A prospective comparison study was carried out at a secondary referral hospital in rural Sri Lanka.
Selection*of*participants*
Consecutive patients admitted with a history of OP self-poisoning between November 2007 and June 2008 were eligible for inclusion. This study was investigating the impact of bedside cholinesterase testing on decisions made by physicians. Local internal medicine physicians were responsible for the management of patients.
Symptomatic OP poisoned patients were initially resuscitated with intravenous atropine using bolus doses and infusions titrated to clinical response. Pralidoxime was
sometimes prescribed depending on the preference of the treating physician (usual dose of 1g pralidoxime chloride IV every 6 hours for 48 hours).
At the time of patient selection we already had 12 months experience in using the Test- mate ChE field kit for the measurement of cholinesterase levels in pesticide poisoned patients. Fourteen patients were selected in whom we expected the ‘reference test’ to be most accurate because of a lower likelihood of ex-vivo reactions.
These patient’s samples were all collected within the 6 months before the shipment to the reference laboratory and had a documented cold chain. Carbamate poisoning was excluded from the comparative sample because of the authors’ experience of rapid ex- vivo reactivation in such cases, which could render the reference test results unreliable (unpublished data).
Interventions*
The protocol was approved by the ethics review committee of Peradeniya University (Sri Lanka). All OP poisoned patients were tested for RBC-AChE and PChE using the Test-mate ChE field kit. Duplicate samples were prepared at the same time to be tested at a later date in a reference laboratory. The frequency of blood tests depended upon the severity of poisoning, and whether oximes were being used (see Figure 4-1).
Figure 4-1 Study protocol showing frequency of blood testing for acetylcholinesterase.
Symptomatic patients were tested more frequently than asymptomatic patients.
Patients who were receiving oxime therapy were tested pre and post pralidoxime dose.
Methods*of*Measurement*
Cholinesterase,measurements,using,the,Test1mate,ChE,field,kit,
This study used a Test-mate ChE field kit [model 400, EQM research] designed for use over an extended temperature range (10-50 degrees centigrade)(118). The field kit consisted of a battery operated portable cholinesterase testing system that used the modified Ellman method. The amount of yellow colour produced as a result of the Ellman reaction is measured using a photo spectrometer indicating the quantity of cholinesterase present (see online appendix for further details). RBC-AChE and PChE were measured within 4 minutes per test using venous blood.
The precision of the Test-mate ChE field kit had previously been established by the manufacturer with analysis of 100 replicate samples of blood to which OP agents had been added(119).
10 microlitres of venous blood was transferred from the EDTA anticoagulated tube and mixed with the buffer solution. The acetylthiocholine or butyrylthiocholine within the buffer solution hydrolyses the RBC-AChE or PChE respectively, producing carboxylic acid and thiocholine, which reacts with the Ellman reagent (dithionitrobenzoic acid) to form a yellow colour. The rate of production of the yellow colour was measured on the photometer at 450nm, and this value indicated the activity of cholinesterase(114). RBC-AChE activity was determined in the presence of a specific inhibitor of PChE and the value is corrected for the amount of haemoglobin by dividing the measured AChE by the haemoglobin concentration (also measured photometrically). The machine was operated in an air-conditioned research room (temperatures between 19C to 22C) within the hospital premises.
The measurement of PChE was similar, also utilising 10 microlitres of whole blood. It simply requires a “mode change” button to be pushed on the Test-mate ChE device, prior to testing.
In cases of very low cholinesterase levels the Test-mate ChE field kit displayed the warning “No Reaction” followed by the level of enzyme activity. In some instances a negative integer (close to zero) was obtained. We converted these values to zero for the purpose of data analysis, as negative enzyme activity is not possible.
Standard deviations of 0.82 (U/g Hb) and 0.15 (U/ml blood) were observed for repeated RBC-AChE and PChE measurements respectively, and from this we
calculated the limits of quantification (LOQ) to be 4.1 U/g Hb for RBC-AChE and 0.75 U/ml blood for PChE. Testing above these limits, we verified the precision of the Test-
mate ChE in our setting by testing replicate cholinesterase measurements in 10 patients with OP self-poisoning. The mean coefficient of variance (CV) was 4.5% for RBC-AChE (range 1.3% to 8.4%) and 6.0% for PChE (range 2.0% to 11.6%). Using regression to extrapolate values where we would expect the CV to be greater than 20%, we re-estimated the LOQ for Test-mate ChE in our setting to be 1.3 U/g Hb (4.1% of normal) for RBC-AChE and 0.26 U/ml blood for PChE.
Cholinesterase,measurements,using,the,reference,test,
Duplicate samples were tested for RBC-AChE and PChE at the Bundeswehr Institute of Pharmacology and Toxicology, in Munich, Germany, and the modified Ellman method was used as previously described(85). The reference laboratory sample was prepared at the same time as testing with the Test-mate field kit. Sample preparation for RBC-AChE measurement consisted of transferring 200 microlitres of whole blood to another collection tube. This sample was immediately diluted and mixed with ice-cold distilled water in a 1:20 dilution, and frozen soon afterwards in order to minimise ex- vivo reactions.
The mean coefficient of variance (CV) measured at the reference laboratory was 2.75% for RBC-AChE (range 0.50% to 4.26%) and 4.00% for PChE (range 2.23% to 5.54%). The limits of quantification were 0.2 U/g Hb and 0.026 U/ml for RBC-AChE and PChE respectively(85).
Samples for PChE measurement were prepared by centrifuging the anticoagulated blood and transferring 1ml of plasma into a separate tube.
All samples were stored in a minus 20 degree centigrade freezer before being transported to the reference laboratory in Germany. The cold chain was strictly maintained during transport.
Data*Collection*and*Processing*
All blood samples were collected by research assistants who were junior doctors trained in venipuncture. They were also trained in the operation of the Test-mate ChE field kit according to the users manual (supplied by EQM research), and with additional power point presentations and a training video specifically created for education of the research protocol. As a quality control measure the principle investigator supervised the performance of each research assistant during the collection, processing and storage of samples in 5 test cases.
Results for RBC-AChE and PChE measurements obtained from the Test-mate ChE field kit were recorded on a datasheet and transferred to a computer spreadsheet program (Microsoft Excel). The results from the reference laboratory were also entered into an Excel spreadsheet. The reference laboratory staff did not have access to the Test-mate results.
Outcome*measure*
Primary outcome measures were the RBC-AChE and PChE levels measured at the bedside using Test-mate ChE field kit, and in the reference laboratory.
Primary*data*analysis**
The ‘limits of agreement’ method (described by Bland and Altman(120, 121)) was used to assess agreement. The differences between the two methods were plotted against their mean for each individual blood sample. This plot is bounded by the 95% limits of agreement (which is equivalent to +/- 1.96 SDs above and below the mean difference) [calculated using GraphPad Prism v5]. The agreement for the different clinical
categories of RBC-AChE inhibition was analysed using the weighted Cohen’s kappa statistic [using STATA 10].
Regarding the clinical categories of RBC-AChE inhibition, we based the cut-off points upon literature findings for the degree of symptoms associated with different levels RBC-AChE activity, which were as follows:- ‘normal’ >75% ( >23.6 U/g Hb), ‘mild inhibition’ 30-74% (9.4-23.5 U/g Hb), ‘moderate inhibition’ 10-29% (3.1-9.4 U/g Hb) and ‘severe inhibition’ <10% (<3.1 U/g Hb) (Table 4-1)(27, 28, 39, 122, 123).