TECO DIAGNOSTICS CHOLINESTERASE (PTC) REAGENT

1268 N. Lakeview Ave. SET

Anaheim, CA 92807 (KINETIC PROCEDURE)
1-800-222-9880

INTENDED USE
Cholinesterase reagent is used for in vitro diagnostic use in the REAGENT DETERIORATION
quantitative kinetic determination of cholinesterase in human serum, The reagent should be discarded if:
plasma, or whole blood. 1. Moisture has penetrated the vial and caking has occurred.
2. The reconstituted reagent has an absorbance against water
INTRODUCTION greater than 1.2 at 405 nm.
Measurement of serum cholinesterase has been used to assess liver
function and monitor excessive exposure to organophosphorus SPECIMEN COLLECTION AND STORAGE
insecticides.1 It is also useful in predicting susceptibility to Cholinesterase activity is stable in undiluted serum for as long as 2
prolonged apnea after the administration of succinylcholine and to weeks at 2 - 8°C and up to 3 months at -20°C.4 Serum should be
investigate the inheritance of variants of cholinesterase enzyme.2,3 removed from clot promptly.5 EDTA does not inhibit cholinesterase
There are many methods for the measurement of cholinesterase activity.
activity including manometric, titrimetric and photometric
procedures. The colorimetric procedure based on the Ellman reaction Specimen preparation for measurement of cholinesterase activity in
is sensitive and simple. Therefore, this technique forms the basis of whole blood is as follows. Draw blood in tubes containing EDTA as
our reagent. anticoagulant. Mix whole blood thoroughly. An aliquot of blood is
removed for hematocrit determination. Prepare blood hemolysate by
PRINCIPLE mixing 0.1 ml blood with 1.9 ml distilled water. Mix until hemolysis
Reactions involved in the cholinesterase assay are as follows: is complete. Centrifuge the remaining blood to obtain clear plasma.
Determine the cholinesterase activities in plasma and hemolysate by
CHOE following the instructions in "Procedure" section. Samples for
Propionythiocholine + H2O Propionic Acid + Thiocholine hemolysate cholinesterase activity are stable for 8 hours when stored
at 2 - 8°C.
Thiocholine + DTNB 5-Thio-2-nitrobenzoate
INTERFERING SUBSTANCES
Cholinesterase hydrolyzes Propionythiocholine (PTC) to form Hemolyzed serum samples should not be used. Certain drugs and
Thiocholine which reacts with 5,5'-dithiobis-2-nitrobenzoic Acid other substances are also known to affect cholinesterase activity.5
(DTNB) to yield yellow 5-thio-2-nitrobenzoate with an absorbance
maximum at 405 nm. Therefore, the rate of change in absorbance at MATERIALS REQUIRED BUT NOT PROVIDED
405 nm is directly proportional to cholinesterase activity. 1. Spectrophotometer, with temperature controlled compartment,
capable of accurately measuring absorbance at 405 nm.
REAGENT COMPOSITION 2. Test tubes with optical properties suitable for use at 405 nm
The Cholinesterase (PTC) Reagent, when reconstituted according to 3. Pipetting devices for the accurate delivery of volumes required
directions, has the following active ingredients. The approximate for the assay
concentration of each component is as follows: 4. Timer
5. Dibucaine and Sodium Fluoride Solutions
Propionythiocholine 4mM, DTNB 0.4 mM, Buffer and non-reactive
stabilizers and fillers AUTOMATED PROCEDURE
Refer to appropriate application manual available.
WARNING AND PRECAUTIONS
The Cholinesterase (PTC) Reagent is for in vitro diagnostic use. MANUAL PROCEDURE
Normal precautions exercised in handling laboratory reagents should 1. Reconstitute reagent according to instructions.
be followed. 2. Pipette 1.0 mL of reagent into appropriate tubes and allow to
equilibrate to 30°C or 37°C.
REAGENT STORAGE AND STABILITY 3. Zero spectrophotometer with water at 405 nm.
Store the dry reagent refrigerated (2 - 8°C). Reagent is stable until the 4. Add 10 µL of sample (serum, plasma or hemolysate). Mix well.
expiration date shown on the label. Reconstituted reagent is stable for 5. After 15 seconds, measure the absorbance (A1). Return tube to
6 hours at room temperature (15 - 30°C) or for 3 days at 2 - 8°C. 30°C or 37°C for another 30 seconds and measure another
absorbance (A2).
REAGENT PREPARATION
6. Calculate the ΔA per 30 seconds by subtracting A1 from A2,
TOTAL CHOLINESTERASE ACTIVITY:
Reconstitute Cholinesterase (PTC) Reagent with volume of deionized Multiply by 2 to obtain ΔA per minute.
water indicated on via label. After addition of water, stopper vial and 7. Calculate cholinesterase activity (U/L) of sample by multiplying
immediately mix several times by inversion. ΔA/min. times 7426.

DIBUCAINE INHIBITOR: ALTERNATE VOLUMES

Reconstitute one vial of Cholinesterase (PTC) Reagent with The cholinesterase activity can also be determined by using 10 µl
Dibucaine Solution, instead of deionized water. sample and 3 mL reagent. Different calibration factors should be used
if sample and reagent volumes are different from the volumes
FLUORIDE INHIBITOR: required in the procedures.
Reconstitute one vial of Cholinesterase (PTC) Reagent with fluoride
solution, instead of deionized water.
DIBUCAINE AND FLUORIDE INHIBITION ASSAYS 2. This procedure does not include Dibucaine or Fluoride for
Following the same steps given in the Procedure, determine resistance studies
cholinesterase activity in the samples using Dibucaine and Fluoride CALIBRATION
containing reagents prepared according to the instructions given in The procedure is calibrated by means of the millimolar absorptivity
Reagent Preparation. To determine percent of inhibition of activity, of 5-thio-2-nitrobenzoic acid, which is 13.6 at 405 rim. The linearity
refer to Calculations. range of this reagent varies depending upon the sample to reagent
ratio. The upper limit of linearity according to this procedure is 8,000
CALCULATIONS (KINETIC) U/L with sample to reagent ratio of 1:100.
Cholinesterase Activity (U/L) = ΔA/min. × TV × 1000
13.6 × LP × SV QUALITY CONTROL
It is recommended that high and low values of cholinesterase are
ΔA/min. = Absorbance change per min. included in each set of assays.
TV = Total Volume (1.01 mL) Commercially available control material with established
SV = Sample Volume (0.01 mL) cholinesterase values may be used for quality control.
13.6 = Millimolar absorptivity of DTNB
LP = Lightpath (1 cm) Temperature Conversion Factor for Human Serum
1000 = Conversion of units per mL to Units per Liter
Desired Temp
Cholinesterase Activity (U/L) = ΔA/min. × TV × 1000 Assay Temp. 25 30 37
13.6 × 1.0 × 0.01 25 1.00 1.20 1.55
30 0.83 1.00 1.29
NOTE: Preparation of hemolysate involves 20-fold dilution of the 37 0.65 0.77 1.00
sample; therefore, the Cholinesterase Activity in hemolysate
calculated by the above formula should be multiplied by 20 to EXPECTED VALUES
compensate for the dilution. The expected range of serum cholinesterase activity at 30°C was
determined to be as follows:
Example: A1 = 0.316 A2= 0.491 Serum 3100 – 7700 U/L
A2 - A1 = 0.491 - 0.316 = 0.175 Plasma 1700 – 4100 U/L
ΔA/min. = ΔA/30 seconds × 2 = 0.350 Whole Blood 3300 – 5500 U/L
Erythrocytes 4400 – 8200 U/L
Cholinesterase Activity (U/L) of sample = 0.350 × 7426= 2599
PERFORMANCE CHARACTERISTICS
DIBUCAINE AND FLUORIDE INHIBITION 1. Linearity: 8000 IU/L at 30°C.
Calculate percent of inhibition of Cholinesterase Activity (U/L) as 2. Sensitivity: An absorbance change of 0.001/min. at 405 nm
follows: corresponds to 7.4 U/L of cholinesterase activity under the
stated conditions of this assay system.
Dibucaine Inhibition of Cholinesterase Activity (%) = 3. Comparison: A study performed between the present procedure
100 - (CHE - D) × 100 and one commercial product on whole blood resulted in a
CHE coefficient of correlation of 0.96 with a regression of y = 1.00 x
-103 (n = 20) and on serum/plasma resulted in a coefficient of
Fluoride Inhibition of Cholinesterase Activity (%) = 0.99 with a regression of y = 0.96 x + 63 (n = 47).
100 - (CHE - F) × 100 4. Precision Studies:
CHE Within Run
Mean (U/L) S.D. C.V.(%)
Where: 4539 173 3.8
CHE= Cholinesterase Activity in sample determined by using reagent 3717 162 4.4
containing neither Dibucaine nor Sodium Fluoride
Run-To-Run
CHE-D= Cholinesterase Activity in sample determined by using Mean (U/L) S.D. C.V.(%)
reagent containing 0.3 mMol/L Dibucaine 4603 153 3.3
3760 151 4.0
CHE-F = Cholinesterase Activity in sample determined by using
reagent containing 40 mMol/L Sodium Fluoride. REFERENCES
1. Silk, E., et al., Ann Clin. Biochem. 16:57 (1979).
Erythrocyte Cholinesterase Activity: 2. Newman, M.A. et al., Clin. Chem. 30:308 (1984).
Erythrocyte Cholinesterase Activity (ECHE) is calculated from the 3. Dietz, A.A., et al., Clin. Chem. 19:1309 (1973).
results obtained for plasma Erythrocyte Cholinesterase Activity 4. Clinical Chemistry-Principles and Techniques, 2nd Ed., R.J.
(PChE), Hemolysate Cholinesterase (HChE) and the hematocrit Henry, D.C. Cannon, J.W. Winkelman, Editors, Harper and
(HCT) value of the sample. Row, Hagerstown (MD), p. 919 (1974).
5. Witter R.F., Arch. Enviro. Health 6:537 (1963).
(HChE) = (EChE x Hct*) + (PChE x (1-Hct*)
C511: 04/2012
EChE = HChE - (PChE x (1 - Hct*)
Hct* TECO DIAGNOSTICS
1268 N. LAKEVIEW AVE.
* Hematocrit value expressed as decimal equivalent. ANAHEIM, CA 92807
U.S.A.
LIMITATIONS
Authorized Representative:
1. Extremely lipemic or icteric serum should have blank correction Emergo Europe
performed. Molenstraat 15
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