GLUCOSE HK Liquiform Ref.

: 137
Instruction for use

Intended Use . Enzyme system for glucose determination by The one-reagent methodology can be applied by using one Working
endpoint ultraviolet photometry in blood, urine, cerebrospinal fluid and Reagent stable for 60 days under refrigeration, obtaining adequate
ascitic fluid, pleural fluid and synovial fluid samples. performance even under low demand testing.

Professional use. The system also allows preparing the required Working Reagent volume
to perform an assay for the determination of glucose concentration.
[For in vitro diagnostic use.]
The system is easily applicable to automated and semiautomatic
analyzers capable of accurately measuring the absorbance at 340 nm.
Principle . Adenosine triphosphate promotes phosphorylation of
glucose in a reaction catalyzed by hexokinase (HK), according to the
following reaction: Methodology . Bondar and Mead modified.

HK Reagents
Glucose + ATP Glucose-6-phosphate + ADP

Glucose-6-phosphate produced in the reaction above is oxidized to

6-phosphogluconate in the presence of nicotinamide adenine
1. ( - Reagent 1 - Store at 2-8ºC.
It contains buffer pH 7.8; ATP ³1.0 mmol/L; NAD ³1.0 mmol/L;
magnesium chloride ³10 mmol/L; and 7.7 mmol/L sodium azide.
dinucleotide (NAD), in reaction specifically catalyzed by glucose-6-
phosphate dehydrogenase (G-6-PDH). It yields one mole of NADH for
each mole of glucose-6-phosphate which is oxidized. The resulting
absorbance measured at 340 nm is directly proportional to the
concentration of glucose in the sample.
2. ) - Reagent 2 - Store at 2-8ºC.
Hexokinase ³1000 U/L; G-6-PDH ³6000 U/L, and 15 mmol/L sodium
azide.

Glucose-6-phosphate + NAD
G-6-PDH
6-Phosphogluconate + NADH
3. < - Standard 100 mg/dL - Store between 2-30ºC.
After handling, store well sealed to prevent evaporation. The standard
stabilizer may precipitate at low temperatures, which does not interfere
System features . Hexokinase is an enzyme that catalyzes the with their performance.
transfer of phosphate from ATP not only to glucose, but also to
D-fructose, D-mannose, D-glucosamine, 2-deoxy-D-glucose. Because The unopened reagents, when stored under the specified conditions, are
G-6-PDH has high specificity for glucose-6-phosphate, other hexose or stable until the expiration date printed on label. During handling, the
pentose esters that are phosphorylated do not participate in the reaction. reagents are subject to contamination of chemical and microbial nature
Thus, the assay is highly specific for glucose. which can cause reduction in stability.

The specificity of the reaction provided by G-6-PDH enables the System traceability . System calibration is traceable to the
determination of urinary glucose with a high degree accuracy, which is Standard Reference Material 917 of the National Institute of Standards and
not achieved when using other systems employing other enzymes as Technology.
reagents.

The data of repeatability and reproducibility demonstrate unequivocally Precautions and warnings
that the Glucose HK Liquiform system has an extremely desirable and
Usual safety care should be applied when handling the reagent.
valuable robustness for an analytical system.
Reagents of Glucose HK Liquiform contain sodium azide, which is toxic.
The substances used in the reaction are properly distributed in two Care should be taken to avoid ingestion, and in case of contact with eyes,
reagents to confer greater stability in the original liquid form and wash it immediately with plenty of water and seek medical advice. Azide
maintenance of optimum reaction conditions allowing the direct use of can form highly explosive compounds when in contact with lead and
reagents into automated systems. copper pipes. Therefore, use large volumes of water to dispose the
reagent.
The use of two-reagent methodology significantly reduces the
interference present in the sample, conferring greater accuracy to the Do not use the Work Reagent when its absorbance at 340 nm against
results. water is higher than 0.350.

01 English - Ref.: 137

Materials required and not provided For one-reagent methodology, bilirubin values up to 10 mg/dL,
hemoglobin up to 50 mg/dL, and triglycerides up to 350 mg/dL do not
1. Water bath maintained at constant temperature (37ºC). produce significant interference.
Bilirubin values up to 20 mg/dL, hemoglobin up to 200 mg/dL, and
2. Photometer able to accurately measure the absorbance at 340 nm. triglycerides up to 1000 mg/dL produce interference that can be
3. Pipettes to measure samples and reagent. minimized by using the blank sample.
4. Chronometer.
To evaluate the approximate concentration of hemoglobin in a
hemolyzed sample, you can proceed as follows: Dilute 0.05 mL of sample
Pre-analytical Influences . In the 24 hours following the acute in 2.0 mL of 150 mmol/L NaCl (0.85%) and measure the absorbance at
ingestion of alcohol, a significant reduction in blood glucose occurs. 405 or 415 nm, hitting the zero with distilled or deionized water.
Reductions may also be significant in individuals undergoing prolonged
fasting or in obese subjects treated with low-calorie diets. Hemoglobin (mg/dL) @ Absorbance405 x 601
Hemoglobin (mg/dL) @ Absorbance415 x 467
Diabetic patients on continued use of chlorpropamide (Diabinese) may
develop significant hypoglycemia, being very difficult to fix. Blank sample: This procedure is applicable when there is interference. Mix
1.0 mL of 150 mmol/L NaCl (0.85%) with 0.01 ml of sample. Measure the
absorbance at 340 nm, hitting the zero with distilled or deionized water.
Sample
Subtract the absorbance thus obtained from the assay absorbance and
Use plasma, serum or urine observing the following precautions. The calculate the concentration.
blood sample should be obtained after fasting for at least 8 hours or
according to doctor's recommendation. Preparation of working reagent . The combination of Reagent 1
and Reagent 2 allows preparing the working reagent. Transfer the
Collect the blood sample by using an anticoagulant containing an inhibitor contents of one vial of Reagent 2 into a vial of Reagent 1 and mix by
of glycolysis. The use of the anticoagulant Glistab (Labtest Ref. 29) allows inversion. Note the expiration date. It is stable for 60 days at 2-8ºC when
the collection of only one sample for the measurement of creatinine, there is no chemical or bacterial contamination. Identify the vial of
glucose and urea. Working Reagent to avoid confusion with other vials of Reagent 1. To
Blood samples not containing antiglycolytic agent should be centrifuged maintain its performance, the reagent must remain outside the refrigerator
immediately after collection and plasma or serum separated from clot or only for the time needed to obtain the volume to be used. Avoid exposure
cells. to direct sunlight.
In blood samples treated with antiglycolytic agent, the glucose Optionally, you can prepare smaller volumes of Working Reagent by using
concentration remains stable up to 8 hours. In plasma, serum and other the ratio of 4 volumes of Reagent 1 by 1 volume of Reagent 2, e.g., to
fluids separated from cells, the glucose remains stable for 3 days at prepare 1 mL of Working Reagent, mix 0.8 ml of R1 and 0.2 mL of R2.
2-8ºC, if no microbial contamination occurs6.

The presence of ascorbic acid in the sample does not interfere with the Procedure
results, which occurs on systems using the Trinder methodology. Take 3 test tubes and proceed as follows:

In other biological fluids (ascitic and cerebrospinal fluid, synovial fluid and
Blank Unknown Standard
pleural fluid), add anticoagulant containing antiglycolytic agent at the
Sample ---- 0.01 mL ----
same ratio used to blood sample and centrifuge before measuring.
Standard ---- ---- 0.01 mL
Working Reagent 1.0 mL 1.0 mL 1.0 mL
Urine samples should be stored between 2-8ºC to avoid interference by
microbial contamination.
Mix and incubate in water bath at 37ºC for 5 minutes. The water level in the
Since no known test can ensure that samples of human biological material bath should be higher than the level of the reagents in the test tubes.
do not transmit infections, all must be considered potentially infectious. Determine the absorbance for test and standard tubes at 340 nm, hitting
Therefore, when handling them, you must follow the rules established for the zero with the blank. The absorbance is stable for 60 minutes.
biosafety.
The suggested procedure for measuring is suitable for photometers whit
For disposal of chemicals and biological material we suggest applying the minimum volume of solution for reading equal to or less than 1.0 mL. It
applicable local, state or federal environmental protection standards. must be checked the need for adjusting the volume for the photometer
used. The sample and reagent volumes can be modified proportionately
without affecting the performance of the test procedure and the
Interference calculations remain unchanged. In case of reduced volumes, it is
For two-reagent methodology, bilirubin values up to 20 mg/dL, essential to observe the minimum volume required for the photometric
hemoglobin up to 200 mg/dL, and triglycerides up to 1000 mg/dL do not reading. Sample volumes smaller than 0.01 mL are critical in manual
produce significant interference. applications and should be used with caution because they increase the
inaccuracy of the measurement.

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Calculations . See linearity. Control materials should be used to assess the calibration inaccuracies
and deviations. It is suggested that the specifications for the coefficient of
Assay Absorbance variation and the total error are based on the components of the biological
Glucose (mg/dL) = x100 variation. (BV)8.9.
Standard Absorbance
It is suggested to use the products from Qualitrol H-Labtest line for internal
quality control in clinical chemistry assays.
Example

A Test = 0.322 A Standard = 0.357 Reference ranges . Ranges should be used only as a guide. It is
recommended that each laboratory establish, in the population serviced,
0.322 its own reference ranges.
Glucose (mg/dL) = x 100 = 90
Plasma (8 hours fasting)
0.357
Age mg/dL
Premature 20 - 60
The result can also be obtained by using the calibration factor.
0 - 1 day 40 - 60
> 1 day 50 - 60
100
Children ad Adults 65 - 99
Calibration Factor =
Standard Absorbance
Diagnostic criteria for diabetes and pre-diabetes can be obtained from:
Glucose (mg/dL) = Test Absorbance x Factor American Diabetes Association. Diabetes Care 2012; (suppl): 1 S64-S71.

Example Cerebrospinal SF: 2/3 of the blood glucose when measurement is

performed on samples taken simultaneously.
100
Factor = = 280 In healthy individuals, the small amount of fluid present in the joint, pleural
0.357 and peritoneal cavities originates from the plasma ultrafiltrate. Therefore,
it can be considered that, practically, the glucose present in these fluids is
Glucose (mg/dL) = 0.322 x 280 = 90 at the same concentration as in plasma.

mg/dL x volume (mL) Urine . less than 20 mg/dL and up to 250 mg/24 hours.
Urine (mg/24 hours) =
100 Conversion . Conventional Units (mg/dL) x 0.0556 = IS units (mmol/L)

Calibration . The standard traceable to Standard Reference Material Performance features12

(SRM) 917 in the National Institute of Standards and Technology (NIST).
Accuracy . In two samples having glucose concentrations equal to 51
Manual Calibrations and 103 mg/dL different quantities of analyte were added, yielding in the
To obtain the calibration factor when using a new batch of reagents or endpoint method recoveries between 98 and 99%. The average
when the internal quality control states. proportional systematic error obtained in value of 120 mg/dL is equal to
1.8 mg/dL or 1.5%.
Automated systems
Reagent blank: water or 150 mmol/L (0.85%) sodium chloride solution;
Specificity . The proposed method was compared with a similar
Standards: use protein calibrators. The concentration of glucose in
method, obtaining the following results:
Calibra H is traceable to NIST SRM 917.

Comparative Labtest
Linearity Method Method
Number of samples 40
The reaction is linear up to 700 mg/dL. For higher values, dilute the sample
Concentration range
with 150 mmol/L NaCl (0.85%), perform a new measurement and 44 - 575
(mg/dL)
multiply the result by the dilution factor. We suggest checking the
Labtest Method (mg/dL) = 0.9965 x
photometric and methodological linearity at least every six months by Regression equation
Comparative + 0.8167
using samples with values up to 700 mg/dL.
Correlation coefficient 0.999

Internal quality control . The laboratory should maintain a Using the regression equation, the total error (constant and proportional)
program of internal quality control, clearly defining applicable regulations, found at levels of decision of 45,120 and 180 mg/dL were equal to 0.66,
objectives, procedures, criteria for quality specifications and tolerance 0.40 and 0.19 mg/dL or 1.47; 0.33 and 0.11%, respectively.
limits, corrective actions and activity recording.

03 English - Ref.: 137

The total systematic error obtained is smaller than the total systematic 2. The deionized or distilled water in the laboratory to prepare reagents,
error of the desirable specification based on the components of Biological use in the measurements and for final glass washing must have resistivity
Variation, which is £±2.2%. ³1 megaohm.cm, or conductivity £1 microsiemens/cm and silicates
concentration must be <0.1mg/L.
As the samples were randomly selected on outpatients and in patients, it
can be inferred that the method has a suitable methodological specificity. References

Imprecision studies . Imprecision studies were conducted using 1. Bergmeyer HU. Methods of Enzimatic Analisys, 3ed, vol 6, Deerfield
80 samples with mean concentrations equal to 46, 123 and 184 mg/dL. Beach: Verlag Chemie,1984;163-172.

2. Bjorkem I, Blomstrand R, Falk O, Ohman G. Clin Chim Acta

Imprecision - within run 1976;72:353-62.

N Mean SD (%) CV
3. Bondar RJL, Mead D. Clin Chem 1974;20:586-90.
Sample 1 80 46 0.46 1.00
Sample 2 80 123 1.01 0.83 4. Inmetro - Boas Práticas de Laboratório Clínico e Listas de Verificação
Sample 3 80 184 1.45 0.79 para Avaliação, Qualitymark eds, Rio de Janeiro, 1997.

5. Kaplan LA, Pesce AJ. Methods in Clinical Chemistry, St. Louis: The C.V.
Imprecision - Run-to-run Mosby Co., 1987;105-11.

N Mean SD CV (%) 6. BURTIS CA, Ashwood ER, Bruns DE. Tietz, Textbook of Clinical
Chemistry and molecular diagnostics, 4th edition 2006. St Luis,
Sample 1 80 46 1.04 1.85
Missouri: Elsevier Saunders. 2006.
Sample 2 80 123 1.47 1.19
Sample 3 80 184 2.46 1.34
7. Westgard JO, Barry PL, Hunt MR, Groth T. Clin Chem 1981;27:493-
501.
The total error (random error + systematic error) estimated at
concentrations equal to 45, 120 and 180 mg/dL are equal to 4.53%, 8. Biological Variation Database specifications - Westgard QC. Disponível
2.30% and 2.32%, respectively. em:
< h t t p : / / w w w. w e s t g a r d . c o m / b i o d a t a b a s e 1 . h t m >
The results indicate that the method meets the specification desirable for (acesso em 28/10/2011).
the total error (£±6.9%) based on desirable components of the biological
variation. 9. Basques JC. Especificações de Qualidade Analítica. Labtest
Diagnóstica 2005.
Methodological sensitivity . A protein sample containing no
glucose was used to calculate the detection limit of the assay, having 10. Burtis CA,Ashwood ER. Textbook of Clinical Chemistry, 2ª. Edição,
found a value of 0.62 mg/dL, equivalent to average concentrations Philadelphia:W.B. Saunders, 1986:2175-2211.
obtained plus two standard deviations. Using the absorbance of the
standard as parameter, the photometric detection limit is 0.28 mg/dL, 11. American Diabetes Association: Point: Impaired Fasting Glucose: The
corresponding to an absorbance equal to 0.001. Case for the New American Diabetes Association Criterion. Diabetes
Care May 2006 vol. 29 no. 5 1170-1172.
Effect of matrix dilution . Two samples with values equal to 821
12. Labtest: Data on file.
and 834 mg/dL were used to evaluate the system response in matrix
diluted with 150 mmol/L NaCl (0.85%). By using dilution factors ranging
from 2 to 8, it was found an average recovery of 100.1%. The total error Presentation
obtained is lower than the total systematic error of the desirable
specification based on components of the Biological Variation, which is Product Reference Contents
£±6.9%. 1 2 X 80 mL
Glucose HK Liquiform 137-2/100 2 2 X 20 mL
Notes 1 X 5 mL
1 10 X 65 mL
Glucose HK Liquiform
1. The material cleaning and drying are fundamental factors to the Labmax 560/400
137-10/82 2 10 X 17 mL
reagent stability and to obtain correct results. 1 X 5 mL

04 English - Ref.: 137

The number of tests in automated systems depends of the programmed Labtest Diagnóstica S.A.
parameters.
CNPJ: 16.516.296 / 0001 - 38
Av. Paulo Ferreira da Costa, 600 - Vista Alegre - CEP 33240-152
Application procedures using Glucose HK Liquiform are available for Lagoa Santa . Minas Gerais Brasil - www.labtest.com.br
various automated systems.
Customer Service e-mail: customerservice@labtest.com.br

Customer information
Edition: November, 1996 Copyright by Labtest Diagnóstica S.A.
Revision: February, 2013 Reproduction under previous autorization
[Warranty conditions]
Ref.: 110422(01)

Labtest Diagnóstica warrants the performance of this product under the

specifications until the expiration date shown in the label since the
application procedures and storage conditions, indicated on the label and
in this insert, have been followed correctly.

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06 English - Ref.: 137