Limitation of Methods

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My Experiences during the development

of quality control system with special


reference to limitations of method
By Dr. S. M. Natu
Ex Professor in Chemical Pathology, Department of Pathology, KGMU,
Lucknow
Senior Clinical Biochemist, 24x7 Lab, SGPGIMS, Lucknow
Introduction
 The present era is era of automation. In today scenario we need not to prepare reagents.

 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.

 When we are using automated instrument. It is most important to evaluate the


performance of automated instrument and we should also be aware about the analytical
sensitivity or detection limits of the methods.

 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.

 After so much of documentation finally the lab gets NABL Accreditation.

 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

1. It’s the most important part.

2. 60-70% errors occur during sample collection.

Unfortunately this is the most neglected part.


B) Order of Draw
 Reason behind a sequence of a certain tubes.
1) first we collect sample in culture bottle because, if the sample is collected in a
coagulation or in other tube the sample may get contaminated by coagulant.

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)

4)fourth we should collect Sodium/Lithium Heparin because, Heparin is a human


anti coagulant.

5)Lastly sample should be collected in EDTA, and in Sodium Fluoride .


C) Sample Transportation

*Manual :

Manpower is needed for this method, Hence more error can


occurred as comparison to second method, because human
being is getting involved in this method and it may vary from
person to person.

*Pneumatic Tube System(Automatic System) :

Error could be low as comparison to the first method. Sufficient

sample should be kept in the bag along with appropriate packing

material . Sample cap should be tight to avoid any leakage.


2)Analytical
 A) Sample receiving in the lab :
When samples are received in the lab we should consider following points

* Temperature – whether cold chain has been maintained or not

* Sample Quantity – sample should be up to the mark, assigned by


the manufacturer to maintain the proper ratio of anticoagulant and blood

* For Anticoagulant tubes – their should be no clot, as microclot can give


false results

* 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

Principle : Enzymatic determination of glucose according to the following reaction

Glucose+O2 Glucose oxidas Gluconic Acid + H2O2

2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinonemine +4H2O


Interference/Limitations :

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)

Principle : Enzymatic determination according to the following reaction

Urea + H2O Urease 2NH4+ + HCO32-

NH4+ + α–Ketoglutarate + NADH GLDH L-Glutamate + NAD+ + H2O

Interference/Limitations : Urea concentration can be measured in plasma serum or urine. If


plasma is collected ammonium ions, high concentration of sodium citrate and sodium flouride
must be avoided because it inhibit urease enzyme.
Protein content of diet may also some time infuence urea concentration
Non haemolysed sample Is recommended
Sample should be analysed with in few hour because urea is susceptible for bacterial
decomposition .
Creatinine :

It is based on Jaffe reaction

Criatinine + alkaline Picrate ion Orange red colour complex λ max at 490nm and 520nm

Exact chemical reaction is not clearly understood.

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.

Interference/Limitations : Bilirubin and Hb may false negative interfernce, Protein, high


concentration of glucose, guanidine., ascorbic acid, ketone bodies, pyruvate, blood
substitution method, cephalosporin also interfere.
Bilirubin Total :
Method: Malloy-Evelyn modified.
Principle : Sulphanilic acid reacts with sodium nitrite to form diazotized
sulfanilic acid. In the presence of accelerator (cetrimide), conjugated and
unconjugated bilirubin react with diazotized sulfanilic acid to form
azobilirubin. In the absence of accelerator, only conjugated bilirubin reacts.
The increase of absorbance at 546 nm is proportional to bilirubin
concentration.

Sulfanilic acid + NaNO2 diazotized sulfanilic acid


Bilirubin + diazotized sulfanilic acid azobilirubin

Interference/Limitations: Prolonged exposure of light cause


photoisomerization increasing direct reacting bilirubin. Hemolysed sample will
cause decrees in bilirubin. Lipemia also interferes in this method. This method has
a negative interference by haemoglobin/turbidity because protein
precipitation.
AST and ALT:

Method: IFCC method without pyridoxal phosphate(P-5’-P)


AST
Principle :
L-Aspartate + α-Ketoglutarate AST Oxaloacetate + L- Glutamate

Oxaloacetate + NADH + H+ Malate dehydrogenase L - Malate + NAD+

ALT

L- alanine + 2oxoglutarate ALT L-glutamate + pyruvate

Pyruvate + NADH + H+ LDH L-lactate + NAD+


 In both reactions conevrsion of NADH to NAD is measured at 340 nm
Interference/Limitations :

 Substrate should be in appropriate amount at least 20% should be left after


the completion of reaction.

Wavelength should be exactly 340 nm hence the instrument should have


monochromator with a narrow band width.

Temperature should be maintaine since the molecular extinction coefficient


of NADH varies with temperature.

The serum should be separated as early as possible since AST activity is many
fold higher in erythrocytes.
Ionic Calcium :

Method : ISE-Indirect

Interference/Limitations: Haemolysed sample

Ca2+pH = Ca2+M - ∆pH


1.89

Where Ca2+ pH is the corrected ionized calcium concentration (mmol/I),


Ca2+M is the measured ionized calcium concentration (mmol/I), ∆pH is
the measured pH of the sample minus 7.40
3) Post Analytical :
 Typographical Errors

 Decimal Error

 Measurement unit error like mg/dL to g/dL

 External remark for the status of sample(Haemolysed, Lipemic and


Icteric)

 Critical Reports

 Telephonic report information should not be provided, if informing in a


critical condition ask for listen value to confirm whether its correct or not.

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