Clinical and laboratory methods -

classification and requirements.

Rules for calibration of quantitative
methods in clinical chemistry.
Calibration curves. Reference
materials. Traceability.
 TYPES OF METHODS IN CLINICAL
LABORATORY

Qualitative and semi-quantitative

Quantitative
 Quality methods

 Methods of proving the presence or absence of a specific
ingredient in the sample to be tested
 Based on two qualities of the method:
1. Detection limit-LLOD
(limit of detection, Ld) -
the lowest concentration of test compound that
can be determined by the analytical method
 Quality methods

2. Analytical specificity-
the ability of the method to indicate the
presence of the test component in the
presence of all other components of the
matrix
 Matrix

 The sum of all the constituents
and structures of the biological
material in which the ingredient
(component) to be tested is located.
 QUALITATIVE METHODS-
continued

The result is expressed as "positive" or
"negative"
Basic question:
at what quantity of the studied component
does the result change from “negative” to
“positive”?

Some of these methods are descriptive - cell

morphology
 SEMI-QUANTITATIVE
METHODS

Examine the component at certain concentration
ranges using an approximate scale;

Example:
urinary protein;
chemical examination of urine with a test strip
Main disadvantage:
Not sensitive and specific enough.
Need for standardization
 QUANTITATIVE METHODS

Definitive

Reference

Routine
 Definitive method

Method of the highest metrological class for the
quantitative determination of a substance of
known physicochemical composition with a
negligible error (bias);
Practically independent of the sample matrix

It is characterized by extremely high accuracy

and specificity of the results (closest to the
"true" value;
 True value

The theoretical concept. It is not possible to
accurately determine it experimentally;

For practical purposes, the term

conventional true value (assigned value)
obtained with a reference method or a
reliable routine method is used.
Definitive method - continuation

Maximum calibration requirements
(primary standard calibration)

We need complex and expensive

equipment, clean reagents and highly
qualified specialists
 Definitive method - continuation

Example:
A definitive method for the determination of urea is
SID GC-MS method based on isotopic dilution with
marked O18 (internal standard-urea with O18),
which has a coefficient of variation (VC) <0.2% and
a negligible deviation from the "true" value
 Reference method

A method with a fully described and studied
analytical procedure, with very good
reproducibility and accuracy, which allows its
use for:

Evaluate the accuracy of other methods (routine);

Certification of reference materials.

ISO Guide 30
 Reference method

 Class А, В и С
 Class А – evaluated by a definitive method and there are
calibration materials for it also evaluated by a definitive
method;
 Class В – it has not been evaluated by a definitive method,
but very high metrological class standards have been used
for it;
 Class С - methods for which assessment by a definitive
method is not possible and there are no standards of high
metrological class (hormones, tumor markers, etc.). They are
calibrated with agreed standards.
 Reference method

Example

The reference method for the determination of urea is the
conjugated enzyme reaction urease / glutamate dehydrogenase
and the Warburg optical test - absorption at 340 nm, based on
NAD (P) H / NAD (P) indicator reaction

NAD (P) H - nicotinamide adenine dinucleotide (phosphate) - reduced

form
NAD (P) - nicotinamide adenine dinucleotide (phosphate) - oxidized
form
 Routine method

Method with analytical reliability sufficient
for practical purposes;

The amount of system bias from the "true"

value must be known.
 Routine method

CLASS A
estimated by a reference method or by
secondary standards, with a value estimated
by a reference method. The influence of
interferences is precisely defined. It can also
be used as a method of comparison (if there is
no reference or definitive method or standard
of a very high metrological class)
 Routine method

CLASS B

THE INFLUENCE OF INTERFERENCES IS NOT

DEFINED!
 CLASSIFICATION OF ANALYTICAL METHODS IN
CLINICAL CHEMISTRY

method characteristics results

Definitive None or with a minor error “True value”

Reference
Class А Evaluated by a definitive Value obtained by reference
method method
Class В Verified with a very high Value obtained with a
metrological class standard reference method(reference
method value)
Class С A and B assessment is not
possible
Routine
Class А Recommended method
with some deviation
Class В The method has an
inexplicable deviation
 Criteria for choosing a routine
method

Medical
 diagnostic specificity
 diagnostic sensitivity
 time to receive a result
 quantity and type of the primary sample

Analytical
 Recommended methods - Medical standard in clinical
laboratory, other requirements
Economically
- cost of one analysis (clinical result)
 REQUIREMENTS FOR THE DESCRIPTION OF
THE ANALYTICAL PROCEDURE (according to
ISO 15189)

The analytical procedure of each method in the clinical
laboratory must be described in detail and precisely,
which includes:

 Description
 Purpose of the study
 Principle of the analytical procedure used; literary
source
 REQUIREMENTS ...

Analytical features of quantitative methods:
 Linearity
 Accuracy (inaccuracy)
 Reproducibility (time and series)
 Sensitivity
 Specificity
 Detection limit (Ld)
 Measurement interval
 Reliability of the measurement
 REQUIREMENTS ...

 Primary sample (eg plasma, serum, urine, blood, etc.)
 Type of sampling vessel and necessary additives
 Necessary technical means (equipment)
 Necessary reagents
 Calibration procedure (metrological traceability)
 REQUIREMENTS ...

 Quality control procedures
 Interferences (eg lipemia, haemolysis, bilirubinemia and
drug interactions)
 Principle of the procedure for calculating the results incl.
and measurement uncertainty
 Biological reference intervals
 REQUIREMENTS ...


Values in life-threatening conditions (critical
values, where appropriate);

Laboratory interpretation !!;

Safety measures;

Potential sources of variation

To achieve high reliability and comparability
of laboratory results, two approaches are used:

 Calibration and standardization - including

calibration of measuring equipment and
standardization of analytical methods;

 Quality control of results

These are two fundamentally different activities!!!

Difference between calibration and
analysis control

 CALIBRATION Ensures THE ACCURACY OF
RESULTS

 QUALITY CONTROL CHECKS THE STABILITY

AND ACCURACY OF THE CALIBRATION, AS
WELL AS OF THE ANALYTICAL PROCEDURE, AS
A WHOLE OF DIFFERENT ORIGINS AND
NATURE
 Types of calibration in the
laboratory

 Technical calibration (of apparatus, means and
instruments ... )

 Calibration of analytical methods

 Technical calibration of apparatus and aids


 Technical modules - dispensers, thermostats

 Measuring and technical modules - e.g.

characteristic of the filters of one photometer

 Technical calibrators - means that serve to certify

certain physical or methodological and technical
characteristics
 Technical calibration - examples

Mechanical micropipettes and dispensers -
is performed gravimetrically, the actual
volume being obtained by multiplying the
volume of tempered distilled water
weighed on an accurate analytical balance
by multiplying the barometric pressure
correction factor for the day.
CALIBRATION OF ANALYTICAL
METHODS

A sequence of procedures that determines the
relationship between specific conditions:

 measured values of the equipment;

 of the test material or reference material;

 and their respective values of the test ingredient

using standards.
THE QUANTITATIVE RELATIONSHIP
BETWEEN:

The measured value (y)

The investigated value (Х)

Calibration value (С)

 CALIBRATION FUNCTION


 The mathematical relationship between the
measured signal and the amount of test component
is expressed by the calibration function of the
equation
y = f (x)
in which f is the calibration factor.

The graphical representation of a

calibration function is the calibration curve.
 CALIBRATION CONDITIONS

 Calibration standards

 Calibration solutions

 Exact description of the analytical method (analytical

procedure)

 Written rules for construction and verification of

calibration curve in photometric analytical methods
TYPES OF STANDARDS - Hierarchy


 Primary standard
 Secondary standard
 International standard
 National standard
 Reference standard
 Working standard
 Conditional standard
 Certified reference material (CRM)
 Calibration standards

Primary standard material - a substance that
has the highest metrological properties
(purity) for specific purposes, the declared
value of which is accepted unconditionally.

Primary standards are most often used for

calibration, in which the concentration of the
substance is determined by weight, and
water with a high degree of purity is used as
a solvent..
 Calibration standards

The primary standard is also the one in
which the concentration of the ingredients is
determined by a definitive method
Example
SRM 909 is a lyophilized product based on
human serum matrix, the declared values of
which are obtained in with definitive methods
from NIST (National Institutes of standards and
Technology) - USA
 Secondary standards

The concentration of the substance is
determined chemically by using methods
with very good analytical reliability -
reference or routine class A.

An important condition is that the declared

value is traceable to a certified product of
high metrological class or international
standard.
 Conditional standards

When the test ingredient is present in a
poorly defined degree of purity, e.g. proteins,
protein hormones, tumor markers, for
calibration products are used in conventional
units, most often provided by international
organizations.
It is necessary to declare which conditional
standard was used in the calibration of a
method
 Certified reference material (CRM)


Reference material accompanied by a
certificate in which one or more
characteristics are certified by a procedure
with proven traceability and in which each
certified value has a declared uncertainty at
a certain confidence interval.
 Certified reference material (CRM)


Some CRMs or RMs have properties that
cannot be attributed to a specific chemical
structure and cannot be accurately determined
by chemical or physical measurement
methods. These are some biological materials
for which international units have been
designated by the World Health Organization
(WHO).
 Reference material

A material or substance in which one or more
ingredients are sufficiently homogeneous or
specified to be used for:

 calibration of apparatus,
 assessment of measurement methods or
 to determine values in test material
 NB!!!!!!


 The value of each standard is

characterized by a certain uncertainty!
 (UNCERTAINTY)

 A range of values in which the "true value" of the test
substance is located, ie. the limits within which a laboratory
result is considered accurate (reliable and reproducible);

 Characterizes the scattering of values that can reasonably be

attributed to the investigated ingredient;

 In the hierarchical order - the highest standard has the least

uncertainty;
 Each of the lower classes leads to an increase in measurement
uncertainty
 Calibration solutions

Primary standard solution - obtained by
weight using a defined solvent. Strictly
follow the rules for weight and volume
analysis.

The application of these solutions in clinical

chemistry is not always possible and
appropriate. A suitable matrix resembling the
biological material for testing (serum, blood,
cerebrospinal fluid, plasma, etc.) is used as
the solvent.
 Calibration solutions

A number of equidistant dilutions are
prepared from the concentrated calibration
solution.

Concentrations are selected to cover the

reference limits and clinically relevant
concentrations of the relevant indicator, as
well as to take into account the sensitivity
of the method.
 CALIBRATION CURVES


They differ:
- simple;
- complex (accurate)
 Simple calibration curve

y
•
• Y=a+bx
•
a = y-intercept
•
b=slope
• Y=bx
0 x
 Complex (accurate) calibration curve

 The five main points of the simple curve are
extended by:
 three points from 0 to the first main point
 the required number of points above the
maximum base point
 Five parallel samples are prepared from each
calibration solution, in contrast to the simple
calibration curve, where three parallel samples are
prepared and determined.
TRACEABILITY- an important condition for
ensuring the reliability of the results

The quality of the result of the survey or the
value of the standard, which shows its
comparability with national or international
standards, assessed under well-defined
conditions / metrological chain /, under
constant conditions of uncertainty, which is
declared "
 Metrological traceability

” „The traceability of the declared
value of the standards and / or
control materials must be ensured
by reference methods and / or
reliable reference materials of
high metrological class “
(Direcive 98/79: Annex 1:Essential requirements)
The relationship between
the three types
 of methods
and the reference materials,
which defines the
traceability of the result, is
expressed schematically as
follows:
 Metrological chain - traceability

material method responsibility

Definitive
WHO
Primary standard
IFCC
Reference NMI,s
Secondary standard
Procedure of
Working Laboratory
manufacturer
Standard of the manufacturer
/ manufacturer /

Pt Routine
Routine lab
sample
Result
 Traceability elements

Calibration of measuring instruments

Validation of test / calibration methods

Control of the accuracy of test / calibration

results

Interlaboratory circular / beam comparisons

(VOC) and proficiency testing)
 Validation of methods

CONFIRMATION BY
INVESTIGATION AND
PROVISION OF OBJECTIVE
EVIDENCE
 Validation of methods

 Non-standardized methods are subject to
validation

 IMPORTANT! All methods in the clinical

laboratory are non-standardized!

 All methods must be described as working

procedures
 VALIDATION PARAMETERS



SELECTIVITY AND SENSITIVITY
 CARRY-OVER
 LLOQ & LLOD, ULOQ (measurement limits)
 CALIBRATION CURVE (linearity, range)
 ACCURACY AND RELIABILITY (bias)
 REPRODUCIBILITY
 OPENNESS
 DILUTION PROTOCOL
 MATRIX EFFECT / INTERFERENCE
 STABILITY
 REFERENCE INTERVALS
 APPROVAL AND DOCUMENTATIONНЕ
THE METHODS ARE APPROVED
THROUGH:

 USE OF CERTIFIED REFERENCE MATERIAL
(CRM)

 PARTICIPATION IN AN EXTERNAL QUALITY

ASSESSMENT SYSTEM (EQAC)