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DIAGNOSTIC IN CLINICAL

CHEMISTRY I
MKEB 2404
Title:
Determination of the accuracy of the assay

Objectives:
 To determine the accuracy of the assay by calculation recovery and
percentage of recovery.
 Develop the calibration curve and determine the value of samples that
have been made
 To assess the accuracy of a method; it, tests whether a method can
measure an analyte in the presence of other substances contained
within the sample matrix

Principle:
Recovery is the ability of an analytical method to correctly measure an
analyte when known amount of it is added to samples. Recovery studies
can also test for interferences by substances contained in the sample; like
competition of protein for calcium in some dye binding methods of
calcium estimation.

There are 2 ways of measuring recovery:

Method 1: using control and standard → equal volume of various


standard (of different concentration) is added to the control

Method 2: using patient samples – analyze a set of samples that consists of


authentic specimens (i.e baseline samples) to which specific quantities of
the analyte in question (i.e test samples / diluted test) have been added;
next calculate the differences between the test and baseline concentrations
or activities to determine the amount recovered. Then, calculate the ratio
of the amount recovered divided by the amount added. Finally, multiply
this value by 100% to obtain the recovery value in percent.

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Calculation of recovery:
• Concentration added = [standard] X standard volume (ml)
Standard vol + serum vol

• Concentration recovered = [diluted test] – [baseline]

• % of recovery = concentration recovered X 100%


Concentration added

Methods:
1. Preparation for standard BSA with concentration 0, 10, 20, 40, 60, 80 100
in mg/dl
Concentration Calculation of volume BSA required
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
0X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (0)(200µl)
V1 = 0 µl
Volume water need: 200µl - 0µl = 200µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
10X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (10)(200µl)
V1 = 20 µl
Volume water need: 200µl - 20µl = 180µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
20X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (20)(200µl)
V1 = 40 µl
Volume water need: 200µl - 40µl = 160µl

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Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
40X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (40)(200µl)
V1 = 80 µl
Volume water need: 200µl - 80µl = 120µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
60X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (60)(200µl)
V1 = 120 µl
Volume water need: 200µl - 120µl = 80µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
M2 = final concentration of BSA
80X
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (80)(200µl)
V1 = 160 µl
Volume water need: 200µl - 160µl = 40µl
Set total volume is 200µl
To determine volume of Bovine Serum Albumin (BSA) need
use M1V1 = M2V2.
M1 = actual concentration of BSA
100X M2 = final concentration of BSA
V1 = volume need
V2 = final volume
(100mg/dl)( V1) = (100)(200µl)
V1 = 200 µl

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20µl of each diluted
BSA
+
1000µl color reagent

Mix and incubate for 5 min at 20-250C

Measure at 540 nm against reagent blank (2 X)

Plot absorbance VS concentration

2. Preparation of samples:
• This method using standard (BSA) with certain concentration that
have been prepared before
• Serum A is supplied for use as base specimen:
• To the baseline specimen, different concentration (but same volume
(25µl)) of standard was added as follows:

Standard Serum
Standard Total
Specimen volume dH2O (µl) volume
(mg/ml) volume (µl)
(µl) (µl)
A0 0 0 25 475 500
A1 100 25 0 475 500
A2 80 25 0 475 500
A3 60 25 0 475 500
A4 40 25 0 475 500
A5 20 25 0 475 500
A6 10 25 0 475 500

Specimen A0 = baseline
Specimen A1 – A5 = diluted test

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3. Make a double dilution technique using above specimen / samples

20µl of each
specimen
+
1000µl color reagent

Mix and incubate for 5 min at 20-250C

Measure at 540 nm against reagent blank

Repeat this method again

Results:
Table below showed the results for standard:

[Standard] mg/dl 1st absorbance 2nd absorbance Mean


0 0 0 0
10 0.02 0.013 0.017
20 0.06 0.081 0.071
40 0.403 0.391 0.397
60 0.276 0.238 0.257
80 0.319 0.307 0.313
100 0.411 0.383 0.397

Table below showed the results for samples:

Samples 1st reading 2nd reading Mean


A0 0.343 0.320 0.332
A1 0.345 0.320 0.333
A2 0.226 0.408 0.317
A3 0.372 0.345 0.359
A4 0.393 0.325 0.359
A5 0.368 0.369 0.369
A6 0.383 0.349 0.366

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From the graph that has been plotted the concentration value for each
sample was listed below:

Samples Concentration (mg/dl)


A0 83
A1 84
A2 80
A3 90
A4 90.5
A5 93
A6 92

Discussion:
Below is the calculation of recovery for each sample:

Concentration Concentration
% of recovery
Samples added (refer recovered (refer
(refer formula)
formula) formula)
A0 Baseline Baseline Baseline
A1 25 84 mg/dl – 83 1
10 × = 0.5 × 100 = 200%
500 mg/dl = 1 mg/dl 0.5
A2 25 80 mg/dl – 83 3
20 × =1 × 100 = 300%
500 mg/dl = -3 mg/dl 1
A3 25 90 mg/dl – 83 7
40 × =2 × 100 = 350%
500 mg/dl = 7 mg/dl 2
A4 25 90.5 mg/dl – 83 7.5
60 × =3 × 100 = 250%
500 mg/dl = 7.5 3
mg/dl
A5 25 93 mg/dl – 83 10
80 × =4 × 100 = 250%
500 mg/dl = 10 mg/dl 4
A6 25 92 mg/dl – 83 9
100 × =5 × 100 = 180%
500 mg/dl = 9 mg/dl 5

From all calculations, the results are invalid. Usually the % of recovery should be
below than 100%. If the results are more or higher than 100% the results are
invalid.

In determine the % of recovery, the percentage that near to 100% showed that
the method that was used is more effective.

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The results from this experiment are invalid because of certain factor:
 Interference that occurs in the samples
 The cuvette that was used are not cleaned properly
 The interference that occur in the spectrophotometer
 The serum that was used are contaminate with other substances
 Error in taking the absorbance value.
 The graph that was plotted are not really accurate

Conclusion:
From this experiment, we can’t make any conclusion about the % of recovery
because the results exceed 100%, therefore it can’t be used. We suggest that
proper in handling and making the method of preparation are important in order
to prevent any error from occur that may cause a false results.

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