Introduction to clinical chemistry

MSC : Seema Alfallah

Clinical Biochemistry Lab – 85326

Lab #1
Contents
✔ Standard operating procedures
✔ Statistics for Laboratorians
✔ Definitions
✔ Control and standard
✔ Quality control
✔ Instrumentation
✔ Spectrophotometer
✔ Mechanical pipettes
What is
clinical
chemistry
?lab
Combination of tests
✔ Diabetic profile (Glucose test and HbA1C ) .
✔ Lipid profile ( Cholesterol , Triglyceride , HDL , LDL )
✔ Liver function test ( GPT , GOT , Alkaline Phosphatase ,
Bilirubin )
✔ Renal Panel ( Urea , BUN , Uric acid )
✔ Cardiac function test ( LDH , CK, Troponin )
✔ Serum Electrolytes
 Standard operating procedures (SOPs)
• Contain detailed descriptions of each analytical method, are essential for
maintaining the same analytical quality over a long period of time.

• Contents of SOP :
1) Introduction
2) Principle of method
3) Specimen types, collection and storage
4) Reagents, standards and control - preparation and storage
5) Equipment, glassware and other accessories
6) Detailed procedure
7) Calculations, calibration curve
8) Analytical reliabilities – (QC and Statistical assessment)
9) Hazardous reagents
10) Reference range and clinical interpretation
11) Limitations of method (e.g. interfering substances and troubleshooting)
12) References
13) Date and signature of authorization
14) (Effective date + Schedule for review)
Definitions :
• Accuracy: is the degree of agreement between a measured value
and its ‘true/consensus’ value. Therefore, good accuracy means
least analytical error.
• Precision : refers to reproducibility. It refers to the agreement
between replicate measurements. It is quantitatively expressed as
the standard deviation (SD) or more precisely as percent
coefficient of variation (CV). Therefore, good precision means
least CV.
• Precision inter assay : refers to the variation
between separate runs of the same assay run on
different days with the same samples and
standards/controls using the same conditions,
instruments, etc.

• Precession intra assay : refers to the variation

between replicate samples in a single run. Intra gives
an indication of the assay's reproducibility.
• Linearity : Is an indicator of the consistency of measurements over
the entire range of measurements. In general, it is a good indicator of
performance quality of a sensor. linearity tells us how well the instrument
measurement corresponds to reality / looks at the accuracy of the
measurements over the full range of the device

• Interferences : Occurs when a substance or process falsely alters

an assay result.

• Reference value : Is the range of values that is deemed normal for

a physiologic measurement in healthy persons

• Sensitivity : The ability of the test to correctly identify those patients

with the disease (TRUE POSITIVE) False negative = low sens.

• Specificity : The ability of the test to correctly identify those patients

without the disease (TRUE NEGATIVE) / False positive = low speci.
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• Controls :

Defined substance, whose physical and chemical properties

resemble the unknown specimen

A control should have the same appearance and consistency as

does the patient samples:

✔ If patient sample is ‘serum’; the control should look like and

have the same consistency as serum.
✔ If the patient sample is ‘urine’, the control is urine, etc

Controls are used to verify the accuracy and acceptability of a run

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• Control Solutions :
A Control specimen is used to monitor Quality Control (QC)

A Control has known acceptable ranges, established either by the

manufacturer (assayed) or the hospital lab itself (un-assayed)

It is usually a serum/plasma based solution that is treated just as if

it were a patient specimen

Control specimens must produce results within established ranges

in order for the ‘run’ to be acceptable.

✔ Normal-level QC serum
✔ High-level QC serum
✔ Low-level QC serum
• Standard deviation (SD): is a measure of precision. It
reflects the spread of values from the mean (average) value.
• Coefficient of variation (%CV): is the standard deviation
expressed as a percent of the mean.
• CV = (SD/Mean) x 100
• Reference interval :
A reference range or reference interval is the range of values for a
physiologic measurement in healthy persons . It is a basis for
comparison (a frame of reference) for a physician or other health
professional to interpret a set of test results for a particular
patient.
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• Standard Solutions

A Standard solution is a highly purified solution that is usually

not serum / plasma based

Have set, listed values that are established by the manufacturer

Are used to “calibrate” instruments, that is to “set”

instruments to measure correctly at known concentration

Are also called “ Calibrators” – if they are biological in nature

These substances do not come in the ‘highly purified state, as

calcium, glucose, etc.

A bilirubin standard is biological based, and technically a

calibrator rather than the purely defined standard.
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Primary Standard : Highly purified solution of known

concentration. These standards are used in the clinical lab to
“calibrate” / “standardize” instruments in order to measure other
solutions of unknown concentration
Primary Standards must be 99.98% pure.

Secondary Standard : Less pure substance whose concentration

was determined by comparison to a Primary Standard.

Standard Reference Material / Calibrator

✔ The name for biological substances used as ‘standards’

✔ Most biological standards cannot be 99.98% pure because
the chemical processes to achieve this level of purity would
destroy the substances.
Use of calibration graphs

A fresh standard curve should be carried out for the

analyses described in this manual whenever:

1. The calibrator is changed

2. New reagents are introduced
3. Problems with QC are encountered
Laboratory errors :
• Classified to :
✔ Preanalytical
✔ Analytical
✔ Postanalytical

• Analytical errors are classified into:

Random errors: ( indicate poor precision) such as :
pipetting error, transcription error, wrong sample numbering
and labelling, and fluctuating readings on the colorimeter.
Systematic errors: ( indicate poor accuracy) occur due to
wrong procedure, incorrect standard and calibration procedure.
Construction of Levey Jennings Chart
Westgard Rules are multi-rule QC rules to help analyze
whether or not an analytical run is in-control or out-of-control.
Levey-Jennings chart is a graph that quality control data is
plotted on to give a visual indication whether a laboratory test
is working well. The distance from the mean is measured in
standard deviations (SD).
It is named from S. Levey and E. R. Jennings who in 1950
suggested the use of control chart in the clinical laboratory.
13s (rejection rule )
12s (warning rules) .
2 2s (rejection rule )
R4s
4 1s
10x
8x
12x
2of32s
31s
6x
9x
7T
External quality assessment
• Participation in External Quality Assessment
Schemes is important for interlaboratory comparison
and the maintenance of long-term accuracy and
precision of the analytical systems in the laboratory.

• This gives the laboratory an opportunity to have an

appraisal of the methods employed and switch over to
better methods.
Spectrophotometer
• It is an instrument used to measure the amount of light
absorbed by the solution. It includes:

1. Light source- gives steady white light

2. Prisms- splits white light to visible colors (red, orange,
yellow, green, violet)
3. Monochromater (wavelength selector)- system for isolating
radiant energy of a desired wave length and excluding others
4. Cuvette – contains sample solution
5. Detector (photoelectric tube)-converts the transmitted light
energy into an equivalent amount of electrical energy.
6. Meter (recording device)-displays the amount of transmitted
light or provides numerical display of absorbance
If the protein is dissolved in water : we prepare another cuvette the “blank.”
with only water in it. the blank is a cuvette which contains everything that is in the
sample (or experimental) cuvette, except the one material whose absorbance we are
measuring.

To use the blank, you could measure your experimental cuvette, then measure
your blank cuvette and find the difference between them. However the
spectrophotometer has a built in calibration system that allows you to avoid this
calculation and its much like zeroing an electronic scale.

All substances absorb some light. Therefore, if we wish to measure the

absorbance (or the transmittance) of a material like protein in solution, we must not
only consider the light absorbed by the protein, but also the light absorbed by the
cuvette and the light absorbed by the solution in which the protein is dissolved.

In order to measure only the absorbance of the protein in the solution, we must
“subtract” out (using BLANK) the absorbance due to the cuvette and the solvent in
which the protein is dissolved.
ENDPOINT REACTIONS
• Can measure the :

a) Creation of a product : the absorbance is higher at

the endpoint than at the start point = called an
end-up reaction.
b) The loss of reactant : the absorbance is lower at the
endpoint than at the start point = called an
end-down reaction .
 RATE REACTIONS
• Enzymatic reaction

• Determination of the enzyme concentration is based on how

fast a fixed amount of substrate is converted to product. The
more enzyme present, the faster the conversion.

a) If measuring the appearance of a product, the absorbance

increases with time = called a rate-up reaction.
b) If measuring the disappearance of a substrate, the
absorbance decreases with time = called a rate-down
reaction .
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• C. REAGENTS; Chemical Grades

✔ Reagent preparation in the clinical lab is decreasing - most

reagents are obtained from commercial manufacturers.

✔ Identify and differentiate the different degrees of

chemical purity.

✔ Common terms that relate to reagent purity:

1. Analytical Grade (purest), also called reagent grade or

ACS grades - best choice for lab work.
2. National Formulary (NF) or US Pharmacopeia (USP) –
used for drugs, may be OK for lab work
3. Chemical Pure (least pure) – not recommended for lab
4. Technical or commercial grade – never for lab use
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• Water Specifications :

Tap water is unsuitable for lab use (too many impurities)

Types of water purification techniques :

✔ Distillation – removes most organic matter

✔ Reverse osmosis
✔ Filtration
✔ Deionization – ions removed

Reagent Grades of water

Type I Purest – Required for sensitive tests

Type II Acceptable for most uses
Type III OK for washing glassware

CAP - QC of water : pH, electrical resistance, bacterial culture