Tan Zhao Rong
Tg01
Pathology/Biochemistry Lab

For the past two weeks in the pathology lab, I've been trained to carried out RBC cholinesterase testing. Here goes..

What is Cholinesterase?
Cholinesterase is an enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. They are important in the proper functioning of the nervous system in human, other vertebrates and insect.

There are mainly two types of cholinesterase in human are Acetylcholinesterase and Pseudocholinesterase. Acetylcholinesterase are mainly found in nervous system, RBCs, lungs, spleen and grey matter of the brain while Pseudocholinesterase are mainly found in heart, liver, pancreas, serum and white matter of the brain.

Why is Cholinesterase (ChE) measured?

1. Monitor pesticide poisoning

Pesticides can be ChE inhibiting and may enter the human body through skin absorption, inhalation and ingestion. When individual is overexposed to ChE inhibiting pesticides, these pesticides will combine with acetylcholinesterase at the nerve endings in the brain and nervous system, resulting in cholinesterase inhibition. Acetylcholine will build up causing symptoms of pesticide poisoning to show. Therefore, ChE measurement is a useful indicator of pesticide poisoning.

2. Test Sensitivity to Succinylcholine

Succinylcholine is a short acting muscle relaxant administered during surgery. It is hydrolyzed and eliminated by ChE. Therefore, individuals without sufficient ChE or with certain genetic enzyme variants may be unable to metabolize the drug quickly, resulting in prolonged apnea.

3. Alzheimer Disease

In Alzheimer’s disease (AD), the production of acetylcholine is decreased. Cholinesterase inhibitors (ChEI) are drugs prescribed to treat symptoms resulting from the early and middle stages of AD. ChEI block the activity of ChE, thereby making more acetylcholine available to nerve cells in the brain. Monitoring cholinesterase levels is often used for therapeutic drug monitoring purposes.

How is RBC cholinesterase measured?

a. Wash EDTA blood 1 time with 0.9% saline.
b. Centrifuge to pack the cells at 3000rpm for 10 minutes.
c. Remove the saline and buffer coat completely
d. Pipette 200ul washed packed cells into 200ul normal saline to obtain 1:1 RBC suspension.
e. Mix the suspension.
f. Pipette 50ul of 1:1 suspension into 500uL of 0.5% saponin for lysis.
g. Mix well and stand at 2-8 degree C for 10 minutes.
h. The haemolysates would then be ready to send for testing for ChE at LX20.
i. The packed cell volume (PCV) is determined by filling a hematocrit tube with the 1:1 suspension from step d.
j. Centrifuge at 12000 rpm for 5 minutes.
k. Read off PVC value from a calibration rule.
l. Divide ChE value by PVC and multiply by 100% to determine the actual ChE value.