INTRODUCTION TO CLINICAL CHEMISTRY

• Clinical chemistry is an applied core MT subject. The major aim of the course is to relate
chemical changes that occur in the body to the health status of the individual. The course
includes lectures, tutorials and practical classes to cover various topics that are important to the
clinical practice and reflect the body chemical status.
• Clinical chemistry covers the scientific, pathophysiological, chemical and biochemical
nature of
the analyte, and analytical aspects.
o Analytical aspects include; specimen requirements, methods of
determination, precautions, normal values and patient correlations. In addition,
quality control aspects that requires awareness of the sources of errors and how to
prevent them are
covered.
• Because the results obtained in clinical chemistry are quantitative, the student is also
required to know how to recognize, interpret and convert units as well as other essential lab
mathematics including reagent preparations which necessitates knowledge and practice of
concentrations, dilutions, specific gravity, water of hydration…etc.
• The human body is made up of various organs and tissues which are made from a
number of biochemical compounds such as proteins, carbohydrates, lipids, nucleic acids,
minerals, electrolytes, enzymes, hormones, growth factors and trace elements. All the
functions of the
body involve chemical interactions.
• Blood represents the major transport system of chemicals to and from all the body
tissues. It is therefore provide valuable information about most of the body chemicals. Other
body fluids such as urine, cerebrospinal fluid (CSF) also contain many types of chemicals
which are mainly specific to that type of fluid.
In all of the body fluids, there is a normal range for the chemicals that it contains.
For example;
Analyte Blood
Glucose 4.1-5.9 mmol/L
Protein 65-83 g/L
Albumin 40-50 g/L
Na 135-145 mmol/L
Urea 2.1-7.1 mmol/L
Creatinine 62-115 µ mol/L
K 3.5-4.5 mmol/L
Cholesterol Sex & age variation
Triglycerides Sex & age variation

• All the chemical substances that can be measured (analyzed) they are called
"analyte"
• Under the various circumstances that the body goes through, it tries to keep the
levels of all
the chemicals within a specified range.
 • If the values fall below the lower end of the range or increase above the upper limit
then the results are considered abnormal and take the prefix (hypo OR hyper); for
example, if the protein value for a patient was = 55 g/L then the condition is known as
hypoproteinemia. On the other hand, if the protein value was = 95 g/L then the condition is
known as
hyperproteinemia.
• Some analytes show variations with sex, age and status of the person.
o Many analytes such as cholesterol and triglycerides are higher in males than
females
o Testosterone is present in much higher concentrations in males than females
o Estrogen, FSH, LH, progesterone are mainly female hormones
o ALP is age dependent with high levels in children and old people
o Not all abnormal results always indicate disease. Diet and medications can affect the
results of many analytes. This is called interference. For example, a person who just ate a meal
high in fat will have falsely increased triglycerides; a person who ate a lot of sweets he will have
a falsely
elevated glucose.
o It is therefore important to obtain carefully controlled sample, for example fasting
blood sample, where the patient is asked to stop eating and drinking for a period 10-14 hours
before the
collection of blood.
o In addition to single analytes, many body functions affect many analytes and any
disturbance to their function will affect those analytes;
 For example, lipids include cholesterol, triglycerides(TG), lipoproteins and
phospholipids
• Increase in TG and cholesterol indicate serious condition that may
affect the heart and blood circulation as the excess fat will deposit on arteries
resulting in blockage of the blood supply to some sections of the heart (heart attack).
o Increase in LDL-Cholesterol is dangerous to men and post-
menopause women
o Increase in TG is dangerous to women especially above 30
years of age.
o Simple methods are also possible to determine whether a
person is normal or fat;
 Body mass index (BMI) = weight (kg)/[height (m)]2
 BMI <18.5 is underweight
 BMI 18.5-25 is normal
 BMI 25-30 is overweight
 BMI > 30 is obese
  Also waist/hip ratio (W/H ratio) indicate the excess
amounts of TG
 For women W/H ratio of <0.8 is normal, > 0.8 is
abnormal
 For men W/H ratio of upto 1 is normal, > 1 is
abnormal
 The liver performs many metabolic functions such as
synthesis, storage and detoxification.
• When there is a liver dysfunction a number of
analytes are affected and measurement of these analytes represent LIVER FUNCTION
TEST(LFT)
o LFT include measurement of albumin,
total protein, bilirubin, ALT, AST, GGT, ALP.
o For example in jaundice, bilirubin will
be high
o In liver biliary obstruction ALP, GGT
and bilirubin will be high
o The kidneys perform removal of water-
soluble waste products especially urea and creatinine. Increase in blood urea and creatinine
indicates abnormal renal function.
o Other aspects of clinical chemistry
involves endocrinology, Enzymology, pH, blood gasses, electrolytes, minerals, trace elements,
tumor markers, vitamins, nucleic acid, RNA& DNA,
thyroid function, porphyrins and other functions.
o It also deals with diseases especially
those that affect chemical balance and metabolism such as diabetes mellitus, Cushings Disease,
Addison's Disease, Hypo & hyperthyroidism, inborn errors of metabolism, acidosis &
alkalosis.

The function of the clinical chemistry laboratory

• To carry out tests and investigations on patients specimens upon the doctors
request and produce meaningful and timely results which help in the diagnosis of disease,
treatment and management of the patient.

Types of results obtained in the clinical laboratories.

o Quantitative (objective) (numerical value + units)
 e.g. glucose = 4.5 mmol/L protein = 78 g/L
Hb= 13.5 g/dL
o Semi-quantitative (subjective)
 Score; -ve, + , ++ , +++ , ++++ , TNC
 Titre; 1/2, 1/4, 1/8, 1/16, 1/32……..1/256
 Range; 0-5, 6-10, 11-20
 Descriptive; clear, hazy, cloudy, turbid
 o Qualitative (objective)
 Abscent, present; -ve, +ve, weakly +ve

o The majority of results obtained in clinical

chemistry are QUANTITATIVE
o Meaningful results: Reliable results which are
Expressed in Acceptable (correct) units
Reliable results
Accurate : close to the real “true” value
Precise : similar results if repeated on same specimen

Acceptable (correct) units

(quantitative: SI, conventional, qualitative and semi-quantitative)

– Timely results: Available to the doctor when needed

– routine
– urgent (LIFE SAVING INVESTIGATIONS)

• Components (Stages) of QA Program

– Pre analytical (~ 68-86% sources of errors)
– Analytical (~ 40 % sources of errors)
– Post analytical (~ 20 % sources of errors)

• To obtain meaningful and timely results ALL components of the QA Program

MUST observe and adhere to the specific instructions set by the lab regarding all the aspects
included in the specific component.

• Pre analytical stage

– Outside the control of the lab
– Clear and accurate instructions must be given to personnel
– Involves: doctors, nurses, patient, phlebotomist, messenger (porter)
– Includes:
• request for investigation by the doctor after examination of the patient,
• request form, filled by doctor or nurse and includes the following
information;
-patient's name -hospital No., File No. or Medical Record No. -age (DOB) -sex
-date of request -specimen type -tests required
-possible diagnosis (or query) -unit/ ward -Doctor's Name
-Doctor's signature -Doctor's Bleep or phone No. -Consultant's Name
-Date & time of collection
-Information about the patient regarding; -medications -diet -physical condition -smoking
-any other relevant information that may be useful for the lab
• patient instruction, the patient must receive clear and accurate instructions
regarding the following;
– importance of the test & how it will help in the diagnosis and treatment of his illness
– Type of specimen to be collected (blood, urine, stool …etc.)
– Fasting or random sample:
• -clinical fasting (10-14 hours)
• Patient usually fasts from the last evening meal (~ 7 pm) until the specimen is
collected in the next morning between 7 & 8 am
• Fasting sample used for diagnostic and confirmatory tests
 • It provides information about the body status for the last 24hours
• Fasting sample also means that the patient observes other restrictions
regarding diet, medications, smoking, physical activities, sleeping habit other life-style
and method of transportation
• Random specimen (on spot sample): taken just before analysis; is mainly
used for screening tests
– Dietary restrictions; some types of diet may affect the analysis and results. For
example;
• lipids intake must be controlled for lipid profile analysis;
• carrots may affect the colour of urine and colourimetric procedures,
• bananas and mango may elevate the level of K in blood
• high intake of salt will increase the levels of Na & Cl
– Use of medications;
• almost all types of medications affect laboratory results
• the type of medication chronically used by the patient must be written on the
request form
• if the type of medication used by the patient affects the required tests, then
the patient may be asked to stop taking the medicine for upto 24hrs until the specimen is
collected
– Physical activities restrictions;
• Strenuous exercises may affect the results of some tests
• Walking under hot or cold climate to the hospital may also affect some
results
• The patient must be clearly informed if his physical activities affect the result
and if he needs to refrain from such activities one or more days before collection of the
specimen.
– Life style
• All types of Smoking interfere non-specifically with many test results
• The patient must be told to refrain from smoking for at least 24hours before
collection of specimen
• sleeping habit; the patient must have enough sleeping hours before collection
of the specimen
• some analytes follow a circadian rhythm (~24 hours; night and day related)
this includes hormone releasing factors (released fro the hypothalamus) & ACTH
• some analytes follow a diurnal rhythm (24 hours exactly)
e.g. cortisol highest level at 7:30 am & lowest level at 7:30 pm
- patient preparation,
• the patient must be properly identified as the person who is mentioned on the request
form
• the patient must be relaxed before, during and after the collection
• stress on the patient can lead to increase levels of adrenalin which will result in
hyperventilation and increased rate of blood flow in the body which may affect many tests
• appropriate patient posture must be observed during collection (sitting, or
recumbent)
- specimen collection & handling
- for every test there is a specific type of specimen
- for every test there are specific conditions for collection & handling of specimen
- specimen is any part of the patient which is collected for specific investigation
- Whole blood, serum and plasma are the most common types of specimen received in
clin chem. Lab
- Also urine & CSF are commonly received in clin chem
- serum is the ideal specimen for clinical chemistry analysis (plain tube) (red top tube)
 - Li heparin is the preferred anticoagulant because; Li exist in insignificant
concentrations in the body & heparin is the physiological anticoagulant in the body, so there
are no interferences on analysis
- EDTA, Citrate & Oxalate should not be used as they lower the concentrations of Ca,
Mg & many enzymes
- NaF/Koxalate (Grey Top tube) is used for glucose analysis only.
o It inhibits glycolysis by inhibiting glycolysis enzymes
o It must always be collected as the last tube.
o NaF is a non-competitive enzyme inhibitor
o The tourniquet must not be applied for more than one minute for most tests
o The specimen in the tube must be gently mixed by inversion 6-7 times to ensure
good mixing of anticoagulant with the blood
o Never shake the tube or mix vigorously as this may cause hemolysis
o Avoid hemolysis of the sample (K & Mg increased, most colourimetric procedures
are affected)
o Avoid collection from an intravenous line site (dilution of all analytes and
exaggerated increase in the infused analytes concentration)

- transportation
- blood samples must be transported within 45 minutes of collection for routine tests
- urgent samples must be sent immediately to the lab (within 10 minutes of collection)
- turn around time for urgent samples is 20 minutes (collection, transportation,
analysis and return of results)
- other specimens must be delivered to the lab as soon as collection is completed
- specimens must be transported in an upright position to prevent spillage,
contamination and minimize the affects of slight hemolysis
- specimens for pH & blood gasses must be transported in syringes immersed in
normal ice immediately to the lab and analyzed within 1hour.
- specimen for bilirubin must be transported in fully covered tube to prevent exposure
to light which will oxidize bilirubin and decrease the result
- avoid exposure of specimen to direct sunlight or high temperature
- samples should be placed in a double pocket (side-pocket) transparent plastic bag
which has keeps the specimen separate from the request form

- storage
- plasma must be separated immediately after receiving the sample
- serum must be separated immediately after clot formation (10-15 minutes after
collection)
- for routine analysis for most tests samples can be analyzed within 2hours after
collection at room temperature
- refrigerate the sample (4-8 oC) if analysis is delayed (upto 8 hours)
- normal freezers (-18 to -22 oC) for upto 3months
- deep freezers (~-70 oC) for months and years
- liquid nitrogen (-140 oC) for months and years
- NEVER FREEZE WHOLE BLOOD -- severe hemolysis
- Never repeat freezing and thawing of the sample. This will denature most proteins
and polypeptide hormones and also affect some analytes.
- If some of the analysis are done on separate days, then the sample is divided into
several aliquots (number of tests +1). Aliquots are frozen. Then one aliquot is used once on
the particular day for the required tests.

– The quality of the result is dependent on the quality of the specimen.

 • Analytical stage
– Within the control of the lab
– Involves technicians, lab aids, technologist, supervisor, lab manager, lab
director, engineers, clerk, secretary, administration
– Policy & Procedure Manuals
– Includes:
• Specimen reception and booking
• Centrifugation, separation, aliquoting, storage
• Equipments and instruments:
• Procedure of analysis
• QC system
– Heart of the QA program
– No QC No results
– Checks for errors
– QC materials (human, animal sera….)
– Systems for quantitative results (Levy-Jennings Chart,
CuSum, Multi Rule, Replicate Analysis, Delta Check)
– Systems for qualitative results
– System for semi-quantitative results
– Automated or manual QC
– Recording of QC and actions taken

• Chemicals, water & kits

• Safety
– Physical -Chemical -Health - Fire -
Radiation
– General -Safety equipments -Waste disposal
– Rules and regulations
– ALWAYS PRACTICE SAFETY
– NEVER EXPOSE YOURSELF OR OTHERS TO HAZARDS
• Personnel

• Post analytical stage

– Mostly controlled by the lab.
– Involves: technologists, clerks, secretary, lab supervisor, lab consultant, lab
director, porter, nurse, doctor
– includes
• Results acquisition
• Use of QC for results validation
• Results reporting (result form, machine printout, manual writing,
routine & urgent requests)
• Filing -Queries and complaints
SOURCES OF ERRORS IN CLINCAL CHEMISTRY ANALYSIS
TYPES OF ERRORS: TWO TYPES:
1) QUALITATIVE (UNMEASURABLE)
o The severity of the error is proportional to the severity of the outcome.
o Upto 5% are considered as human errors
o Four types exist:
a)-Administrative & managerial ERRORS: errors concerning the following;
organizations, appointments, locations, instructions, wrong decision, wrong order, contradictions,
 misplacement, errors in assignment, lack of knowledge, lack of experience
b)-Clerical/ Secretarial ERRORS:- errors concerning the following; mis-matching of request form
and specimen, mis-placing of forms or specimens, lost results, wrong filing, late filing, patient
name, file No., patient age or D.O.B, patient sex, date of request, date & time of collection,
specimen type, tests requested, doctor's name, doctor's bleep, unit or ward, photocopying,
c)-Typographical ERRORS:- errors concerning the following; typing errors, unclear writing, untidy
writing, mis-spelling, improper writing of numbers, improper placement of decimal point
d)-Communication ERRORS:- errors concerning the following; lack of communication, language
problems, phoning of results, mis-communication, contacting the wrong person, no
communication rules present.
2) QUANTATIVE (MEASURABLE) (ANALYTICAL)

-Analytical: relates to errors in the analysis.

1- Random error (RE)
• Occurs by chance
• In every procedure
• Fluctuates about the mean
• It shifts the SD
• Affects the precision
• No magnitude
• No direction
• Value= ±1.96 SD (2SD)
• Values ± 2SD - ± 3SD indicate the presence of RE
• Values ≥ ± 3SD indicate the presence of large RE & considered as outliers (result
rejected)
• cv gives an estimate about the precision & RE
o smaller cv small RE & high precision
o bigger cv big RE & low precision
sources: Normal: vibration, pipetting, temperature, air current, humidity, skill, experience
2-Systematic error (SE)
o should not occur in the procedure
o it has magnitude
o it has direction
o it shifts the mean
o affects accuracy

Two types:
a- Constant SE (CSE)
The size of the error is the same regardless of the causative factor.
Due to:
1-interference: Endogenous (haemolysis, ictraemia, lipaemia)
Exogenous (wrong anticoagulant, diet, medications, smoking, activites)
2-permenant damage (lamp, electrode, tubes, electronic circuit …..)
3- expired reagents, standards
4- extreme variations in vibration, temperature, air current, humidity
5- inadequate skill
6- lack of experience
 b- Proportional SE (PSE)
The size of the error depends on the size of the causative factor.
Due to; deteriorating reagents, lamps, diaphragam, electrodes, transformers, components

Limits of Precision & Accuracy

• For every procedure, instrument, equipments and analytes there are limits which
determine the acceptable levels of accuracy and precision
• The acceptable precision for all analytes, equipments and instruments is a cv upto
5% EXCEPT for enzymes and polypepetide hormones where the acceptable cv is upto 10%
• The acceptable accuracy for all analytes, equipments and instruments is upto 2%
deviation from the "true" or "expected" value.
• Reporting of results depend on;
o Clinical relevance of the values : glucose = 6.5 mmol/L (one decimal place)
Ca = 2.54 mmol/L (two decimal places)
pH = 7.45 (two decimal places)
o Minimum detectable limits of the analyte e.g. Mg = 0.35 mmol/L
 Values lower than the minimum detectable level are reported as : Less
than the detectable limit ( e.g. Mg <0.35 mmol/L)
o Maximum (upper) detectable limit: For values higher than the upper detectable
limit, the sample should be diluted until the result fall within the detectable range of the
analyte which is usually a straight line (on a plain graph paper) when the reaction obeys
Beer's Law. The actual result is then obtained by multiplying by the dilution factor (df).
o Limit of accuracy of the measuring device. Generally, the measuring device level
of accuracy has to be one decimal place more than the clinically acceptable number of
decimal places. For example the pH measuring machine must be able to measure pH
upto 3 decimal places so that the pH can be accurately reported in 2 decimal places; i.e
if the true pH of the patient is 7.38; then the machine must measure it as 7.380 (7.375-
7.385)

o Reporting of volumes, weights or lengths depend on;

a) the minimum amount that can be accurately measured
b) the required level of measurement
c) the stated level of accuracy

 e.g the minimum volume that can be measured manually and

automatically by many measuring devices is in µ L quantities.
So when writing a volume of solution in mL; then we must write to three decimal places
e.g 12.345 mL or 150.250 mL

o the required level of measurement will also determine the level of accuracy. For
example if the level of weight is in g quantities, then we write the weight to one decimal
place (1/10g); 15.5 g.
o sometimes a specific limit of accuracy is stated in such measurements (e.g. to three
decimal places or to the nearest mg) then the device used must fulfill this requirement