Introductory topics in chemical pathology

1. GENERAL INTRODUCTION TO
CHEMICAL PATHOLOGY.
DR. EMMANUEL KUNLE ABUDU
MBBS, M.Sc, FMCPath

2. Lecture Outlines
• Common terminologies in Chemical Pathology.
• Concept of Reference Values and Units of
Biochemical Quantities.
• Collection, Handling, Registration and
Preservation of Specimens in Chemical Pathology
Laboratories.

3. Introduction to clinical laboratories:
• Clinical laboratories are important in diseases diagnosis,
determination its severity and patient response to specific
treatment.
• Diagnosis of any disease is first done by physical examination by
physician and confirmed by laboratory diagnostic tests.
• Laboratory values are very important in determination of disease
severity, drug doses and in follow up.

4. Introduction
The sections of clinical laboratory are:
• Clinical pathology including Histology and cytology
• Haematology including Serology and Blood bank
• Clinical biochemistry/Chemical Pathology
• Clinical microbiology

5. Purpose of Clinical Chemistry Tests
– It deals with the applications of biochemistry laboratory
to find out the cause of a disease.
– Measure levels of substances found normally in
human blood that have biological functions.
Examples: Glucose, Calcium
– Detect or measure non-functional metabolites or
waste products. Examples: Creatinine, Blood
Urea Nitrogen (BUN)
– Detect or measure substances that indicate cell
damage or disease. Examples: Liver enzymes,
such as ALT, Cardiac enzymes, such as CK-MB
– Detect or measure drugs or toxic substances.
Examples: Dilantin, Drugs of abuse screen

6. Types of Specimens for Chemical Analysis
– Whole blood, serum or plasma. The most
common specimen is serum, collected in a tube
with no anticoagulant so that the blood will
clot.
– Urine – often 24 hour collections
– Others – Cerebrospinal Spinal Fluid (CSF),
joint aspirate fluid, pleural, pericardial or
asctific fluid, faeces, and other body fluids or
tissues

7. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Collection and Handling of Blood
Specimens for Chemical Analysis
– Blood collection tubes for obtaining serum -
Serum Separator Tubes (SST) do not have
an anticoagulant but do contain a gel
substance which will form an interface
between the clot and the serum when the
blood specimen is centrifuged. These tubes
are sometimes referred to as “Tiger Tops”.

8. Common terminologies in Chemical
Pathology:
Phlebotomy refers to blood draw from a vein, artery, or the capillary
bed for lab analysis or blood transfusion.
Veinpuncture:
Normal or Reference Values – range of values for a
particular chemistry test from healthy individuals.
Controls:

9. Laboratory request and laboratory report
forms
Laboratory request form: it fills computerize or paper filled by the doctor then send it to the
lab. The lab request contains a list of tests to be performed on specimen of patient. Each lab
has its specific request; for example, chemistry request, hematology request… etc.
Laboratory report form: it contains the result of patient.
Laboratory work flow cycle:
The flow cycle includes the entire steps of laboratory test, starting from test ordering by a
doctor until reporting the results.
Three phases of laboratory testing:
Pre-analytical: test ordering, specimen collection, transport and processing
Analytical-testing
Post-analytical: testing results transmission, interpretation, follow-up, retesting.

10. Patient preparation for sample collection
– time of collection;
– effects of eating on chemistry analysis
– Some specimens are increased or decreased
after eating (ex. Glucose, triglycerides), so it
is important to know what the test and
collection method call for. Specimens for
these tests are usually collected in a fasting
state.

11. Patient preparation for sample
collection
• Sometimes serum or plasma
appears lipemia (milky)
after a patient has eaten a
fatty meal.
• Lipemia affects most
chemistry analyses. The
blood must be recollected
when the patient is fasting.
• A*

12. Normal or Reference Values
• Clinical Chemistry Tests
– Normal or Reference Values – range of values for a particular chemistry test
from healthy individuals
– A reference range is usually defined as the set of values 95 percent of the normal
population falls within (that is, 95% prediction interval).
– It is determined by collecting data from vast numbers of laboratory tests
• Units
• Mass concentration (g/dL or g/L) is the most common measurement unit in the
United States. Is usually given with dL (decilitres) as the denominator in the United
States, and usually with L (litres) in, for example, Sweden.
• Molar concentration (mol/L) is used to a higher degree in most of the rest of the
world, including the United Kingdom and other parts of Europe and Australia and
New Zealand.
• International units (IU) are based on measured biological activity or effect, or for
some substances, a specified equivalent mass.
• Enzyme activity (kat) is commonly used for e.g. liver function tests like
AST, ALT, LD and γ-GT in Sweden.
• Percentages and time-dependent units (mol/s) are used for calculated derived
parameters, e.g. for beta cell function in homeostasis model
assessment or thyroid's secretory capacity

13. Normal or Reference Values
• Arterial or venous
• If not otherwise specified, a reference range for a
blood test is generally the venous range, as the
standard process of obtaining a sample is
by venipuncture.
• An exception is for acid-base and blood gases, which
are generally given for arterial blood.
• Still, the blood values are approximately equal
between the arterial and venous sides for most
substances, with the exception of acid-base, blood
gases and drugs (used intherapeutic drug
monitoring (TDM) assays).
• Arterial levels for drugs are generally higher than
venous levels because of extraction while passing
through tissues

14. Chemistry Panel grouping
• – some tests are “bundled” according to the system or organ
targeted. Examples: thyroid panel, liver panel, cardiac panel,
kidney panel, basic metabolic panel, etc
• Lipid profile
• Diabetic profile
• Kidney profile
• Liver profile
• Bone profile
• Electrolyte profile

15. Disposable syringes Vacationer systems Disposable lancets
Gauze pads absorbent cotton Tourniquet
Alcohol swap Plastic bandage Waste container
Phlebotomy or blood collection:
The term phlebotomy refers to blood
draw from a vein, artery, or the
capillary bed for lab analysis or blood
transfusion.
The phlebotomy equipments:
For specimen collection, the
following materials will be required:
Phlebotomy

16. Usually vein is used to collect blood
by veinpuncture procedure.
In adults: most venipuncture
procedure use arm vein.
On arm, one of three arm veins is
used: median cubital vein "located
on the middle", cephalic vein or
basilic vein "located on both sides".
Median cubital vein is the best
choice (why?) because it has good
blood flow than cephalic and basilica
which has slower blood flow.
However if veinpuncture procedure is
unsuccessful in median capital;
cephalic or basilica is used.
Artery blood is rarely used in special
cases as when blood gases, pH,
PCO2, PO2 and bicarbonate is
requested. It is usually performed by
physicians.
Selecting vein site

17. Preparation of Blood Sample
One of three different specimens may be used:
• Whole blood
• Serum
• Plasma
First: Whole-blood specimen:
It must be analyzed within limited time (why?)
– Over time, cells will lyse in whole-blood which will change the
concentration of some analytes as potassium, phosphate and
lactate dehydrogenase.
– Some cellular metabolic processes will continue which will alter
analytes concentration like glucose and lactate.

18. Serum
Second Serum:
Difference between Serum and plasma:
• Serum is the same as plasma except it doesn't contain clotting
factors (as fibrin).
• Plasma contains all clotting factors.
• So, serum and plasma all has the same contents of electrolytes,
enzymes proteins, hormones except clotting factors.
• Serum is mainly use in chemistry laboratory and serology.

19. Procedure of Serum preparation
• Draw blood from patient.
• Select vacutainer with no anticoagulant.
• Allow to stand for 20-30 minutes for clot formation.
• Centrifuge the sample to speed separation and affect a greater
packing of cells.
• Clot and cells will separate from clean serum and settle to the
bottom of the vessel.
• The supernatant is the serum which can be now collected by
Dropper or pipette for testing purposes or stored (-20°C to -80°C) for
subsequent analysis or use.

20. Plasma
Third Plasma:
• The tube will have anti-coagulation
• After centrifugation the blood sample got
separated into three layers

21. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Blood collection
tubes for obtaining
plasma

22. Procedure of plasma preparation
• Draw blood from patient.
• Select vacutainer with an appropriate anticoagulant.
• Mix well with anticoagulant.
• Allow to stand for 10 minutes.
• Centrifuge the sample to speed separation and affect a greater
packing of cells.
• The supernatant is the plasma which can be now collected for testing
purposes or stored (-20°C to -80°C) for subsequent analysis or use.

23. In the laboratory
Specimen rejection criteria:
• Specimen improperly labeled or unlabeled.
• Specimen improperly collected or preserved.
• Specimen submitted without properly completed request
form.
• Haemolyzed sample (show tubes).
• Lipeamic sample

24. Haemolysis
Haemolysis :
• It means liberation of haemoglobin due to rupture of red blood cells (RBCs).
• Due to haemolysis plasma or serum appears pink to red color.
• It causes elevation in: K+, Ca2+, phosphate, SGOT, SLDH and acid
phosphatase.
• Haemolysis is occurred due to sampling, transporting and storage (too hot
or too cold).
• According to the degree of haemolysis it is classified as H+, H++ and H+++.
H+ may be accepted for some tests that are not affected by RBCs contents
as glucose and lactate, H++ and H+++ not acceptable for any test.
Changes in the serum color indicate one of the following:
• Haemolyzed: serum appears pink to red due to rupture of RBCs
• Icteric: serum appears yellow due to high bilirubin.
• Lipemic: serum appears milky or turbid due to high lipid.

25. Blood collection tubes:

26. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry

27. Blood collection tubes:
Two major types of blood collecting tubes:
• Serum separating tubes (SST)
• Plasma separating tubes (PST)

28. Top Color Additives Principle Uses
Lavender EDTA -The strongest anti-coagulant
- Ca+2 chelating agent
- To preserve blood cells components
- Hematology
- Blood bank (ABO)
- HbA1C
(Glycosylated Hb)
Light Blue Sodium Citrate Ca+2 chelating agent - PT: Prothrombin Time
- PTT: Partial
Thromboplastin Time
( in case of unexplained
bleeding and liver
disease)
Green Sodium Heparin
or Lithium
Heparin
Heparin binds to Thrombin and inhibits
the second step in the coagulation
cascade
(Prothrombin Thrombin)
Fibrinogen Fibrin
Enzymes
Hormones
Electrolytes (Na+, K+,
Mg+, Cl-
Heparin
Plasma Separating Tubes (PST)

29. Top Color Additives Principle Uses
Black Sodium Citrate Ca+2 chelating
agent
ESR ( Erythrocyte Sedimentation Rate)
to test how much inflammation in the
patient, unexplained fever, Arthritis,
Autoimmune Disorder
Gray -Sodium Fluoride
-Potassium Oxalate
Glycolysis
inhibitor
Anti-Coagulant
Glucose tests
Royal Blue Heparin
Na-EDTA
Anti-Coagulant
Tube should not
be contaminated
with metals
Toxicology
Trace Elements and metals
Yellow ACD ( Acid-Citrate
Dextrose)
Anti-Coagulant DNA Studies
Paternity Test
HLA Tissue Typing
(Human Leukocyte Antigen)
The body used this protein to
differentiate the self-cells from non-self
cells

30. Top Tubes Additives Principle Uses
Red ------
Sometimes it has gel
or silicon at the
bottom of tube to
reduce hemolysis
Enhancing the
formation of blood
clot
Serology
-Antibodies
-Hormones
-Drugs
Virology
Chemistry
Blood cross
matching before
blood transfusion
Gold -------
It has gel at the
bottom of the tube
to separate serum
from the blood
Serum separating
from the blood
through the gel in
the tube
Serology
Chemistry
Serum Separating Tubes (SST)

31. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Commonly Performed Chemistry Tests
or Analytes
– Proteins – essential components of cells and
body fluids. Some made by body, others
acquired from diet. Provides information
about state of hydration, nutrition and liver
function, since most serum proteins are
made in the liver.

32. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Electrolytes – sometimes called “lytes”
• Includes sodium (Na), potassium (K), chloride
(Cl) and bicarbonate (HCO3-)
• Collectively these have a great effect on
hydration, acid-base balance and osmotic
pressure as well as pH and heart and muscle
contraction
• Levels differ depending on if inside vs. outside
cells
• Important in transport of substances into and
out of cells

33. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Minerals
• Calcium
– Used in coagulation and muscle contraction
– 99% is in skeleton and is not metabolically active
– Influenced by vitamin D, parathyroid hormone,
estrogen and calcitonin
– Hypercalcemia – occurs in parathyroidism, bone
malignancies, hormone disorders, excessive vitamin D,
and acidosis; may cause kidney stones
– Hypocalcemia – can cause tetany; occurs in
hypoparathyroidism, vitamin D deficiency, poor
dietary absorption and kidney disease

34. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Phosphorus
– 80% in bone and rest in energy compounds such as
ATP
– Influenced by calcium and certain hormones
• Iron
– Essential for hemoglobin
– Deficiency results in anemia; may be caused by lack of
iron in diet, poor absorption, poor release of stored
iron or loss due to bleeding
– Increased in hemolytic anemia, increased iron intake
or blocked synthesis of iron-containing compounds,
such as in lead poisoning

35. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Kidney Function Tests
• Serum Creatinine
– Best test for overall kidney function; not affected by
diet or hormone levels
– Waste product of muscle metabolism
– Serum creatinine rises when kidney function is
impaired

36. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• BUN (Blood Urea Nitrogen)
– BUN is surplus amino acids that are converted to urea
and excreted by kidneys as a waste product
– BUN influenced by diet and hormones, so it is NOT as
good an indicator of renal function as serum
creatinine levels
– BUN increased in kidney disease, high protein diet,
and after administration of steroids
– BUN decreased in starvation, pregnancy and in
persons on a low protein diet

37. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Uric Acid
– Formed from breakdown of nucleic acids and
excreted as a waste product by kidneys
– Increased in kidney disease, but most often used to
diagnosis gout (pain in joints, mainly big toe, due to
precipitated uric acid crystals)
– Also increased in increased cell destruction, such as
after massive radiation or chemotherapy

38. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Liver Function Tests
• Liver functions:
– Synthesizes glycogen from glucose
– Makes plasma proteins (albumin, lipoproteins,
coagulation proteins)
– Forms cholesterol and degrades it into bile acids,
which emulsifies fats for absorption
– Stores iron, glycogen, vitamins and other substances
– Destroys old blood cells and recycles components of
hemoglobin

39. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry

40. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Total Bilirubin
– Waste production of hemoglobin breakdown
– Increased in excessive RBC breakdown, such as
hemolytic anemia, or impaired liver function or some
sort of obstruction, such as a tumor or gall stone

41. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Liver Enzymes – levels increase following
damage to liver tissues
– Alkaline Phosphatase (ALP or AP) - Greatly increased
in liver tumors and lesions; moderately increased in
diseases such as hepatitis
– Alanine Aminotransferase (ALT; formerly called
SGPT) - Increases up to 10x in cirrhosis, infections or
tumors and up to 100x in viral or toxic hepatitis

42. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Asparate Aminotransferase (AST; formerly called
SGOT) - Increased in liver disease, but also in heart
attacks
– Gamma Glutamyl Transferase (GGT) - Often used to
monitor patients recovering from hepatitis and
cirrhosis
– Lactate Dehydrogenase (LD) - Increased in liver
disease and following heart attacks

43. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Cardiac Function Tests
– Creatine Kinase (CK) - Widely used to diagnosis and
monitor heart attacks
– Troponins
» Only present in heart muscle, making it a more
accurate indicator of heart attack than CK
» Cardiac Troponin T (cTnT)
» Cardiac Troponin I (cTnI)

44. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Lipid Metabolism Tests
– Cholesterol
» Present in all tissues
» Serves as the skeleton for many hormones
» Recommended to be less than 200 mg/dL in
adults)
» LDL = “bad” cholesterol; HDL = “good”
cholesterol

45. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
– Triglycerides
» Main storage form of lipids, comprising 95% of
fat tissue
» Hyperlipidemia – having high blood levels of
triglycerides – may increase risk of heart attack
• Carbohydrate Metabolism Tests
– Glucose - Largely regulated by insulin

46. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry

47. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• Thyroid Function Tests
– Thyroid Stimulating Hormone (TSH) - Inverse
relationship to thyroid function (the higher the TSH,
the lower the thyroid function and vice versa)
– Other less common thyroid tests include T3 and T4
– Hypothyroidism – underactive thyroid gland
– Hyperthyroidism – overactive thyroid gland

48. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
• For more information on most clinical
laboratory tests, visit:
http://www.labtestsonline.org/
Click on any lab test or condition or
disease and find all sorts of
information!

49. Unit #6D – Clinical Laboratory
Testing - Basic Clinical Chemistry
Photos of some clinical chemistry
laboratories

50. Thanks for your attention
Any questions??

51. ASSIGNMENTS
• 1) Write the reference values for all variables/analyte
that can be measured in the Chemical Pathology
Laboratory.
2) Tabulate the effects of environmental factors,
genetic factors and cultural behaviours on the
analysis of analytes in the Chemical Pathology
laboratory emphasizing whether they are increased,
decreased or unchanged.