Chapter 2  |  Chapter 4
 
CHAPTER 3

VERIFICATION OF NERVE AGENT EXPOSURE--MONITORING BLOOD CHOLINESTERASE ACTIVITY WITH THE TEST-MATETM OP KIT

 
3-1. Background

a. Various pesticides, drugs, and chemical agents inhibit cholinesterase (ChE) activity. Determining depression of ChE activity provides an indication of exposure to nerve agents.1 Plasma ChE recovers in 30 to 40 days and red blood cell (RBC) acetylcholinesterase (AChE) recovers in 90 to 100 days after exposure to organophosphorus nerve agents.1 It is recommended that the Test-MateTM OP Kit (EQM Research Incorporated, 2585 Montana Avenue, Cincinnati, OH 45211) be used to establish ChE levels when exposure to nerve agents is suspected.

b. The chemistry employed in the ChE activity determination is that described by Ellman2 in which thiocholine, the product of substrate acetylthiocholine (ATCh) hydrolyses, is detected by reacting with 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB). This kit can determine RBC AChE and plasma butyrylcholinesterase (BChE) activities within minutes, requiring 10 µl of blood per determination.

3-2. Materials and methods

a. Equipment. The equipment and reagents for the Test-MateTM OP Kit are contained in a plastic, airtight case measuring 26x23x16 centimeters (cm). The kit consists of the following items:

(1) Alcohol swabs.

(2) Battery, 9 volt.

(3) Biohazard disposal bags.

(4) Biopsy punch.

(5) Blood lancets.

(6) Buffer dropper.

(7) Capillary bulb assembly.

(8) Capillary pipettes.

(9) Erythrocyte AChE reagent.

(10) Gauze pads.

(11) Vinyl gloves.

(12) Graduated cuvette, 2 ml.

(13) Plasma ChE reagent.

(14) Sample buffer.

(15) Stirring paddle.

(16) Test-MateTM OP blood analyzer.

(17) Transfer pipettes.

(18) Test rack for droppers, paddle and cuvette.

(19) Waste bottle.

(20) Water dropper.

(21) Water reservoir bottle.

(22) Water dispenser.

b. Sample collection. Blood samples are drawn from a pricked finger tip into a 10 ml, heparinized microcapillary tube. If analysis can not be done immediately, blood should be drawn into VacutainersTM containing ethylenediaminetetraacetic acid (EDTA) as anticoagulant and kept refrigerated or on ice. Tests should be performed within a week for stored samples.

c. Reagents. The reagents in the Test-MateTM OP Kit are either in a concentrated aqueous form or lyophilized in 96-well microplates. The reagent concentrations reported here are those in the total reaction mixture.

(1) In the erythrocyte AChE assay are:

(a) ATCh, 1 millimole (mM).

(b) Potassium phosphate, 20 mM, pH 7.4.

(c) Triton X-100, 0.1 percent.

(d) DTNB, 0.3 mM.

(e) 10-(a-diethylaminopropionyl)-phenothiazine hydrochloride (Astra 1397), 21
µ M.

(2) In the plasma BChE assay are:

(a) Butyrylthiocholine (BTCh), 2 mM.

(b) Potassium phosphate, 20 mM, pH 7.4.

(c) Triton X-100, 0.1 percent.

(d) DTNB, 0.3 mM.

d. Analyzer.

(1) The Test-MateTM blood analyzer is a battery powered colorimeter with an LED light source and 450 nanometer (nm) filter. It is slightly larger than a hand-held scientific calculator. On the upper face of the colorimeter are function keys, a liquid crystal display screen and the cuvette compartment. The function keys are used to initiate successive steps in the procedure and to recall the results at the end of analyses.

(2) The LED screen displays instructions prompting the user through the analyses procedure and displays the results at the conclusion. In addition, the results are compared to normal mean values preprogrammed in the colorimeter computer and displayed as a percent of the normal mean value.

WARNING

To avoid the transmission of disease, follow all safety precautions listed below
.

e. Safety. Precautions must be taken to eliminate hazards either to the operator or the person being tested. The following rules must be followed during the testing procedure:

(1) Do not reuse blood lancets or capillaries.

(2) Put used lancets and capillaries in a hazard bag.

(3) The operator must wear gloves and safety glasses.

f. Erythrocyte AChE activity analyses. Remove the Test-MateTM components from the carrying case and lay them out, within easy reach, in front of you on a flat surface. In the field it could be the lid of a car trunk, the back of a pickup truck, a flat stone, etc. Before going out in the field, fill the water reservoir bottle with deionized or distilled water and pack an extra battery. Procedures are as follows:

(1) Press the "Power" key. The display screen indicates a 15 second warmup countdown.

(2) Press the "Mode" key until "AChE mode" is displayed.

(3) Press the "Test" key. The display will read "Add buffer" followed by "Insert cuvette" and "Press test." Add four drops of buffer to the cuvette, add water to the 2 ml mark, using the water dispenser, and mix with the stirring paddle. Insert the cuvette into the colorimeter.

(4) Press the "Test" key. A 10 second "Blanking" is displayed ending in a "beep." "Remove cuvette" followed by "Press test" is displayed.

(5) Press the "Test" key. The display reads "Add blood" followed by "Insert cuvette" and "Press test."

(6) Wipe the fingertip of the person being tested with the alcohol swab. Twist off the cap of the blood lancet and prick the finger at its inside tip, off-center. Wipe off the first drop of blood with the gauze pad and squeeze out a second drop. Fill the capillary tube with blood and dispense into the buffer filled cuvette. Mix the solution with the stirring paddle and insert the cuvette into the sample chamber.

(7) Press the "Test" key. A 30 second "Reading" is displayed followed by a "beep." During this time Hgb has been determined. The display reads "Remove cuvette" followed by "Press test."

(8) Press the "Test" key. The display reads "Get ready" followed by "With reagent" and "Press test." Remove the plastic covering of one of the reagent wells of the microplate with the biopsy punch. Add four drops of water with the water dropper to the well and dissolve the reagent by aspirating it three times using the transfer pipette. Fill the transfer pipette with the dissolved reagent.

(9) Press the "Test" key. The display reads "AT BEEP" followed by "Add reagent." At the "beep" add the reagent in the transfer pipette to the diluted blood-filled cuvette and mix the contents with the stirring paddle. The display reads "Insert cuvette" followed by "Press test." Insert the cuvette into the sample chamber.

(10) Press the "Test" key. The display shows an 80 second pre-incubation countdown followed by a 50 second reading phase ending in a "beep." The analysis of erythrocyte AChE is concluded. The display reads "Remove cuvette" followed by "Press test." Remove the cuvette and pour the solution into the waste bottle. Use the water dispenser to rinse the cuvette.

(11) Press the "Test" key to initiate results recovery. After the first result is displayed, keep pressing the "Disp." key to recover the rest of the data.

g. Plasma BChE activity analyses. Procedures are as follows:

(1) Press the "Power" key. The display screen indicates a 15 second warmup countdown.

(2) Press the "Mode" key until "PChE mode" is displayed. From this point on, the procedure is the same as for AChE activity determination except that there is no Hgb assay.

3-3. Results and discussion

The Test-MateTM kit uses blood as sample for both RBC AChE and plasma BChE activity measurement by using two different modes to distinguish the two enzymes. In the RBC AChE procedure, a specific inhibitor of BChE, Astra 1397,3 has been incorporated into the lyophilized reagent, eliminating interference due to the plasma enzyme. In the plasma BChE activity analysis, the substrate ATCh has been replaced by BTCh, which is hydrolyzed at a much faster rate by BChE than AChE.

a. Reproducibility. A blood specimen was analyzed repetitively six times to establish the reproducibility of the kit. The results are shown in table 3-1.

b. Intrapersonal variability of ChE activity. It is desirable that the pre-exposure baseline values of ChE are available for each individual to serve as control. Of the two enzymes, RBC AChE and plasma BChE, the former has the least intrapersonal variability4 and therefore is the enzyme of choice to be monitored for possible exposure. The kit provides RBC AChE results in two units; U/ml and U/gHgb. The RBC AChE expressed as U/ml will increase or decrease with the Hgb level which will vary due to blood transfusion, hemorrhage, or blood sampling techniques. Therefore, the RBC AChE value expressed as U/gHgb is recommended. A previous study indicated that the intrapersonal variability of RBC AChE (U/ml RBC) within a year is around 8 percent.4 With the addition of 1 percent variability of the method, a total of 9 percent variation would be expected. When a test value shows a drop of more than 9 percent from the baseline value, exposure to nerve agent would be suspected.

c. Interpersonal variability of ChE activity. If a baseline value is not available, possible RBC AChE inhibition has to be estimated from the population mean or normal range. Thirty U.S. Army personnel volunteered to participate in the Test-MateTM kit evaluation study. AChE activity in the blood obtained from a finger stick and BChE activity obtained from blood collected in an EDTA VacutainerTM were determined on groups of six volunteers per week for 5 weeks. Five volunteers were females and twenty five were males. As shown in table 3-2, RBC AChE values ranged from 22.5 to 33.3 U/gHgb. A test value below 22.5 U/gHgb will indicate a possibility of exposure to nerve agents.
 


Table 3-1. Reproducibility of the Test-MateTM kit
 
  Blood BChE Blood AChE Blood Hgb Blood AChE
  U/ml U/ml g/dl U/gHgb
MEAN 2.73 4.5 16.2 29.3
SD 0.10 0.07 0.3 0.3
CV% 3.70 1.50 1.9 1.0

Table 3-2. Cholinesterase activities of the volunteer population
 
  Blood BChE Blood AChE Blood Hgb Blood AChE
  U/ml U/ml g/dl U/gHgb
N 30.00 30.00 30.00 30.00
MEAN 2.51 2.51 15.6 27.9
SD 0.46 0.46 1.3 2.7
CV% 18.30 18.30 8.3 9.7
MEAN - 2SD 1.59 3.54 13.0 22.5
MEAN + 2SD 3.43 5.18 18.2 33.3

Footnotes

1 Grob, D. and A.M. Harvey. "The Effects and Treatment of Nerve Gas Poisoning," Amer. J. Med. 14 (1953), pp. 52-63.

2 Ellman, G.L., K.D. Courtney, J. Andres, Jr., and R.M. Featherstone. "A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity," Biochem. Pharmacol. 7 (1961), pp. 88-95.

3 Augustinsson, K.B. "A Titrometric Method for the Determination of Plasma and Red Blood Cell Cholinesterase Activity Using Thiocholine Esters as Substrates," Scan. J. Clin. and Lab. Investigation 7 (1955), pp. 284-290.

4 Sidell, F.R. and A. Kaminskis. "Temporal Intrapersonal Physiological Variability of Cholinesterase in Human Plasma and Erythrocytes," Clin. Chem. 21 (1975), pp. 1961-1963.


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