Optical density is a measure of absorbance in the photometer. This is the basic principle for the measurement of blood and urine parameters in a semi-auto biochemistry analyzer. All of these parameters, either may depend upon end-point, kinetic, or two-point kinetic chemistry.
Absorbance (Optical Density )
Absorbance (A) = log10 (1/T) = log10 (I0/I)
where T = I/I0; transmittance of the light through the sample
I= Transmitted light (Output)
I0 = Incident light (Input)
In reality, a biochemistry analyzer measures the amount of transmitted light through the sample. But the graph between transmittance and the concentration of the sample becomes non-linear. So, to get the linear graph, the absorbance of the sample is calculated by the machine.
The term end-point suggests that the single measurement of the absorbance of the sample occurs at the end of the reaction. When the sample reacts with the working reagent, there will be the formation of the color. After a certain period of time, all the substrates present in the reaction convert into a new product. Thus, increasing the incubation period for the end-point does not cause any fluctuation in the absorbance in the sample for a certain time period.
Some of the common examples of the end-point method are uric acid, glucose, and albumin measurement.
If you are using a semi-auto biochemistry analyzer, make sure that you set the delay time to 5 seconds. Do the incubation in an incubator, water bath, or dry bath. Doing an incubation inside the flowcell or the cuvette will reduce the lifespan of the machine and delay the process of measurement.
During the delay time, the measurement of the absorbance of the sample does not take place. It is the time for incubation and the stabilization of the sample.
Since UV ray is used during kinetic tests, it is also known as UV kinetics.
If the no. of readings becomes more than one, then it is known as kinetic chemistry. It is useful for enzyme estimation. The enzyme does not take part in a chemical reaction. It acts as a catalyst. Some of the common examples of kinetic chemistry include ALP, GPT, GOT, etc.
In the case of a semi-auto biochemistry analyzer, take about 3-4 readings. Since the graph of the kinetic test is linear the change in the absorbance per minute remains almost the same. In other words, there will not be any change in the slope of the curve. You need to take around 3-4 readings for a better result.
Fixed Time Kinetics
Fixed time kinetics (two-point method) is a mixture of an end-point and kinetic method. Some of the common examples of fixed-time kinetics include creatinine and urea measurement. In this method, there will be a measurement of absorbance at two different points.
Sometimes, in the case of kinetic chemistry, you may be given a standard solution as well. In that case, you can use the standard solution for the calibration process. Then save the new factor. However, you will generally be provided with a factor. Just put the factor value in the programming of a semi-auto biochemistry analyzer.
Programming in the Semi-Auto Biochemistry Analyzer
Different reagent kits can have different programmings which will be given in the manual of the reagent. So you need to look into the manual and program it in the semi-auto biochemistry analyzer.
- Total reading time= delay time + reading time
During delay time there will not be any reading of any absorbance
- Reading time = time interval (T1 – T0 )X (n-1)
where n= no. of readings
1. End-point Method
- No. of readings (n) = 1 (always)
- Time interval (T1 – T0) = 0 (because there is no T1 )
- Delay time = 3 to 5 seconds (I prefer to set it at 5 seconds )
Thus reading time= 0 and total reading time = delay time
2. Two-point Method
- No. of readings (n) = 2 (always)
- Time interval (T1 – T0) = 80 seconds (This is just an assumption. You need to verify the time interval between two absorbance readings by looking in the manual given in your reagent kit )
- Delay time = 30 seconds (This value is also an assumption )
Then reading time = 1 X 80 seconds = 80 seconds, Total reading time = 110 seconds.
3. Kinetic Method
- No. of readings = 4 (This is just an assumption. You need to verify the no. of readings of the absorbance by looking in the manual given in your reagent kit )
- Time interval (T1 – T0) = 60 seconds (This is just an assumption )
- Delay time = 60 seconds (This is just an assumption )
Then, reading time = 3 X 60 seconds = 180 seconds, total reading time = 60 seconds + 180 seconds = 240 seconds.