Clinical Laboratory
San Frnacisco State University
1600 Holloway Avenue
San Francisco, CA
CLS Laboratory Procedure #900.099
Section: Chemistry
Date of Implementation: April 4, 2011
Revision:_______________________
Glucose Hexokinase
Procedure for the quantitative determination of glucose in serum
Policy
The laboratory’s compliance to CLSI GP2A5 guidelines shall identify
work processes in the laboratory’s operational path of workflow and
write procedures for pre-analytical, analytical, and post-analytical
laboratory activities.
Purpose
This procedure provides instructions for in vitro testing for the
quantitative determination of glucose in human serum and plasma on
the Pointe 180 QT analyzer.
Responsibility
The CLS internship Program Director shall review and approve
procedural documents, appoint designees for the implementation and
to maintain document control and record.
This procedure follows the CLS SOP template # 900.099 to implement
SOP and the designated author will perform document revisions.
Clinical
Significance
Glucose is the major carbohydrate present in the peripheral blood.
Oxidation of glucose is the major source of cellular energy in the body.
Glucose derived from dietary sources is converted to glycogen for
storage in the liver or to fatty acids for storage in adipose tissue. The
concentration of glucose in blood is controlled within narrow limits by
many hormones, the most important of which are produced by the
pancreas.
The most frequent cause of hyperglycemia is diabetes mellitus resulting
from a deficiency in insulin secretion or action. A number of
secondary factors also contribute to elevated blood glucose levels.
These include pancreatitis, thyroid dysfunction, renal failure and liver
disease.
Hypoglycemia is less frequently observed. A variety of conditions may cause
low blood glucose levels such as insulinoma, hypopituitarism or insulin
induced hypoglycemia. Glucose measurement in urine is used as a diabetes
screening procedure and to aid in the evaluation of glycosuria, to detect renal
tubular defects, and in the management of diabetes mellitus. Glucose
measurement in cerebrospinal fluid is used for evaluation of meningitis,
neoplastic involvement of meninges and other neurological disorders.
CPT code
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99214
Clinical Laboratory
San Frnacisco State University
1600 Holloway Avenue
San Francisco, CA
Principle
CLS Laboratory Procedure #900.099
Section: Chemistry
Date of Implementation: April 4, 2011
Revision:_______________________
UV Test
Enzymatic reference method with hexokinase1
HK
Glucose + ATP----------------------->G6P + ADP
G6PDH
G6P + NAD------------------------> 6-Phosphogluconate + NADH + H+
Glucose is phosphorylated with adenosine triphosphate (ATP) in the
reaction catalyzed by hexokinase (HK). The product, glucose-6phosphate (G6P) is then oxidized with the concomitant reduction of
nicotinamide adenine
dinucleotide (NAD) to NADH in the reaction catalyzed by glucose-6phosphate-dehygrogenase (G6PDH). The formation of NADH causes
an increase in absorbance at 340 nm. The increase is directly
proportional to the amount of glucose in the sample.
Specimen
Requirements
For specimen collection and preparation, only use suitable tubes or
collection containers.
Serum: Use fresh, unhemolyzed serum.
Plasma: Unhemolyzed samples from tubes containing oxalate, citrate,
EDTA, fluoride or heparin may be used.
Serum and plasma must be separated from the red cells promptly to
prevent glycolysis. Glucose will decrease approximately 7% per hour
when left in contact with red cells.10 The addition of sodium fluoride
to the specimen may prevent glycolysis.2
Glucose in serum of plasma is stable for 8 hours at room temperature
and 24 hours refrigerated at 2-8°C.
Specimen Rejection: Any unlabelled or improperly labeled specimen
or sample not collected in accordance with requirements above will be
rejected.
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Clinical Laboratory
San Frnacisco State University
1600 Holloway Avenue
San Francisco, CA
Materials/
Equipment
CLS Laboratory Procedure #900.099
Section: Chemistry
Date of Implementation: April 4, 2011
Revision:_______________________
Equipment
 Accurate pipetting
devices
 Spectrophotomet er
with ability to read at
340nm
 Heat block
Procedure
Calculations
Reagents
 Glucose
Hexokinase
reagent
Supplies
 Timer
 Deionized water
 Test tubes/rack
Step
Action
1.
Reconstitute Glucose Hexokinase reagent with distilled water
using the volume stated on the vial label. Swirl gently to
dissolve lyophilized material. Do not shake.
2
Pipet 1.0 ml of reagent into the required number of labeled test
tubes (eight tubes in total), and incubate for 3-5 minutes.
3
Turn on the spectrophotometer and when asked if you’d like to
use a “Stored Curve” select NO.
4
Read a blank tube at 340 nm containing 1.0 ml of Glucose
Hexokinase. Remove tube.
5
Add 10 ul each of control and patient to their respective tubes.
Mix well and set the timer to incubate in the heat block for
three minutes.
6
Read and record the absorbance values of all tubes.
7
If the result exceeds linearity or an “R” flag appears, dilute the
flagged sample 1:1 with normal saline, repeat the
measurement, and multiply the result by two.
8
To determine results, see “Calculations” section.
Standard method to determine concentration of unknown values:
Abs. = Absorbance
Conc. = Concentration
Sample Abs. X Conc. of Standard = Glucose (mg/dL)
Standard Abs.
Example: If Sample Abs. = 0.150
Standard Conc. = 100 mg/dL
Standard Abs. = 0.200
Then: 0.150 X 100 (mg/dL) = 75 mg/dL
0.200
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Clinical Laboratory
San Frnacisco State University
1600 Holloway Avenue
San Francisco, CA
Interpretation
of Results
Clinical
Significance
Serum
Normal Reference intervals
Abnormal results
Critical statements
CLS Laboratory Procedure #900.099
Section: Chemistry
Date of Implementation: April 4, 2011
Revision:_______________________
74-100 mg/dL (adults, fasting)
135-140 mg/dL (nonfasting)
Greater than 180-200 mg/dL
Less than 60-65 mg/dL
< 40 mg/dL or > 400 mg/dL
Abnormal results greater than 180-200 mg/dL indicates
hyperglycemia. Results less than 60-65 mg/dL is indicative of
hypoglycemia.
Interferences
Serum/plasma
Hemolysis: No significant Interference up to a hemoglobin
concentration 1000 mg/dL
Lipemia: Extremely lipemic samples may give a falsely elevated
glucose value for an L index of 1000.
Icterus: No significant interference up to an I index of 60 (60 mg/dL)
In very rare cases gammopathy, in particular type IgM (Waldenström's
macroglobulinemia), may cause unreliable results.
Drugs: No interference was found using common drug panels.
Expected
Values
Reported normal range: 65-110 mg/dL
It is recommended that each laboratory establish its own reference
range.
Clinical
Applications
Urine is not specified as an appropriate sample type per reagent
package insert. However, there are many other reagent applications
that do accept and give reference ranges for urine.
Reporting
Analytical test result calculated manually. See “Calculation” section for
a sample calculation.
Critical Values
Serum - less than 40 mg/dL or greater than 400 mg/dL
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Clinical Laboratory
San Frnacisco State University
1600 Holloway Avenue
San Francisco, CA
CLS Laboratory Procedure #900.099
Section: Chemistry
Date of Implementation: April 4, 2011
Revision:_______________________
Protocol to follow if critical value result is obtained:
Immediately contact attending physician. See Critical Values Policy.
Notes
For calibrator use an aqueous glucose standard of 100 mg/dl and quality
controls please reference the control procedure.
References
References and MSDS associated references
1. Tietz NW, ed. Clinical Guide to Laboratory Tests. 3rd ed.
Philadelphia: WB Saunders 1995:268-273.
2. Sacks DB. Carbohydrates. In: Tietz NW, ed. Fundamentals of
Clinical Chemistry. 4th ed. Philadelphia: WB Saunders
1996:351-374.
Author:_Alaina Whitaker, CLS Trainee
Date:_April 4, 2011__________________
Approved by:_______________________
Date:______________________________
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