Email: henrysamk@live.

com

CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL

CHEMICAL PATHOLOGY – PART 1
INTRODUCTION:
Chemical Pathology is also known as clinical biochemistry, medical biochemistry or clinical chemistry.
The Chemical Pathology Unit provides diagnostic and consultations services for patient management.
Our services cover analysis and interpretation of biochemical changes in body fluids for screening, diagnostics
and monitoring of diseases.
Chemical pathology – its involves the biochemical investigation of the bodily fluids such as blood, urine and
cerebrospinal fluid. By discovering how and where the body's chemistry has changed, diseases can be
diagnosed and monitored.
Chemical Pathology laboratory carries out routine and special blood analysis. Most of the tests are performed
using fully automated or semi-automated analyzers to produce higher level of efficiency, accuracy, precision
and testing throughout.
To ensure reliable and accurate results, the division participates in both national and international quality
control programmes in addition to using internal quality control sera daily to minimize errors related to the
reagents, instruments or operator.
As the demands for biochemical tests are heavy, users of the service are urged to be selective when requesting
for Chemical Pathology tests.
Requests should be confined only to those tests that are useful for the diagnosis or control of treatment of the
patients.
IMPORTANCE OF STUDYING CHEMICAL PATHOLOGY
Chemical pathology brings together science and medicine. By understanding the chemistry of bodily fluids and
monitoring these, laboratory professionals can tell whether a patient's organs are working properly, diagnose
diseases and recommend treatment.
For example high glucose levels in blood may be a sign of diabetes.
Pathology is a branch of medical science that is focused on the studying and diagnosis of disease. Clinical
pathology involves the examination of surgically removed organs, tissues (biopsy samples), bodily fluids, and in
some cases the whole body (autopsy).
Many illnesses are reflected in a disturbance in the body’s chemistry. Chemical pathology brings together
science and medicine. By understanding the chemistry of the bodily fluids and monitoring these, laboratory
professionals can tell whether a patient’s organs are working properly, diagnose diseases and recommend
treatment. For example high glucose levels in the blood may be a sign of diabetes. Glucose is a sugar that
provides fuel for the body. The blood glucose level is regulated by the hormone, insulin.
If the body doesn’t produce enough insulin, diabetes may develop. Diabetes can cause eye and kidney disease,
and can cause blood vessels to narrow, resulting in heart disease and poor circulation. Some women develop
diabetes during pregnancy.
ROLE OF CHEMICAL PATHOLOGIST
Clinical scientist, medical consultants and biomedical scientist are all involved in providing a clinical
biochemistry service in hospitals. Skilled biomedical scientists carry out most of the day-to-day analytical work.
DR.SH COKER 1
Email: henrysamk@live.com

CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL

Both clinical scientists and biomedical scientist may carry out complex analytical work. Clinical scientist and
medical staff are responsible for the appropriate use of investigation and interpretation of results as well as the
development of the service. Medical staff may also be involved in direct patient care. For example running lipid,
diabetic, endocrine and bone disease clinics.
Chemical Pathology is the branch of pathology dealing with the biochemical basis of disease and the use
of biochemical tests for diagnosis and management. Doctors in the specialty have dual responsibilities.
First there is the provision of a reliable analytical service, for example measuring serum electrolytes,
indices of liver function, hormones, drugs and tumour markers in hundreds of patient samples every day.
Many of these analyses are performed on automated analysers, usually operated by technologists, but the
management of the process (and the staff), assurance of quality and provision of guidance on the
selection of tests and assessment of the significance of the results (particularly with some of the less
generally familiar tests) are the province of the chemical pathologist.

Secondly, Chemical Pathologists have an important clinical role, not only advising on the management
of patients with metabolic disturbances but in several countries now, they are increasingly having direct
responsibility for such patients in out-patient clinics and on the wards.

Chemical Pathology not only brings together science and medicine, it relates to all the medical
specialties. Chemical Pathologists are frequently consulted about further investigation or management of
patients found to have biochemical abnormalities on 'routine' testing. They frequently have to deal with
investigating patients with dyslipidaemias, diabetes and hypertension, review ward patients receiving
artificial nutrition, discuss the introduction of a new diagnostic test with consultant colleagues, review
the quality of the laboratory's analytical service and manage research projects of trainees.

Under application of biochemical techniques can be listed the following:

 Diagnosis: tests can be used to help differentiate between various possibilities in the differential
diagnosis based on the initial history and examination when the patient first presents.

 Screening: detection of disease before it is clinically evident, e.g. testing all infants at birth for a
specific inherited disease (phenylketonuria, thyroid deficiency)
 Monitoring: following the progression of disease processes, checking against adverse drug
effects (e.g. hypokalaemia with diuretic therapy), or response to therapy (glucose levels in
diabetes mellitus).
 Prognosis: providing information on disease susceptibility, e.g. cholesterol to predict heart disease.

DR.SH COKER 2
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

MEASUREMENTS IN CHEMICAL PATHOLOGY: concentration of a substance

A. Units, Amounts and Concentrations

Units and abbreviations - The common units, and their respective abbreviations, used in
biomedical sciences are based on the International System of Measurements (SI units): grams
(g), metres (m) etc. Often it is necessary to deal with large quantities e.g. 1500 g or, more
commonly, very small quantities, e.g. 0.0000015 g. In such cases, such numbers are either
expressed by use of powers of 10 (positive or negative) or by use of the appropriate prefix.

Thus: 1500 g = 1.5 x 103 g = 1.5 kg (kilograms)

and 0.0000015 g = 1.5 x 10-6 g = 1.5 µg (micrograms)

Laboratory scientists most commonly use the prefix notation, particularly those that differ by a
factor of 1000 in magnitude, so you should try to become familiar with them. Use of the prefixes
can greatly simplify calculations, especially mental ones.

Note also that biomedical scientists normally express volumes and concentrations in terms of
litres rather than in cubic measurements:

e.g. 1 litre, rather than 1 dm3

1 millilitre (1 ml) rather than 1 cm3

A concentration of 1 milligram per litre is also commonly expressed in the form 1 mg/l rather
than 1 mg.l-1 or 1 mg.dm-3.

The commonly used prefixes are:

Prefix Name Which modifies an amount by Examples

m milli 1/1000th, i.e. by 10-3 mmol, mg, ml
µ micro 1/1000 000th, i.e. by 10-6 µmol, µg, µl
n nano by 10-9 nmol, ng
p pico by 10-12 pmol, pg

DR.SH COKER 3
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
f femto by 10-15 fmol, fg

k kilo by 1000 times, i.e. by 103 kg

M mega by 106 MPa

The prefixes centi (10-2) and deci (10-1) are only commonly used in specific cases e.g. cm

Concentrations

The determination of the concentration of a substance in a biological fluid is central to many

areas of medical and dental practice (e.g. electrolytes in serum or glucose in urine). It is
important, therefore, that concentrations are expressed in clear unambiguous terms. There are
several ways of doing this. The simplest is to express the concentration as the weight or mass of
the substance per unit volume:

e.g. 10 g/l or 20 mg/ml or 2 µg/ml

Another way is to express the concentration of a solution or mixture in terms of per cent (%).
This is a somewhat outdated method but you may still come across it, particularly if you read the
older medical literature, so you should know what it means. Note that there are different types of
% concentration:

% (v/v) (volume by volume)

% (w/v) (weight by volume)

% (w/w) (weight by weight)

 A 1% (v/v) concentration is obtained by diluting 1 volume of a substance into 100

volumes (total) of solution, e.g. 1 ml ethanol diluted with water to a final volume of 100
ml gives a 1% (v/v) ethanol solution.

 A 1% (w/v) concentration is obtained by dissolving 1 g of substance in a final volume of

100 ml solution, e.g. 1 g glucose dissolved in water to a final volume of 100 ml solution
gives a 1% (w/v) glucose solution.

DR.SH COKER 4
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
 A 1% (w/w) concentration is obtained by mixing 1 g of substance with something else in
a total weight of 100 g, e.g. 1% (w/w) salt in sand.

Another old method of expressing concentration that you may still see (just look at the side of a
tube of toothpaste) is "parts per million (ppm)". One ppm is one part of anything in one million
parts of total material, e.g. 1 g of compound X in a million g total, or 1 litre of Y in a million
litres total.

Moles and molarity

By far the most important unit defining an amount of a biological substance is the mole, with
the corresponding concentration being the molarity. As far as possible, the concentrations of
specific compounds in serum, urine etc. are now expressed as molarities in clinical laboratories.

One mole of a substance is the molecular weight of that substance expressed in grams. Thus, the
molecular weight of glucose is 180, so:

1 mole of glucose = 180 g

1 mmol glucose = 180 mg

1 µmol glucose = 180 µg

100 µmol glucose = 18,000 µg = 18 mg

Note the abbreviations: 1 mmol = 1 millimole; 2 mmol = 2 millimoles; 5 µmol = 5 micromoles.

Concentrations in molarities are given by expressing the number of moles of the substance
present in a defined volume of solution:

A 1 molar (1 M) solution contains 1 mole per litre (1 mol/l)

a 1 millimolar (1 mM) solution contains 1 millimole per litre (1 mmol/l)

Note: mol and moles mean the same thing (an amount) and moles/litre (long winded but correct)
is a concentration and can be expressed as mol/l, or mol.l-1, or (best and simplest of all)
— M. Ensure you know the distinction between concentrations and amounts.

So, if you dissolve 0.5 mol of a compound in one litre of solvent the concentration of the
compound is 0.5 mol/l, or 0.5 M. 100ml of the solution contains 0.05 mol.

Prefix notation

The value of the prefix notation can now be seen as it allows rapid mental calculations to be
performed (after much practice!). The following concentrations are all the same:

DR.SH COKER 5
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
0.5 M, 0.5 mol/l, 0.5 mmol/ml, 0.5 µmol/µl, 0.5 pmol/pl

You see that by scaling both the units (amount and volume) in the concentration up or down by a
factor of 1000, the value of the concentration remains the same. If you only scale one of the
terms (amount or volume), then you can express the same concentration in yet more ways:

0.5 mol/l = 0.5 mmol/ml = 500 µmol/ml = 500,000 pmol/µl

Now, let’s say you wanted to determine the amount of cholesterol in the blood of a newborn
infant. You know it will be around 5 mM. The detection limit of the method you are going to use
is about 20 nmol and you can only take 20 µl of blood. Will this be enough?

5 mM cholesterol contains 5 mmol/l, or 5 µmol/ml, or 5 nmol/µl.

It is easy to see now that 20µl contains 100 nmol of cholesterol

- ENOUGH.

There are 2 types of units of concentration, molar units, and mass units. The former is
preferable, since it is a better comparative descriptor of the concentration of a substance,
but unfortunately many countries still cling to the older mass units, usually grams/100 ml,
and it is often necessary to convert.
I mole of a compound corresponds to a mass (in grams) equal to the molecular weight of that
compound,

e.g. 1 mole of NaCl = (23+35)

=58 grams of the salt or,

1mole/liter (mol/l) of NaCl = 58

g/l or 5.8 g/100ml

Abbreviations:
1 mol/l = 1 M = 1 molar
1 mmol/l = 1 mM = 1 millimolar = 10-3 M
1 mol/l = 1 M = 1 micromolar = 10-6 M
1 nM = 1 nanomolar = 10-9 M
1 pM = 1 picomolar = 10-12 M

DR.SH COKER 6
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
Concentrations of some analytes (to demonstrate the range of concentrations in clinical
chemistry)

serum Na+ 140 mM serum glucose 8 mM

serum Mg2+ 1 mM serum albumin 0.6
mM
serum Fe3+ 20 M serum cortisol 500
nM
serum [H+] 40 nM plasma ACTH 50 pM

INTRODUCTION TO CLINICAL LABORATORIES – PART 2

Diagnosis of any disease is first done by physical examination by physician and confirmed
by lab diagnostic tests. Lab values are very important in determination of disease severity, drug
doses and in follow up.
The sections of clinical laboratory are:
 Clinical biochemistry
 Clinical micro biology and paracytology.
 Hematology
 Serology
 Blood bank
 Histologyand cytology

Clinical biochemistry:
In this lab the concentration of one or more substances in biological specimen of patient are
measured and compared with reference value obtained from healthy subjects.
Types of samples that are used in testing:
Body fluids: blood, serum, plasma, urine, cerebrospinal fluid(CSF), feces, and other body
fluids or tissues.
Biochemical tests in clinical medicine
 Lipid profile
 Diabeticprofile
 Kidneyprofile
 Liver profile
 Boneprofile
 Electrolyte profile

Methods Used In The Chemical Pathology Laboratory

• Colorimetric methods: Analyte reacts with a dye, changing its absorption spectrum (colour).
Measured with a spectrophotometer. Rapid & easily automated. Cheap. Concentration range:
mM to μM.

DR.SH COKER 7
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
Examples: urea, creatinine, phosphate, albumin, total Ca2+

Ion-selective electrodes: Membrane selectively permeable to an ion generates a membrane

potential proportional to concentration of free ion. Rapid & easily automated. Cheap.
Concentration range: mM to nM Examples: Na+ , H+ (pH meter), free Ca2+

Enzymatic: Example: lactate dehydrogenase (LDH) catalyses the reaction

Radio-Immuno-Assay(RIA)and related techniques:

In the competitive RIA, the analyte or antigen (Ag) in the sample competes with radio actively
labeled analyte (Ag*) for binding to a limiting number of antibody sites (Ab) in the test tube:

Ag*-Ab can be easily separated from the free Ag* and the amount of labeled Ag* bound is
determined by using a radio activity counter. The more unlabelled Ag in the specimen, the
lessAg* will be bound .

The technique has high sensitivity i.e. is able to measure low concentration so fAg (nM to pM
range). Automation has been achieved with some but not all immunoassays in current use, so it
can be a time-consuming method.

Chromatographic methods
Electrophoresis and other chromatographic method can be used to separate compounds in
plasma or urine, e.g.
-serum proteins (electrophoresis)
-amino acids (ion exchange chromatography)
-organic acids (gas chromatography, mass spectrometry)

-isoenzymes (electrophoresis)

•DNA techniques
Analysis of patient’s DNA for specific mutations, or linked polymorphisms, by molecular
biological techniques, usually involving use of the versatile polymerase chain reaction (PCR)

Meaning Of Normal And Reference Ranges:

Normal Range” should be avoided because it implies health and/or a norma distribution and
neither might represent the significance of a test result or the distribution of test results.

Reference Values: A value or set of values used to interpret a laboratory result

DR.SH COKER 8
CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
The reference values can be a single cut-off, a setof cut-offs, or a range of values containing
95%of the results from a reference population.
-The most common definition of the Reference Range is the range of values containing the
central 95% of the “healthy” population.
This definition results in 5% of the “healthy” population being classified as “abnormal” or
“positive”

Test Reliability:
Four indicators are most commonly used to determine the reliability of a clinical laboratory test.
Two of these, accuracy and precision, reflect how well the test method performs day to day in a
laboratory. The other two, sensitivity and specificity, deal with how well the test is able to
distinguish disease from absence of disease.

Precision (Repeatability)

A test method is said to be precise when repeated analyses on the same sample give similar
results. When a test method is precise, the amount of random variation is small. The test method
can be trusted because results reliably reproduced time after time.

Accuracy (Trueness)
A test method is said to be accurate when the test value approaches the absolute “true” value of
the substance (analyte) being measured. Results from every test performed are compared to
known "control specimens" that have undergone multiple evaluations and compared to the
"gold" standard for that assay, thus analyzed to the best testing standards available.

Sensitivity:
Sensitivity is the ability of a test to correctly identify individuals who have a given disease or
condition. For example, a certain test may have proven to be 90% sensitive. If 100 people are
known to have a certain disease, the test that identifies that disease will correctly do so for 90 of
those 100 cases (90%). The other 10 people (10%) tested will not show the expected result for

DR.SH COKER 9
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
this test. For that 10%, the finding of a "normal" result can be misleading and is termed false-
negative. The more sensitive a test, the fewer false-negative results will be produced.

Specificity:
Specificity is the ability of a test to correctly exclude individuals who do not have a given
disease or condition. For example, a certain test may have proven to be 90% specific. If 100
healthy individuals are tested with that method, only 90 of those 100 healthy people (90%) will
be found "normal" (disease-free) by the test. The other 10 people (who do not have the disease)
will appear to be positive for that test. For that 10%, their "abnormal" findings are a misleading
false-positive result. The more specific a test, the fewer false-positive results it produces.

For reference, sensitivity (the ability of a test to detect a disease when it is present), and
specificity ( the ability of a test to reflect the absence of the disease in those disease-free)
can be calculated as follows:

Sensitivity = 100 x TN /

(TN + FP) %

Specificity = 100 x

TP / (TP + FN)%

There is one other statistic which can be useful in deciding either what test to use in a
particular context (screening, confirmation etc), or what reference range to apply in
such a context, and that is the predictive value (PV) of a test, which can be positive
or negative. The PV for a positive result is dependent on the prevalence of the disease,
and is the % of all positive results which are true positives, e.g. PV(+) = 100 x
TP/(TP+FP) %

If the test is less than 100% specific and the condition has a low prevalence (such as
an inherited metabolic disease), then many FPs will result. A high predictive value is
important if the (unnecessary) treatment of a false positive had significant dangerous
consequences or side effects. The PV for a negative result should be maximised if
one does not want to miss a patient who has the disease, it being the proportion
DR.SH COKER 10
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
of all negative results which are true negatives. It is given by
PV(-) = 100 x TN/(TN+FN) % and implies that the test should maximize sensitivity.

Specimen Handling In Chemical Pathology:

If the physician is to have confidence that the laboratory result is correct and meaningful, he/she
needs to ensure that the specimen is taken in an appropriate way and that it gets to the laboratory
in optimum condition and in good time.

Preparation of Blood Sample:

One of three different specimens may be used: whole blood, serum, or plasma. Serum and
plasma are prepared from whole blood by centrifugation.

After centrifugation of blood, it is separated into

three layers (see the below figure).

Whole blood:
Whole-blood specimen must be analyzed within limited time (why?)
1- Over time, cells will lyse in whole-blood which will change the conc. of some analytes as
potassium, phosphate and lactate dehydrogenase.
2- Some cellular metabolic processes will continuo which will alter analytes conc. like glucose
and lactate.
Serum or plasma:
Differences between serum and plasma:
1- Serum is the same as plasma except it doesn't contain clotting factors (as fibrin).
2- Plasma contains all clotting factors.
3- So, serum and plasma all has the same contents of electrolytes, enzymes proteins,
hormones except clotting factors.
4- Serum is mainly use in chemistry lab & serology.

Blood anticoagulants:
They are substances which prevent blood coagulation. They inhibit coagulation process by
eliminating Ca2+ ions which are important in coagulation or by binding with thrombin and
prevent conversion of fibrinogen to fibrin.

EDTA, citrate and oxalate: inhibits coagulation by binding with Ca2+ and prevent the
activation of prothrombin to thrombin.
Heparin: inhibits coagulation by binding with thrombin which is important in activation of
fibrinogen to fibrin.

DR.SH COKER 11
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
Contamination
1. In the patient. Taking blood from a vein where the patient has a drip installed peripherally can
give very strange results ("drip arm").
2. In the tube (wrong additive)

Separation of red cells from serum/plasma

For most tests delay of a few hours does not matter. After a 12 h delay the specimen is
classified as a "non separated specimen" (NSS). NSS typically shows false high K+, Pi and
LDH (leakage from RBCs).

Haemolysis is the liberation of hemoglobin due to rupture of RBCs. Plasmaor serum of

haemolyzed sample appears pink to red color.

Labile analytes
Special precautions have to be taken when measuring labile constituents. EXAMPLES:
1- Blood gases : blood has to be taken anaerobically and into a stoppered tube (to prevent
CO2 escaping) and placed on ice to prevent lactic acid production.
2- Peptide hormones are susceptible to protease degradation, and require addition of
protease inhibitors.
3- Plasma ammonia rapidly rises after sampling due to breakdown of glutamine.

Identification of Normal Physical and Chemical Urine Constituents

Urine is ultrafiltration of plasma which contains waste products and chemical
substances excreted by the kidney. Normally, urine is clear, transparent and contains
many constituents. From urine sample you can determine all metabolites in body.

Normal constituents of urine:

In general, urine consists of:
95 % water and 5% other dissolved chemicals.

DR.SH COKER 12
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

Factors affect on urine constituents are:

dietary intake, physical activity, body metabolism, endocrine function and others.
Urine Sample collection:
Types of urine specimens:
The types of specimen and collection procedure are depending on the test to be
performed.
 Qualitative “Para + microbiology”
 Quantitative “conc.” in chemistry lab.
- First morning.
- Random
- 24 hour urine collection container
- Timed collection after use of medication.
Routine urinalysis:
It includes both macroscopic and microscopic analysis.

Routine urinalysis

Macroscopic Microscopic

Physical Chemical

Macroscopic Examination:
A. Physical Characteristics:
The first part of a urinalysis is direct visual observation like color, smell, volume,
transparency, pH, and specific gravity.
Appearance
1. Color:
Normally, Urine color ranges from pale yellow to deep amber depends on how diluted
or concentrated it is, this color is due to pigment called urochrome.

Abnormal colors:
 Silvery sheen or milky appearance ------> Pus, bacteria or epithelial cells
 Reddish brown ------> Blood (Hemoglobinuria)
 Yellow foam -------> due to Bile pigments in jaundice disease or medications.
 Orange, green, blue or red ------> due to some medications.

2. Transparency or clarity:
 This is classified as clear or turbid.

DR.SH COKER 13
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
 The degree of cloudiness of urine depends on both its pH and dissolved solids.
 In normal urine, the main cause of cloudiness is crystals and epithelial cells.
3. Odour:
 Odour has a little diagnostic significance.
 Aromatic odour------> Normal urine due to aromatic acids.
 Ammonical odour ---------> On standing due to decomposition of urea.
 Fruity odour --------> Diabetes due to the presence of ketones.
4. Volume:
 Urine volume measurements are part of the assessment for fluid balance and
kidney functions.
 Urine volume in adult: 750ml-2500ml/ 24h, (average: 1.5L/ 24h)

5. Specific Gravity (SG):

 It measures the amount of dissolved substances in urine (urine density.

Urine SG refractometer
6. Reaction (pH):
 The pH measure how acidity or alkalinity (basic) of urine
 Normal urine pH (~6).

DR.SH COKER 14
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

Name Procedure Observation

Urea 1ml urine + 3ml NaOCl (sodium hypochlorite) Evolution of N

2 gas
Uric acid 1ml urine + 0.5 ml 10% NaOH + 1ml Folin`s reagent Blue colour.

Creatinine 1ml urine + drops of sat. picric acid + drops of NaOH 10% Deep red color of
creatinine ppt.
Cl- + AgNO =========> AgCl + NO
Chloride 3 3 White ppt. of AgCl

1 ml urine + 1 ml conc. HNO + 1 ml ammonium molybdate

Phosphate 1 ml urine + drops conc. HCl Yellow colour
3
Carbonate Na CO + 2HCl =======> H O + 2NaCl + CO Effervescence
2 2 2 2

Ammonia 1ml urine + 1ml phenol + 1ml NaBr Blue color

Sulphates 4 2 4 White ppt. of BaSO4

=======> SO + BaCl =====> BaSO + 2Cl-

DR.SH COKER 15
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
Identification of Pathological Physical and
Chemical Urine Constituents
Pathological urine constituents are substances which are not usually present in urine such as
glucose, protein, ketones, RBCs, Hb, bilirubin…. etc.
Abnormal urine constituents include:
1- Proteinurea:
Proteinuria is the presence of abnormal amount of protein in urine.

 Urine of healthy individual contains no protein or only traces amounts, due to:
: In normal physiology, protein is reabsorbed by kidney tubules (proximal
tubule). Protein has large molecular weight so it can't pass through kidney tubule to urine
unless kidney tubule has damage.
 The main protein in urine is albumin; therefore, proteinurea=albumin urea.
Microalbumin urea: Is the presence of small amounts of albumin in urine. It is very important
in detection of early stage of nephronpathy.
 High protein in urine makes urine looks foamy.

2- Glucoseurea:
Glucosuria is the presence of abnormal concentration of glucose in urine.
 Normally, glucose is reabsorbed by active transport in proximal tubule and
therefore it doesn't appear in urine.
 If the blood glucose level exceeds the reabsorption capacity of kidney tubules
(renal threshold), glucose will appear in urine. (this is indication for high
blood glucose).
 Renal threshold of glucose: is around 160 mg/100 ml.
Glucosuria indicates that glucose concentration in blood exceeds this amount and
the kidneys are unable to reabsorb it efficiently.
 Glucosuria occurs in diabetes mellitus, which characterized by:
hyperglycemia, polyurea (increased volume of urine), high SG and urine may
DR.SH COKER 16
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
be light in color.

3- Ketourea:
Ketourea is the presence of abnormal amount of ketone bodies in urine.
 Body normally uses carbohydrates as source of energy. If carbohydrate source
is depleted or if there is a defect in carbohydrate metabolism; body use fat as a
source of energy.

 Fat metabolism is occurred for certain time. At certain point, fatty acid
utilization occurs incompletely results in production of intermediate
substances (keton bodies : acetone, acetoacetate and ß- hydroxybutayric acid).
 Elevated ketone bodies in blood and urine cause acidosis which leads to coma
and death.
 Ketourea is common in uncontrolled DM (why?) because uncontrolled
diabetic patient use lipids as source of energy although they have high blood
glucose but they can't use it (body cells can't uptake glucose).
 Causes of ketourea: Some diseases, diet low in carbohydrates and high in
lipids and proteins or vomiting for long time.
 Results effected by: diet and drugs.

4- Bilirubin (Bile) and Urobilinogen:

 Urine normally does not contain detectable amounts of bilirubin. Presence of
high concentration of bilirubin in urine indicates liver dysfunction.
 Normal urine contains only small amounts of urobilinogen. Hemolysis and
hepatocellular disease can elevate urobilinogen levels.
7- Haematourea:
 It is the presence of red blood cells (RBCs) in urine it can be either gross
(visible) or microscopic.
 It is caused when blood passed into the urine through some lesions of the kidney
or the urinary tract.
 When the red blood cells enter the urine, hemolysis usually occurs after a short
time.

DR.SH COKER 17
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

8- Hemoglobinuria:
 Presence of heamoglobin in urine due to rupturing of RBCs.
 Intravascular hemolysis (lysis of red blood corpuscles) from any cause
liberates hemoglobin into the circulation.
 This may occur in malaria, typhoid, yellow fever, hemolytic jaundice or other hemolytic
diseases.

Routine Urine Examination

1- Proteinuria ( heat coagulation test):

Put 5ml urine in the test tube & Heat at urine segmented part for 1 min
if coagulum formed on the side of the tube, it may be either proteins or phosphate . To
differentiate add 2-3 drop of 33% acetic acid.

If coagulum dissolve → phosphate.

If coagulum dose not dissolve → protein

2- Glucosuria

Add 5 drops of urine to 5ml benedicts qualitative reagent boil and record the color developed

Observation
Result
Blue Nil *
Green color Trace

Green ppt +

Yellow ppt ++

Orange ppt +++

Red ppt ++++

*Nil = there is no suger

DR.SH COKER 18
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com
3- Ketonuria (Rothera's test):
In a test tube add 1g of Rothera's reagent + a few drops of urine just to wet the powder→
purple color.
4- Bile salts (Hay's test):
Add sprinkles of sulfur powder to 5 ml urine :
 If S-particles sink →bile acid are present
 If S-particles float →bile acid are absent

5- Bile pigments:
 Bilirubin (Fouchet's test) : 2ml of urine + 1ml of 10% BaCl , mix well, stand for 5
min, then filter. Unfold the filter paper and add 1 drop of Fouchet's reagent to the
paper→ blue green color around the drop.
 urobilinogen (Ehrlich test): 5ml fresh urine + 0.5 ml Ehrilch's reagent, allow to stand
for 5 min →
pink color on cold → normal trace.
red color on cold → increased amounts.
red color after heating → normal traces.

ACIDIC URINE ALKALINE URINE

Uric acid Triple phosphate
Amorphous urates Amorphous phosphates
Calcium oxalate Calcium oxalate

Urine Microscopic Examination

Microscopic analysis of urine
1- Crystals:
 The crystal is formed when the urine sample stands and cools before
examination.
 The type of crystal depends on the pH, temperature, and constituents of the urine.
 There are common crystals and pathological crystals.
 Presence of large amounts of crystals in urine for long period of time large may lead to
kidney stone formation.
 Can identified by morphology alone but knowing urine pH is important (provide
supporting information)

DR.SH COKER 19
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

a. Common crystals:
1. Calcium oxalate dehydrate
 Colourless squares resembling an envelope.
 It is formed in urine of any pH.
 It takes different sizes from very small to quite large.

2. Triple phosphate (magnesium ammonium phosphate)

 It is formed in urine whose pH is neutral to alkaline.
 The primary factor of the triple phosphate crystals formation is the ammonia
concentration.
 It can indicate urinary tract infection.
 Triple phosphates are usually associated with bacterial growth. With a first- morning
fresh specimen.
Calcium oxalate dehydrate Triple phosphate

3. Amorphous (irregularly shaped crystals)

It appears as aggregates of finely granular.
a. Amorphous urates
 It is salts of Na, K, Mg, or Ca.
 It is formed in acidic urine.
 They have a yellow or yellow-brown color.
b. Amorphous phosphates
 It contains precipitate of calcium and phosphate.
 It is formed in alkaline urine. It caused by the diet and an also represent a pathological
situation.
 The different between amorphous urates and amorphous phosphates is pH
and the color of the centrifuge pellet (the precipitate of calcium phosphate is
white, but the amorphous urate is pink).

DR.SH COKER 20
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

Amorphous urates Amorphous phosphates

2- Cells:
Epithelial cell, RBCs, and WBCs (pus cells) are the most common types of cells found
in urine.
a- Pus cells:
Pus cells are polymorphs leukocytes which have come from the blood to fight bacteria
invading the urinary tract.
Normal urine contain up to 5/HPF. More than five WBCs may indicate an acute
infection in the urinary tract. It needs more conformation test like uine culture test.
Bacteria may be insignificant contaminants, but the pathologic bacteria usually lead
to increased numbers of leukocytes that are referred to pyuria.
b- Red blood cells (Erythrocyte):
 Red cells do not contain granules, and so can be distinguished from pus cells.
 They are usually smaller, red cells are not normally seen in urine.
 Normal urine contain up to 5/HPF. More than five RBCs is termed hematuria.

c- Epithelial cells:
These are flat large cells with a nucleus that can usually be seen quite easily urethra. A
few epithelial cells are found in normal urine and in female due to reproductive period.

3- Casts:
solid substances blocking the kidney tubule and becomes loose and goes

down into the urine.

4- Micro organisms:
a- Bacteria:
Normal urine contains no bacteria. Finding bacteria in old urine means nothing but in
fresh urine means that the patient has a urinary infection. A common kind of bacteria
found in the urine is called E.coli. It is a thin motile rod like a very small pencil.

DR.SH COKER 21
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

b- Yeast and Fungi:

The threads or mycelium of a mold or a fungus are longer and thicker than a bacterium
and they branch.

Procedure:
1. centrifuged well-mixed urine 10 ml at 3000 r.p.m for 10 min until precipitate.
2. supernatant is decanted and 0.2 -0.5 ml is left inside the tube.
3. The sediment is resuspended in the remaining supernatant.
4. drop of sediment is poured in slide and cover-slipped.
5. The sediment is first examined under low power to identify most crystals, casts,
cells and other large objects.
6. Next, examination is carried out at high power to identify crystals, cells, and
bacteria.
7. Record the results in the lab report of urinalysis

Blood film
Composition of blood:
The average person circulates about 5L of blood, of which 3L is plasma and 2L
is cells. Blood cells are classified as white cells (leukocytes), red cells
(erythrocytes), and platelets (thrombocytes).

Complete blood picture or complete blood count (CBC), includes:

 White blood count (WBC)

 Red blood count (RBC)
 Differential white blood count
 Reticulocyte count
 ESR (erythrocyte sedimentation rate)
 Bleeding time, clotting time
 Prothrombin time (PT)
 Partial thrombin time (PTT)

DR.SH COKER 22
 CHEMICAL/CLINICAL PATHOLOGY 1&2 NOTES – SCHOOL OF CLINICAL SCIENCES SL
Email: henrysamk@live.com

A-CBC (complete blood count):

The CBC is a basic screening test and is one of the most frequently ordered
laboratory procedures. The CBC consists of a series of tests that determine
number, variety, percentage, concentration, and quality of blood cells.

1. Red blood cells (RBC):

The main function of the red blood cell (RBC or erythrocyte) is to carry oxygen
from the lungs to the body tissues and transfer carbon dioxide from the tissues to
the lungs. This process is achieved by means of the hemoglobin in the RBCs.
The RBC tests an important in the evaluation of anemia.

Pathophysiology:
 RBCs count is elevated after muscular exercise, emotional excitement and
increased temperature.
 RBCs count is lowest during sleep, rises on awakening and continuous to rise during
the rest of the day.
 Person living at high altitudes have higher RBCs count than those living at sea level.

2. White blood cell (WBC):

Leukocytes fight infection and defend the body by a process called phagocytosis, in which the
leukocyte actually encapsulate foreign organisms and destroy them.

Pathophysiology:
 It increases in leukemia inflammation, other malignancy and also in infectious
diseases.
 It decreases in fever, bone marrow depression and in some type of anemia.
Differential white blood cell :
White blood cells or (leukocytes) are divided into two main groups:
granulocytes and agranulocytes. The granulocytes receive their name from the
distinctive granules that are present in the cytoplasm of neutrophils, basophils,
eosinophils. The nongranulocytes, which consist of the lymphocytes and
monocytes, do not contain distinctive granules. Each type has specific function
as follow:

DR.SH COKER 23