtain blood serum, the native
cipally, to be a suitable measurement parameter so that it was blood plasma was immediately separated from the cellular
employed in subsequent investigations. components of native full blood by means of centrifugation
The proof of the above-mentioned assumption was implic- (10,000 g0 for 2 min, g0 = 9.81 m/s2 ), incubated for 1 h at
itly ensured by the following orientating evaluation of the 37 C (in order to obtain a complete coagulation) and subse-
assessment of blood glucose content using refractometry in quently centrifuged (15,000 g0 for 10 min) to remove the
healthy (in particular non-diabetic) persons. fibrin clots which had formed.
A further important quantity which can also influence Ultrafiltrates of blood serum were obtained with ultra-
the accuracy (i.e. correctness and precision) of the value filtration stirring cells (Omegacell, Pall Gelman, D) with
measured is the temperature. Both the refractive index nominal molecular weight limits (NMWL) of 3, 30, 300 and
and the electrical conductance are temperature-dependent 1000 kDa. Compressed technical grade gases (nitrogen or
[5,6]. The temperature does of course not need to be oxygen) served as propellants.
taken into account if the temperature of the sample to Water was added to the blood serum whenever a dilu-
be measured (ultrafiltered serum) is held at a constant tion of the latter was required. More concentrated serum
prescribed value during refractometrical assessment of was obtained by storage in the presence of desiccating beads
the glucose concentration. This however only applies to (Alumosilicatgel mit Farbindikator, Roth, D) in an exsic-
measurements being made outside of the body (i.e. in cator. Preliminary investigations showed that when serum
vitro). was rendered more concentrated in this way, no measur-
In vivo, a sufficiently accurate temperature control of the able changes in the composition of the dissolved constituents
measurement system would appear to be complicated and to occurred and that a re-dilution of the serum to the initial con-
require a considerable technical effort. Instead, it would be centration resulted in a restoration of the original values for
technically advantageous if the influence of the prevailing osmolality, electrical conductivity and glucose concentration.
temperature could be compensated for by its measurement As a measure of dilution or concentration, the relative
and a direct correction of the other parameters measure- mass change (RMC) of the serum samples were calculated
ment signals. For this, the temperature coefficients of the using
measurement parameters (the changes in the measurement  
ma mb
values as a function of temperature) would need to be con- RMC = 100%
firmed to be constant. Only if this is the case can temperature mb
be ruled out as a further (third) independent variable in the whereby ma and mb are the serum masses obtained gravi-
assessment of blood glucose which needs not be taken into metrically after (ma ) or before (mb ) adjustment for the water
account in the calibration required for the measurement of content. Positive values indicate a dilution, and negative val-
glucose contents. ues a concentration.
452 K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458

3.2. Analytical methods and measurement devices The described values obtained using the test-strip analyser,
precision and contact refractometers, the osmometer and the
For the determination of the glucose content of ultrafil- conductance monitor were generally obtained from repeated
trated blood serum (as a reference method for the refractomet- measurements. Values are presented as the arithmetic mean
rical estimation), a commercially available test strip analyser (x), and a measurement of error (seen in the graphs in the form
(Accutrend Sensor, Boehringer Mannheim, D) was used, of error bars) is provided by the standard error of the mean
the measurement principle of which is based on the oxida- (SEM). Linear fits for functional associations were calculated
tion of -d-glucose (enzymatically catalysed by means of from the Gaussian principle of the smallest sum of the square
glucose dehydrogenase), with the reaction rate being quanti- proximity.
fied amperometrically. An estimation of the maximum error for the refractometri-
A calibration of the measurement results from this device cal assessment of glucose content was made according to the
was carried out by the construction of individual calibration Gaussian law of error propagation using known or estimated
curves for the different serum ultrafiltrates. In conjunction, maximum errors (x). The error obtained for the coefficient
gravimetry served as the reference for further additional function from the measurement values was also calculated
amounts of the analyte glucose (with the native glucose using the Gaussian error propagation law.
content being obtained by reverse extrapolation).
In this way, a range from approximately 80 up to more 3.3. Measurement scheme
than 300 mg/dL was calibrated. The calibration curves were
generally linear and readings (averages of five single mea- For the estimation of substance-dependent refractive
surements for each specific glucose content) showed a relative indices from ultrafiltration fractions of blood fluids, blood
(one-sided) imprecision of less than 5%, corresponding well serum samples from a volunteer (K.Z.) were filtered
to the values published by Wahl [7]. using ultrafiltration membranes with various different nom-
For the determination of optical refractive indices, a com- inal molecular weight separation ranges (3, 30, 300 and
mercially available precision refractometer D U R (Schmidt 1000 kDa). Subsequently, the refractive indices were esti-
& Haensch, Berlin, D) was used and practically all mea- mated at 25 C (precision refractometer D U R).
surements were carried out at a temperature of 25.0 C (set For all further experiments, ultrafiltrated (<3 kDa) blood
with a circulating thermostat). The display was accordingly serum was exclusively used.
adjusted to the value for the refractive index of distilled Where no appropriate values could be found in the lit-
water at 25.0 C (1.33245). The imprecision of this refrac- erature for an estimation of the contribution of individual
tometer is 105 (0.00001). Alternatively, measurements were dissolved substances or substance groups to the refractive
also made with a newly developed contact refractometer index of the blood fluids, the refractive index increments
[8] at a temperature of 25.0 C (set using an incubation (measured using the precision refractometer D U R) of
cabinet). aqueous (low molecular weight) glucose, saline, albumin,
Real (i.e. measurable) osmolalities taken as the sum amino acid and urea solutions were assessed to determine
of the particular (real) molalities of all dissolved osmot- the dependence of the refractive index on the mass concen-
ically active solute components were determined using tration of the individual solutes.
an osmometer (Halbmikro-Osmometer Typ M, Knauer, In order to test whether the in vivo quantity ratio between
D), the measurement principle of which is the determina- serum electrolytes (ions) and uncharged molecules (neu-
tion of the depression of the freezing point. The device tral particles) remains identical in concentrated (hypertonic)
was calibrated with a NaCl calibration solution (Eichlosung or diluted (hypotonic) serum samples (i.e. whether purely
400 Milliosmol/kg, Knauer; containing 12.687 g NaCl/kg a concentration or dilution is taking place), measurements
H2 O, and the mole of osmotically active dissolved par- of the specific electrical conductivity and osmolality were
ticles here Na+ and Cl ions is referred to here as made (and plotted against each other) in ultrafiltrated serum
osmol, abbreviated as osm). According to the manufacturers samples which had been obtained from a fasting male non-
operating instructions the inaccuracy of the measurements diabetic volunteer (H.P.) who was in a state of physiological
is 2%. haemoconcentration (dehydration) or haemodilution (hyper-
The specific electrical conductivity at 25.0 C was mea- hydration or hydraemia). In order to obtain the greatest pos-
sured using a flow-through conductometer (Conductivity sible variation in the state of hydration, the blood probe used
Monitor, Pharmacia Biotech, D) with an integrated temper- for the production of the necessary serum samples was taken
ature sensor. The display on this apparatus was permanently shortly after the consumption of 2.5 L water or after a 26 h
adjusted by the manufacturer, according to whom the relative abstinence from fluid intake.
imprecision of the individual measurements is less than 2%. A serum sample obtained from a venous blood sample
The device was calibrated using highly purified WFI (with taken from a fasting male non-diabetic (K.Z.) was used to
a specific conductivity of below 1.1 S/cm) and an aqueous establish a calibration matrix of the serum glucose content
NaCl calibration solution (Eichlosung 400 Milliosmol/kg with the refractive index and the specific electrical conduc-
in ampoules, Knauer). tivity as independent variables. For this, the serum sample
K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458 453

underwent ultrafiltration (3 kDa) and was divided into four


parts, two of which were diluted with WFI (the relative dilu-
tions (RMC) being 7.5 and 13.8%) and one of which was con-
centrated (a 5% relative concentration; RMC = 5%), which
resulted in the respective variations in the (real) osmolalities
and the electrical conductivity. The variations bordered on
the possible physiological changes seen in vivo [3].
Subsequently, each of these portions (of varying osmo-
lality) was divided into four further portions, to three of
which different amounts of -glucose were added and incu-
bated at 36 C for 20 min so that a mutarotation equilibrium
between - and -glucose could be obtained. This was fol-
lowed by the measurement or estimation of the glucose con-
tent, the (real) osmolality, the conductivity and the refractive
indices of the samples and the construction of the calibration
functions. Fig. 1. Organisation of the most important plasma components into classes
To determine the influence of temperature on the refractive according to molecular weight, together with the dependency of the refractive
index n of ultrafiltrated human serum from a non-diabetic on the pore size of
index and the conductivity, these parameters were assessed in the filter used. Each point of the refractive index of the ultrafiltrated plasma
an ultrafiltrated serum sample (K.Z.) at 20.0, 25.0, 30.0, 35.0 () represents a mean value from five individual measurements, and the
and 40.0 C (temperature adjustment made using a circulating values indicate the nominal molecular weight separation limit of the used
thermostat). The measurement of conductivity for the above- ultrafilter in kDa. cm X is the mass concentration of solute X in the serum
mentioned temperatures was carried out with the help of (the heights of the marked areas), and the areas width specifies the range of
molar masses within each group of substances.
the temperature compensation function already stored in the
device used. For the assessment of the refractive index, Water
for Injection was used for comparisons, and for the mea- the different ranges of the molecular weights present and the
surement of conductivity, a saline calibration solution (Cal- (physiological) contents of the main solute groups found in
ibration solution Eichlosung 400 Milliosmol/kg12,687 g the blood fluids (proteins, low molecular ions and uncharged
NaCl/kg H2 O, Knauer) served this purpose. particles) are shown, as well as the most important substances
For the purpose of comparing the methods used for the in each of these groups.
estimation of glucose content (either by refractometry, or the The remaining components of the matrix (and poten-
chosen reference method Accutrend Sensor) ultrafiltrated tial disturbing factors) following the respective ultrafiltration
(3 kDa) serum samples were obtained from the blood of eight were therefore on the whole presumably salts (sodium and
non-fasting, non-diabetic volunteers (four females and four chloride ions, etc.) together with amino acids and other low
males), and divided into two or three portions. In each case, molecular organic substances (urea, lactate, etc. [4]) with
one portion was diluted by the addition of WFI and/or a given their generally very similar specific refractive index incre-
amount of -glucose added (with a 20 min incubation at 36 C ments (dn/dc) of approximately 1.42.0 104 L/g ([9] or
to ensure that a mutarotation equilibrium between - and - as measured personally). Table 1 provides an overview of
glucose had been reached). These samples were then used for the refractive index increments for possible disturbing low-
a comparison of the glucose contents as estimated using the molecular substances.
described refractometry (contact refractometry) and conduc-
tivity techniques, together with values obtained directly with
the reference method. Table 1
Refractive index increments for low molecular weight substances present in
an ultrafiltrate of body fluids. Lit.: Literature, O.m.: Own measurement
4. Results Dissolved substance Solvent Refractive index
increment, 104 L/g
4.1. Constituents of the measurement sample as Lit. O.m.
disturbing factors d-(+)-Glucose (C6 H12 O6 ) Water 1.4 1.4
Urea (NH2 CONH2 ) Water 1.4 1.4
The ultrafiltrates of a volunteers blood serum sample, Sodium lactate (C3 H5 O3 Na) Water 1.5
which had been prepared using ultrafilters with varying pore Sodium chloride (NaCl) Water 1.7 1.7
size, were seen to possess the same refractive index when Amino acids (physiological Water 2.0
they were passed through an ultrafilter with a nominal molec- standard mixture for an
ular weight limit of 30 kDa maximum. The refractive indices amino acid analyser)
measured are shown as a function of the nominal molecular Bovine serum albumin 9.0 g/L aqueous 1.8
NaCl solution
weight limit of the various ultrafilters in Fig. 1. In addition,
454 K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458

content was seen to be increased or decreased depending on


the hydration state of the serum. Even so, all measurement
values lay (within the realms of the measurement errors) on
a straight line passing through the origin, with changes in
the contents of the two groups of substances being directly
proportional to one another (the relationship between the two
being constant) indicating the occurrence purely of a dilution
or concentration.

4.3. Determination of the calibration coefcients

As the methodological basis for a correct determination of


the glucose content of ultrafiltrated (NMWL = 3 kDa) blood
serum samples, a determination of the functional relation-
ships between the refractive index, electrical conductivity
and glucose concentration was necessary. For this, the above-
named parameters were varied by the dilution or concentra-
tion of the serum as well as by addition of -glucose.
Following the addition of glucose, the conductivity
remained constant within the scope of measurement errors
(as might be expected considering that glucose in solution
is electrically neutral and does not dissociate into ions). Fur-
thermore, the osmolality values were practically constant (the
contribution of the added glucose to the total osmolality was
very small, being maximally approximately 4%).
Fig. 3A shows the refractive index as a function of glu-
cose concentration (assessed using the reference method
Accutrend Sensor), at different conductivity (and as a result,
osmolality) values. Changing the osmolality (by means of
dilution or concentration) resulted in a parallel shift of the
linear function of the graphs, with the gradient remaining
Fig. 2. (A) Specific electrical conductivity as a function of the osmolal-
ity qn Osm of aqueous electrolyte solutions (: calibration value for NaCl constant.
solution of qn Osm = 400 mmol/kg) and ultrafiltrated human blood serum Fig. 3B shows the refractive indices (obtained by extrapo-
(NMWL = 3 kDa) from a non-diabetic male volunteer (, SE-UF), together lation from Fig. 3A) of glucose-free serum ultrafiltrates (the
with values for this serum after arithmetical subtraction of the glucose con- ordinate axis) as a function of their conductivity, whereby
tent (, red-SE-UF). (B) Detailed excerpt from (A) showing the (most
as would be expected a linear dependency is recognisable.
probable) functional dependence indicated by straight lines passing through
the origin. Each closed symbol () represents a mean value from three indi- Therefore, the conditions for an indirect assessment of the
vidual measurements. glucose concentration by means of the measured refractive
index and conductance are fulfilled. The basic procedure for
4.2. The total ion concentration as a measure of such a measure is graphically illustrated in Fig. 4A and B.
osmolality Fig. 4A shows essentially the same as Fig. 3A (the refractive
index of serum ultrafiltrates at different conductance values
Fig. 2A and B shows the specific electrical conductiv- as a function of their glucose concentrations) in addition to
ity as a function of osmolality both for ultrafiltrated serum the determination and position of the fulcrum for the con-
(SE-UF) and for a solution of a strong electrolyte (EL). struction of Fig. 4B in which the (parallel) linear functions
In addition, reduced osmolalities of the serum ultrafiltrates for a given constant refractive index are shown in a depiction
(red-SE-UF, calculated by subtraction of the molality of of the glucose content as a function of specific conductance.
glucose from the measured osmolality) are also shown. In addition, the indirect determination of the glucose content
The (measured) concentration of glucose in the extracel- from possible measurements for a specific case is exemplarily
lular space remained almost unchanged (due to an active demonstrated.
regulation by the body) despite fluid restriction or the con- In order to obtain a mathematical description of the depen-
sumption of substantial quantities of water, its contribution dencies between the individual parameters, the following
to the osmolality amounting to a maximum of approximately relationships were taken from Fig. 3A and B:
2%.
This was in contrast to the molar contents of the remaining dn
n = n0 + S1 cm Glu, with S1 = (1)
substances, the electrolytes and uncharged molecules. Their dcm Glu
K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458 455

Fig. 3. (A) Dependency of the refractive index n of human diluted, native-


concentrated and concentrated blood serum samples from a non-diabetic on
the glucose concentration cm Glu together with (B) the dependency of the
refractive index in glucose-free serum samples obtained by extrapolation Fig. 4. (A) Dependency of the refractive index n of human diluted, native-
n0 on the conductivity . The dotted line separates the regions for serum concentrated and concentrated blood serum samples from a non-diabetic on
samples with and without added -glucose. The increasing indices on the the glucose concentration cm Glu (see also Fig. 1A) and (B) the resulting func-
line for conductivity indicate the increasing degree of concentration. Each tional relation between glucose concentration cm Glu and the conductivity
symbol represents a mean value from five individual measurements. for a series of different refractive indices. The dotted lines show examples of
the indirectly determined glucose contents for possible measurement value
dn0 pairs ( = 13.6 and n = 1.33435). The rising indices on the formula symbol
n0 = n0,0 + S2 , with S2 = (2) for the refractive index indicate the rising value of the parameter. The open
d
symbols in (A) served as supporting points for the development of the series
By insertion of Eq. (2) in Eq. (1), and solving this for of lines in (B).
cm Glu, the (indirectly) measured glucose concentration can
the values determined from the measured values. The max-
be obtained:
imum error cm Glu for the defining quantity cm Glu can be
n n0,0 S2 estimated using the Gaussian law of error propagation [10].
cm Glu = (3)
S1 Thus:
   
 cm Glu   cm Glu 
This dependency of the glucose concentration of ultrafiltrates cm Glu =  n +  n0,0 
(obtained using filters with a nominal molecular mass separa- n n0,0
tion limit of 3 kDa) of human blood serum on their respective    
 cm Glu   cm Glu 
refractive indices and specific electrical conductance values +   +  S1 
S1
can be used as a defining equation for the refractometrical  
determination of the glucose content.  cm Glu 
+  S2 
Since this defining equation was obtained using measure- S2
ment values which themselves show a margin of error, the
accuracy of the new method is dependent on the accuracy of      
1   1   S2 
the measurement methods employed. Therefore:  
cm Glu =  n +    
n0,0  +  
S1 S1 S1
cm Glu = cm Glu cm Glu    
 n n0,0 S2   
+  
S1  +  S2 
(n n) (n0,0 n0,0 ) (S2 S2 ) ( ) S12 S1
=
S1 S1 (5)
(4)
After insertion of the errors n and  provided for the
whereby n and  are the maximum errors of the measured measurement devices as well as the errors determined by
values and n0,0 , S1 and S2 are the maximum errors for the Gaussian law of error propagation n0,0 , S1 and S2 ,
456 K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458

a maximum possible error of 60 mg/dL is obtained for the ductivity in a sodium chloride solution and in ultrafiltrated
defining quantity cm Glu. Thus, it can be seen that the accu- serum samples are considered. The temperature coefficients
racy of the measured values n and has a distinct influence of the diverse media differ from one another, although the
on the correctness of the indirectly determined glucose con- gradients for the concentrated and diluted serum samples are
tents, with a relative deviation of approximately 20% with the same.
respect to a measurement of 300 mg/dL.
A further important parameter which can influence the 4.4. Calibration and comparison with the reference
measurement accuracy (in terms of correctness and preci- method
sion) is the temperature. In the form presented, the defining
equation applies only when the temperature is constant since For an experimental determination of the correctness of
both the refractive index (n) and the specific conductance () the refractometrical assessment of the glucose content (car-
are functions of temperature [5,6]. In order to estimate this ried out here with the newly developed contact refractome-
influence, the temperature-dependence of the refractive index ter), the refractive index n in Eq. (3) was first of all expressed
was determined for highly purified water and an ultrafiltrated using the proportional output voltage of the contact refrac-
serum sample (3 kDa), and the temperature-dependence of tometer [8]:
the specific conductivity was assessed in a sodium chloride
n = f (VA ) = 66.22 106 V1 (V0 + VA ) (6)
calibration solution and an ultrafiltrated serum sample.
Fig. 5A and B depicts the interdependencies measured. Following insertion in Eq. (3):
As might be expected, the (differential) slopes (temperature 
1 1
coefficients) in Fig. 5A for the refractive index are within the cm Glu = 66.22 106 (VA + V0 )
range of measurement error the same for water and the ultra- S1 V

filtrated serum; only the absolute values differ. A different
picture is seen if the slopes (temperature coefficient) depicted n0,0 S2 (7)
in Fig. 5B for the dependency of the specific electrical con-
Inclusion of the measured values for S1 , S2 and n0,0 (accord-
ing to the procedure described in Fig. 3) gives:
cm Glu = 838.0 103 (mg/dL)

1
66.22 106 (VA + V0 ) 1.332449
V

6
125.2 10 (cm/mS) (8)

For the calibration of the glucose content determination, the


equation is solved for V0 :
V0 = f (, cm Glu, VA )

= 18.02 103 (dL/mg) cm Glu + 1.891

(cm/mS) + 20121.55 V VA (9)

Following the insertion of values for and VA , as well as


an appropriate reference value for cm Glu, a compensation
voltage can be calculated. After a corresponding electronic
adjustment of the display value, the glucose content values
shown on the contact refractometers display correspond to
those determined using the reference method. Thus, using a
reference value of approximately 100 mg/dL (1.00 g/L), the
refractometrical determination of glucose content was cali-
brated.
Fig. 5. (A) Dependency of the refractive index n on temperature T in a For an assessment of the correctness (or incorrectness)
highly purified water () and in ultrafiltrated serum samples (), and (B) of the refractometrical analysis of glucose, ultrafiltrated
the dependency of the conductivity of a NaCl calibration solution () and
(NMWL = 3 kDa) blood serum samples obtained from eight
ultrafiltrated serum samples () on temperature. The rising indices on the
formula symbol for the osmolality qn Osm indicate the increasing degree of non-diabetics (four males and four female) were divided into
concentration. Each symbol represents a mean value from five individual two or three portions. One of these parts was diluted with
measurements. water, and glucose was added to a further part. Subsequently,
K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458 457

dency of the refractive index on the ultrafiltrate decreased


with each reduction in the pore size of the filters used (Fig. 1).
At the same time, the demonstrated minimal necessary sepa-
ration limit of approximately 30 kDa points in particular
to large plasma proteins. The major portion of the serum
proteins possess molecular masses starting at 60 kDa (the
albumins, amounting to more than 60% of the serum proteins
and the members of the globulin group possessing, without
exception, even greater masses). The list of the serum pro-
teins with molecular masses below 30 kDa is, in contrast, very
limited (microglobulins, some hormones) [4,11].
Ultrafilters with a separation limit of 30 kDa only allow the
passage of very small proteins and polypeptides and these are
apparently present either in a small enough concentration or
in inter-individually comparable amounts, so that they do not
substantially disturb the refractometrical glucose analysis.
Fig. 6. Method comparison of glucose concentration (cm Glu) determina- The low molecular components (except for glucose) also
tions using the new refractometry method (ordinate) with the Accutrend do not appear to present a relevant disturbance, even though
Sensor analyser reference method (abscissa). The correlation coefficient of in particular amino acids, urea, lactic acid and above all
the linear smoothing function (best fit straight line) has a value of 0.9923, the
arrows indicate the experimental adjustment of refractometry at 100 mg/dL
sodium chloride are present in similar or even consider-
(arbitrarily chosen). ably greater quantities than glucose in ultrafiltrated serum
[4,11]. The initial assumption, that the refractive index of
the conductance and the refractive index (using the contact ultrafiltrates of body fluids (with the exception of the glu-
refractometer) were determined in the ultrafiltrated serum cose content) is practically only a function of the subjects
samples, both at 25 C. hydration state, could be indirectly confirmed by the correct
The values obtained were entered together with the pre- estimation of the glucose content demonstrated in this study.
viously determined compensation voltage V0 in Eq. (8) That the sum of the contributions of the low molecular
and are shown in Fig. 6, which depicts the refractometri- components to the total refractive index provides compara-
cally determined value against the glucose value (expected ble values is presumably due to the fact that their respective
values) obtained using the analyser Accutrend Sensor (as refractive index increments are very similar (Table 1)and
the reference method). The maximal deviation is seen to their total content is obtained from the measurement of the
occur in the highest concentration range, with a maximal electrical conductivity.
relative deviation of 10% over the complete concentration Since the glucose concentration is indirectly determined
range investigated. from the two measured parameters (conductance and refrac-
tive index), its accuracy is dependent on the accuracy of the
two individual measurement methods. In the estimation of
5. Discussion the maximum possible error using the law of error prop-
agation, the partial derivative of the determined function
The initial aim of this study was to assess the possibility cm Glu = f(n, n0,0 , , S1 , S2 ) from the slope S2 (cm Glu/S2 ,
of using a refractometrical procedure (in combination with S2 = dn0 /d) could be identified as the greatest disturbing fac-
the necessary estimation of the degree of dilution) to deter- tor (Figs. 3 and 4). This is quite understandable, since the
mine the glucose content of blood fluids with an accuracy slope S2 is indirectly constructed from the error-flawed mea-
which is adequate for medicaltechnical purposes. With the surement values n, cm Glu and .
described materials and equipment the following could be The fact that the error of the reference method used
shown: the blood glucose content in healthy subjects can be (glucose-analyser Accutrend Sensor) was disregarded in the
assessed over a range of 80300 mg/dL (0.803.00 g/L) with consideration of the degree of error of the refractometrical
an experimental inaccuracy (compared to a reference method) method should also be borne in mind. This error adds to the
of less than 10% using refractometry and electrical conduc- deviation from the respective true value. Because of this,
tivity. Furthermore, a clear improvement of the measurement changes in the glucose concentration can, for example, be
accuracy appears to be possible with the use of more precise determined even more exactly with the measurement devices
measurement devices. used than the absolute values.
In the future, these orientating results will obviously need The calibration, i.e. the assignment of a reference value
to be verified in a larger number of subjects, in particular in for the glucose content to the refractometrical determin-
diabetic patients. ing equation, occurs purely electronically, with the dc volt-
After high molecular weight substances were removed age component V0 being selected so that after entering the
from human serum using a variety of ultrafilters, the depen- measurement value in the determining equation, this pro-
458 K. Zirk, H. Poetzschke / Medical Engineering & Physics 29 (2007) 449458

duces the same glucose value. In order to ensure that the ultrafiltrated serum. Although sodium chloride makes up 75%
deviation of the output signal of the contact refractometer of the electrolytes in serum, the temperature coefficients for
[8] is as small as possible within the normal physiological the calibration solution and serum differ (whereby in both
range, the measurement calibration should also be carried cases a temperature compensation factor of 2% was applied).
out within this normal range, as was the case in the present According to this finding, the remaining electrolytes in serum
study. must have a substantial influence, which becomes evident in
A further aim of this study was to assess whether the influ- the reduction of the temperature coefficient. A dilution or
ence of temperature could be immediately compensated a relatively small concentration of the serum led within
for by a correction of the primary measurement signal, or the selected concentration range to a parallel shift of the
whether temperature is a quantity which needs to be regarded graphs linear function, since the total temperature coefficient
as a further (third) independent variable in the refractomet- is independent of the concentration within this range [12].
rical assessment of blood glucose, thus needing to be taken This also contributes to the possibility of a greatly simplified
into consideration in the corresponding calibration. temperature compensation.
If the temperature of the measurement sample is kept con-
stant (as it was for the evaluation of the glucose content with
the refractometry method), its measurement and subsequent References
compensation become unnecessary. The measurement value
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This differs somewhat to what is seen in Fig. 5B for the berg: Huthig; 1983.
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