The effect of any compound of the sample on the accuracy of measurement of the desired analyte.

1. INTERFERENCES
Tapeshwar Yadav
(Lecturer)
BMLT, DNHE,
M.Sc. Medical Biochemistry

2. The effect of any compound of the sample on the
accuracy of measurement of the desired analyte.

3. 4 basic types :-
• Arise from the limitation of detectors
• Chemical subs in the sample that directly interfere with
the analytical method.
• Disease state or exogenous agents that modify certain
physiological process
• As a result of sample processing

4. Limitation of detectors

5. • Methods for quantitative answers usually employ a
detector, such as spectrophotometer.
• There is a relationship between the detector response
and conc.of the analyte in the sample.
• Two interrelated types of error may occur
- mathematical relationship between the
absorbency & percentage of transmittancy.
- limitation of instrument

6. Absorbance variance
• There is a logarithmic relationship between the percentage
of transmittance (%T) & the absorbance.
• Small change in %T at a very low transmittance will result in
disproportionately large change in the calculated absorbance
that leads to a increase in error of analysis.
• Spectrophotometric measurements at absorbances between
0.1 to 1.1 will minimize the errors

7. Instrument limitation
• In spectrophotometer at 100 %T or 0 absorbance the
entire light signal is converted to an electrical signal.
• At high absorbances the limitations are caused by the
inability of the detection system to measure the small
difference between the high levels of absorbance
accurately.
• Thus in analyse with relatively high levels of absorbing
compounds or interferents ,a large spectrophotometric
error occurs.
• An initial dilution to lower the total absorbtion in addition
to a sample blank can eliminate this problem.

8. Fluorescence Spectrophotometer
• Different from transmission spectrophotometer.
• Fluorescence signal is linearly related to concentration if
very little light is absorbed by the sample.
• If a significant portion of light passing through a
fluorescence is absorbed the relationship is no more linear.
• To minimize this problem fluorescence analysis should be
performed with relatively diluted solutions,the absorbance
of which is < 0.1.
• A blank limits the accuracy of fluorescence measurment.
• Blank must be considered in relation to the total signal.

9. In Vitro Interferences

10. • Arise from the fact that biochemical analyses are
performed in the complex matrices that make up the
biological fluids.
• This fluids are having hundreds of compounds that have
chemical groups capable either of reacting to some extent
with the test reagents or mimicking the
physical,chromatographic or spectral properties of the
desired analyte.
• This situation is further complicated becoz of chemical
composition of the body fluid can vary with the nature and
the extent of the disease process.
• This possibility is increased by the presence of large number
of drugs.
• Each of this factors,alone or in combination ,can result in a
possible interferance.

11. • The in vitro interferents can be sub-classified into
Spectral nature
Competing chemical reaction.
• Most commonly observed interferences are
Hemolysis
Icterus
Lipemia

12. Spectral interferences

13. Absorbance
• Spectral interferences are observed when a compound
causes a response in spectrophotometer similar to that of
the analyte of interest, though the interferents themselves
do not necessarily undergoes any chemical change during the
analyte reaction.
ex - Hb

14. Turbidity
• Common type of spectral interference is turbidity of the
sample.
• Turbidity is most commonly caused by large lipoprotein
molecule e.g. VLDL which is suspended in plasma.
• When a turbid specimen is analyzed in a colorimetric
reaction ,the lipoprotein causes the incident light to scatter.
• light Scattering tends to ↓ transmitted light thereby
increase the apparent absorbance of the specimen causes
falsely elevated results.
• Best method to eliminate the interferences due to turbidity
is dilution of the sample.

15. Fluorescence
• It effects the fluorescence measurement similarly.
• Some scattered light will reach the detector set at 90
degree to the incident light, thus giving an apparent increase
in fluorenscence and falsely elevated conc.
• Reducing turbidity problem is more difficult here.
• Best approach is the elimination of the source of light
scattering by filtration or centrifugation.

16. Correction of the spectral
interferences

17. Sample blank
• Spectral interferences can be minimized by measuring the
absorbance of the assay against the sample blank.
• Simplest sample blank is obtained by a mixture of a sample
and a diluent.
• Correction for the interferences is made by subtracting the
absorbance value of blank from the absorbance of complete
reaction mixture.
• In case of fluorescence sample blank allows for the
correction of non-specific fluorescence.
Reagent blank
• Reagent blanks(diluent+reagent) are used in the similar
fashion to correct for high absorbance of the reagent.

18. Kinetic measurement
• Measurement of a typical end point reaction as a two-point
kinetic reaction.
• In two point kinetic measurement absorbance is measured at
two different time points.
1) final colour development has not occurred
2) the response of absorbance versus time
response is still linear.
• Delta absorbance is caused only by specific colour formed by
the analytical reaction.
• Two-point kinetic reaction is self blanking.
• This technique plays an important part in performing
automated chemical analysis on large number of specimens.

19. Biometric analysis
• Many currently used instruments employ a spectral
interferent correction technique that involves measurment
of the absorbance of the reaction mixture simultaneously at
2 different wavelengths δ₁ & δ₂.
• δ₁ -minimum absorbance of the chomogen.
• δ₂ -maximum absorbance of the chomogen.
• As the reaction is analysed simultaneously at two weve-
lengths, known as bio-chromatic analysis.
• This technique also correct the for instrument correction
as dirt on the cell which causes light scattering or
reflectance.
• Allen correction- 3 measuring wavelengths.

20. Dilution
• Dilution of a sample containing spectral interferences can
reduce the error.
• Carefull not to over dilute the desired analyte or chromogen
to a conc. below the minimum detectable level.
• Several dilution should assayed simultaneously to get the
most effective one.

21. Instrumental indices
• Many automated chemistry analyzers give 3 different
indices each for Hb,bilirubin &turbidity.
• The operator may choose to dilute the sample according to
interference or can inform the physician about it.

22. Chemical
interferences

23. • This kind of interferences reacts with the chemical of the
analytical reactions.
• Reaction products of the analytical reactions usually results in
positive interferences though negative interferences are also
there.
• The types of non-specific ,chemically reacting interferents can vary
greatly.
Ex- uric acid –(-)interference in glucose oxidase
bilirubin & ascorbic acid – (-)interference method

24. Correction of chemical
interferences

25. Elimination by following techniques
• Diluting the interferents
• Increasing the specificity of the reaction
• Removing the interferent
• Monitoring an assay by kinetic measurements
• Monitoring an assay by bichromatic measurements

26. Dilution
• Dilution of the sample is an effective method.

27. Specificity
• Specific enzymes as reagents can increase the specificity of
the reaction.
Ex – glucose by hexokinase or glucose oxidase.
• Immunochemical reaction can also increase the specificity.
Ex- measurment of theophylline by enzyme immunoassay.

28. Removing The Interferent
• Protein free sample
• Liquid-liquid extraction
• Adsorption or partition chromatography.

29. Kinetic measurements
• By two point kinetic reaction is the fact that many
interferent reacts at different rate than the analyte of
interest.

30. Chromatographic interferences
• No single set of chromatography conditions can possibly
prevent interferences from co-chromatographing or closely
chromatographing compounds specially if the patient is
receiving several potentially interfering drugs.
• Two primary modes are used to minimize chromatographic
interferences :
Increasing the specificity of the detector
Removing the interferences from the analyte.

31. Immunochemical Interference

32. • Hyperlipidemia can greatly affect immunochemical
reactions that use turbiditometric or nephlometric
measurements becoz of the increased sample turbidity.
• Most difficult interference in which the patient has
antibodies to the test reagent antibodies (heterophile
antibodies) or to the actual test antigen.
Ex- HAMA
• Methods for reducing the interferents include adding
specific animal sera.

33. In Vivo Interferences
• Drugs
• Alcohol
• Disease process

34. Source Reference material
• Development of the interferogram allows the laboratory to
estimate the effect of hemolysis,icterus & lipemia on the
analyses performed on frequently used instruments.
• Newer automated chemistry analyzer can be calibrated to
detect increased levels of hemolysis,lipemia & icterus.
• The laboratory must determine both the in vivo & in vitro
effects exerted by each of these drugs on clinical
laboratory analysis.

35. Allowable Interferences
• Some interferences is potentially present for every assay,
clinical chemist must determine how much interference is
allowable.
• A statistically significant interferent may not be a clinically
significant one.

36. Thank U