Limitation of Methods
Limitation of Methods
Limitation of Methods
Most of the instruments manufacturers also make reagents in the form of readily available
kit and only addition of water or buffer is required to prepare the reagent.
It is very true that the automation saves our time, manpower and make work easy, but
method as well as instrument has certain inbuilt errors.
Hence to give very accurate and correct reports. All the things should be done by yourself
with consideration of all the possible limit.
Previous speakers gave a very good information about NABL and quality control of
the results.
For QC we generally use Internal Quality Control and External quality Control
samples and also participated in EQAS program.
Now my question is….When a Lab is NABL Accredited, even than some reports are
challenged. There are so many contributing factors for the same, which can be
understood under following of headings:-
1) Pre Analytical
2) Analytical
3) Post Analytical
Pre-Analytical
A) Sample Collection
2) After that we collect sample in Sodium citrate because, after prick clotting
factors get activated in human body. After mixing with sodium Citrate blood
clotting stopped
3) Third we should collect sample in gel tube/ Yellow top or clot activator tube ,
because some serum parameters get affected if tourniquet get tight more than 1
min, so we need to collect sample in plain tube as soon as possible(but not breaking sequence
of order of draw)
*Manual :
* Leakage
* Receiving time should be noted on the form and we should also maintain
the TAT
B) Centrifugation:
We should centrifuge plain or anticoagulant sample as per norms of the test at
appropriate RPM with appropriate balance.
C) Grossing of sample :
If we are getting blood sample for Biochemistry and other investigation, it is very
important to look at the sample whether it is grossly or slightly haemolysed,
Lipemic or icteric. This will help you to co-relate the results properly. If the sample is
haemolysed it should be immediately informed to the ward, requesting for fresh
sample.
D) Limitations of methodology :
Since I am a biochemist, I will discuss basic principle of very few methods and their
limitations. Substances interfering in the methodology whether it is positive or
negative interference.
Glucose :
Methods :
1. Glucose oxidase peroxidase (GOD-POD)
2. Glucose hexokinase method
Peroxidase is less specific than glucose oxidase reaction, various substances such
as:-
Uric Acid
Ascorbic Acid
Bilirubin
Heamoglobin
Tetracylclin
Glutathione
Inhibit the reaction. This inhibition is by competition with chromogen for hydrogen peroxide. Thus ultimate result is
reporting towards lower side.
Some glucose oxidase preparation contains catalase as a contaminant. Catalase actively decompose peroxide
and thus there is a decrease in the final colour.
This method is not suitable for urine because urine contains high interfering substances which interfere with
peroxidase such as uric acid.
Urea :
Method : Enzymatic –UV-Kinetic (Uricase UV)
Criatinine + alkaline Picrate ion Orange red colour complex λ max at 490nm and 520nm
Jaffe reaction is not specific for criatinine estimation. Many attempts have been made to
improve the specificity of Jaffe reaction but unfortunately these modifications can not be used
in automation
Method : Kinetic method: Two kinds of non creatinine chromgen have been identified
1. Those which react within 20 sec after mixing the reagent and sample( for ex. acetoacetate)
2. Those whose rates did not become rapid until 80-100 sec after mixing (for ex. Protein)
Window period 20 and 80 sec is predominately suitable for creatinine pictrate reaction,
however this window period may vary from individual to individual depending upon the
interfering substances.
ALT
The serum should be separated as early as possible since AST activity is many
fold higher in erythrocytes.
Ionic Calcium :
Method : ISE-Indirect
Decimal Error
Critical Reports