- It can be used to detect and diagnose organophosphate pesticide exposure and/or poisoning. It may also be used to monitor those who may be at increased risk of exposure to organophosphate compounds, such as those who work in agricultural and chemical industries, and to monitor those who are being treated for exposure. Typically, tests for red blood cell acetylcholinesterase (AChE) and serum pseudocholinesterase (PChE) are used for this purpose.
- It can be used several days prior to a surgical procedure to determine if someone with a history of or family history of post-operative paralysis following the use of succinylcholine, a common muscle relaxant used for anesthesia, is at risk of having this reaction. In these cases, the test for pseudocholinesterase is usually used. A second test, referred to as a dibucaine inhibition test, may be done to help determine the extent to which the activity of the enzyme is decreased.
- Headache, dizziness
- Excessive tearing in the eyes, sweating and/or salivation
- Vomiting, diarrhea
- Dark or blurred vision due to constricted pupils
- Muscle weakness, twitching, lack of coordination
- Slowed breathing leading to respiratory failure, requiring lifesaving ventilation
- In serious cases, seizures, coma, and death
• Manual or microscopic method
• Automated method
Free-flowing capillary or well-mixed anticoagulated venous blood is added to a diluent at a specific volume in the Unopette reservoir. The diluents (1% ammonium oxalate) lyses the erythrocytes but preserves leukocytes and platelets. A 20 µL pipette is used with 1.98 ml of diluents to make a 1:100 dilution. The diluted blood is added to the hemacytometer chamber. Cells are allowed to settle for 10 minutes before leukocytes and platelets are counted. (Always refer to the manufacturer’s instructions for the procedure.)
Hemocytometer with cover glass, compound microscope. Unopette capillary pipette, lint-free wipe, alcohol pads, hand counter, petri dish with moist filter paper.
Ammonium oxalate: 11.45 gm
Sorensen’s phosphate buffer: 1.0 gm
Thimerosal: 0.1 gm
Distilled water: 1000 ml
EDTA-anticoagulated blood or capillary blood is preferred.
(1) Using the protective shield on the capillary pipette, puncture diaphragm of Unopette reservoir.
(2) Remove shield from pipette assembly by twisting. Holding pipette almost horizontally, touch tip of pipette to blood. Pipette will fill by capillary action. Filling will cease automatically when the blood reaches the end of the capillary bore in the neck of the pipette.
(4) Squeeze reservoir slightly to force out some air while simultaneously maintaining pressure on reservoir.
(5) Cover opening of overflow chamber of pipette with index finger and seat pipet securely in reservoir neck.
(6) Release pressure on reservoir. Then remove finger from pipette opening. At this time negative pressure will draw blood into reservoir.
(7) Squeeze reservoir gently two or three times to rinse capillary bore forcing diluent up int, but not out of, overflow chamber, releasing pressure each time to return mixture to reservoir.
(8) Place index finger over upper opening and gently invert several times to thoroughly mix blood with diuent.
(9) Cover overflow chamber with pipette shield and incubate at room temperature for 10 minutes before charging the hemacytometer.
(10) Meticulously clean the hemacytometer with alcohol or other cleaning solution. This is important because dust particles and other debris can be mistaken for platelets especially on a light microscope. Allow to dry completely before charging with diluted specimen.
(11) To charge the hemacyto-meter, convert to dropper assembly by withdrawing pipette from reservoir and reseating securely in reverse position.
(12) Invert reservoir and discard the first 3 or 4 drops of mixture.
(13) Carefully charge hemacyto-meter with diluted blood by gently squeezing sides of reservoir to expel contents until chamber is properly filled.
(14) Place hemacytometer in moist Petri dish for 10 minutes to allow platelets to settle. (Moistened filter paper retains evaporation of diluted specimen while standing.)
(15) Mount the hemacytometer on the microscope and lower its condenser.
(16) Procedure for counting platelets:
• Under 40x magnification, scan to ensure even distribution. Platelets are counted in all twenty-five small squares within the large center square. Platelets appear greenish, not refractile.
• Count cells starting in the upper left of the large middle square. Continue counting to the right hand square, drop down to the next row; continue counting in this fashion until the total area in that middle square (all 25 squares) have been counted.
• Count all cells that touch any of the upper and left lines, do not count any cell that touches a lower or right line.
• Count both sides of the hemocyt-ometer and take the average.
Ns x As x Ds
Total number of cells= 230
Number of squares counted: 1
Area of each square: 1 mm3
Depth of solution: 0.1mm
cells/mm3 = 230 x 100
1 x 1 mm2 x 0.01 mm
= 230 x 103/L
• 150 - 450 x 109/L
1. Brown, B.A., Haemotology, Principles and Procedures, Lea & Febiger, U.S.A., 1976.
2. Hoffbrand, A. V. and Pettit, 1. E., Essential Haemotology, Blackwell Scientific Publication, U.S.A., 1980.
3. Kassirsky, I. and Alexeev, G., Clinical Haemotology, Mir Publishers, U.S.S.R., 1972.
4. Widmann, F.K., Clinical interpretation of Laboratory tests, F.A. Davis Company, U.S.A., 1985.
5. Kirk, C.J.C. et al, Basic Medical Laboratory Technology, Pitman Book Ltd., U.K. 1982.
6. Green, J.H., An Introduction to human Physiology, Oxford University Press, U.K., 1980.
- A chronic cough or shortness of breath
- Red, watery eyes
- Joint pain
- Manual or microscopic method
- Automated method
(4) Pasteur pipette
(5) Test tube (75 × 12 mm).
WBC diluting fluid (Turk’s fluid) consists of a weak acid solution (which hemolyzes red cells) and gentian violet (which stains leucocyte nuclei deep violet). Diluting fluid also suspends and disperses the cells and facilitates counting. Its composition is as follows:
• Acetic acid, glacial 2 ml
• Gentian violet, 1% aqueous 1 ml
• Distilled water to make 100 ml
EDTA anticoagulated venous blood or blood obtained by skin puncture is used. (Heparin should not be used since it causes leukocyte clumping). While collecting capillary blood from the finger, excess squeezing should be avoided so as not to dilute blood with tissue fluid.
(1) Dilution of blood: Take 0.38 ml of diluting fluid in a test tube. To this, add exactly 20 μl of blood and mix. This produces 1:20 dilution. Alternatively, 0.1 ml of blood can be added to 1.9 ml of diluting fluid to get the same dilution.
(2) Charging the counting chamber: Place a coverglass over the hemocytometer. Draw some of the diluted blood in a Pasteur pipette. Holding the Pasteur pipette at an angle of 45° and placing its tip between the coverglass and the chamber, fill one of the ruled areas of the hemocytometer with the sample. The sample should cover the entire ruled area, should not contain air bubbles, and should not flow into the side channels. Allow 2 minutes for settling of cells.
= Nw x 20 x 10
= Nw x 50
Where Nw is the number of WBCs counted, Cd is the correction of dilution, Cv is the correction of volume and NLS is the number of large squares counted.
If TLC is more than 50,000/ml, then dilution of blood should be increased to 1:40 to increase the accuracy of the result.
If TLC is less than 2,000/ml then lesser dilution should be used.
Expression of TLC: Conventionally, TLC is expressed as cells/μl or cells/cmm or cells/mm3. In SI units, TLC is expressed as cells × 109/liter. Conversion factors for conventional to SI units is 0.001 and SI to conventional units is 1000.
NRBC + 100
• At birth: 10,000-26000/μl
• 1 year: 6,000-16,000/μl
• 6-12 years: 5,000-13,000/μl
• Pregnancy: up to 15,000/μl
2. Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Haematology (9th Ed). London. Churchill Livingstone, 2001.
3. The Expert Panel on Cytometry of the International Council for Standardization in Haematology: Recommended methods for the visual determination of white blood cell count and platelet count. World Health Organization. WHO/DIL/00.3. 2000.
4. World Health Organization. Manual of Basic Techniques for a Health Laboratory (2nd Ed). Geneva: World Health Organization, 2003.
- "Binding" antibodies attach to the acetylcholine receptors on nerve cells and may initiate an inflammatory reaction that destroys them.
- "Blocking" antibodies may sit on the receptors, preventing acetylcholine from binding.
- "Modulating" antibodies may cross-link the receptors, causing them to be taken up into the muscle cell and removed from the neuromuscular junction.
- Drooping eyelid
- Double vision
- Decreased eye movement control
- Difficulty swallowing, chewing, with choking, drooling and gagging
- Slurred speech
- Weak neck muscles
- Trouble holding up head
- Difficulty breathing
- Difficulty walking and an altered gait
- Specific muscle weakness but normal feelings/sensations
- Muscle weakness that worsens with sustained effort and improves with rest