Clinical Chemistry

Glucose Metabolism
Impairment of glucose homeostasis can be studied in terms of hyperglycemia and
hypoglycemia.
Temporary hyperglycemia may develop in cases of:
1- Excessive secretion of adrenaline (e.g. emotional stress, asphyxia and anaesthesia)
2- Following cerebral injury and increased intracranial pressure.
3- Rapid absorption of glucose from the gut e.g. after mastectomy, gastroenterostomy or
pyloroplasty.
4- Following meal in severe liver disease.
Fasting hyperglycemia:
1- Insulin deficiency:
a) Primary (idiopathic) insulin insufficiency that may be due to:
1) Insulin antagonizing factors.
2) Abnormal insulin or.
3) Insufficient insulin secretion.
b) Secondary insulin insufficiency that may be due to:
1) Inflammation: acute and chronic pancreatitis.
2) Autoimmune: with islet cell antibodies.
3) Infiltration (hemochromatosis and tumors).
4) Trauma
5) Pancreatectomy.
2- Excessive insulin-counter regulatory hormones:
1- Hyperpituitarism (ACTH, GH or TSH excess).
2- Cushing's syndrome.
3- Pheochromocytoma.
4- Thyrotoxicosis.
3- Miscellaneous:
In acute and chronic infections, acidosis, salicylate poisoning, smoking, pregnancy,
and drugs (e.g. glucocorticoids & contraceptive pills).
Diabetes mellitus (D. M.):
D. M. may be:
1) Potential: normal glucose tolerance test (GTT) with potential risk of developing D. M.
as in cases with family history, obesity, old age.
2) Latent: GTT is only abnormal under stressful condition (infection, pregnancy and
steroid therapy).
3) Asymptomatic: G.T.T is abnormal but the patient has no symptoms.
4) Clinical: G.T.T is abnormal and the patient suffers from D. M. symptoms.
Diagnosis of D. M:
1) Plasma glucose:
According to the WHO criteria, a fasting venous plasma glucose of 7.4 mmol/L (126
mg/dl) or more in two occasions is diagnostic of D.M. Alternatively a random venous plasma
glucose of 11 mmol/L (200mg/dl) or more establishes the diagnosis. Blood glucose 2 hours
after meal is considered the simplest screening test for D.M. Normally it should be 6.7
mmol/L. The oral glucose tolerance test is regarded as a definitive test for confirming a
provisional diagnosis of D. M. in borderline cases. Post prandial levels above 11 mol/L are
diagnostic.
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Indication: The diagnosis of (1) asymptomatic or mild D. M. (2) Impaired glucose tolerance
especially in pregnancy.
The test: should not be performed in patients suffering from trauma, recovery from a serious
illness, intercurrent infection. Corticosteroids and diuretics should be stopped if possible. The
patient should be on diet containing 150 g carbohydrate / day for at least 3 days. The patient
drinks 1 g/kg body weight glucose in water. Blood and urine glucose is determined in fasting
and thereafter at 30 minutes intervals for 2-3 hours.
Diagnosis of D.M.: Fasting blood glucose above 7.4 mmol/L (126 mg /dl), a peak blood
glucose more than 10 mmol/L (180 mg/dl) with glucosuria and the return to fasting level is
delayed.
Other abnormal glucose tolerance curve (GTC):
- Flat curve: plasma glucose fails to rise after glucose intake. It may be due to
malabsorption, hypopituitarism, insulinoma and Addison's disease.
- Lag-storage curve: After glucose intake there is a sharp rise of plasma glucose with a
peak that appears early and exceeds 11 mmol/L (200 mg/dl). There may be
glucosuria. It is found after gastric surgery, thyrotoxicosis or severe liver disease.
2) Glucosuria:
It occurs when plasma glucose concentration exceeds its renal tubular absorptive
capacity [renal glucose threshold: 10 mmol/L (l80 mg/d)]. Hyperglycemia without glucosuria
occurs when the renal threshold for glucose is raised. There may be glucosuria without
hyperglycemia as in low renal threshold for glucose or renal tubular defects (e.g. Fanconi
syndrome).
3) Plasma insulin:
The normal insulin level does not exceed 20 µU/mol. It is increased in patients with
insulin or due to functional or therapeutic causes.
4) C- Peptide:
It is formed by splitting of proinsulin in equimolar amounts with insulin, so it is an
index of endogenous insulin secretion. Normal fasting level is 2 ng/ml and 5-10 ng/ml
postprandial.
5) Glycosylated hemoglobin (HbA1c):
Reference range 4.5-8%. Its level indicates the average degree of blood glucose control
for the previous 1-2 months.

Metabolic complications of D. M.:

1) Hyperglycemic coma:
 Ketotic hyperglycemic coma: hyperglycemia, glucosuria and urinary loss of water and Na,
ketonemia, ketonuria, metabolic acidosis and hyperkalemia. Mostly occurs in IDDM.
 Non-ketotic hyperglycemic coma: It is due to poor renal function in elderly patients with
NIDDM and greater loss of water and electrolytes. The residual insulin can correct fat but
not glucose homeostasis.
2) Lactic acidosis:
Extreme dehydration and tissue hypoxia may lead to lactic acidosis, due to over
production of H+ caused by anaerobic metabolism.
3) Uremia:
Due to diabetic nephropathy, polyuria and decreased renal blood flow.

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Hypoglycemia
Venous plasma glucose level below 2.2 mmol/L. (40 mg /dl). It may be:
1) Stimulative (reactive) hypoglycemia:
- Drugs (e.g. insulin overdose) or poisons, inborn error of metabolism (e.g. galactosemia)
or idiopathic.
2) Fasting hypoglycemia: It may be due to:
a) Enhanced glucose utilization: physiological in neonates, insulinoma, pancreatitis, and
pancreatic tumors.
b) Defective glucose production:
 Endocrine disorders: adrenocortical insufficiency, hypothyroidism, growth
hormone deficiency.
 Liver diseases: severe cirrhosis, acute hepatic necrosis, tumors.
 Renal diseases: end stage renal failure.
 Miscellaneous: malnutrition or severe starvation.

Plasma Lipid Disorders

Lipids act as energy stores and as structural components of cells. In addition, they have
specialized functions (e.g. as hormones):
- Neutral fats (triglycerides - TG): they are glycerol esters of long chain fatty acids. The
normal fasting plasma level is 0.3-1.8 mmol/L (50-150 mg/dl). An increase of its level
causes an opalescent plasma (lipemia).
- Phospholipids: They are compounds which contain a nitrogenous base, a phosphoric acid
residue, one or more fatty acids and often glycerol e.g. lecithin. The normal plasma level is
150-250 mg/dl.
- Cholesterol: Its concentration is l40 - 250 mg/dl varying with the population, increasing
with age, higher in men. The ester cholesterol is 65-75% of the total.
- Fatty acids: are mostly straight chains with an even number of carbon atoms. They are
alternative or additional energy source to glucose.
Lipoproteins:
They are relatively insoluble in water, transported in body fluids by apolipoproteins.

Different analytical procedures divide the plasma lipoproteins into different fractions:
Ultracentrifugation: separates plasma lipoproteins according to their density and size into:
a) Two large triglyceride-rich complexes:
- Chylomicrons: which transport exogenous lipids from the intestine to the systemic
circulation via thoracic duct, they are not detected in fasting plasma.
- VLDL (very low-density lipoproteins): which transport endogenous lipids mainly from the
liver.
b) Two smaller lipoproteins contain mostly cholesterol:
- LDL (low-density lipoproteins): which transport cholesterol to the cells. LDL arises from
VLDL removed from circulation by the live receptor dependent mechanism (LDL -
receptor) or receptor independent mechanism in the macrophages.
- HDL (high-density lipoproteins): it is the main route whereby cholesterol can return from
peripheral tissues to the liver.
A fifth class, IDL (intermediate density lipoproteins) is a transient lipoprotein formed
during conversion of VLDL to LDL.

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A sixth type of lipoprotein particle [Lp (a)] has the same lipid composition as LDL but contains
also apolipoprotein (a). It competes with plasminogen for tissue plasminogen receptors,
thereby inhibiting thrombolysis.
* Electrophoretic mobility:
- -Lipoproteins: correspond approximately to HDL.
- -Lipoproteins: correspond approximately to LDL.
- pre-Lipoproteins: correspond approximately to VLDL.
Apolipoproteins:
They are glycoproteins: there are four major groups (apo A, B, C and E), while the
minor groups include (apo D and apo F) and apo (a), they have the following functions:
- They promote the solubility of lipids in plasma and enhance the stability of lipoprotein
particles.
- Some of them activate the key enzymes of lipid metabolism e.g. apo-CII activates
lipoprotein lipase and apo-AI activates LCAT.
Hyperlipoproteinemias:
Table (1): It is either primary due to purely genetic factors or secondary to other diseases.
* Primary hyperlipoproteinemia:
Conc. in plasma Lipoproteins IHD
Hyperlipoproteinemia
Chol. TG affected Risk
- Familial hypercholesterolemia
(WHO type IIa, IIb): may be
 N or  LDL 
dietary or due to defect in apo B
receptors.
- Familial hypertriglyceridemia
VLDL and
(WHO type IV, V): defect in either  or N  
Chylomicrons
production or catabolism of VLDL.
- Familial combined hyperlipidemia
LDL and / or
(WHO type IIa or type IV): the  or N  or N 
VLDL
underlying defect is unclear.
- Remnant hyperlipoproteinemia
IDL and
(WHO type III): defect in   
Chylomicrons
conversion of VLDL to LDL.
- Lipoprotein lipase deficiency
Chylomicrons and
(WHO type I): deficiency of LPL or  or N  
VLDL
its activator apo C-II.
- Hyper -lipoproteinemia inherited
abnormality, need no treatment for  -- HDL 
raised cholesterol.
N: normal, :increased, : much increased IHD: ischemic heart disease

* Secondary hyperlipidemia:
- Hypercholesterolemia is a marked feature of hypothyroidism and nephrotic syndrome with
increased plasma LDL. It also occurs in cholestatic jaundice with the presence of
lipoprotein X.
- Hypertriglyceridemia is most commonly due to diabetes mellitus where VLDL is usually
increased; LDL is often increased whereas HDL may be reduced. In addition, it occurs in

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 alcoholism, chronic renal disease and in patients on estrogen therapy including estrogen
containing oral contraceptives.
Hyperlipidemia and atherosclerosis:
The following associations are now clearly established:
1- Increased plasma total cholesterol and LDL-C is positively correlated with the incidence of
ischemic heart disease (IHD).
2- Increased HDL- cholesterol is negatively correlated with IHD.
3- Increased triglycerides are positively correlated with IHD but weaker association than that
with cholesterol.
4- Plasma Lp (a) is positively associated with IHD.

Plasma Proteins Disorders

Plasma proteins are a heterogeneous mixture with variable origins and functions. The
main functions are:
1- Maintenance of plasma osmotic pressure, mainly due to albumin.
2- Transport of biologically important substances such as hormones, metals, free fatty acids,
bilirubin, and drugs.
3- Defense: through circulating antibodies.
4- Coagulation and fibrinolysis.
5- Enzyme activity and enzyme precursors e.g. angiotensin II and renin.
The main components of proteins are albumin, prealbumin, globulins, and fibrinogen.
Albumin:
The major component of plasma proteins.
Causes of hypoalbuminemia:
1- Increased loss through the skin (e.g. extensive burns), and GIT (e.g. protein loosing
enteropathy).
2- Increased catabolism as in infections, trauma, thyrotoxicosis and Cushing's syndrome.
3- Decreased synthesis: malabsorption, malnutrition, chronic liver disease.
4- Artefactual: hemodilution during intravenous fluid therapy.
Hyperproteinemia:
1- Increased immunoglobulin synthesis as in some collagen diseases chronic inflammatory
conditions and multiple myeloma.
2- Hemoconcentration.
Prealbumin:
This protein has a role in transport of thyroxine and vitamin A; it is a marker of protein-
energy malnutrition.
Globulins:
 -globulins: levels of 1 and 2 globulins are raised in states of tissue necrosis or cell
multiplication. The 2 globulin is specifically raised in nephrotic syndrome.
 -globulin: this is usually elevated in hyperlipidemias such as those associating
obstructive jaundice and nephrotic syndrome.
 -globulin: this contains the immunoglobulins, all of which possess antibody activity.
Acute-phase proteins:
This group comprises plasma proteins whose concentration alters following
inflammations (e.g. rheumatoid arthritis and systemic lupus erythematosus), tissue necrosis
(e.g. myocardial infarction) or trauma (e.g. surgery). Commonly measured fractions include:
1- C-reactive protein: It increases early in acute inflammation and reflects disease activity.

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2- Complement: the most important fractions are C3 and C4 that comprise 95% of total
complement and are easily measured. Low levels indicate increased consumption or
decreased synthesis. Important diseases associated with immune complex formation are
systemic lupus erythematosus, glomerulonephritis, rheumatoid arthritis and chronic active
hepatitis.
3- 1-antitrypsin: comprises 80-90% of the 1-globulin. It is protective due to its anti-
proteolytic activity. Deficiency is inherited as a recessive character.
4- Fibrinogen: sensitive indicators of connective tissue disorders.
5- Haptoglobins: are 2 globulins that bind with free hemoglobins and play an important role
in its degradation. Its concentration falls in hemolytic states. Plasma haptoglobins increase
in acute infection and trauma.

Serum Enzymes
Changes in plasma enzyme activity can be attributed to:
1) Necrosis or severe damage by ischemia or by toxic substances e.g. AST, ALT.
2) Increased rate of cell turnover e.g. alkaline phosphatase during active bone growth.
3) Increased conc. of enzymes within cells as result of disease or drugs e.g. GT by
ethanol, and barbiturates.
4) Common bile duct obstruction and regurgitation into the blood e.g. amylase.

* Lactate dehydrogenase (LD):

5 isoenzymes are present according to the polypeptide chain present (H or M) H4, H3M,
H2M2, HM3, M4. They are distributed in tissues as follows:
 Predominant LD1 (H4) and LD2 (H3M) as in heart and red cells.
 Predominant LD5 (M4) and LD4 (HM3) as in liver and some skeletal muscles.

Total LD activity is increased in:

- Myocardial infarction.
- Hepatocellular damage.
- Hemolytic anemia.
- Skeletal muscle disease
- Various malignant diseases
* Creatine kinase (CK)
3 isoenzymes are present, each composed of two polypeptide chains (B & M):
 Skeletal muscle has 98% CK-MM and the rest CK-MB
 Cardiac muscle has 70-80% CK-MM and 20-30% CK-MB
 Brain and thyroid CK-BB predominates.
Total CK activity is increased in:
- Myocardial infarction.
- Skeletal muscle diseases.
- Trauma, intramuscular injection.
- In comatosed patients, diabetic ketoacidosis.
- Acute renal failure and hypothyroidism.

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Table (2): Plasma enzyme tests in myocardial infarction:
Enzyme Abnormal activity Peak value of Duration of
detectable (hours) abnormality abnormality (days)
(hours)
CK-MB 4-6 12-24 2-3
Total CK 3-8 10-30 3-4
AST 6-12 20-36 2-4
LD 24-48 48-72 7-10

Troponin I:
A regulatory protein in striated muscle. It starts to rise 4-8hr. after AMI, remains
elevated and may be detected for 3-6 days following AMI. It is the most specific.

Liver Function Tests

The liver has a great reserve capacity; nearly four fifths of the liver can be removed
without affecting its function. Some functions may be affected more than others in liver
diseases. So, a group of tests is selected for diagnosis of specific liver dysfunctions.
I. Excretory Function:
A- Excretion of Endogenous Substances:
(1): Serum bilirubin:
Bilirubin exists in the serum in two forms, indirect reacting unconjugated form, carried
on albumin, and a conjugated form (chiefly as glucuronides). The later is direct-reacting, more
water soluble, and excreted in urine. The normal value for the direct bilirubin is 0.1-0.4 mg/dl,
the indirect bilirubin is 0.2-0.8 mg/dl and so the total bilirubin is 0.3-1.2 mg/dl (1.7-17.0
mmol/L).
An increase in serum bilirubin concentration to above 2.5 mg/dl results in clinical
jaundice. This increase may be due to:
a- Hemolytic causes (prehepatic):
The normal liver is unable to cope with the great load of circulating unconjugated
bilirubin resulting from the marked intravascular hemolysis.
b- Hepatic Causes:
(i) Defective transport of bilirubin through hepatocytes. A congenital malfunction of this
active transport system produces a mild jaundice (serum bilirubin around 2 mg/dl) that
is harmless and usually detected accidentally. This transport defect is known as
Gilbert’s disease.
(ii) Damage of the liver parenchyma by toxic, infectious causes produces decrease of
bilirubin excretion, which leads to increase of both direct and indirect bilirubin.
c- Obstructive causes (intra or extra hepatic):
The proper flow of bile is inhibited by obstruction in the biliary tract. The bile flows
back into the bloodstream leading to rise of direct bilirubin.

Delta bilirubin:
As jaundice develops during acquired liver diseases, delta-bilirubin (conjugated
bilirubin covalently bound to albumin) is increased in parallel with other fractions. However,
as jaundice resolves the delta fraction, which is not filtered in urine, decreases relatively
slowly, and so urine bilirubin testing may become negative in spite of high serum level.

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(2) Urinary Bile Pigment (see Table 3).
(3) Bile Acids:
Hepatitis and cirrhosis are associated with decreased hepatic bile acid excretion and
subsequent elevation of its serum levels.

Table (3): LABORATORY FINDINGS IN JAUNDICE

Item Hemolytic Hepatocellular Obstructive

I. Bile Pigment:
1- Serum direct bilirubin Normal Increase Marked increase
2- Serum indirect bilirubin Increase Increase Normal
3- Urinary urobilinogen Marked Variable Decreased or
increase absent
4- Urinary bilirubin Absent Increase Marked increase
II. Serum Enzymes:
1- ALT, AST Mild increase Marked increase Increase
(interahepatic)
2- Alkaline phosphatase Normal Moderate increase Marked increase
III. Other tests:
1- Cholesterol Normal Decrease (ester Increase
fraction)
2- Albumin Normal Normal or Normal or
decrease decrease
(interahepatic)
3- Prothrombin time No change No change from Normalization of
response to vitamin K from normal prolonged basal the prolonged
injection basal value value basal value

II- Metabolic Functions:

A) Protein Metabolism:
Plasma Proteins:
Normal values of plasma proteins are total: 6.5-8.0 gm/dl, plasma albumin (A): 3.5-5.0
gm/dl, plasma globulins (G): 1.5-2.5 g/dl. In advanced hepatic affections, serum albumin is
decreased and globulins are increased so that the A/G ratio may be reversed. Increased plasma
IgA is moderate in cirrhosis, however in primary biliary cirrhosis plasma IgM and in chronic
active hepatitis, plasma IgG tend to be mostly increased.
B) Lipid Metabolism:
In cholestatic syndromes, the concentrations of all plasma lipid fractions are frequently
raised. An abnormal lipoprotein X is present in nearly all cases of cholestasis. In
parenchymatous liver disease, the cholesterol level is either low or normal.

C) Carbohydrate Metabolism:
Fasting hypoglycemia may occur in severe liver disease due to impaired
gluconeogenesis or glycogen breakdown, or both. However, postprandial hyperglycemia may
occur in chronic liver diseases.

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III- Enzyme Changes in Liver diseases:
1- Parenchymal Enzymes: The aminotransferases (alanine aminotransferase ALT (GPT) and
aspartate aminotransferase AST (GOT) :
In the preicteric phase of hepatitis, serum aminotransferases are markedly elevated and
progressively increase during the course of the disease. Both enzymes return to normal 2-3
months after onset of the illness in uncomplicated cases. Maintained rise or exacerbation after
normalization is a sensitive marker of developing chronic liver disease. In cirrhosis of the liver,
the ALT level is usually high normal or moderately elevated (≤ 80 units) while AST is always
elevated (≤150 units).
Permanent elevations of enzyme activity reflect the decompensation of the cirrhotic
liver.
2- Enzymes reflecting impaired excretory function of the liver: Alkaline Phosphatase (ALP):
Normal range in adult is 3-13 King Armstrong units (KAU) per dl. In obstructive
jaundice the level is markedly elevated to above 35 KAU and is moderately raised (less than 30
KAU) in hepatocellular form.

The specificity of ALP determination can be enhanced considerably by the investigation of ALP
(isoenzymes) which are:
1-Liver 2- Bone 3- Intestinal
4- Placental (Last trimester of Pregnancy) 5-Biliary
 5`- Nucleotidase [5` NT]:
High values are found in post hepatic obstructive jaundice while moderate elevation is
observed in hepatocellular conditions. However, unlike ALP, no significant changes occur with
bone disease.
 Gamma Glutamyl Transferase (GGT):
It rises in liver disease but the change is marked in cholestasis. A number of drugs and
chemicals are known to increase serum GGT activity e.g., contraceptives barbiturates and
alcohol abuse.

3- Enzymes reflecting diminished synthesizing capacity of the liver:

Cholinesterase (ChE):
Abnormally low values are encountered in cirrhosis of various etiology, and liver
malignancies.
Blood coagulation factors:-
With the exception of factor VIII all coagulation factors and fibrinolytic enzymes are
synthesized by the liver. The so-called 'routine' tests of blood coagulation (prothrombin time
[PT], partial thromboplastin time [PTT] antithrombin III, etc.) may therefore be considered as
liver function tests.

Diagnosis of Liver Diseases

Acute Hepatitis:
1- Gross elevation in serum transaminases (AST and ALT). ALT rise is an early marker.
(the only abnormal chemical finding in the pre-icteric phase)
2- Serum bilirubin is elevated.
3- Urine contains excess urobilinogen if intrahepatic obstruction develops.

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 4- Serum alkaline phosphatase may be normal or slightly raised while serum gamma GT is
raised.
Chronic Persistent Hepatitis:-
1- Moderately elevated serum bilirubin.
2- Persistent elevation of ALT & AST for months.
3- Elevated bile acids.
Chronic Active Hepatitis:
1- Low serum albumin and high globulins.
2- High values of transaminases especially AST and ALP.
3- Marked increase in bilirubin.
Cirrhosis:
a) Mild or latent (compensated):
1- Elevated postprandial plasma bile acid levels.
2- Increased GGT (alcohol drinking must be excluded).
3- Less often increase in transaminases and ALP.
b) Severe (decompansated)
1- Increased serum bilirubin.
2- Fall in plasma albumin with elevated globulins.
3- Prolonged prothrombin time.
4- Aminoaciduria, increased plasma ammonia and reduced blood urea associated with
development of acute hepatic failure.

Hepatic Coma:
The liver is concerned with carboxylation of the neurotoxic blood ammonia to form the
physiologically inert substance urea, which is excreted by the kidney. Failure of this process
underlies the neurological manifestations. The upper limit of normal blood ammonia level is 3
ug/ml (varies according to the method used). Gastrointestinal hemorrhage or protein feeding
accentuates the porto-systemic encephalopathy.
Cholestasis:
1- Markedly raised serum bilirubin especially the direct form.
2- Urobilinogen is absent from urine and stool unless the obstruction is intermittent.
3- Serum ALP, GGT & 5`-NT (cholestatic or biliary markers) are markedly raised.
4- Variable increase in cholesterol, triglycerides and phospholipids.
5- Increased IgM in primary biliary cirrhosis.
Acute Hepatic Necrosis:
1- High serum bilirubin.
2- Serum ALT & AST are high.
3- Low plasma albumin levels and prolonged prothrombin time.
4- Hypoglycemia, high lactate and pyruvate levels and acidosis.
5- High plasma ammonia and low blood urea levels.

Hepatic infiltrations:
1- Marked increase in serum activities of GGT, ALP, 5`-NT and LAP (leucinaminopeptidase).
2- Slight increase in bilirubin.
3- Marked increase in alpha feto protein in cases of hepatic carcinoma.
4- Other findings: hypoglycemia, hypofibrinogenemia, and increased enzymes (AST, ALT,
GT, LDH).

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 Stomach and Pancreas
Gastric Function Tests
The gastrointestinal tract is both a major endocrine organ and a major target for many
hormones released locally and from other sites.
Gastrointestinal hormones influence motility, secretion, digestion, and absorption in the
gut; they regulate bile flow and secretion of pancreatic hormones.
Normal stomach produces 1000 ml/24 hours of gastric juice, which contains intrinsic
factor, pepsinogen and others. Its pH is 1.5 – 3.5.
Gastric secretion is stimulated by vagus nerve and gastrin. Gastric secretion is inhibited by
secretin (except in Zollinger Ellison Syndrome), cholecystokinin-pancreozymin, and gastric
inhibitory polypeptide.
Examination of stimulated gastric acidity:
The stimulus for gastric secretion may be:
1- Simple test meal.
2- Histamine test: 0 – 10 mg/kg (unpleasant).
3- Pentagastrin test: the test of choice.
- In normal individuals, the total basal acid output (BAO) is less than 5 mmol/hour and
maximum acid output (MAO) is 20 mmol/hour.
- BAO and MAO are increased in duodenal ulcer and Z.E. syndrome.
- In carcinoma of the stomach, there may be achlorhydria or normal secretion.
- Absolute achlorhydria is found in pernicious anemia.
4-Insulin Test “Hollander’s Test”:
This test is performed to confirm the completeness of vagotomy three to six months
after the operation. With incomplete vagotomy, acid concentration is more than 20 mmol/L, or
total acid output is more than 2 mmol/hour.
5- Tubeless Test:
Indirect and less sensitive method to test for gastric acidity without intubation. The
patient ingests a capsule formed of a dye linked to a resin. The dye is released if HCl present in
gastric juice and can be detected in urine (the test depends on normal intestinal absorption and
renal function).

6- Other gastric function tests:

a. Serum gastrin: increases in Zollinger-Ellison syndrome.” Duodenal or pancreatic tumor”
producing a large amount of gastrin, which in turn causes fulminant peptic ulcer and
sometimes diarrhea and fat malabsorption.
b. Schilling test: in pernicious anemia due to intrinsic factor deficiency which is important for
absorption of vitamin B12.

The pancreas
Acute pancreatitis:
Clinical diagnosis is confirmed by measurement of:
1- Plasma amylase: activity arises from both the pancreas (P-isoenzyme) and the salivary
glands (S-isoenzyme). P-isoamylase is more sensitive and more specific for the diagnosis
of acute pancreatitis. Total amylase activity greater than 10 times normal is diagnostic.
High values also occur in acute parotitis, acute biliary tract disease and mesenteric
infarction. Moderate increase occurs in diabetic ketoacidosis. Small and transient increase
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 occurs in any acute abdominal condition and after injection of drugs causing spasm of the
sphincter of Oddi.
2- Decrease of serum calcium occurs due to the formation of insoluble calcium salts of fatty
acids in areas of fat necrosis.
Chronic pancreatitis:
Impaired secretion of pancreatic enzymes leads to malabsorption and steatorrhea.
Compared to radiology and endoscopy, chemical tests are of limited value.

Investigations of Kidney Functions

I- Blood Estimates
(1) Total Plasma non-protein nitrogen:
Amino acids, ammonia, urea, uric acid, creatine and creatinine.
(a) Blood Urea: The normal level is 25-40 mg/dl (12-20 mg. Urea N2/dl). High blood urea is
expected in a variety of conditions including: kidney damage, retention of urine as in prostatic
obstruction. Cardiac failure and dehydration. The kidneys however, may still be working
efficiently as evidenced by a high urea concentration in the urine.
(b) Creatinine: Its normal concentration is less than 1.4 mg/dl. In advanced renal failure,
plasma creatinine level is significantly raised.
(c) Uric acid: Its normal concentration in plasma varies from 3 to 7 mg/dl. Hyperuricemia
occurs in renal insufficiency, gout, and myeloid leukemias.
(2) Plasma proteins:
Type II nephritis is accompanied by a marked lowering of the plasma protein
concentration particularly albumin (less than 2.5g/dl).
(3) Serum cholesterol:
The blood cholesterol is generally raised in nephrotic syndrome.

II- Combined blood and urine tests (Clearance Tests)

The normal glomerular filtration rate (GFR range is 100-125 ml/min. for a body surface
area of 1.74 m2).
It is decreased when:
1- The renal plasma flow is diminished.
2- The filtration pressure is decreased.
3- The glomerular filter is defective.
Clearance of a substance is the volume of plasma in ml. cleared of this substance in one
minute through urine. Clearance = U x V.
P
U= Urine concentration of the cleared substance in mg/dl.
V = Urine volume/minute (more than 2.0 ml/min).
P = Concentration of the substance in the blood in mg/dl.
The clearance is related to the active renal mass, which is proportional to the body
surface area. So, for emaciated, obese, unusually tall adults or children, correct the result by
multiplying it by:
1.74 (Mean Adult Body Surface Area)
Patient Surface Area in Sq.m.
1- Inulin clearance test:
Inulin is the most accurate of all GFR assays, but as a non-endogenous substance, it
requires intravenous infusion under constant medical surveillance.

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2- Creatinine Clearance:
Creatinine is a nearly ideal substance for the measurement of clearance since it is an
endogenous metabolic product. Specimen collection must include both a 24- hour urine
specimen and a serum creatinine sample (ideally taken at the midpoint of the 24 - hour urine
collection). Reference range for the creatinine clearance is 97 to 137 ml/min/ 1.73 m2 and 88 to
128 ml/min/ 1.73 m2 for male and female respectively. Creatinine clearance normally decreases
with age. It is related to muscle mass and is less affected by diet.

III- Urine Analysis (UA)

A - Physical Examination
1- Volume: The minimal 24-hour output of urine needed to remove the waste products of
normal metabolism is about 500 ml. in 24 hours. Anuria occurs when 24-hour urine volume is
below 100 ml.
Causes of Polyuria: Hysterical polydepsia, diuretics, chronic renal insufficiency, diabetes
mellitus and diabetes insipidus.
Causes of Oliguria: Severe diarrhea or vomiting, shock and severe hypotension, acute
glomerulonephritis, tubular necrosis and bilateral obstruction of the ureters.
2- Color:
Yellowish brown to green (bile pigment oxidation). Red and brown after standing
(porphyrins). Reddish brown in fresh specimens (hemoglobin and red cells). Brownish black
after standing (alkaptonuria). Drugs and some foods such as beets also may alter urine color.
3- Reaction: Normal urine pH is 4.5 to 8.0. Alkaline urine is found after vegetarian diet and on
standing due to formation of ammonia by bacteria. High acidic urine is found after high protein
diet, febrile conditions and ketonuria.
5- Specific gravity: The normal specific gravity is between 1.005 and
1.030 (osmolality 60-1250 mosmo1/kg.)
High specific gravity occurs in the presence of glucosuria, proteinuria, and disorders
associated with oliguria.
Low specific gravity occurs in cases of polyuria (except in D. M.), cases of oliguria with low
specific gravity as in acute tubular necrosis.
Fixed specific gravity (Isothenuria): Around 1.010 (that of protein free plasma) occurs in
severe chronic renal disease.

B- Chemical Examination
The analytes routinely tested are glucose, protein, ketones, nitrites, bilirubin and
urobilinogen.
* Proteins: normal 24 hours urine contains 100-150 mg proteins.
Causes of proteinuria:
A. Renal disease: acute glomerulonephritis, nephrotic syndrome, chronic glomerulonephritis,
B. Prerenal: heart failure, pressure on the renal vein, fever, anemias, nephrotoxic drugs,
jaundice and orthostatic (postural) albuminuria.
* Glucose: normal 24 hours urine contains 20-200 mg. of glucose.
Causes of glucosuria:
Hyperglycemia and low renal threshold for glucose (renal glucosuria).
Causes of ketonuria:
(1) Over fasting.
(2) Severe vomiting or diarrhea.
(3) Diabetic ketoacidosis (uncontrolled diabetes mellitus).
13
 (4) Uncontrolled D.M.
C. Microscopic Examination
1- Red cells (Hematuria): in acute glomerulonephritis, other infections, stones, parasites
(Schistosoma hematobium), trauma and malignancy of the urinary tract.
2- Pus cells: in urinary tract infections. Aseptic pyuria occurs in T. B. and mycoplasma
infection.
3- Casts: red cell casts in acute glomerulonephritis, pus cell casts in pyogenic kidney
affections. Granular casts: they are degenerated cellular casts, found in chronic
glomerulonephritis, and chronic renal failure. Fatty casts: found in late nephrosis of different
types.

Laboratory Finding in Kidney Diseases

Acute glomerulonephritis (type I Nephritis)
1- Rapid onset of hematuria and proteinuria (< 3.0 g/day).
2- Rapid development of decreased GFR with elevated blood urea nitrogen (BUN) and serum
creatinine, oliguria and water retention.
3- The urine is blood stained "smoky", contain numerous hyaline and pus cell casts, the
presence of red cell casts is highly suggestive of this disease.
4- Decreased complement: C3, which returns to normal within 6 weeks.
Chronic glomerulonephritis:-
- This disease is undetected for lengthy periods.
- Minor decrease in renal function at first with slight proteinuria and hematuria.
- Gradual development of impaired kidney functions.
Nephrotic syndrome (Type II nephritis):
An abnormally increased permeability of the glomerular basement membrane which result in:
1- Extremely massive proteinuria (2-5g/day and even up to 20.0 g/day).
2- Hypoalbuminemia which leads to decreased oncotic pressure and development of edema.
3- Hyperlipidemia (secondary to lipoproteins alterations).
4- Loss of anti-thrombin-3 with development of hypercoagulability state.
5- Marked decrease in C3 and C4 is suggestive of SLE (ANA and LE cells helps in its
diagnosis).
A similar picture may be produced by renal amyloidosis, nephropathy of diabetes
(Kimmelsteil-Wilson syndrome), poisoning by heavy metals and compression or occlusion of
renal veins. Many of these cases gradually develop progressive renal failure.
Acute renal failure:
A few patients maintain a normal urine volume throughout the course of the illness, but
usually oliguric, diuretic and recovery phases can be recognized. Chemical investigations help
to determine the severity of the disease and to follow its course, but do not help much in
determining the cause.
Oliguric phase:
Less than 40 ml urine is produced each day. For monitoring patients, plasma creatinine
or urea, and K+ are particularly important and need to be determined at least once daily.
Diuretic phase:
Urine volume increases but the clearance of urea, creatinine and other waste products
may not improve to the same extent. Large losses of electrolytes may occur in the urine and
require to be replaced orally or parentrally.

14
Chronic renal failure:
The GFR is invariably reduced, associated with retention of urea, creatinine, urate,
phosphates, various phenolic and indolic acids and other organic substances. The progress and
severity of the disease are usually monitored by measuring plasma (creatinine) or (Urea) or
both periodically.

Acid-base balance
The normal plasma pH is 7.42 (range 7.38 – 7.44) corresponding to H+ concentration of
40 nmol/L (range 36 - 43 nmol/L). It is maintained within these narrow limits through strict
regulations by respiratory mechanisms, renal tubular mechanisms and body buffers (carbonic
acid, dihydrogen phosphate, organic acids and proteins).
Acidosis results when hydrogen ion is greater than 44 nmol/L (pH < 7.35). Alkalosis
exists when the hydrogen ion of arterial blood is less than 35 nmol/L (pH > 7.45).
Compensatory mechanisms start immediately through alteration of the rate of ventilation,
followed by activation of body buffer system, lastly by the renal mechanism.

Biochemical changes in acid-base imbalance:

a) Respiratory alkalosis (CO2 deficit):
- Causes: over breathing e.g. hysterical, over ventilation in a mechanical respirator or
injury to the respiratory center e.g. trauma.
- Compensatory mechanisms; body buffer systems, followed by loss of bicarbonate in
the urine and decrease in H+ excretion.
b) Respiratory acidosis (CO2 retention):
 Non- respiratory (metabolic) disturbances:
a) Non-respiratory alkalosis:
Causes: ingestion of large amounts of alkali for treatment of peptic ulcer and loss of
acid from the stomach (prolonged vomiting). Compensatory mechanism:
hypoventilation followed by renal loss of bicarbonate
b) Non-respiratory acidosis:
Causes: renal disease leading to failure to excrete hydrogen ions e.g. renal tubular
acidosis, acute and chronic renal failure, failure to excrete an excessive load of
hydrogen ions as in ketoacidosis, lactic acidosis and uretero-colic anastomosis, and loss
of base by excessive loss of GIT secretions.
Compensatory mechanisms: hyperventillation followed by renal excretion of H+ (if the
kidney is healthy) and fall in plasma bicarbonate due to its elimination with H+  H2 CO3 
CO2, which is washed by the lungs.
Laboratory assessment of acid -base balance:
Arterial or capillary blood samples are used. Samples are withdrawn in a syringe using
heparin as anticoagulant and transferred immediately to the laboratory. If there is some delay
(15-30 min.), samples must be kept in iced containers. For the precise investigation of acid-
base balance, the three parameters: [H+], bicarbonate concentration and PCO2 must be
determined.

15
 Laboratory Endocrinology
The hypothalamus and pituitary form a functional unit concerned with the control of
most of the endocrine system.
Secretion of the anterior pituitary hormones is controlled by the secretion of
hypothalamic factors, which may be stimulatory or inhibitory to each specific hormone. The
control is by feed back loops, which "turn - off " the trophic hormone when the level of the
target hormone rises.
Anterior pituitary hormones:
- Basal plasma GH levels are of little value in diagnosis. Increases can be caused by stress or
exercise and this forms the basis of the insulin-hypoglycemia stress response test.
- Hyperfunction of GH-secreting cells (adenomas of the anterior pituitary) leads to gigantism
if the disorder occurs before closure of the epiphyses of long bones, and to acromegaly if it
occurs after their closure. Diagnosis of these disorders is based on:
* High basal plasma (GH) levels
* Response to oral glucose tolerance test: normally, plasma (GH) falls to less than 2 mU/L at
some time during the test. In patients with acromegaly and gigantism, plasma GH does not fall
in response to the stimulus of hyperglycemia.
- Hypofunction of the anterior pituitary and short stature in children can be diagnosed by the
insulin-hypoglycemia test (0.10 - 0.15 insulin unit /Kg). Normally, hypoglycemia causes a
marked increase in plasma GH to more than 2O mU/L, but this does not occur in pituitary
dysfunction. Care must be taken against the occurrence of dangerous hypoglycemia. Induction
of hypoglycemia can be achieved by strenuous exercise.
Prolactin, FSH and LH:
Measurements are indicated in cases of amenorrhea, oligomenorrhea, subfertility and
galactorrhea.

Posterior Pituitary Hormones

The posterior pituitary is an integral part of the neurohypophysis. It produces mainly
two hormones, oxytocin and arginine vasopressin. (AVP) often called anti-diuretic hormone
(ADH). This peptide is released in response to a rise in plasma osmolality, or to a fall in
extracellular fluid (ECF) volume. It increases the permeability of the distal tubules and
collecting ducts of the kidney to water.
- Over production of AVP may occur with head injury, cerebral tumor, and ectopic hormone
secreting tumors, [inappropriate secretion of ADH (SIADH)].
- Deficiency of AVP gives rise to diabetes insipidus. It may be primary (idiopathic, familial) or
secondary (pituitary disease or injury)

Thyroid Function Tests

The thyroid gland is directly controlled by the pituitary gland in a feed back
mechanism. The hypothalamus secretes TRH (thyrotropin-releasing hormone) that steadily acts
on the pituitary gland to produce and release TSH (thyroid-stimulating hormone).
TSH acts on the thyroid follicles to synthesize and release T4 and T3 into the blood
stream; they are rapidly bound and transported by circulating thyroid binding globulin (TBG),
albumin and prealbumin.
An equilibrium exchange occurs between free and bound hormone. The relatively small
quantity of free T4 (0.05 % of plasma T4) and T3 (0.2 % of plasma T3) has a major biological

16
effect on the rate of oxygen consumption and heat production in the tissues. They also play a
critical role in growth, development and sexual maturation.
Tri-iodothyronine (T3) is the biologically active hormone. A small fraction of T3 is
formed in the thyroid, but the major portion is formed by peripheral deiodination of T4, mainly
by the liver, kidneys and muscle.
Investigations for thyroid status:
The tests used to investigate thyroid dysfunction can be grouped into:
1- Tests which establish the presence thyroid dysfunction e.g. Plasma TSH and thyroid
hormone (T4 and T3) measurements.
* Plasma total T4 and total T3: Conditions causing elevation of plasma TBG e.g. pregnancy or
administration of oral contraceptives lead to false elevation of T4 and T3, while lowering of
TBG levels e.g. protein loosing states, cause a false lowering of T4 and T3.
* Plasma free thyroxine (free T4 and free T3): Plasma free T4 provides a more sensitive and a
more specific index of thyroid status than plasma total T4 and T3. Plasma free T3 is of limited
value in the diagnosis of hypothyroidism as its level is often normal in these patients.
* TSH assay: High levels are found in primary hypothyroidism and low or very low levels are
found in primary hyperthyroidism (below 0.1 mU/L). Low and high levels may be found with
euthyroid status in cases of non-thyroidal illness.

2- Tests for the integrity of hypothalamo pituitary axis. These include:

* TSH stimulation tests can be performed by injection of TSH, which corrects secondary
hypothyroidism but not primary hypothyroidism.
* The TRH test: In normal subjects, after I.V. TRH, serum TSH increases by more than 2
mIU/L above the basal level at 20 min. and returns towards the basal values at 60 min. Patients
with T3-receptor defect (end organ resistance) show TSH response to TRH test. Absent
response is found in:
(i) Hyperthyroidism due to TSH secreting tumor.
(ii) Primary hyperthyroidism.
(iii) Hypopituitarism.

3- Tests to elucidate the cause of thyroid dysfunction i.e. thyroid auto-antibodies and
serum thyroglobulin measurements.
* Thyroid auto-antibodies: Several types of antibodies to thyroid tissue have been detected in
serum of patients with thyroid disease, examples are:
- Complement-fixing antibodies in 80% of patients with Hashimoto’s thyroiditis.
- Anti-thyroglobulin antibodies and antimicrosomal antibodies (anti-thyroid peroxidase) in
patients with Hashimoto's thyroiditis and (long acting thyroid stimulator).
- Thyrotrophin-receptor antibodies present in the serum of patients with Grave's disease.

Laboratory findings in thyroid disorders

* Hyperthyroidism (thyrotoxicosis)
- TSH is usually very low, very rarely, it may be elevated (TSH secreting tumor).
- Total and free T4 and T3 are elevated.
- Occasionally, only T3 is elevated (T3 toxicosis).
Plasma T3, T4 can be used to follow the treatment with antithyroid drugs. Measurement of
plasma TSH is not a reliable guide for thyroid status during the first 4 to 6 months of treatment
(TSH still be suppressed).
* Adult hypothyroidism:
17
- TSH is high in primary hypothyroidism but low in secondary hypothyroidism.
- Plasma total and free T4, T3 and FTI are low.
- An elevated TSH with normal T4, T3 occur early in the course of the disease (premyxedema).
- Hypercholesterolemia
* Neonatal hypothyroidism (cretinism):
- Raised TSH levels above 30 mu/L with low T4 (< 6.0 μg/dl) on the third day of life (in a
normal neonate). These tests should be performed as parameter of neonatal screening
programs.

Parathyroid Function Tests

Parathyroid hormone is secreted in response to a fall in plasma Ca ++. The rise of
plasma Ca is achieved through the following:
- Direct effect on bone by stimulating bone turnover.
- Direct effect on the renal tubules, to enhance Ca ++ reabsorption.
- Indirect effect on the small intestine: PTH stimulates the formation of 1, 25-dihydroxy
cholecalciferol in the kidney, and this increases Ca ++ absorption from the small intestine.
Hyperparathyroidism:
Primary hyerparathyroidism may be caused by parathyroid hyperplasia adenoma, or
carcinoma. These patients usually present with renal calculi or with metabolic bone disease.
Laboratory findings in this condition are:
- Raised serum calcium.
- Decreased fasting serum phosphate.
- Increased serum alkaline phosphatase activity.
- Elevated PTH levels
Secondary hyperparathyroidism develops when long standing hypocalcemia results in
stimulation of parathyroid with development of hyperplasia of the gland. Tertiary
hyperparathyroidism refers to the development of a functioning parathyroid adenoma as a
complication of secondary hyperparathyroidism.
Hypoparathyroidism:
Failure to secrete PTH may be familial or sporadic. It may be a complication of
surgery, or autoimmune process or as a result of infiltration by carcinoma of the thyroid or
other neoplasms.
Laboratory findings in hypoparathyroidism:
- Low serum calcium and increased serum phosphate in a patient who does not have renal
disease.
- Serum alkaline phosphatase activity is usually normal.
- Serum PTH is reduced, and sometimes is undetectable.

Plasma calcium: Calcium is the most abundant mineral in the body. About 99% of the body's
calcium is present in bone where it acts as a reservoir which helps to stabilize Ca++ in extra
cellular fluid.
In the plasma, calcium is present in three forms, ionized calcium (Ca++), calcium bound
to plasma proteins, and calcium complexed with citrate. The first is the physiologically active
component (50-65% of total calcium), and is regulated mainly by parathyroid hormone.
Measurements of total plasma calcium (2.12 – 2.62 mmol/L) is often misleading due to the
effect of changes in plasma albumin, since both are closely associated. It is better to measure
plasma ionized calcium (Ca2+) which is maintained constant by PTH.

18
Other causes of hyperca1cemia are:
- Malignant disease e.g. multiple myeloma.
- Excessive calcium absorption e g vit. D overdose.
- Drugs e.g. thiazides.
- Artifact e.g. excessive venous stasis during venepuncture.
Other causes of hypocalcemia are:
- Renal disease (impaired hydroxylation of 25 -HCC, acidosis).
- Dietary deficiency of calcium or vitamin D.
- Hypoproteinemia.

Supra-renal gland
The cortex of the gland secretes mainly cortisol, aldosterone and androgens. The
medulla secrets epinephrine and not epinephrine.
Control of cortisol secretion:
- Higher centre Negative feedback control: through corticotropin-releasing hormone (CRH)
from the hypothalamus and ACTH from the anterior pituitary.
- Response to stress: e.g. physical and emotional stress lead to sudden large increase in CRH
and ACTH.
- Diurnal rhythm: values of plasma cortisol are higher in the morning than at midnight. This
depends also on CRH and ACTH secretions which are related to the rhythm of an individual’s
sleeping-waking cycle.
In the circulation, cortisol is mainly bound (90%) to cortisol-binding globulin (CBG).
The biologically active fraction, free cortisol (10%) is in equilibrium with the protein-bound
cortisol.
Aldosterone:
This is the principal mineralocorticoid, its functions is to conserve Na+ mainly by
enhancing the reabsorption of Na+ and reciprocal K+ or H+ secretion in distal renal tubule and
in other epithelial cells.
Regulation of aldosterone secretion is achieved by:
- Renin-angiotensin system: renin is released in response to fall in blood volume or renal
perfusion pressure, and by loss of Na+.
- Potassium: increase in plasma K+ stimulates aldosterone Secretion.
- ACTH has a little role in aldosterone regulation.
Suprarenal androgens:
Their production is determined by the rate of synthesis of precursors and is controlled
by ACTH.

Disorders of supra-renal cortical functions

A) Hyperfunction: Cushing's syndrome:
This results from pituitary hyperplasia (Cushing disease), adenoma, carcinoma of the
suprarenal, iatrogenic or ectopic production of ACTH or CRH.
Laboratory diagnosis:
* Screening tests:
- Elevation of urinary free cortisol.
- Low dose dexomethasone suppression test: normally dexamethasone causes suppression of
CRH, ACTH and cortisol secretion by negative feedback. Failure of this suppression occurs in
Cushing's disease.

19
* Confirmatory tests:
- Elevation of serum cortisol with loss of diurnal rhythm.
- Insulin hypoglycemia test: insulin induced hypoglycemia is a sensitive stress test for the
integrity of the hypotholamo-pituitary-adrenal axis. Induction of sufficient hypoglycemia
causes marked elevation of serum cortisol in normal subjects. In Cushing's syndrome, basal
cortisol levels are high with no further increase after adequate hypoglycemia. This test should
be performed under careful observation in hospital.
Tests to assess the cause:
- Plasma ACTH: low in cases due to primary suprarenal pathology e.g. tumor, raised in cases
of pituitary origin. Very high plasma ACTH, in ectopic production.
- High-dose dexamethasone suppression test: Patients with Cushing’s disease due to
hyperplasia respond to this test by a fall of serum cortisol or urinary free cortisol to less than
50% of the basal level. Patients with suprarenal tumors or ectopic ACTH production do not
show suppression.
Other laboratory finding:
- Hypokalemic alkalosis.
- Impaired glucose tolerance (steroid diabetes).
- Tumor markers may be positive in ectopic ACTH production.
B) Hypofunction:
Primary chronic suprarenal cortex insufficiency (Addison's disease) arises due to
autoimmune disorder, infections especially T.B, CMV, congenital adrenal hypoplasia or
hyperplasia or to post-partum hemorrhage secondary hypofunction due to pituitary disease.
Diagnosis is based on:
 Screening tests: serum cortisol is usually low and fails to rise thirty min. after I.M.
injections of synacthen (Synthetic ACTH short synacthen test).
 Tendency for hyponatremia and hyperkalemia.
 Depot (long) synacthen test: serum cortisol is measured on basal sample and on further
samples taken between five and eight hours after I.M. injection of 1.0 mg synacthen for
three successive days. The cortisol fails to rise in Addison’s disease while stepwise
increase in cortisol response occurs in secondary adrenocortical insufficiency.
 Tests to assess the cause: ACTH values are high in primary and low in secondary
hypofunction.
 Tests for adrenal antibodies.
Disorders of suprarenal medulla
The suprarenal medulla secretes the catecholamines (adrenaline and noradrenaline).
Tumours at this site secrete excessive amounts of either noradrenaline or adrenaline, together
with increased excretion of their metabolites in urine.
Tumours causing these effects are pheochromocytoma and neuroblastoma.
Laboratory diagnosis is based on:
- Measurement of plasma catecholamines.
- Measurement of urinary catecholamines and their metabolites: metadrenaline
normetadrenaline and 4-hydroxy, 3-methoxymandelic acid (HMMA), which are commonly
referred to as vanilyl mandelic acid (VMA). These analytes are sensitive to drugs
(azothioprine) and certain dietary constituents (vanilline and bannana). In patients with
paroxysmal attacks of hypertension basal measurements should be made at the time of the
attack and in-between.

20
 Gonads and Infertility
Investigations of male hypogonadism and infertility:
1- Semen analysis should be performed. Normal seminogram on two separate occasions
requires no further endocrine functions testing.
2- Low sperm count necessitates measurements of plasma FSH, LH testosterone and prolactin.
a) Cases with primary (testicular, hypergonadotophic) hypogonadism show low plasma
testosterone levels with increased FSH and LH.
b) Cases with secondary (pituitary - hypothalamic hypogonadotrophic) hypogonadism show
reduction of plasma testosterone and selective reduction of FSH, LH or both.
Injection of human chorionic gonadotrophin corrects secondary hypogonadism.
Investigations for female subfertility:
1- In patients with normal menstruation: Serum progesterone should be determined daily
between days 19 and 23 of the cycle. Values more than 30 nmol/L indicate ovulatory
cycle, those between 10 and 30 nmol/L suggest ovulatory cycle with luteal phase defect,
while values below 10 nmol/L indicate anovulatory cycle.
2- In patients with amenorrhea or anovulatory cycle, it is important to measure plasma levels of
prolactin, FSH, LH, estradiol and sometimes TSH and free T4.
- If prolactin level is high: specific therapy with bromocriptine is started.
- If prolactin is normal: measure FSH, LH and estradiol. When FSH and LH are high and
estradiol is low, there is primary ovarian failure. When LH is high with low FSH and
oestradiol, the patient may have the polycystic ovary syndrome (PCOS). When all
hormonal values are low, there may be hypothalamic, pituitary or other endocrine disease.

Some laboratory aspects of pregnancy

The clinical laboratory has an important role in diagnosis, management and evaluation
of pregnancy, both normal and abnormal.
 Diagnosis of pregnancy and ectopic pregnancy: The diagnosis of pregnancy is based on
the detection of hCG in serum about 10 days after conception, with a marked and gradual
increase thereafter. In ectopic pregnancy plasma hCG fails to rise at the normal rate.
 Maternal serum screening for fetal defects: Prenatal diagnosis of fetal abnormalities is
advised for pregnancies at risk because of either advanced maternal age or family history.
- Maternal serum -fetoprotein (AFP) is used to screen for neural tube defects
(anencephaly and spina bifida) at 18-20 week gestation. If elevated levels are found
on two occasions, ultrasound and amniocentesis may be indicated.
- Screening for the presence of a fetus with Down’s syndrome can be performed using
measurement of the following analytes in maternal serum at 16 wk gestation:
* AFP: values are approximately 30% lower in mothers having trisomy 21 or
trisomy 18.
* Chorionic gonadotropin: values are approximalety 2 times higher when fetal
Down syndrome is present.
* Unconjugated Estriol: values are approximately 0.7 time lower when fetal
Down syndrome is present.
* Values of these analytes (triple markers) should be compared to median values
reported for women of matched age, body weight and gestational age in
weeks.

21
 Tumor Markers
A tumor marker is a substance sometimes found in an increased amount in the blood,
other body fluids or tissues that may suggest the presence of a type of cancer. They are
measured qualitatively or quantitatively by chemical, immunological or molecular biological
methods to identify the presence of cancer.
Potential uses of tumor markers:
- Screening in general population (of limited value).
- Differential diagnosis in symptomatic individuals in conjunction with clinical and
radiological evidences.
- Prognostic indicator of disease progression when the plasma concentration
correlates with the tumour mass.
- Evaluation of success of treatment.
- Monitoring of response to therapy and detecting recurrence.
Tumor markers include:
 Enzymes: elevated levels can serve as tumor markers, examples are:
- Alkaline phosphatase (ALP): primary or secondary liver cancer, metastatic cancer
with bone or liver involvement, specially with osteoblastic lesions.
- Lactate dehydrogenase (LDH): liver cancer, non-Hodgkin’s lymphoma, acute
leukemia, non-Seminomatous germ-cell testicular cancer, seminoma,
neuroblastoma, etc ……
- Prostatic acid phosphatase (PAP): prostatic cancer, osteogenic sarcoma, multiple
myeloma, and some benign conditions such as benign prostatic hyperplasia,
osteoporosis and hyperparathyroidism.
- Prostate-specific antigen (PSA): it is extremely useful tumor marker to detect, stage,
and monitor treatment of prostate cancer. Serum PSA rises also with age, benign
prostatic hypertrophy and lower urinary tract infection
 Hormones: production of hormones in cancer involves either production of hormones in
excessive amounts by its original gland or production at a distant site by a non endocrine
tissue (ectopic syndrome), e.g. production of ACTH by the small cell carcinoma of the
lung.
 Oncofetal antigens: are proteins produced during fetal life and decrease to low levels or
disappear after birth. They reappear in individuals with cancer.
- Alpha-fetoprotein (AFP): a marker for hepatocellular and germ-cell carcinoma (non-
seminoma) its measurement can also used to monitor treatment. Level in healthy adults is
less than 10 ug/L. [During pregnancy, maternal AFP levels start to show progressive
increase]. Levels can also be increased in non-cancerous liver diseases such as hepatitis
and cirrhosis, but the increase is mild.
- Carcinoembryonic antigen (CEA): this is a marker for colorectal, gastrointestinal, lung
and breast carcinoma. The upper limit in the healthy population is about 3 ug/L for non-
smokers and 5 ug/L for smokers. Because of the false-positive and false-negative results,
CEA testing is better used as an adjunct to clinical staging and together with other
markers. CEA levels decline after successful therapy. Rising values may indicate
recurrence.
 Carbohydrate markers: These are either antigens on the tumor cell surface or secreted by
the tumor cells. They tend to be more specific than enzymes or hormones and are
abbreviated as CA. Examples are CA 15-3, CA 549 (for breast and ovary), and CA 125 (for
ovary and endometrium).

22
 Blood group antigens: These include CA 19-9 (colorectal and pancreatic cancer) and CA
72-4 (cancer of GIT and ovary).
In addition, proteins, hormone receptors and genetic markers are now used with various
degrees of success.

23
 Cerebrospinal fluid
It is the fluid that occupies the spaces of the C.N.S, it circulates upward over the
cerebral hemispheres and downward over the spinal cord and nerve roots.
Formation:-
70% by the ventricular choroid plexus and 30% by epindymal lining of the ventricles as
well as cerebral and arachinoid space. Its normal adult volume is 100-200 ml. It functions as an
excretory vehicle, circulate nutrients and lubricates CNS.
Function.
C.S.F. examination:-
C.S.F is obtained by lumbar puncture, lateral cervical puncture or through ventricular
cannula or shunts under aseptic conditions. Indications for C.S.F examination are meningeal
infection, subarachinoid hemorrhage, CNS malignancy and demylinating diseases .
Appearance: -
Normal C.S.F is clear, colorless, clot free and has the viscosity of water. Turbidity in
fresh CSF may be due to erythrocytes, leukocytes or bacteria. Colored CSF is due to oxy-
hemoglobin, bilirubin or drugs. Bloody CSF sample may be due to a traumatic tap (clear
supernatant after centrifugation) or hemorrhage (yellow supernatant, xanthochromia). Clot
formation may be due to its traumatic tap, complete spinal block (Froin`s syndrome) and
suppurative or T.B meningitis.
Chemical examination:-
Total protein (normal range 20 – 40 mg/dl): -
Increased CSF proteins occur in diseases of the CNS, general toxic states as uremia, acute
meningitis, below spinal block and traumatic tap and myelomatosis. Increased levels of IgG
CSF protein may be derived from either diffusion across blood CSF barrier or increased
synthesis. As albumin is not produced by CNS, so CSF/plasma albumin ratio reflect the
functional integrity of the barrier.
Decrease in CSF protein occur due to leakage of CSF, removal of large amount of CSF
during radiographic technique, increased intracranial pressure and hyperthyroidism.
Glucose (normal CSF level 40-80 mg/dl):-
Increased level (relative to plasma glucose) is an evidence of hyperglycemia 2-4 hour prior to
lumbar puncture. Decreased level of CSF glucose < 40 mg/dl in fasting patients with normal
blood glucose may be due to bacterial meningitis, T.B meningitis, fungal meningitis, amaebic
meningo-encephalitis and subarachinoid hoemorrhge. In viral meningitis CSF glucose is
usually normal.
In Rhinorrhea : The fluid is tested for glucose, if glucose is present the fluid is CSF.
Chloride :- (Normal 120 – 130 mmol/L,is higher than plasma level).
Its level is low in pyogenic meningitis and much lower in T.B meningitis.
Increased level of lactic acid occurs in case of traumatic brain injury and bacterial meningitis.
Enzymes:
Lactic dehydrogenase (LDH): Increased in bacterial meningitis, viral meningitis, leukemia,
lymphoma, metastatic and subarachinoid hemorrhage.
Creatine kinase (CK):-
Increased CK is sensitive but not specific for CNS diseases as hemorrhage, thrombosis,
multiple sclerosis.
Aspartate amino transferases (AST):- Increased in neurological disease with bad prognosis.

24
 Reference Values

Analyte** Reference Interval

Alanine Aminotransferase (ALT/SGPT) 0 – 38 U/L (37 C)
Albumin 3.8 – 5.1 g/dl
551 – 1.9 mol/L*
Alkaline Phosphatase (ALP) 34 – 114 U/L (37 C)
Amylase 16 – 108 U/L (37 C)
Aspartate Aminotransferase (AST/SGOT) 0 – 40 U/L (37 C)
Bilirubin, Total Upper limit
1.2 mg/dl (Adult)
20.5 mol/L*
12.0 mg/dl (Newborn)
205 mol/L*
Calcium, Total 9.2 – 11.0 mg/dl
2.3 – 2.75 mol/L*
Chloride 98 – 106 mEq/L (Serum)
118 – 132 mEq/L (CSF)
Cholesterol, Total 140 – 200 mg/dl (Adult)
3.6 – 5.2 mmol/L*
Creatine Phosphokinase (CPK) 25 – 192 U/L (37 C)
Creatinine 0.8 – 1.5 mg/dl (Serum)
7 – 133 mol/L*
Glucose 70 – 105 mg/dl (Serum)
3.85 – 5.78 mol/L*
40 – 75 mg/dl (CSF)
2.2 – 4.13 mol/L*
Gamma-GT (GGT) 0 – 45 U/L (Males)
0 – 30 U/L (Females)
Iron, Total 72 – 186 g/dl (Adult)
12.9 – 186 mol/L*
53 – 119 g/dl (Children)
9.5 – 21.3 mol/L*
* Systemic International Unit

25
 Analyte** Reference Interval
Iron, TIBC 25 – 400 g/dl (Adult)
4.5 – 71.6 mol/L*
Lactate Dehydrogenase (LDH) 80 – 285 U/L (Males)
103 – 227 U/L (Males)
Magnesium 1.3 – 2.1 mEq/L (Adult)
0.8 – 1.1 mmol/L*
1.4 – 2.1 mEq/L (Newborn)
0.7 – 1.1 mmol/L*
Phosphorus, Inorganic 2.5 – 4.8 mg/dl (Adult)
0.8 – 1.6 mmol/L*
4.5 – 6.5 mg/dl (Children)
1.5 – 2.1 mmol/L*
Potassium 3.6 – 5.5 mmol/L (Serum)
Sodium 135 – 155 mmol/L (Serum)
Total protein 6.6 – 8.3 g/dl
66 – 83 g/L*
Triglycerides 30 – 150 mg/dl
0.3 – 1.7 mmol/L*
Uric Acid 3.1 – 7.0 mg/dl (Males)
184 – 415 mmol/L*
2.4 – 5.7 mg/dl (Females)
143 – 339 mmol/L*
Urea Nitrogen (BUN) 8 – 23 mg/dl

* SI Units. ** Measured in plasma or serum

26
 Hematology
HEMOPOIESIS

Blood consists of three formed elements namely the red cells (erythrocytes), white cells
(Leukocytes), and platelet (thrombocytes) susbended in a fluid medium (Plasma). These cells
are continually being destroyed and replaced by newly formed cells. In healthy subjects, there
is a balance between the rat of formation and destruction, thus the number of each cell type
remains remarkably constant, although there are the minor daily physiological fluctuation.
Sites of hemopoiesis
Embryogenesis of blood cells
o Mesoblastic phase: occurs in the 3rd – 4th week in the yolk sac.
o Hepatic phase: occurs in the liver at the 6th week of intrauterine life.
o Spleen: starts at the 7th week.
o Lymph nodes: develops, monocytes and plasma cells.
o Thymus: starts at the 12th week of pregnancy forming lymphocytes.
o Bone marrow or myeloid phase: starts at the 12th week of intrauterine life and form
granulocytes, normoblasts and megakaryocytes. Lymphocytes are formedin the lymph
nodes.
(NB): Sometimes under stress of life such as hemorrhage, the liver and splene revert to the
embryonic state where a pool of more blood cells production is needed ( extra-
medullary hematopoiesis or myeloid metaplasia ).
Hemopoiesis in adults:
o Hemopoiesis begins in the bone marrow at the 12th w. of foetal life reaching full
activity at 30th w. with decline of liver sources that stop compeletly at birth.
o At birth, there is transitional fall in the erthropoiesis for 4-6 w., that returns to birth
level at the 13th w.
o Red marrow occupies whole skeletal system which gradually shrinks untel the 18th
years of age where the red marrow is restricted to the end of long bones, and flat
bones where it is replaced by fatty marrow.
Hematopoietic growth factors:
o HGFs are glycoprotein hormones that regulate the proliferation and differentiation of
hemopoietic progenitor cells and the function of mature blood cells.
o The biological effects of the growth factors are mediated through specific receptors
on target cells.
o Lymphocytes, monocytes and stromal cells are the sources of growth factors ( G-
CSF, GM-CSF, Interleukin – 3, Interleukin – 1 ) except for erythropoietin ( kidney )
and thrombopoietin ( liver ).

Steps in blood formations:

Three steps are involved in formation in blood cells:
o Multiplication of the devloping cells which takes place by mitotic division.
o Maturation, may be defined as the progressive devlopment of full characteristics
both structural and functional.
o Relase into the blood stream. The factors controlling the relase are not fully
Understood. The spleen probably plays a role; this is suggested by the fact that
Immediately following splenoctomy there is a rise in leukocytes and platelets in
the pripheral blood.

27
Origin and fate of the blood cells:
All blood cells are derived form the undifferentiated primitive cells known as stem
cells. The formed blood elements, erythrocytes, granulocytes and platelets have
definite life span. It is 4 monthes for the erythrocytes and few days in platelets and
granulocytes. The monocytes have special cycle as they rapidly pass to tissue and
change into tissue macrophages where they remain for months. The lymphocytes have a
very long life span at least 100 days up to 5 years.

NORMAL RED CELL VALUES

Red cells
Men 4.5 – 6.5 million per cmm
Women 3.9 – 5.6 million per cmm
Infants(full-term, cord blood) 4.0 – 5.6 million per cmm
Children 1 year 3.6 – 5.0 million per cmm
Children 10-12 years 4.2 – 5.2 million per cmm
Hemoglobin
Men 12.5 – 6.0 g per 100 ml
Women 11.0 – 14.5 g per 100 ml
Infants(full-term, cord blood) 13.6 – 19.6 g per 100 ml
Children 1 year 11.0 – 13.0 g per 100 ml
Children 10-12 years 11.5 – 14.8 g per 100 ml
Packed volume (hematocrit value)
Men 40 – 54 %
Women 35 47 %
Infants(full-term, cord blood) 44 – 62 %
Children 1 year 36 – 44 %
Children 10-12 years 37 – 44 %
Mean corpuscular volume (M.C.V) 76 – 96 c. μ
Mean corpuscular hemoglobin (M.C.H) 27 – 32 μμg (pg)
Mean corpuscular hemoglobin concentration 30 – 35 %
(M.C.H.C)
Mean corpuscular diameter 6.6 – 7.7 μ
Reticulocytes: (Adults) 0.2 – 2.0 %
(Ifants, Full term, cord blood) 2–6%

WHITE BLOOD CELL

Granulocyte series :
Granulocyte are produced by prolifration and maturation of precursors from earlest
stage , myeloblast produce promylocyte then myelocyte then metamyelocyte then staff
form then matur granulocyte (Neutrophils , Eoseniphils and Basophils)
Monocyte macrophage series :
Monoblasts (are immature stage locted in B.M. ) produced promonocytes then
monocytes ( in pripheral blood ).
Lymphoid series:
B-cell maturation: pleuripotent stem cell in B.M. produce B-lymphopoietic progenitors
then mature B-lymphocytes (15- 20% of lymphocytes in pripheral blood )

28
 T-cell maturation : primitive T-cell from B.M. are seeded into thymic cortex and
differentiate into fully mature T- lymphocytes. They represent 70 – 85 % of blood
lymphocytes.

Table (4): Total and differential leukocytic count

Adults Infants At 1 year At 4-7 year At 8-12 year

TLCX 4 - 11 10 - 25 6 - 18 5 - 15 4.5 – 13.5

3
10 /cmm
Absolute Neutrophils Eosinophils Basophils Monocytes Lymphocytes
count X 2 – 7.5 0.04 – 0.4 0.01 – 0.1 0.2 – 0.8 1.5 – 4
103 /cmm
% 40 - 65 1-6 <1 2 - 10 20 - 45

PATHOLOGICAL CHANGES IN LEUCOCYTIC COUNT

Leucopenia: Decreased total white cell count below 4 x 103

Causes:
o Infection : Bacterial : brucellosis, typhoid and paratyphoid miliary TB.
o Viral: infectious hepatitis, influenza, measels, rubella, infectious mononucleosis.
o Rickettsial: Scrub typhus.
o Portozoal: malaria.
o Chemicals: cytotoxic drugs, analgesics, hypnotics, antithyroid drugs, antiepileptic drugs
tranquilizers, antibiotics, gold salts, antimalarial drugs, benzene derivatives, ionizing
radiations.
o Hemopoietic disorders: megaloblastic anemia, hypersplenism, aplastic anemia,
paroxysmal nocturnal hemoglobinuria and agranulocytosis.
Neutropenia: Decreased of neutrophil count below normal absolutelevel ( < 2.000/cmm )
Causes of selective neutropenia:
o Idiopathic acute, chronic
o Drug induced: anti-inflammatory, anti-TB, anti bacterial ( cloramphinecol and
sulphonamides ), tranquilizers, diuretics, some anticonvulsants, antidiabetics,
antithyroid, antimalarial, anticoagulants, antihistaminics, antipyretics, and analgesics (
optalidon ).
o Infections: bacterial; brucellosis, thyroid, and military TB. Some viral, protozoal and
fungal infections.
o Immune neutropenia: SLE and Felty's syndrome.
o Megaloblastic anemia.
o Miscellaneous: hyperthyroidism, cyclical neutropenia, and familial neutropenia.
o As a part of aplastic anemia and pancytopenia.
Agranulocytosis: Extreme reducation in neutrophils usually < 500 / cmm .
Lymphopenia: Decreased lymphocyte count ( < 1.5 x 103 ).
Causes:
o Corticosteroid therapy.
o Uremia ( chronic ).
o High dose radiotherapy.
o Hodgkin's disease.
29
 o Acquired immune deficiency syndrome ( AIDS ).
o Cytotoxic drugs.
o Myeloid leukemia.

Leucocytosis: Increase in the number of circulating leucocytes more than 11 x 103 /cmm.
Causes:
o Acute infections e.g. abscesses and septicemia.
o Tissue damage e.g. myocardial infarction, burns, gangrene etc….
o Neoplasia.
o Hemorrhage and hemolysis.
o Acute and chronic leukemia & myelofibrosis.
o Drugs e.g. prednisone.
o Metabolic disease e.g. diabtic coma, gout etc…
Eosinophilia: (>0.44 x l03)
Causes:
o Allergy e.g. hay fever, asthma.
o Parasitic infestations: Bilharziasis, ascaris, hydatid cyst.
o Eczema, dermatitis, psoriasis, familial eosinophilia.
o Myeloproliferative diseases- postsplenectomy.
o Hodgkin’s disease.
o Pulmonary eosinophilia
o Hyper-eosinophilic syndrome.
o Eosinophilic leukemia.
o Drugs: liver extract, penicillin, streptomycin, chiorpromazine.
Monocytosis:> 0.8 x 109/L
Causes:
o Chronic infections e.g. TB, brucellosis, malaria, kala-azar, trypanosomiasis, and
ulcerative colitis.
o Inflammatory disorders e.g. rheumatoid arthritis and SLE.
o Hodgkin’s disease.
o Hematological malignancy e.g. myelomonocytic (MML) leukemia, acute myeloblastic
leukemia.
Basophilia : >0.1 x 103 IL
Causes:
o Myxoedema.
o Chicken pox
o Myeloproliferative disorders (polycythemia, chronic granulocytic leukemia).
o Chronic ulcerative colitis.
Lymphocytosis: >4 x109 /L
Causes:
o Viral infections; infectious mononucleosis, infectious. lymphocytosis, cytomegalovirus,
rubella and infective hepatitis.
o Bacterial infections; pertussis, TB, brucellosis
o Chronic lymphocytic leukemia
o Lymphomas
Leukemoid Reaction (LR):
It is an extremely high leucocytic count (50 x 103 / cmm) seen in
nonleukemic conditions, simulating myeloid or lyrnphatic leukemia.
30
It is associated with infections.
Types:
o Myeloid LR. must be differentiated from CML
o Lymphatic LR

LEUKEMIAS
Definition:
A heterogeneous group of malignant neoplasm of the precursors of blood cells
resulting in appearance of blastcells in the blood and bone marrow.
Aetiology
It is unknown but theories are implicated:
o Ionizing radiation.
o Drugs and chemicals; prolonged chemotherapy and immunosuppressive agents,
benzene and its derivatives.
o Genetics; familial incidence, chromosomal abnormalities in leukemic patients.
o Retrovirus.
o Immune status; increased incidence of immunosupressed individuals.
Classification:
FAB classification of acute Ieiikemia depending on morphological and cytological
differences..
Now the diagnosis of acute leukemia based on morphology, cytochemistry,
immunophenotyping and cytogentic.
a)Acute lymphoblastic leukemia (ALL):
Cytochemistry: MPO: Negative, PAS : Positive
Immunophenotyping: B-cell marker ( CD19, CD20 and CD22 ),
T-cell marker ( CD2, CD3, CD5, CD7 )
Cytogentic: t(8;21), t(8;14)
FAB classification
o LI (small homogenous blasts)
o L2 (large heterogeneous blasts)
o L3 (large homogenous blasts).
b)Acute non lymphoblastic leukemia (ANLL)or (AML)
Cytochemistry: MPO: Positive, PAS : Negative
Immunophenotyping: ( CD13, CD33 ) and others
Cytogentic: t(15;17 ) in M3 and other cytogentic abnormalities in diefferent FAB
Classification
FAB classification
o Ml: Acute myelobla~tic leukemia without maturation.
o M2: Acute mycloblastic leukemia with maturation.
o M3 :Hypergranular promyelocytic leukemia.
o M4:Myelomonocytic leukemia.
o M5:Monoeytic leukemia.
o M6: Erythroleukemia.
o M7: Megakaryocytic leukemia.
Clinical Features of acute leukemia:
o ALL is common in children while ANLL is common in adults
o It is of acute onset with pallor, fever, various infections.
31
 o Bone tenderness.
o Mild hepatosplenomegaly.
o Enlarged lymph nodes (in ALL).
o Infiltration of organs (in M4 and M5).
o Meningeal and testicular infiltration in ALL.
o Symptoms of hyperviscosity (due to increased blasts).
o Bleeding tendency in M3.

Hematological findings:
o Severe anemia (normocytic normochromic).
o WBCs; usually increased, but may be within normal or even below the normal range..
o Blood film; blasts are present or absent as in aleukemic leukemia.
o Thrombocytopenia.
o B.M.; hypercellular and infiltrated with blast cells (>20%).
o Biochemical fmdings:
 Increased serum uric acid.
 Increased serum LDH.
N.B.: By treatment with chemotherapy (< 5% blasts in B.M. indicates remission).

Lymphoproliferative Disorders
o It is a group of both malignant ( clonal ) and reactive ( polyclonal ) lymphoid disorders.
o It includes:
1. Acute lymphoblastic leukemia.
2. Chronic lymphocytic leukemia.
3. Prolymphocytic leukemia.
4. Hairy cell leukemia.
5. Plasma cell leukemia.
6. Non Hodgkin's lymphoma.
7. Large granular lymphocytosis.
8. reactive lymphocytosis.
o The above mentioned disorders are either :
1. B – cell disorders.
2. T - cell disorders.
3. MK cell disorders.
Chronic lymphocytic leukemia
o Affect older people ( > 50 years old )
o Male predominance.
o Diagnostic criteria :
1. Peripheral blood : absolute lymphocytosis > 5.000 / mm3 usually > 10.000 /
mm3 .
2. Bone marrow: lymphocytosis > 20% of total bone marrow cells.
3. Mature B – cell markers ( CD19, CD20, and single type light chain ).
4. Express CD5.
5. Certain karyotypic abnormalities.
6. Hairy cell leukemia.
o Age > 50 years.
o Male prodominance.
o Characterized by : splenomegaly / pancytopenia.
32
 o Diagnostic criteria :
1. Megaloblastic cell have fine hair – like cytoplasmic projection.
2. Cytochemical stains : tartrateresistant acid phosphatase positive.
3. Clonal disorders of mature B – cell markers ( CD19, CD20, sIg, and its specific
markers CD103 / CD 11c.
Plasma cell dyscrises:
o It is a groupe of disorders characterized by expasion of a single clone of
Immunoglobulin – selecting cells.
o Resultant of increase in serum level of a single homogenous Ig or Ig chain
o It includes:
1. Multiple Myeloma.
2. Solitary Plasmocytoma.
3. Waldenstom's macroglobulinemia.
4. Heavy chain disease.
5. Primary Amyloidosis.
6. Monoclonal Gammopathy of undetermined significance.
Multiple Myeloma
o Criteria for diagnosis of plasma cell myeloma:
1. Major criteria:
 Bone marrow plasmocytosis > 20 % (provided clonal markers either K or L)
 Plasmocytosis on tissue biopsy.
 Monoclonal globulin sipke on serum electrophoresis (> 3.5 gm / l for IgG /
2.0 g / L for IgA and > / g / 24 hrs for kappa or lambda light chain excretion
in urine )
o Minor criteria :
 Marrow plasmacytosis 10 – 20 %
 Lytic bone lesion.
 Monoclonal globulin spike less than defined in mejor criteria.
 IgM, IgA or IgG decreased.
Myeloproliferative disorders
o Related hematopoietic stem cell malignancies – classified by lineage as following:
1. Chronic myeloid leukemia ( CML ) predominanthy myeloid.
2. Polycythemia vera ( pv ) predominanthy erythroid.
3. Essential thrombocytobenia ( ET) platelets.
4. Myelofibrosis with myeloid metaplasiafibrosis.
5. Myelodysplasia.
6. Paroxysmal nocturnal hemoglobinurea
7. Dysplastic anemias
o Middle aged – eldery individual
o Chronic process – More indulent than acute leukemia
Chronic myeloid leukemia
Diagnostic criteria:
o Pripheral blood and bone marrow: increased total leukocytic count more than 50.000
with immature myeloid cells, basophilis and eosinophilis
o Decresead leukocytic alkaline phosphatase.
o Unique chromosomal abnormality philadelphia chromosom which is reciprocal
translocation t(9;22) resulting in bcr / ab / gene fusion.

33
Polycythemia rubera vera:
Increased erythrocyte cell mass lead to increased hematocrit blood volume and blood viscosity
with subsequent thrombotic or hemorrhagic problems.
Primary criteria:
o Increased hemoglobin / Hematocrit 18 gm / d1 in men and 16 gm in women or red
cell mass increase ( > 36 ml / kg in male and > 32 ml / kg in female )
o Normal arterial oxygen saturation.
o Palpable splenomegaly
o No other explenation – renal disease, hypocia, tumours …
Secondary criteria :
o Leukocytosis (> 12.000 / mm3 )
o Thrombocytosis (> 400.000 / mm3 )
o Abnormal marrow karyotyping
o Elevoted leukocytic alkaline phosphatase
o Elevoted serum β 12 / transcoblamin

Diagnosis of polycythemia vera is usually mode by presence of all primary criteria with
some of secondary criteria.

Myeloid metaplasia ( myelofibrosis )

o Abnormal stem cells leads to deranged hematopoietic cells (esp. megakaryocytes )
which stimulate fibroblast.
o Originally agnogenic ( etiology unknown ) with myeloid metaplasia ( extramedullary
spleen )

o The natural history is in two phases

 Initial cellular phase
 Progressive bone marrow faliure / fibrosis
o Diagnostic criteria
 Diamorphic blood picture with pancytopenia or not depend on the phase.
 Bone marrow biopsy show increase reticulin and collagen.
 Biggest spleen in medicine.
Essenthial thrombocythemia:
o The increased blood plateletes leads to episodicsymptoms: Bleeding thrombosis,
unrelated or acute leukemia in < 1 %
o Diagnostic criteria:
 Mainly increased platelets more than one million and half / mm3 .
 Exculsion of all other secondary ( reactive causes )
 Defective platelets aggregation to epinophrine collagen, or Abp.

34
 ANEMIA
Definition:
Reduction of the red cell mass due to a lowering in the Hb, conc. and or the RBC count below
the normal for age & sex.
Classification
According to the morphological feature and red cell indices:
I- Microcytic, Hypo chromic (MCV< 80 fl, MCH< 26pg ).
1. Iron deficiency.
2. Thalassemia.
3. Sideroblastic anemia.
4. Anemia of chronic diseases

II- Normocytic, Mormochromic( MCV:85 -97fl, MCH 28-34 pg).

 A plastic anemia.
 Acute hemorrhage
 Hemolytic anemia.

III- Macrocytic Anemia (MCV> 100fl).

 Megaloblastic anemia
 Non megaloblastic anemia.
 Liver disease
 Acute hemolysis.
 Acute blood loss
 Hypothyroidism.

Iron deficiency anemia

Basic metabolic features:
 Sources: red meat, liver, vegetables.
- Mainly ferric.
- Daily minimum requirements 12 mg
 Absorption:
-Preliminary reduction into ferrous in the stomach.
- Absorbed in the duodenum.
 Transport:
Attached to the protein transferrin.
 Utilization:

Incorporation into hemoglobin, myoglobin, enzymes, (cytochromes, catalases, peroxidases)

 Storage:
- Tissue iron stores : about 1gm .
- Sites: liver, bone marrow, spleen, muscles.
- Forms :
Ferritin: protein (apoferritin) shell surrounding a crystalline
core of ferric oxide-phosphate. Iron can be mobilized from this
molecule for erythropoiesis.

35
 Causes of iron deficiency
Iron imbalance when requirements outstrip the available iron because of:
1- Inadequate dietary intake.
2- Failure of absorption.
3- Excessive iron loss particularly chronic blood loss (commonest cause).

Pathogenesis:
 As the availability of iron stores becomes critical erythropoiesis becomes
abnormal, resulting in:
1- Reduction in red cell size.
2- Reduction in Hb, conc in the RBCs.
3- Fall in Hb level & PCV in the blood

 With established iron deficiency, tissue changes may occur.

Laboratory diagnosis:
1- Hemogram:
 Anemia typically microcytic, hypochromic (decreased MCV, MCH& MCHC).
 Increased red cell distribution width (RDW).
 Blood film: hypochromia, microcytosis, anisocytosis, poikilocytosis, pencil
RBCs, target cells.
 WBCs & platelet: variable.

2- Bone marrow exam:

 Erythroid hyperplasia
 Absent iron stores.

3- Iron studies:
 Decreased serum iron.
 Low % saturation.
 Increased total iron binding capacity ( TIBC).

4- Serum ferritin :
 Low, generally reflects the mobilisable iron stores& more sensitive than serum iron&
TIBC.
 May be falsely raised e.g. Malignancy or chronic disease.
5- Increased erythrocyte free protoporphyrin.
Assessment of response to treatment:
1- HB: rise by 0.1 to 0.2 g/dl/day.
2- Reticulocytic count: rises 5-11 days of treatment.
Megaloblastic Anemia
- Due to
- Vitamin B12 deficiency.
- Folic acid deficiency.

Megaloblastic anemia due to B12 deficiency

 Basic features of B12
- dietary vitamin B12
36
  Animal sources only as meat and dairy
 synthesized in the gut ( not absorbed )
 Daily requirement is 1-2 mg.
 Absorption:
- Liberated from food stuffs in the stomach.
 Bound by intrinsic factor ( synthesized by gastric parietal cell) at an acid
PH
 Absorbed through the terminal ileal mucosa.
Transport:
* Mainly by Transcobalamins.
* Important in nucleic acid synthesis.
Storage:
Primarily hepatic (about 1.5 mg).
Causes of B12 deficiency:
1-Dietary (poverty, vegetarians).
2-Gastric (pernicious anemia).
- Atrophic gastritis with production of anti parietal and anti- in intrinsic factor
anti bodies.
- Failure of intrinsic factor production.
- Gastric surgery.
- Achlorhydria
3- Intestinal: (sequestration).
a-Blind-loop syndrome
*stricture diverticule's, anastomoses.
* Abnormal gut flora competes for the available B12
b- Diphylobothrium latum infestation which is avid for B12.
C- Post – resection of terminal ileum e.g. chron's disease.
4-Congenital deficiency of transcobalamin (rare).
Pathogenesis :
 B12 deficiency →impaired nucleic acid synthesis →defective nuclear maturation of
cells having high growth rates mainly erythroblasts →Megaloblastic erythropoiesis and
neurological damage.

Laboratory investigations
Hematological:
1-Macrocytic anemia (MCV>120 fl).
2- Moderate neutropenia may occur.
3- Thrombocytopenia.
4- Blood film: contains Howel -Jolly bodies, oval macrocytes,aniso-poikilocytosis,large
hypersegmented neutrophils and giant platelet.
5- Bone marrow:
 Hypercellular due to erythroid hyperplasia.
 Megaloblasts : large than normoblasts, with large nuclei, with open stippled chromatin
and nuclear cytoplasmic asynchronism.
 Defective maturation of erythropoiesis.
 Giant metamyelocytes with hypersegmented large polymorphs.
 Atypical megakaryocytes with hypersegmented nuclei.

37
6- Serum B12 assay:
low level (<160 ng/L).
7-Biochemical tests:
a) Increased excretion of methyl-malonic acid in urine.
b) Increased serum bilirubin reflecting mild hemolysis & ineffective erythropoiesis
c) High LDH.
8- Immunological:
a) Parietal cell antibodies in 80% of patients (not specific).
b) Intrinsic factor antibodies in 60% of patients(more specific).
9- Shilling's test:
 Malabsorotion of B12 corrected by addition of intrinsic factor
 One version is the differential absorption and excretion of B12 with and without
IF, using 57 Co and 58Co labeled B12.
10- Other investigations :
Gastric endoscopy , biopsy.

Aplastic /Hypoplastic Anemia

Definition :

Reduction in hemopoietic tissue (not a consequence of bone marrow fibrosis or infilteration ),

resulting in peripheral blood cytopenia .
Basic features:
There are two fundamentall different types :
1- predictable, often reversible hypoplasia:
a) Usually a consequence of ionizing radiation and / or cytotoxic drugs, dose dependent
b) Affecting the rapidly dividing maturing hematopoietic cells rather than the
pluripotent cells .
c) Repeated or prolonged therapy may deplete the sten cells.
2- Unpredictaple, often ivrreversible hypoplasia:
a) Primary "idiopathic" or secondry to an idiosyncratic reaction to drugs or viral
infections.
b) Mainly due to hematopoietic stem cell defect / damage .
c) Sometimes related to an autoimmune or changes in the micro-environment of stem
cells.
Pathogenesis:
1- Reduction in hematopoietic tissue and increase in fat spaces of the marrow.
2- Deficient cell production, usually reflected firstly on granulocyte and platelet counts.
3- Typically development of pancytopenia.
Associations:
a) Drug ingestuion (other than anti-cancer drugs):
1- Antibiotic: chloramphanicol, sulphonanides.
2- Anti- inflamm atory : phenylbutazone, gold.
3- Anti- epileptic : hydantion.
4- Anti- diabetic : chlorporpamide,tolbutamide.
5- Anti- thyroid
6- Psychotropic
b) Exposure to chemicals:

38
 1- Hydrocerbons- benzene.
2- Insectsides.
3- Infection:
1- Viral:specially viral hepatitis(autoimmune reaction).
2- Bacterial: mainly tyberculosis.
Laboratory investigations:
a) Hematological:
(1) CBC:
- Normocytic normochromic anemia.
- Granylosytopenia (< 1.5 X 109/L).
- Reticulocytopenia
(2) Bone marrow (aspiration and biopsy):
- May reveal blood tap.
- Hypocellular, widening of fat spaces.
-Iron srores are increased especially in multi-transfused patients.
(3) Hb F may be increased
(4) Plasma iron often increased, TIBC is normal or decreased and % saturation is increased.
(5) Plasma 55 Fe clearance is prolonged and utilization for Hb synthesis is reduced.

Course and prognasis:

1- 1/3 of cases: Rapid progression to death .
2- 1/3 of cases: Chronic cytopenia →death within a year
3- Spontaneous partial or complete remission.

Special Types of Aphasia

1- Fanconi's anemia:
 Hereditary, autosomal recessive.
 Anemia is macrocytic, presenting before the age of 5years
 Associated with cytogenetic anomalies e.g. skeletal defects, skin pigmentation.
 Poor prognosis.
2- Associated with paroxysmal nocturnal Hemoglobinuria (PNH)
 PNH usually develops a variable time ofter marrow hypoplasia due to emergence of
mutant stem cell clone.
 Alternatively, an episode of hypoplasia may occur in a patient who has PNH with
hemolysis.
3-Red cell aplasia:
Selective depression of the erythroid series.
a) Blackfan –Diamond syndrome:
 Congenital, macrocytic anemia.
 Present typically within the first year.
 Spontantaneous remission, responds to corticosteroids.
b) Acquired:
i) Acute- aplastic crisis- in hemolytic anemia and post- infection,
may respond to folic acid.
ii) Chronic
 Majority assoicated with thymoma or SLE.

39
 Hemolytic anemias
- Anemia due to shortened red cell life span
- General feature of hemolysis
- Reticulocytosis (n=0.5 – 2 %)
- Indirect hyperbilirubinemia
- Increase urobilinogen
- Decrease Hemopexin

- Causes of Hemolytic anemia

- Intracorpuscular causes
- Red cell membrane defect
- Structural hemoglobin variants (Hemoglobin (Hb) S,C,D,E,O,)
- Thalassemia syndrome
- Red cell enzyme defect (G6PD deficieney, pyruvate kinase deficiency)

- Extracorpusular causes
- Nonimmune
- Immune - Isoimmune (antibodies from outside the body) e.g. in compatible
blood transfusion, erythroblastosis fetalis
- Autoimmune hemolytic anemia (self autoantibodies)
also classified into → Cold reacting Ab
→ Hot reacting Ab
Types of Hemolysis
Intravascular : Destruction of RBCs inside the blood vessels
Specific features: Hemoglobinemia, Hemoglobinuria
Occur In : -ABO incompatible blood transfusion
-G6PD enzyme deficiency
-Microangiopathic hemolytic anemia

Extravascular: - occur in reticuloendothelial cells eg: spherocytsis

Intravascular causes
1- Thalassemia
Inherited, autosomal recessive, chronic hemolytic anemia, due to globin chain imbalance

Classificatien:
According to the type of globin chain that is produced at reduced rate
- Alph thalassemia
- Beta thalassemia
Presentation :
β- thalassemia at 9-12 months (the time of globin chain switch)
α- Thalassemia:
Hb- Barts intrauterine
Hb- H Postnatal

40
 Lab. diagnosis of β- thalassemia major:
- CBC: -↓ Hb
-↓ MCV, MCH, ↑ RDW
- Frequent target RBCs
- Frequent schistocytes (fragmented RBCs )ٌ
- May be leucocytosis, thrombocytosis
- Normoblastemia
- Reticulocyte↑ (Inappropriate ↑)

- BM : Intense erythroid hyperplasia with marked ineffective erythropoiesis

- Definitive tests:
1- Hb electrophoresis→↑ HbF (10-90%)
2- quantitation of HbF
3- Molecular characterization of globin gene mutation
Hb-H:
- ↓ MCV, ↓MCH
- Hemoglobin H inclusion bodies in retics (incubation for 60 min).
- Hb-electrophresis→ Hb- H band faster than Hb-A

Lab.dignosis of thalassemia carriers

-↓ MCV , MCH normal RDW
- β thalassemia: Hb A2 ≥ 3.5
-α thalassemia: Hb – Barts 1-5% in cold blood
Definitive diagnosis by DNA study

2- Sickle Cell anemia

- Due to amino acid substitution in β globin chain
- Autosomal recessive
- Carrier has ββs ( one β globin chain affected)
- Disease has βsβs (2 β globin chain affected)
- Carrier are symptom free
- Patient suffer from many type of crises (Hemolytic, occlusive, aplastic, sequesteration )

Diagnosis:

- CBC: Normocytic normochromic anemia,

- ↓ osmotie fragilily
- Definitive Diagnosis by hemoglobin electropresis → Hbs (< 50% in carrier, > 50%
disease).
2- Sickling test : Positive
3- Solubility test : Positive

3- Congenital Spherocytosis
- Autosomal recessive hemolytic anemia
- Due to abnormality in membrane cytoskeleton →↓ Red Cell deformability
- Presentation : at neonatal, adult or even old age

41
- CBC:
- Normal MCV, MCH, and ↑ MCHC (>36 g/L)
- ↑Spherocytes in blood smear (> 15%)
- ↑Osmotic fragility (specific tests)
- Marked ↑ in retics ( up to 80%)
- Corrected by splenectomy

4- Enzymopathies
The red cell enzymes are important in the RBCs for either
1- Energy production
2- Protection of RBCs from oxidant stress (G6PD).

Type:
1- G6PD enzyme deficiency
- The most common hemolytic anemia
- Presention neonatal or infancy period
- Enzyme correction may occur at 14 ys in more than 90% of cases
- In between attacks the child is completely normal.
- Precipitating agents of hemolytic crisis
- Drugs - Infection
- Acidosis - Fave beans or their pollens
Laboratory diagnosis:
- ↓Hb level (usually 3-5 g/dl) normocytic normochromic
- ↑WBCs count.
- ↑ Platelet
- Reticulocytosis

Specific test:
Assay for G6PD enzyme activity.

2- Pyrnuate kinas deficiency :

- Congenital autosomal recessive nonspherocytic hemolytie anemia
- Hb usually 7-10 g/dl.
- Blood smear no characteristic changes
- Autohemolysis test is poorly corrected by glucose

Definitive: determination of pyruvate kinase enzyme activity

42
 Blood transfusion
BLOOD GROUPS
There are a total of 23 blood group systems and a total of over 400 diifferent
individually recognized antigenic differences. Some of the blood group systems (e.g. ABO, Rh,
Kell, Duffy and Kidd) are recognized to be more clinically important than others, i.e. in
relation to transfusion and /or pregnancy.
The ABO and RhD blood groups are so clinically important as to require prospective
testing and matching prior to transfusion.

ABO Blood Groups and ABO Typing

The blood group substances are generally determined and inherited as Mendelian
characteristics. There are three allelic genes (A, B and O) that determine four blood groups:
group O, genotype OO, group A, genotypes AA and AO, group B, genotypes BB, BO and AB,
genotype AB. Depending on which blood antigen is on the red blood cell. The reciprocal
antibody is found in the plasma (serum). The groups are named according to the antigen
present on the red cell. The serum always contains the isoagglutinins for which there are no
corresponding agglutiniogens in the red cells.
Blood Group Antigens:
There are composed of:
1- Major (ABO and Rh) which are of high clinical importance.
2- Minor (MNSs, P, Lewis, Lutheran, Kell, Kidd, Duffy, Ii…..) which are relatively of low
clinical importance (in multi-transfused patients, the corresponding antibodies are potential
causes of reactions in subsequent transfusion).
3- Pubic antigens (Vel, Gerbich, Lan); are of very high incidence i.e. present in almost all
individuals.
4- Private antigens (Wright a and Berrens a) are of very low incidence.
ABO Blood Grouping:
The following two tests should be made:
1- Determination of cell group by testing red cells with antisera of known type.
2- Determination of serum group by testing serum with red cells of known type.
Rh Blood group System:
Rh system is the most complex system in terms of number of antigens, relationship
among antigens and nomenclature proposed by different investigators.
Basic Rh System:
Basic information is derived from 5 antisera: anti-Rho (D), anti-Rh (C), anti-rh’’ (E),
anti-hr’ (C) and anti-hr’’ (C).
The Rh antibodies:
1- They differ from those of the ABO system in lacking naturally occurring antibodies.
2- They are immune in origin, which few exceptions (anti- Cw & anti-E).
3- Usually IgG.
4- They are of clinical importance in recipients, pregnant women and donors.
Blood Donor Selection:
1- Donor’s age should be 20 – 60 years old. Fasting of 3 to 4 hours. Body weight more than
50 kg.
2- None of the following should be present: Anemia, malaria, Syphilis, Viral hepatitis,
Brucellosis, coronary artery disease, Sever hypertension, Active T.B, Rheumatic fever,

43
 Diabetes mellitus, Pregnancy, Infections, Bleeding tendency, Allergies, Immunizations or
AIDS, Blood pressure and Pulse rate should be within normal.
Blood Collection:
Blood is obtained under sterile conditions by phlebotomy and is collected in plastic
bags that contain about 63 ml of CPD for 21 days storage or 70 ml CPDA-1 up to 35 days.
Cross matching:
The purpose is to detect any possible incompatibility between the recipient’s serum and
the donor’s red cells (major cross match) and between the donor’s serum and the recipient’s
erythrocytes (minor cross match) that might lead to a transfusion reaction.
An incompatible cross match is recognized by agglutination or lysis of the donor’s or
the recipient’s red cells at any phase of the cross match. An incompatible recipient, an error in
the identification of the specimen, or atypical antibodies in either donor or recipient blood.
The cross match is preceded by the serologic tests necessary to type ABO, Rh and any
other factor that appears to be indicated in the donor and the recipient.
Screening of donor and recipient for atypical antibodies is not a substitute for the cross
matches that uncovers errors in the ABO typing which screening fails to do.
Screening does not replace the minor cross match when O blood is administered to AB
patients since the minor cross match will always be incompatible.
ABO and Rh type-specific blood compatible by all cross matching procedures should
be chosen.
The Universal Donor:
Group O blood should only be used for group O recipients. In emergencies, for
exchange transfusion in the newborn infant, and if the patient’s group cannot be ascertained,
group O blood may be issued, provided that there is no high-titre antibody present. Low titre O
blood may be issued to A or B recipients, but it should not be given to group AB individuals.
Generally, O blood is considered safe if the titre of both isoagglutins is less than 1: 50. Group
O Rh-negative red cells should be used when the patient’s blood is unknown.
Transfusion Reactions:
The untoward reactions occurring during a transfusion or shortly afterwards and does
not include the long-term deleterious effects as the transmission of hepatitis or the immediate
results as acute congestive heart failure.
Whenever a transfusion reaction occurs, the transfusion must be discontinued at once.
Transfusion reactions can be divided into hemolytic and non-hemolytic.
1- Non-hemolytic Reactions include:
a- Allergic reaction: the most common and the least severe reaction. It may be due to some
soluble substance in donor plasma. Antihistaminics and cortisone should not be added to
the blood, but rather administered to the patient directly.
b- Febrile reactions: the use of disposable equipment and strict adherence to blood bank
regulations eliminate febrile reactions. Some reactions may be due to antileukocyte or
antiplatelet antibodies. Infusion of cytokines performed by leucocytes in the donor stored
blood or provocation of cytokine release in the recipient may lead to reactions.
c- Bacterial reactions: the bacteria are usually gram-negative bacilli. The patient’s reaction is
often severe and anaphylactoid in nature or characterized by profuse bleeding.
2- Hemolytic Reactions:
The most serious, are due to the destruction of red blood cells (donor) by antibodies in
the plasma of the recipient. The severity of the reaction will depend on the amount of blood
transfused, the type and avidity of the antibody. In a patient under anesthesia, unexplained
oozing from the operative wound may be the first sign of a reaction.
44
 There are some hemolytic transfusion reactions that are latent or delayed, other
reactions are nevertheless due to an antigen-antibody reaction. Serious hemolytic transfusion
reaction result in hemoglobinemia, methemoglobinemia, hemoglobinuria, hyperbilirubinemia
(3 – 6 hours after transfusion).
Dangers of Transfusion:
a- After a single transfusion:
1- Transfusion reactions.
2- Overloading of circulation due to speed or excessive volume of transfusion.
3- Temporary arrest of erythropoiesis.
4- Formation of immune antibodies to red cells, white cells, platelets and / or clot factors.
5- Thrombophlebitis.
6- Transmission of disease e.g. hepatitis, malaria, infectious, mononucleosis (syphilis can not
be transmitted by stored blood) and AIDS.
7- Hemosiderosis and post transfusional hemochromatosis.

b- During Massive Transfusion:

1- Fall in level of clotting factors (fibrinogen, calcium, platelets).
2- Increase in anticoagulants (heparin, fibrinolysins).
3- Increase in potassium as donor’s cells release their potassium.
4- Citrate toxicity and fall in ionized calcium.
5- Overloading of circulation.

Table (5): Infectious agents transmitted by blood transfusion:

PATHOGEN TRANSMITTED ILLNESS

Hepatitis B virus (HBV) Hepatitis
Hepatitis C virus (HCV) Hepatitis
Cytomegalovirus (CMV) Pneumonia/ hepatitis
Human immunodeficiency Virus (HIV) AIDA
Human T lymphotrophic virus (HTLV) T cell leukemia
Treponema pallidum Syphilis
Plasmodium sp Malaria
Trypanosomes cruzi Chaga 's disease
Various bacterial infections Bacteraemic shock

Table (6): Virus transmission risk of blood components

NO RISK SLIGHT BUT POSSIBLE LOW BUT FINITE

RISK
RISK

Albumin Pooled plasma products (e.g. Red cells platelets

immunoglobulin Factor VIII, etc. FFP/Cryo Granulocytes
products

45
Indication for direct coomb’s test:
1- Diagnosis of erythroblastosis foetalis.
2- Diagnosis of autoimmune hemolytic anemias.
3- Diagnosis of transfusion reaction due to incompatible blood.
Indication for Indirect coomb’s test:
1- Cross matching to detect incompatibility.
2- Detection and identification of irregular antibodies.
3- Detection of antigens such as Rh, Kell, Duffy and Kidd.

Common Blood Components Used in Hemotherapy

Blood component Indications
* Whole blood Massive transfusion, brisk bleeding
* Packed RBCs Chronic anemia
*Washed RBCs AS packed RBCs, useful for preventing febrile and allergic
(leukocyte- poor RBCs) reactions due to leukocytic or plasma proteins & preventing
anaphylactic reaction in IgA deficient recipient
* Frozen deglycerolized Rare blood group and auto transfusion, prevention of HLA
RBCs sensitization and also as indications of washed RBCs
Random donor platelet Quantitative or qualitative platelet disorders, bleeding (slow
concentrate ooze) due to severe thrombocytopenia, or prophylactic
therapy
Single donor platelet As in random-donor platelets where there are no enough
concentrate by apheresis random-donor platelets. Immunological refractory patient,
when given as HLA match with recipient
* Single-donor Septic, severely granulocytopenic patient unresponsive to 48
granulocyte concentrate hours of antibiotic therapy
by apheresis
* Fresh frozen plasma Bleeding patient with multiple coagulation deficiency
problems secondary to liver disease, DIC, dilutional changes
from massive transfusions, for treatment of factor V, VII, IX
and XI deficiency
* Cryoprecipitate Hemophilia A, Von Willibrand disease, factor XIII
deficiency, hypofibrinogenemia

46
 Clinical Microbiology

It is the study of inter-actions between human and microorganisms that include

bacteria, viruses, chlamydia, rickettsia, mycoplasma and fungi. Basic features of them are
included in table 7.

Table (7): Types of microbes

Nucleic Multiplication Seen by Cell wall Sensitivity to
acid LM* antibiotics
Viruses DNA or Intracellular (only) No No No
RNA
Chlamydia DNA + Intracellular (only) No No Yes
RNA
Rickettsia DNA + Intracellular maybe Rudimentary Yes
RNA (mainly)
Mycoplas DNA + Intra & Yes No Yes
ma RNA extracellular
Bacteria DNA + Intra & Yes Yes Yes
RNA extracellular
Fungi DNA + Intra & Yes Yes No
RNA extracellular
* Light Microscopy

Sampling
Collection of good quality specimens:
1- Optimal time of collection:
- Specimens for culture must be collected before start of antibiotics.
- Blood cultures and blood films for malaria are best collected just as the temperature starts
to rise.
- Specimens for electron microscopy or isolation of viruses are best collected in the most
acute stages of the disease.
2- Correct types of specimen:
- In bacterial meningitis, collect blood cultures as well as CSF.
- In gonorrhea, cervical, urethral and rectal swabs should be collected rather than high
vaginal swabs.
- Pus is always preferable to swabs.
3- Specimens with minimum contamination from normal flora:
- Make sure to collect sputum not saliva from a patient with pneumonia and three
consecutive morning samples for T.B.
- Mid-stream specimens of urine need careful collection to avoid contamination from
perianal or genital flora.
- Throat swab should not touch the buccal mucosa or tongue.
- A high vaginal swab should be collected using vaginal speculum.
- Blood or CSF cultures can be contaminated by skin flora unless aseptic techniques are
used.

47
4- Clearly labeled and safe specimens:
- Samples must be placed in leak-proof containers.
- T.B, enteric infections, HBV and HIV are examples of microbiological hazards to lab.
personnelle.
- Any specimen from a patient with suspected hepatitis or HIV needs to have a biohazard or
inoculation risk label clearly attached.
5- Transport to lab:
- Specimen should be fresh as possible.
- Many organisms do not survive for long time e.g. Gonococci, Hemophilus, Bacteroids,
anaerobic cocci and most viruses.
- Some organisms contaminate specimens from normal flora e.g., coliforms or coagulase-
negative staphylococci may rapidly grow at room temperature.
- Urine or sputum samples should reach lab. within 2 hours. If any delay, refrigerate.
- Transport media e.g. Stuart's transport media should be used for transport of pus or swabs
for bacterial cultures when delay of more than 1/2 hour or when Neisseria is suspected.
- For eye, genital or pertussis infections, bedside direct culture is done.
- Few specimens should be fresh enough to still be warm e.g. CSF from patient with
suspected meningitis, otherwise Meningococci may rapidly die.
- Viral transport media should be used for transporting swabs for viral cultures. CSF for
virological investigations should be examined within 2 hours or kept at -70 C.
- For obtaining good quality sample for microbiologic examination, certain precautions
should be considered:
- Collection of specimens before antibiotic therapy.
- Sample collection as early as possible in the disease.
- Containers used for collection should be sterile, tightly capped, and clearly labeled.
- Cleaning the site of infection.
- Rapid transporting of the specimens to the lab.
Urine Sample:
- Morning sample.
- Washing the site with soap &water three times, and in female from front to backward.
- Mid stream sample for routine culture & 24h for T.B. (3 times).
- In children:
 Suprapubic aspiration.
 Adhesive bag
- If the patient is already catheterized, catheter sample can be used.
Blood Sample:
- During the peak of fever.
- Sterilize the site of venipuncture with alcohol 70 % & iodine.
- 5 ml of blood are collected in adult and 2 ml in infants.
Body Fluids:
- Sterilize the site with alcohol 70 %.
- Transport rapidly to the lab, especially for CSF.
Sputum:
- Morning sample is preferred.
- Wash the mouth with water and brush the teeth.
- Sputum is preferred rather than saliva.
- In infants, gastric lavage can be used.
- Samples for T.B. are 3 consecutive morning samples
48
Throat swab:
- Wash the mouth with water.
- Depress the tongue with a tongue depressor.
- Take the sample from the posterior pharynx or tonsils.
- The swab should not touch the buccal mucosa.
Wounds:
- Clean the wound with sterile gauze soaked in sterile saline.
- Squeeze the wound.
- Swab pus or squeezed exudate.
Eye:
- Wash the eyes by water and soap.
- Apply swab from lower conjunctival fornix upwards.
Ear:
- Clean the site with sterile saline.
- Dry the area.
- Apply swab to get the specimen.
Stool sample:
- Stool is collected in leak proof sterile container into which the excreta can be passed
directly.
- Alternatively, rectal swab may be taken.
- For Cholera, the faeces is sent in bile peptone medium to prevent death of vibrio Cholera.
Gynecological Sampling:
- Cervical swab is preferred rather than vaginal ones.
- Dry, sterile speculum should be used.
- Bimanual examination is not preferred.
Sampling for Mycological examination:
- Clean the site with alcohol 70 %.
- Sterile scalpel is used for scrapping.
- Sterile scissor is used for trimming the nails.
- Scales are transferred in clean dark paper.
Sampling for Clinical Virology:
- Sample should be collected during the most active stages of the disease.
- Samples as urine, stool, C.S.F or sputum are transported directly to the lab, while swabs
from throat, nose, vesicles, and cervix are transported in viral transport medium.
- Biohazard label should be used for containers of HBV, HCV and HIV.

Procedures in Microbiological laboratory

I- Macroscopic examination:
Naked eye inspection of specimens helps in diagnosis.
- Saliva instead of sputum should be discarded.
- Greenish pus  Pseudomonas.
- Foul smelling of pus  anaerobic infections.
- Clot formation in CSF  T.B infection.
- Presence of parasitic segments in stool  diagnostic for parasitic infestation.
- Rice water stool  Vibrio cholera.
II- Microscopic examination:
Provides immediate data in life threatening infections e.g. pneumonia and
49
 meningitis, as the cultures takes days or cannot be performed in some conditions e.g.
Treponema pallidum.
 Wet mount examination: Saline preparation e.g. Trichomonas vaginalis, KOH for fungal
examination, Indian ink for Cryptococcal capsule.
 Gram stain more widely used:
- Differentiate between G -ve & G +ve (table 8).
- Judge adequacy of samples e.g. pure sputum samples contains less than 10 squamous
epithelial cells with low magnification lens.
- Purulence of samples.
- Rough guide for quantitation of bacteria.
 Zeihl-Nelson’s (Z.N.) stain: for acid-fast bacilli (Mycobacteria).
 Auramine-Rhodamine stain: for mycobactria.
 Silver stain: for T. Pallidum, pneumocystis carinii.
 Periodic Acid Schiff (PAS): for fungi.
 Acridine Orange Vital dye: for DNA of bacteria.
 Giemsa stain.
 Dark ground illumination microscopy: for Treponema pallidum.
 Immunofluorescent microscopy and Electron microscopy: for rapid viral diagnosis.
III- Culture:
- Aerobic.
- Anaerobic.
- Microaerophilic.
- Mycobacterial culture.
- Mycoplasma culture.
- Chlamydia culture.
- Viral culture.
Identification of culture includes:
- Biochemical reactions  manual, semi automated and automated.
- Serotyping.
- Molecular biological techniques.
IV- Direct antigen detection:
- Latex tests: latex coated with antibody for pneumococci.
- Co-agglutination, protein A of Staph-aureus labeled with specific IgG for direct antigen
detection.
- Counter immune electrophoresis e.g. for Cryptococcal antigen.
- Immuno-fluorescence and immuno-cytochemistry.
- ELISA.
- RIA.
V- Molecular biology:
a) Direct nucleic acid techniques:
 Plasmid profile e.g. for epidemiological study and antibiotic resistance.
 Restriction endonuclease enzyme.
b) Nucleic acid probe assay:
 Filter.
 Liquid.
 Southern blot.
50
 Northern blot.
 In situ hybridization technique e.g. FISH.
VI- Serodiagnosis and immune status tests:
- Tests for detection of specific antibody for infectious agents including IgM, IgG, IgA.
- Rising antibody titre (at least 4 folds) is diagnostic.
- Precaution: samples are withdrawn at acute and convalescent stages (within 14 days).
Techniques: e.g.:
Enzyme Linked Immunosorbent Assay (ELISA)
Radio-immuno-Assay (RIA)
Recombinant Immuno-Blotting Assay (RIBA) test for HCV
Latex techniques
Hemagglutination inhibition test
VII- Skin tests: For detection of hypersensitivity type, e.g. tuberculin test.
VIII- Laboratory guidance for anti-microbial therapy:
In vitro:
 Disc diffusion method.
 Dilution method.
* Broth
Macro dilution (tube method).
Microdilution  ready to read plates, semi automated or automated, it can be used for
aerobic & anaerobic bacteria, Mycobacterium T.B. and fungi.
It is valuable in determining minimal inhibitory concentration for antibiotics and
determine its action whether bacteriostatic or bactericidal.
B) In vivo:
Determination of the therapeutic level of antibiotics in biological fluids to avoid
toxicity, to achieve good therapeutic level and to know the compliance of the patient by
immunological and bacterial tests.

Clinical Bacteriology
Pyrexia of unknown origin "PUO"
Definition:
A case presented with pyrexia as a predominant clinical feature of 10 days or longer
duration without an obvious cause. It may be acute (if pyrexia persists for few days) or chronic
(if pyrexia persists for 3 weeks or longer).
Causes:
I- Infective:
A) Non specific e.g.:
- Cryptic abscesses in liver, abdomen, pelvis and retroperitoneal or mediastinal sites.
- Infective endocarditis.
- Urinary tract infection.
- Ear, sinus or dental infections.
- Osteomyelitis.
B) Specific e.g.:
- Bacterial: T.B., brucellosis, typhoid F., leptospirosis (Weil’s disease), secondary syphilis.
- Viral: viral hepatitis, glandular fever, yellow fever, CMV, HIV.
- Rickettsial: typhus and Q fever.
- Chlamydial and Bortonella: psittacosis and cat scratch fever.
51
- Fungal: candidiasis, histoplasmosis, cryptococcosis and aspergillosis.
- Protozoal: malaria, amaebiasis, toxoplasmosis, trypanosomiasis and leishmaniasis.
- Helminthic: filariasis and fasciola.
II- Non-infective:
A) Hematological: e.g. leukemia, purpura, hemolytic anemia and lymphoma.
B) Autoimmune and collagen: e.g. rheumatic fever, rheumatoid arthritis, SLE,
polyarteritis nodosa, dermatomyositis and ulcerative colitis.
C) Endocrine: e.g. thyrotoxicosis and familial mediterranean fever.
D) Malignancy: sarcoma, carcinoma, hepatoma and hypernephroma.
E) Miscellaneous:
- Liver cirrhosis and alcoholic hepatitis.
- Gout (rare).
- Granulomas e.g. sarcoidosis, Crohn's disease.
- Drug reaction.
- CNS abnormalities e.g. infiltration of heat regulating center in
hypothalamus by neoplasm or granuloma (rare).
- Malingering.
Diagnosis of PUO:
I) History:
- Age: Child: suspect urinary and respiratory- tract infection.
Young adult: suspect glandular fever, T.B.
Elderly: suspect neoplasia.
- Duration: to categorize whether acute or chronic.
- Occupation: farmers, veterinarians are exposed to brucellosis, Q fever and leptospirosis
(Sewers).
Laboratory personells and surgeons are exposed to viral hepatitis B & C and HIV.
- Recent travel to endemic area e.g. malaria, typhoid, visceral leishmaniasis, brucellosis,
viral hemorrhagic fever, filariasis.
- Contact with animals e.g.: toxoplasma (cats), psittacosis (birds).
- Drinking of unpasteurized milk: brucellosis, bovine T.B, Q fever.
- Drug intake may cause febrile drug reaction.
- Contact with infectious case: e.g. T.B, AIDS, hepatitis, typhoid.
II) Physical examination:
Splenomegaly, lymphadenopathy, skin rash, PV, PR examination are done.
* Temperature Chart:
- Intermittent: regular (may be with rigors) in malaria.
irregular in cholangitis.
- Remittent in patient with collection of pus.
- Relapsing in brucellosis.
- Stepladder (Hectic) in typhoid.
- Non specific pattern in T.B, infective endocarditis.
III) Investigations:
A) Laboratory:
1) Hematological:
- Hb for anemia.
- Platelets for purpura.
- WBCs: total and differential count.
 Neutrophilia in pyrogenic infection.
52
  Neutropenia in malaria; typhoid; leishmaniasis and SLE.
 Lymphocytosis in viral infection, typhoid and brucellosis.
 Monocytosis in TB; atypical monocytes in IMN.
 Blast cells in leukemia.
- Thin and thick blood film in malaria; filaria; trypanosomiasis.
- ESR: > 100 mm/h in T.B; collagen and malignancy.
2- Microbiological:
- Blood culture for typhoid, brucellosis, leptospirosis and infective endocarditis.
- Urine and stool culture for UTI, gastrointestinal infections, salmonellosis, brucellosis,
leptospirosis. In sterile pyuria: T.B. of genitourinary tract is suspected.
- Throat swab if rheumatic fever is suspected, negative culture not exclude rheumatic fever.
- Bone marrow culture for typhoid, brucellosis, T.B.
- Serology:
Paired serum samples are required to look for rising antibody titre. Occasionally, a
single high titre maybe suggestive of recent infection e.g. IgM for toxoplasma.
Some serological tests for diagnosing PUO:
- Widal test for typhoid (diagnostic titre > 1/80).
- Brucella agglutination and CFT (diagnostic titre >1/80).
- ASO titre for rheumatic fever (diagnostic titre > 250 Todds U/ml).
- Latex co-agglutination to detect Ag as Streptococcal, Staph. species, Neisseria, Candida
and Rota viruses.
- ELISA techniques for detection of microbial antigens e.g. Chlamydial Ag, HB Ag & HIV
Ag and microbial antibodies e.g. CMV Ab, HBAb and T.B. (IgA, IgG, IgM).
- Fluorescent treponemal antibody, fluorescent amaebic antibody and fluorescent leishmanial
antibody test.
- PCR technique for HCV-RNA, HBV-DNA, T.B-DNA, CMV & HSV.
3) Biochemical:
- Liver function tests.
- Thyroid function tests.
- Alpha Feto Protein (AFP) for hepatoma.
- Uric acid for gout.
B) Radiological and imaging:
X-rays, IVP, US, ECHO, MRI and CT.
C) Biopsy:
Bone marrow, lymph nodes, liver and transbronchial lung biopsies for culture and
cytology.
D) Skin tests: e.g.
- Mantoux test for TB.
- Kveim test for sarcoidosis.
- Histoplasmin test for Histoplasmosis.
- Frei test for Chlamydia (lymphogranuloma venereum).

53
 Septicemia
The blood is normally sterile.
- Septicemia denotes bacterial invasion of the blood stream from a focus of infection (urinary
tract, chest… etc) resulting in fever, rigors, mental confusion, tachycardia and hypotension.
Causative organisms:
* Gram negative septicemia:
- Mainly caused by E. coli. Others (klebsiella spp., Proteus spp., Enterobacter spp.,
Salmonella spp., Hemophilus influenza, Neisseria Meningitidis, Pseudomonas aeruginosa,
Serratia, Neisseria gonorrhoea).
* Gram positive septicemia:
- Staphylococci: Staph. aureus, Staph. epidermidis
- Streptococci: Strept. pneumoniae, Strept. pyogenes.
* Anaerobic septicemia:
- Bacteroid fragilis and other gram negative non-sporing anaerobic species.
* Others:
 Fungi.
- Candida albicans.
- Aspergillus.
 Rickettsia & Coxiella causing typhus.
 Viruses:
- Arboviruses. -Lassa fever Virus.
Microbiological investigation of speticemia
- Blood Cultures: conventional, automated.
- Culture of other infected sites e.g.: sputum, urine, faeces and infected burns.
- Serological tests.
Meningitis
 Main clinical features:
Headache, irritability, fever, neck stiffiness with +ve kering's sign, nausea, vomiting
and coma.
 Causative organisms:
A) Bacterial:
- N. meningitidis.
- Hemophilus influenza. Three Iry pathogens
- Strept. pneumoniae.

- Mycobacterium T.B.
- Coliforms.
- B. hemolytic streptococci.
- Listeria monocytogenes. In neonates
- Salmonella.
- Pseudomonas.

* Spirochaetes:
- Leptospira.
- Treponema pallidum.
- Borrelia.
B) Viral:
54
- Entero-viruses: Echo, Polio.
- Herpes, Varicella-zoster.
- Adeno-viruses.
- HIV.
C) Fungal:
- Cryptococcus neoformans.
- Candida.
- Aspergillus.
- Mucor.
D) Amoeba:
Naegleria.

Table (8): CSF changes in meningitis and the differential diagnosis.

Appearance Cytology Protein Glucose D.D
0-5 40 -50
Normal Clear <50 mg%
lymphocytes mg% or ----
100-2000 1.Bacterial
Purulent Turbid 90%   2. Amoebic.
polymorphs 3. Brain Abscess
1. Viral.
2. Partially
Clear or antibiotic
15-500
Aseptic slightly  Normal treated
lymphocytes
turbid bacterial
3. Leptospiral
4. TB/Fungal
Clear or
25-500 1. TB.
slightly
lymphocytes 2. Cryptococcal
T.B turbid  or  
+ 3. Brain
coagulum
polymorphs abscess
on standing

* Investigations:
1. CSF and blood culture on chocolate or blood agar in presence of 5 – 10% Co2, before start
of antibiotics. The organsim maybe isolated from blood when CSF culture is negative or
when there is contraindication to lumbar puncture.
2. Nasopharyngeal swab culture: may be the only way to isolate meningeococci.
3. Antibiotic sensitivity tests.
4. Latex agglutination.
5. PCR-DNA of microorganisms e.g. TB DNA.

Rheumatic fever
Rheumatic fever and acute glomerulonephritis are abnormal immunological reaction to
streptococcus, B-hemolyticus, group A infection. The most helpful tests are:
- Throat swab: Negative culture dose not exclude rheumatic fever.
- Antistreptolysin-O (ASO): Starts to appear in the second week after infection, reaches
maximum titre after 4 – 6 weeks. Rising titre is better in diagnosis than single test.
55
 Measurement of the antistreptolysin-O level is of diagnostic importance in streptococcal
infections and their sequelae.
> 400 T.Us/ml  definitely elevated.
> 250 T.Us/ml  diagnostic.
< 50 T.Us/ml  exclude Rh. Fever.
- C-reactive protein (CRP) is an acute phase reactant protein and it is found in low
concentrations in the serum of healthy subjects (up to 5 mg/l). Elevation of CRP is non
specific, similar to the erythrocyte sedimentation rate (ESR), it rises early and returns to
normal before the ESR.
- CRP is raised in active inflammations (acute rheumatic fever, pneumonia, active TB., acute
tonsillitis, scarlet fever, and mumps), tumours, liver cirrhosis and tissue destruction such as
myocardial infarction. CRP is important differential diagnosis of coronary insufficiency
(CRP is –ve) from myocardial infarction (CRP is +ve).
- Also CRP is important than ESR in following the course of rheumatic fever as ESR may be
normal in rheumatic activity in the presence of congestive heart failure. ESR may increase
without inflammation e.g. anemia pregnancy etc… In chorea, CRP is –ve, if +ve it indicates
carditis.

Opportunistic infections
Infections caused by opportunistic organisms which are organisms characterized by low
pathogenicity causing infection in immunocompromized persons.
Examples:
Bacterial:
- Pseudomonas aeruginosa.
- Staph. epidermidis.
Viral:
- Cytomegalo-virus.
Fungal:
- Candida albicans.
Protozoal:
- Pneumocystis carinii
Diagnosis
1- Clinical history
2- Full clinical examination
3- Laboratory
 General
 Microbiological
Culture of organism and identification.

DIPHTHERIA
It is caused by corynebacterium diphtheriae, its exotoxin produces the clinical
manifestations. Infection occurs by droplet transmission from patients or carriers. The
organism is of three strains differing in virulence, diphtheria gravis, intermedius and mitis.
Diagnosis:

56
- Swab: Bacteriological examination detects the organism. Negative results may be obtained
in some cases if antiseptics were applied on the throat in the previous four hours, or there is
secondary infection. If you suspect diphtheria, start treatment immediately and do not wait
for result of the swab. However, swab should be done to decide management of patient’s
contacts.
- Identification of Diphtheria bacilli:
(a) Gram,s stained smear: they are gram-positive rods which are usually club-shaped
and show metachromatic granules by methylene blue stain.
(b) Culture on loffler’s serum and blood tellurite media.
(c) Fermentation tests: Diphtheria bacilli are able to produce acid from glucose and
levulose but are unable to do so from sucrose.
- Schick test: Intradermal injection of 0.2 cc. of saline solution of toxin containing 1/50
M.L.D. A positive result indicates susceptibility; a negative one indicates immunity. All
cases of diphtheria are positive in the beginning of the attack and become negative during
the illness. Susceptibles among patient’s contact should be managed to prevent their
infection.

Pneumonia
The characteristic symptoms of pneumonia include cough, pleuritic chest pain and fever.
Types of pneumonia:
- Bacterial: Strept. pneumoniae, Staph. aureus, klebsiella, Pseudomonas aeroginosa.
- Legionnaire’s disease: Legionella pneumophila
- Tuberculosis.
- Atypical which may be caused by Virus, Chlamydia, or Mycoplasma.
- Rare types caused by fungi, protozoa or worms.
Investigations
- Sample collection should follow the previously described precautions.
- Sputum culture for diagnosis and antibiotic susceptibility .
- Sputum, blood and urine for pneumococcal or Legionella antigen are carried out specially
when antibiotics have already been started.
- Blood culture for one or two sets from all patients with pneumonia should be performed.
- Serum sample should be collected during acute stage and antibodies convalescent stage for
Mycoplasma, Chlamydia ….etc.
- Blood for total and differential count may be useful for distinguishing severe bacterial from
non bacterial causes of pneumonia.

TUBERCULOSIS
Tuberculosis continues to be a very major problem throughout the world causing
serious morbidity and mortality.
Causative organism:
Mycobacteria specially Mycobacterium tuberculosis .
Methods of infection:
- Droplet infection is the most common.
- Ingestion of contaminated food (specially milk) is another important route.
57
- The lungs are the most commonly affected site (pulmonary tuberculosis) but any system of
body may be affected e.g. . kidney, bone, prostate, peritoneal cavity, brain …etc.
Laboratory diagnosis:
Collection of specimens:
- For pulmonary infection: at least 3 successive morning sputum samples should be obtained.
- For suspected urinary tract infection, 3 consecutive early morning urine samples should be
collected.
- For suspected peritoneal infections, as much as possible is taken from peritoneal fluid.
(Ι) Direct methods:
 Zeihl-Neelsen’s stain (Z.N.).
Advantages:
 Easy to perform.
 Cheep & rapidly diagnostic.
 Has very high specificity (about 100%).
Disadvantages:
 Low sensitivity (30% - 40%).
 Cultures: The gold standard for diagnosis and more sensitive method than ZN. They are
either conventional culture on lowenstein Jhensen medium or culture using automated
system (Bactec system).
** Culture on lowenstein Jhensen:
Essential for species identification & susceptibility.
Advantages:
 Good sensitivity (60%-70%).
 Important to perform species identification and susceptibility.
Disadvantages:
 Time consuming taking 4-6 weeks.
** Culture on automated system (Bactec):
 Can be used for detection, identification and susceptibility. It is not time
consuming.
** Polymerase Chain Reaction (PCR):
Depends on the detection of DNA of Mycobacteria after its amplification.
Advantages:
 Very sensitive (up to 95%).
 Result may be obtained in the same day.
Disadvantages:
 Can’t differentiate between living and dead organisms.
 May have a false positive results.
(II) Indirect method:
* Serodiagnosis:
For detection of antibodies e.g. TB IgG, TB IgM &TB IgA.
Advantages:
 Easy and rapid method.
 Can help in diagnosis specially in closed pulmonary lesions.
Disadvantages:
 Has false positive and false negative results.

* Tuberculin test:
58
- Measures delayed type hypersensitivity to the tubercle bacilli.
- Carried out by intradermal injection of purified protein derivatives (PPD) in the forearm.
- Positive reaction is characterized by induration of 10 mm or more (after 48 –72 hours).
Advantages:
 Detection of previously infected persons.
 Detection of newly infected persons when there is a positive reaction in
a previously negative person.
 Failure to react has a value in excluding T.B.
Disadvantages :
 False negative results are seen after faulty technique , vaccination
against measles, poliomyelitis and uptake of drugs e.g. steroids &
alcohol . Also there is a negative reaction in military tuberculosis , old
age, advanced carcinoma , leukemia , syphilis and hyperthyroidism.

Gastroenteritis and food poisoning

Acute condition occurring singly or in a group of patients sharing the same food and
characterized by vomiting and diarrhea except in botulism in which there is constipation.
Causes:
1- GIT infection:
- Salmonella. - E-coli.
- Staph aureus. - Shigella.
- Clostridium. - Yersinia.
- Campylobacter. - Rota viruse.
- Vibrio para heamolyticus. - Bacillus cerus.
- Clostridium botulism.
2- Other site infections:
- Otitis media. - RTI.
- UTI. - Septicemia.
3- Non infectious causes:
- Drugs such as tetracyclins. - Coeliac disease.
- Malabsorption. - Allergic conditions.
Diagnosis:
- Symptoms:
 Acute diarrhea in children.
 Acute diarrhea with or without vomiting usually due to G IT infections,
and may also occur due to other diseases affecting other sites.
- Lab.:
 Samples include vomitus and 3 stool samples.
 Direct microscopic examination on ordinary and selective culture media.
 Identification of the isolated organisms by biochemical reaction, phage
typing, specific antisera or automated systems.
* Infantile gastroenteritis:
The important causative agents are Rota virus, Echo virus, and Adeno virus.
*Bloody diarrhea:
The important causative agents are the following.
- Salmonella.
- Shigella (bacillary dysentry).
59
 - Vibrio parahemolyticus
- Campylobacter jejuni.
* Cholera:
It is caused by Vibrio cholera. Organism is present in intestine & stools and symptoms
are due to absorption of its toxins.
Diagnosis:
1- Detection in stools of vibrio (Gram negative comma shaped bacilli with characteristic
jumping motility) on TCBs medium.
2- Identification by agglutination with cholera antiserum.
* Campylobacter jejuni:
- Gram negative curved microaerophilic bacilli which have been found to be responsible for
about 7 – 10 % of all cases of gastroenteritis can be cultured from stool on selective
Skirow’s medium.
* Yersinia entrocolitica:
- Gram –ve aerobic and facultative anaerobic bacilli affecting human by ingestion of infected
milk or meat giving rise to acute terminal ileitis, mesentric lymphadenitis, and appendicitis.

Enteric fever
- Includes typhoid fever caused by Salmonella typhi.
- Para typhoid fever caused by Salmonella para typhi A,B and C.
Diagnosis:
(1) Isolation of the organism:
- Blood culture which is positive in the first week in 90% of cases. It is also positive in 1st
week, also still +ve in 2nd an 3r wks.
- Stool and urine culture: The organism can be isolated from stool in 2nd and 3rd wks and
from urine in the 2nd week of disease.
- Urine examination may be repeated.
(2) Serologic method (widal test):
- Ab appears in patient serum starting from 7th to 10th day of illness and can be detected by
latex or tube agglutination test.
- The test is considered positive when there is rising of the titer 3-4 folds, however a single
raised O Ab titre of 1/160 or more during the first 2 weeks of illness is considered positive.
- The important antigens used in the test are:
 O Ag which is common for both Salmonella typhi and para typhi.
 Salmonella typhi H Ag for Salmonella typhi.
 Salmonella A for Salmonella para typhi A.
 Salmonella B for Salmonella para typhi B.

Brucellosis
Members of this genus are pathogenic to animals from which they are transmitted to
man causing brucellosis or undulant fever.
Br. melitensis affect goat & sheep.
Br. abortus affect cattle.
Br. suis affect pigs.

60
No man to man spread of infection.
Morphology: Gr –ve short bacilli.

Diagnosis:
- Cultures: Blood culture is positive in the first week, urine culture is positive in 10% of
cases after 2 weeks but stool culture is not successful.
- Blood count: Normal or leucopenia with relative lymphocytosis or monocytosis.
- Agglutination reaction: Positive from the second week in titres of 1 : 80 reach maximum 3-
6 weeks and disappears few months or years after recovery.
- Complement fixation test to detect IgG is useful in chronic cases.
- RIA and ELISA to detect specific IgM antibodies.

Helicobacter–pylori
- Gram negative curved microaerophilic bacilli, proved to be a causative agent for chronic
gastritis, gastric ulcer, duodenal ulcer and even gastric cancer.
Diagnosis:
- Gastric biopsy specimen is used for direct microscopic examination and cultivation.
- Serologic diagnosis including latex test, and ELISA.
- Molecular biological tests such as PCR.

URINARY TRACT INFECTIONS

Diagnosis of urinary tract infection is based on the detection of bacteria (bacteriuria)
and pus cells (pyuria) in urine.
ORGANISMS:
- Escherichia coli - account for about 90%.
- Proteus species - predispose to stone formation (urease render urine alkaline).
- Klebseilla, Enterobacter, Serratia, Citrobacter and Pseudomonas: These organisms are
common in hospital, following catheterization. They are often resistant to popular
antibiotics.
- Streptococcus faecalis.
- Staphylococci (epidermidis, aureus).
- Candida species (follow catheterization-occasionally blood invasion).
- Adenovirus (in children).
Methods of urine collection:
- Voided (midstream - clean catch urine).
- Suprapubic bladder puncture (in neonates).
- Urethral Catheter.
 If the patient is already catheterized.
 Not from the bag.
- Ureteric catheter (during urological surgery).

61
Determination of white cell count:
- Centrifuged deposit (less than 5-10 cells/HPF).
- Counting chamber (less than 10 cells/uL).
- Leucocytes estrase (paper strip).
Methods of quantitating bacteria in urine: (significant bacteriuria > 105/mm3):
- Calibrated wire loop.
- Blotting paper strips.
- Flooded slopes (dip slides).
- Drops from calibrated pipetts.
- Counting chamber.
- Presence of nitrate in urine (paper strip).
Plating media for bacterial culture:
* Sterile pyuria: is defined as pyuria in the absence of bacteriuria.
Causes:
1. Extra urinary infection (vaginal contamination, prostatitis, perinephric abscess).
2. Urinary tract infection: The organism may fail to grow because of:
 Antibiotic or antiseptics in urine.
 Intermittent excretion of organisms in chronic cases.
 Infections due to organisms not cultured on ordinary media (Adeno virus,
Schistosoma, Leptospira, renal turberculosis) .
3. Non infectious diseases e.g. glomerulonephritis.

SYPHILIS
Diagnosis:
- Demonstrating the organism: Serous fluid from the primary ulcerative lesion (chancre) is
inspected by dark ground microscopy for Treponema pallidum. However, the patient is not
usually seen during the primary stage.
- Serologic diagnosis: This consists of two groups of procedures:
1- Non Treponemal Antigen Tests (Cardiolipin antibody tests):
The antigen consists of cardiolipin, purified lecithin, cholesterol and alcohol.
a- Wasserman and Kahn are no longer widely used.
b- The VDRL slide test.
c- Rapid plasma reagin tests (R.P.R.) as they are more sensitive but less specific
than VDRL used as screening test.
Any positive test must be followed by other more specific procedure.
2- Treponemal antibody tests: more specific and more sensitive
In this group an antigen specific for the organism is used. According to the origin of the
antigen:
a- Treponema Pallidum Hemagglutination Test (T.P.H.A): In this test, Tr.
Pallidum Ags. are attached to tanned red cells which are agglutinated by
antibodies in the patient serum. It is almost sensitive as FTA Abs. However, the
test is not a good monitor of therapy and VDRL is recommended for this
purpose.
b- Treponema Pallidum Immobilization (TPI) Method: a strain of living
Treponemae pallidum cultured in rabbit testis are mixed with patient's serum
62
 and complement. Determine after incubation the proportion of immobilized
Treponemas relative to the control. It is technically difficult and not well
standardized.
c- Reiter Protein Complement Fixation Test (RPCFT): The antigen is of the
nonpathogenic Reiter Treponema which are easily cultivated in the laboratory.
It is more widely available than the first but only 50 percent of sera from late
syphilis give a positive test.
d- Fluorescent Treponemal Antibody Absorbed (FTA-ABS) method: The antigen
consists of Nicholes strain of T. pallidium (grown in the rabbit testis). The test is
always positive case of current infection except in very early primary stage.

Gonorrhoeae
Organism: Neisseria gonorrhoea.
Diagnosis:
* Acute stage:
- Sample  urethral discharge from male.
 urethral discharge & cervical secretion in female.
Then a film stained with Gram  intracellular G-ve diplococci is diagnostic.
* Chronic stage:
- Sample:
 Morning drop.
 Prostatic secretion after massage.
in male
 Cervical discharge in female.
- Film stained with Gram may not reveal G-ve diplococci so, culture is necessary.
- Culture on Thayer-Martin medium.
- ELISA which detects gonococcal antigens.
- DNA probe which detects gonococcal ribosomal genes.
Hospital acquired infection (nosocomial infection)

* Definition:
It is any infection acquired while in hospital.
* Sources of infection:
 Exogenous e.g. from another patients or from environment (dust, ventillators, endoscopy).
 Endogenous e.g. self or auto infection.
* Major infections:
Surgical wound infection, UTI, RTI, bacteremia.
Organisms:
Gram negative bacilli (E. coli.), Staph. aureus, MRSA., Candida and viruses (Influenza,
Respiratory syncytial viruses, Measles, Rubella, Herpes, Hepatitis and HIV).
Host factors predisposing for infections:
Extremes of age, impaired host blood supply, immobility, invasive procedure, D.M.,
immunosuppressive therapy.
Consequences of hospital infection:
1- Prolong hospital stay  costs money.
63
2- Additional antimicrobial therapy  increase selective pressure for resistance to emerge
among hospital pathogens.
3- Infected patient becoming a source from which others may become infected in hospital and
in community.
Prevention of hospital infection:
1- Education of hospital staff in:
- Hygiene in theatre, wards, kitchen …. ect.
- Good surgical technique.
- Frequent hand washing.
2- Proper sterilization and disinfection.
3- Special precautious and isolation of infective patients.
4- Protective precautions for high risk patients, e.g. immunosuppressed.
5- Conservative antibiotic use.
6- Surveillance of infections in the hospital by infection control staff.
Investigations of hospital infection:
Responsibility falls on the infection control committe including physician, microbiologist and
at least one nurse:
1- Surveillance:
The source of date maybe:
1- Microbiology laboratory reports.
2- Ward rounds.
3- Other e.g. staff health records, surveys of patients after discharge from
hospital, etc.
2- Investigation of out breaks:
The role of microbiology laboratory is to isolate the causative organisms and to show
that all the patients in the out break are infected with same strain. The type of organism
provides clues to the possible source e.g.
Staph. aureus  contact spread from staff in theatre.
Salmonella gastroenteritis  kitchen.
Methods:
1- Antibiotic susceptibility patterns.
2- Biotyping: typing according to difference in biochemical reaction.
3- Serotyping: This classical technique distinguishes between strains by difference in their
antigenic structure, recognized by reaction with specific antisera. The established schemes
use polyclonal antisera but newer schemes based on monoclonals are being developed.
4- Bacteriophage (phage typing): This technique compares the pattern of lysis when isolates
are exposed to a standard series of phage suspensions. This is important for typing Staph.
aureus, Staph. epidermidis and Salmonella typhi and species such as Pseudomonas
aeruginosa.
5- Bacteriocins: There are small protein molecules produced by species of bacteria and are
lethal to other strains of the same or closely related species. The pattern of inhibition of
growth by the test strains can be used to assign the strain.
6- Molecular typing technique: e.g. plasmid, DNA restriction endonuclease and ribotyping.

64
 Clinical virology
Laboratory diagnosis of viral Infections
Hepatitis viruses
HIV1/HIV2
Clinical Mycology
Definition of virus
Virus is a small infectious agent (10-30 nm) in diameter. It is metabolically inert, since
it cannot replicate outside of a living cell I,e needs a living media (tissue culture) for isolation.
Because viruses are non-motile, they are entirely dependent on external physical factors for
chance movement and spread to infect other susceptible cellsIt is composed of nucleic acid
either DNA or RNA and a capsid. Some viruses acquire envelope from the host.

CLASSIFICATION
Viruses are broadly classified primarily upon the type of genomic nucleic acid, eg.
DNA or RNA, and then further by the number of strands of nucleic acid (eg. double-stranded
DNA, double-stranded RNA or single-stranded RNA, with a positive or negative "sense" of
that single strand).
Retroviruses are a special category of RNA viruses that require reverse transcription of
their RNA to DNA and then integration of that DNA into the host cell genome before
replication can take place. They carry a reverse transcriptase enzyme as part of the virion.

A) DNA viruses
 -Adenovirus, Upper and lower respiratory infections, hemorrhagic cystitis,
keratoconjunctivitis.
 -Herpes Viruses

 Human herpesvirus 1 (herpes simplex virus type 1)

 Human herpesvirus 2 (herpes simplex virus type 2)

These cause:
gingiva stomatitis - vesicles and ulcers in the mouth
herpes genitalis - vesicles and ulcers on genitalia
herpes labialis (cold sores, fever blisters) - vesicles and ulcers of lips
herpes gladiatorum - clusters of vesicles and ulcers on skin
encephalitis
keratoconjunctivitis
whitlow (felon) a purulent infection involving the pulp of the distal phalanx of a finger

 Human herpesvirus 3 (varicella-zoster virus) - this virus causes:

 chickenpox (varicella) - epithelial cell infection resulting in an
exanthem of macules, papules, pustules, vesicles and shallow ulcers
 shingles (zoster) - peripheral nerve cell infection with an eruption in
the overlying epidermis
 Human herpesvirus 4 (Epstein-Barr virus) - This virus causes:
 infectious mononucleosis (b-lymphocyte infection)
 Burkitt's lymphoma
 oropharyngeal carcinoma

65
  Human herpesvirus 5 (cytomegalovirus) - this is the agent of:
 cytomegalovirus mononucleosis - similar to, but milder than,
infectious mononucleosis
 cytomegalic inclusion disease (salivary gland disease) - a generalized
and often fatal disease of the newborn.
 Human herpesvirus 6 (human b-lymphotrophic virus) - causes exanthem subitum
(fourth disease, Duke disease or roseola infantum and possibly multiple sclerosis)
 Human herpesvirus 8 - causes Kaposi's sarcoma in AIDS patients
 Human herpesvirus 7 - causes a cryptic infection of the T-helper cell
 -EBV---Mononucleosis, associated with Burrkitt s lymphoma and
nasopharyngeal carcinoma.
 -HBV—Hepatitis B
 -B19 virus - causes fifth disease (erythema infectiosum), bone marrow aplasia
and polyarthralgia. Target tissue is the erythroid cell

B) RNA viruses
 Influenza virus---Influenza
 Measles virus---measles
 Mumps virus---mumps and orcitis
 Rubella virus---Rubella and congenital malformation as cardio vascular and
neurological.
 Parainfluenza-----Bronchiolitis in infants and croups inyoung children
 RSV------Bronchiolitis and pneumonia in infants
 HIV---AIDS
 HCV----Hepatitis C and complications
 HDV----hepatitis
 HAV---Hepatitis A in children
 Coxsachie viruses------aseptic meningitis, myocarditis and pericarditis
 Rota virus----Diarrhea in young children
 Rhinovirus------common cold

Laboratory Diagnosis OF VIRUSES

Collection of specimens for viral disease diagnosis:

-Swabs of the lesion sites and transport in viral transport media

-Aspiration of secretions or exudates
-Excreta such as urine or stool.
-Biopsy samples obtained by needle aspirations, open exploration or endoscopy.
-Blood samples for antigen detection of some viruses, for serological tests, and for PCR.

Diagnosis of viral infections

Viruses can be studied in a number of direct and indirect ways and all these methods can be
applied in a diagnostic situation, ie. is this patient infected with a particular virus? There are
two approaches:

66
 Detection and demonstration of the virus itself; and
The study of the host's response to that virus
Detection of virus :

I- Culture.
II- Detection of viral antigen
III-Detection of viral DNA or RNA.

I-Culture.
The types of cell culture are
-Monolayer cultures Primary
Semiconscious
Continuous line
- Tissue culture
- Organ culture
Detection of viral growth by:
1- Characteristic cytopathic effect.
2- Hemadsorption (attachment of RBCS to the surface of virus infected cells) Some viruses
(eg. myxo- and paramyxoviruses, including influenza) have the property of
hemagglutination (causing red blood cells to stick together ) which can be used to detect
and quantitate the virus (by hemagglutination).
3- Interference with the formation of cytopathic effect e.g. neutralisation of viral infectivity by
antibodies can be used to detect and quantify either virus or specific antibody to that virus.
4- Adecrease in acid production by infected and dying cells.
5- Recent method depend on use of fluorescent antibodies to the infective virus antigen, or by
use of enzyme linked immunosorbant assay (ELISA).

II-Viral antigens can be detected by a wide range of serological techniques utilizing

polyclonal or monoclonal antibodies. Techniques include precipitation, agglutination,
immunofluorescence, ELISA, complement fixation and radio immuno assays.

III-Molecular techniques of both protein chemistry and nucleic acid biochemistry have
greatly improved the specificity of virus diagnostic procedures. Methods include:-

 polyacrylamide gel electrophoresis (PAGE) of protein fragments

 western blotting, and identification of specific proteins with labelled probes
 polymerase chain reaction (PCR), to amplify specific segments of viral nucleic acid
 Southern blotting, and DNA hybridisation with labelled probes
 sequencing of portions of the viral genome
 restriction fragment length polymorphisms of viral nucleic acid

VIRAL HEPATITIS
The term VIRAL HEPATITIS is usually used to describe infections caused by agents whose
primary tissue tropism is the liver.
To date, at least five hepatitis viruses have been recognised, and these have been named:-
Hepatitis A, B, C, D and E.
Acute hepatitis may also occur as part of the clinical course of a number of viral infections,
67
including
human cytomegalovirus, Epstein-Barr virus, herpes simplex virus, yellow fever virus and
rubella.
Viral hepatitis can be classified according to mode of transmission

ENTERICALLY TRANSMITTED HEPATITIS: A and E

PARENTERALLY TRANSMITTED HEPATITIS B , C , D and G

Hepatitis A Hepatitis B Hepatitis C

Virus HAV(RNA) HBV(DNA) from Hepadna HCV(RNA) from
member of virus Togavirus related to the
picornavirus Flavi and Pesti viruses
Incubation peroid 2-6 weeks 6 weeks-6 months 1-3 months
Mode of transmission Faeco-oral Parenreral,sexual,vertical Parenteral
Clinical course Short-mild Prolonged and more severe Mild, chronic in 50% of
than A cases
Carrier & Chronicity No Yes Yes
Laboratory diagnosis Elevated Elevated ALT,AST from 10- Mild elevation of
ALT,AST 100 folds Acute infection ALT,AST
up to 10 with resolution
folds With fluctuation in
Viral antigens: AST(surrogate
Virus 1) Surface antigen (HBsAg)
cannot be is secreted in excess into the Marker of chronic
cultured in blood as 22 nm spheres and hepatitis C).
vitro from tubules. Its presence in
clinical serum indicates that virus 1) Serology
material, replication is occurring in the .
and liver 1-HCV-specific IgG
diagnosis is 2) 'e' antigen (HBeAg) indicates exposure, not
made on the secreted protein is shed in infectivity
presence of small amounts into the
HAV- blood. Its presence in serum 2) PCR detects viral
specific indicates that a high level of genome in patient's serum
IgM in the viral replication is occurring
patient's in the liver 3) Quantitative PCR to
blood. 3) core antigen (HBcAg) detect viral load response
core protein is not found in to therapy.
blood

Antibody response:
1) Surface antibody (anti-
HBs) becomes detectable
late in convalescence, and
indicates immunity following
infection. It remains
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 detectable for life and is not
found in chronic carriers (see
below).
2) e antibody (anti-HBe)
becomes detectable as viral
replication falls. It indicates
low infectivity in a carrier.
3) Core IgM rises early in
infection and indicates recent
infection
4) Core IgG rises soon after
IgM, and remains present for
life in both chronic carriers
as well as those who clear
the infection. Its presence
indicates exposure to HBV.

Chronic hepatitis B

Persistance of surface
antigen and prolonged
persitance of e antigen.

Hepatitis E
Recently identified cause of enterically transmitted non-A, non-B (NANB) hepatitis

Clinical Features
Incubation period 30-40 days
Acute, self limiting hepatitis, no chronic carrier state
Age: predominantly young adults, 15-40 years

Complications
Fulminant hepatitis in pregnant women. Mortality rate is high (up to 40%).

Virus cannot be cultured in vitro.

1) Calicivirus-like particles in the stool, by electron microscopy

2) Specific IgM in serum
3) PCR HEV-specific sequences in stool

Delta Agent
Defective virus which requires Hepatitis B as a helper virus in order to replicate. Infection
therefore only occurs in patients who are already infected with Hepatitis B.

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Clinial Features
Increased severity of liver disease in Hepatitis B carriers.

Hepatitis G (HGV)
A virus originally cloned from the serum of a surgeon with non-A, non-B, non-C hepatitis, has
been called Hepatitis G virus. It was implicated as a cause of parenterally transmitted hepatitis,
but is no longer believed to be a major agent of liver disease. It has been classified as a
Flavivirus and is distantly related to HCV.

HIV 1 and 2
Human Immunodeficiency Viruses
Transmission
Infection is transmitted in a manner identical to that of hepatitis B.
1.) Blood products

 Blood transfusions
 Intra-venous drug abusers - sharing of needles
 Health care workers:
needlestick injuries - risk approximately 0.36% (depends on extent of the injury)
muco-cutaneous exposure - no sero-conversion incidents have been reported

2.) Organ transplants

3.) Sexual intercourse
Both homosexual and hetero-sexual exposure
Increased risk of transmission if partners have other sexually transmitted diseases

4.) Vertical Transmission

10-40% of babies born of HIV-infected mothers will be infected.

Infection may occur

in utero
during birth
post-natally, through breast feeding

Primary infection
About 90% of patients develop a flu-like illness which co-incides with seroconversion,
between 2 and 4 weeks post exposure. Symptoms include, fever, night sweats, sore throat,
lymphadenopathy, diarrhoea. The illness is self limiting.

Asymptomatic phase
Of variable duration, from 2 to 10 years. Patients are clinically well, but infectious.

Prodromal phase
This period is heralded by the insidious onset of a variety of prodromal disorders, including:

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weight loss, fever, persistant lymphadenopathy, oral candidiasis and diarrhoea. These
symptoms precede the progression to AIDS.

Acquired Immunodeficiency Syndrome (AIDS)

Syndrome with the following features:

1) Constitutional disease: fever, diarrhoea, weight loss, skin rashes
2) Neurological disease: dementia, myelopathy, peripheral neuropathy
3) Immunodeficiency: Increased susceptibility to opportunistic infections.
4) Rare malignancies: Kaposi sarcoma, oral hairy leukoplakia, lymphomas.

Kaposi sarcoma - is a tumour of endothelial cells. Prior to the AIDS epidemic, this tumuor
was rare and only found in middle aged African and Mediterranean Jewish men, in whom it
was an indolent condition. AIDS patients develop a disseminated highly aggressive form of the
disease.

Paediatric Infection
Following infection in the perinatal period, babies may develop a progressive illness in the first
few months of life (No latent period). Clinical features include: Failure to thrive, diarrhoea,
lymphadenopathy, susceptibility to opportunistic infections hepato-splenomegaly, lymphoid
interstitial pneumonia and parotitis.

Pathogenesis

HIV infects CD4 cells

Disseminated infection

Specific immune Response

Antibody
Cell mediated immunity

Clearance of most virus

Some persistence
a) Gradual loss of CD4 cells
b) Destruction of microenvironment of lymphoid tissue
IMMUNO-DEFICIENCY

Cell tropism: CD4+ T cells, Macrophages

Destruction of CD4 cells occurs through the following mechanisms:

direct lysis of infected cells
syncytium formation

Infection of precursor cells and destruction of microenvironment

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Impairment of CD4 cell function

LABORATORY DIAGNOSIS

Serology
IgG develops 4-6 weeks post exposure and remains detectable for life. Its presence in serum
therefore indicates infection.
Exception: Uninfected infants of HIV positive mothers

Direct detection of virus

p24 antigen ELISA
culture from PBMC's
PCR

VACCINE PROSPECTS

There is no effective vaccine available for HIV. Attempts have been made to develop a
vaccine, using:

 purified viral envelope glycoproteins, gp120 or 160

 whole inactivated virus
 live attenuated HIV strains (lacking certain genes)
 live recombinant virus vectors, expressing HIV proteins.

A major difficulty is the fact that neutralizing antibody in the serum does not protect the host
from infection with HIV: Possible reasons for this include:

 Antibody enhancement of infection

 Rapid virus mutation may result in variation of envelope antigens (escape mutants)
 HIV can infect cells in sites that are sequestered from antibody
 Host may be infected by whole virus-infected cells

Clinical Mycology
Introduction:
Fungi constitute a group of non-motile, eukaryotic organisms that have definite cell
walls, devoid of chlorophyll and reproduce by means of spores either sexual or asexual.
Morphology:
Fungi may take the following forms: (Fig. 3).
1- Yeast: uni-cellular spherical or ovoid in shape.
2- Mold: multicellular, formed of:
- Hyphae: long filaments, maybe septated or non septated – A mass of hyphae is called
mycelium.
- Conidiophore or sporangiophore is specialized hyphae that bears the reproductive
structure of the mold.

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 - The vesicle is the bulbous tip of the condiophore.
- Stregmita are flask shaped structure on the vesicle which bears the spores.
- Spores are sexual reproductive structure.
- Conidia are asexual spores.
3- Dimorphism: is a character of some pathogenic fungi which grow as molds in natural
environment and in lab. culture and as yeasts in tissues. e.g. Histoplasma, Blastomyces.

Fig. (3): Mold with microconidia.

Fungal disease:
They are classified into: cutaneous, sub cutaneous, deep and opportunistic mycosis Table (10).
Table (10): classification of fungal diseases.
Type Causative fungus Disease and important clinical
findings
A) Cutaneous Malassezia Tinea versicolor of skin

Dermatophytes Ring worm of skin, nails and

(Epidermophyton, Tricophyton hair.
& Microsporum)
B) Subcutaneous Sporothrix Sporotrichosis of lymph
vessels and lymph nodes
Several genera Mycetoma (Madura foot)
chronic granuloma
discharging pus especially of
leg and foot
C) Systemic 1- Histoplasma Pulmonary or disseminated
histoplasmosis
2- Coccoidoides Pulmonary or Erythema
Nodosum
3- Blastomyces Pulmonary or disseminated

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 4- Paracoccoidiodes Pulmonary, disseminated
D) Opportunistic Candida Candidiasis: oral thrush,
vaginities or disseminated
Aspergillus Aspergillosis: pulmonary,
aspergilloma, toxicosis (ASP.
Flavus produce aflatoxin
which is hepato carcinogen
Cryptococcus Cryptococcal meningitis or
pulmonary cryptococcosis
Mucor Mucor mycosis of blood
vessels esp. of paranasal sinus,
lung, gut.
Diabetic ketoacidosis, organ
transplant and leukemic
patients are mostly
susceptible.

Diagnosis:
A) Collection of samples: according to site of infection cutaneous (hair, nail, skin),
subcutaneous (abscess, sinus, fistula), systemic (blood, sputum, CSF, bone marrow,
urine, faeces).
B) Diagnostic methods:
1- Direct microscopic examination using KOH-indian ink-Giemsa-periodic acid
schiff (PAS).
2- Fungal culture: commonest media is sabouraud’s agar (SAB), antimicrobials
may be added.
3- Direct Ag detection by immunoelectrophoresis or latex.
4- Serology useful for diagnosis and prognosis of systemic fungal infections.

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 Immunology
Antigen recognition by T and B cells
Protein antigens have been cleaved into peptides in a process called antigen processing
followed by antigen presentation on the surface of antigen presenting cells ( APCs ) beside the
MHC class I or class II.

The source of protein antigens can occur through two major pathways:
I- Endocytic Pathway :
Extra-cellular microorganisms or particles engulfed by phagocytosis, pinocytosis or
receptor mediated endocytosis, proteins are captured by APC by any of these routes. They are
taken into membranous endosomal vesicles ,they broken down by acidic PH and cellular
proteolytic enzymes . Many short peptides produced from this process and transported to the
cell surface beside MHC class II for presentation to CD4 T lymphocytes.

II- Cytosolic Pathway :

Antigenic proteins can be derived from pathogens lived inside infected
host cells i.e. viruses, intracellular bacteria and parasites. These proteins are processed in the
cytosol within proteasomes, then via TAP1, and TAP2 they enter the rough endoplasmic
reticulum some of these peptides then associate with MHC class I proteins and delivered to the
cell surface for presentation to CD8 T lymphocytes .

The T lymphocytes recognize peptide – MHC complex on the surface of APCs by the
T cell receptor complex ( TCR / CD3 complex ).

The TCR is formed of heterodimer α and β polypeptide chains present on about 95 %

of T Lymphocytes , while 5% of them carry γδ TCR .

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 The TCR has variable and constant regions, at the chromosomal level; the variable domains
comprised numbers of V, J gene segments and one C segment for the α chain and V, D, J
and two C segments for the β chain.

• Diversity of TCR is generated through :

- Combinatorial Joining of these segments.
- Insertion or deletion of nucleotides at the joining sites.
- Association of α and β chains .
- No somatic hypermutation as in immunoglobulins.

The sequelae of TCR engagement is activation of T lymphocytes resulting in

proliferation, division and cytokine production.

• Subpopulations of T cells:
1- T helper cells (CD4+) represent 65 % of peripheral blood lymphocytes. .
2- T cytotoxic cells (CD8+) represent 35 % of them.

According to lymphokines they produce, the T helper cells is divided into :

* THO produce IFN γ and IL.4.
* TH1 produce IL – 2 , IFN γ & TNF β .
* TH2 produce IL - 4 , IL- 5 , IL- 6 , IL - 10 & IL.13.
* TH3 regulatory T cells , produce TGF β that suppress the TH1 and
TH2 immune response .

The T helper cell is the orchesterator of the immune response, as they help the B
lymphocytes to secrete immunoglobulins, the cytotoxic T lymphocytes, the NK cells and
macrophages to increase their cytotoxic activities.
TH1 cells are responsible for cell mediated immunity , while TH2 cells are responsible
for humoral immunity.

• Interaction of TCR with Superantigens :

 Superantigens are a class of bacterial toxins and retroviral proteins
 They are not processed nor presented by APCs.
 they interact with MHC- Class II outside the peptide binding groove, bind only to
Vβ segment of the TCR.
 Superantigens have the ability of activating 1-10 % of peripheral T cells.
 Exposure to superantigen leads to massive T cell activation and lymphokine release
e.g. Toxic shock syndrome induced by staph . aureus toxins (TSSI – 1).

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The B lymphocytes recognize polymeric proteins, lipids and polysaccharides by their surface
membrane immunoglobulins IgM or IgD ( BCR ).

Activation of B cells is produced following engagement of BCR and require help from TH
(CD4 +) cells such as IL - 2 , IL - 4 , IL - 6.

This activation is followed by proliferation, division and development of plasma cells that
secrete antibodies in the plasma (IgM , IgG , IgA , IgE and IgD).

Functions of B lymphocytes:
1- Humoral immunity:
Secretion of antibodies that are responsible for defense against foreign invaders
(opsonization, phagocytosis, complement activation, ADCC, toxin neutralization & virus
neutralization).
2- Antigen presentation .
В cells act as APC: by processing & presenting antigen to T lymphocytes .
3- Lymphokine secretion (IL - 10, IL – 12 and TNFα).

Hypersensitivity Reactions
Definition: immune mechanisms causing tissue damage .
Type I : Anaphylatic
1- Antigen or allergen : antisera, hormones , enzymes , pollen extract, food antibiotics
polysacchauides.
2- Reaginic antibodies : IgE (Serum concentration is 0.004 % that of IgG).

3- Tissue target cells : blood basophils and tissue mast cells, having receptors for Fc
portion of IgE .
4- Vasoactive amines :

 Histamine : exist in preformed state.

 Esinophil chemotactic factors: exist in preformed state.
77
  Leukotrienes : not exist in a preformed state.
 Prostaglandins : not exist in preformed state.
 Serotonin .
 Heparin : anaphylaxis in dogs
 Kinins.
Their actions are smooth muscle contraction , increased capillary dilation , increased
vascular permeability , and bronchial constriction.

Examples :
1- Allergic bronchial asthma.
2- Allergic rhinitis .
3- Atopic dermatitis .
4- Immediate drug allergy .
5- Acute urticaria .

 Laboratory diagnosis : Serum total IgE and specific IgE.

Skin prick test.

Type II : Cytotoxic reaction:

Antigen : antigenic determinants on cell membrane or a free antigen or hapten that may be
adsorbed to a cell membrane .
Antibody : IgG or IgM .
Antigen – antibody reaction lead to cell damage by the following :
a- Target cell Lysis with or without activation of the complement resulting in RBCS ,
lymphocytes , platelets Lysis.
b- Phagocytosis of target cell by phagocytic cells (macrophages and PMNs).
c- Stimulation of target cell by:
LATS , anti Ig antisera, anti-lymphocyte antisera.
d- ADCC: the mechanism of antibody dependent cell mediated cytotoxicity is mediated
by :
 Cells in the monocyte / macrophage series .
 Killer cells .
 Natural killer cells.
 Null cells ( lack T and B cell markers ).

Examples :
a. Transfusion reaction ABO, Rh. incompatibility.
b. Autoimmune hemolytic anemia.
c. Idiopathic thrombocytopenic purpura (ITP).
d. Hashimoto's thyroiditis.

Type III: Immune Complex Reaction:

Antigen: tissue antigen, drug as heterologous serum.
Antibodies: IgG or IgM.
o Formation of antigen – antibody complexes with fixation of the complement .
o Release of complement components that attract leukocytes .
o Damage of platelets with release of vasoactive amines

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 o Increased vascular permeability and localization of antigen-antibody complexes
in blood vessels .
o Infiltration of PMN leukocytes, ingestion of immune complexes and release of
lysosomal enzymes.
o Damage of tissues , deposition of fibrin as healing of lesion.

Arthus reaction: Local tissue interaction between antigen and antibodies following
experimental intradermal injection of antigen:

Clinical presentation:
 Hypersensitivity pneumonitis.
 Extrinsic allergic alveolitis .

Serum sickness: after injection of heterologous serum (now obsolete)

Examples of immune complex disorders :

 Acute glomerulonephritis .
 Subacute bacterial endocarditis .
 Syphilis, leprosy, leishmaniasis, schistosomiasis.
 Lymphoma and leukemias.
 Carcinoma of lung , breast and colon .
 S L E & Rheumatoid arthritis .
 Celiac disease, ulcerative colitis and hepatic cirrhosis.

Type IV : cell mediated reaction :

 These reactions are due to interactions between specific antigens and sensitized
lymphocytes .
 Mediated by lymphokines , direct cytotoxicity or both .
 The lymphokines amplify the initial cellular response by recruitment of other T and B
lymphocytes, attract PMN leukocytes and activate macrophages.
Examples:
 Chronic infections (viral, fungal, TB and protozoa).
 Graft rejection.
 Graft versus host disease.
 Tumor immunology.

Tuberculin test: ID injection of 0.1 ml PPD in the volar aspect of forearm. reaction appears
after 48 - 72 hours in the form of induration of more than 10 mm in diameter & erythema .
* Interpretation of positive tuberculin test:
1- Good cell mediated immunity
2- B C G vaccination
3- Tuberculosis either recent or past-infection .

* Negative tuberculin test:.

1- Anergy, depressed cell mediated immunity.
2- Miliary tuberculosis .
3- In child below 5 years, he must takes BCG vaccine.
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 Immunodeficiency disorders
I-Antibody or B cell immunodeficiency:
1- X - linked infantile hypogammaglobulinemia (Bruton type ) .
2- Transient infantile hypogammaglobulinemia.
3- Acquired hypogammaglobulinemia.
4- Immunodeficiency with hyper-IgM .
5- Selective IgA deficiency .
6- Selective IgM deficiency .
7- Selective deficiency of IgG subclasses .

II -T cell immunodeficiency :
1- Congenital thymic aplasia (Di George syndrome ) .
2- Chronic mucocutaneous candidiasis .

III- Combined T and B cell immunodeficiency:

1- Severe combined immunodeficiency.
2-Nezelofs syndrome .
3- Wiskott – Aldrich syndrome .
4- Immunodeficiency with ataxia telangectasia .
5- Bare lymphocyte syndrome.

IV- Phagocytic dysfunction :

• Defects either in antibodies or complement (opsonins).
• Decreased total number of phagocytic cells .
• Deficiency of NADPH or NADH oxidase (chronic granulomatous disease ) .
• Deficiency of myeloperoxidase or glucose 6 phosphate dehydrogenase .

V- Complement deficiency :
Associated with increased susceptibility to infection & autoimmune diseases
- Decreased C1 esterase inhibitor leads to hereditary angioneurotic
edema

IV- Secondary immunodeficiency :

- May be associated with tuberculosis and leprosy .
- May be associated with viral infections as HIV leading to acquired mmunodeficiency
syndrome AIDS .

Immunologic features:
1- Decrease helper T cells & H/S ratio .
2- Decrease lymphocyte response to mitogens and antigens .
3- Decrease antibody response after immunization (or absent ).
4- Decrease NK cell function.
5- Decrease IL -2 production .
6- Increase immunoglobulin levels .
7- Increase circulating immune complexes .

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 * laboratory assessment of immunodeficiency:
I- B cell immunodeficiency .
1- Protein electrophoresis to detect hypogammaglobulinemia .
2- Radial immunodiffusion , Nephlometry and ELISA to quantitate immunoglobulin
levels.

Normal mean concentration :

I g M level: 120 mg / dl
I g G level: 1000 mg / dl
I g A level: 200 mg /dl
I g E level: 150 Iu / ml

3- B cell quantitation with MAB (CD19 , CD 20 ).

B cells 10 - 25 % of total lymphocytes .

4- In vitro stimulation of B cells by mitogen and assessment of immunoglobulin synthesis.

5- Immunization with TAB vaccine or DT toxoid and assessment of specific antibody titer
after 2 weeks .

II- T cell immunodeficiency :

1- Total lymphocyte count (Normal: 1200 / µl ).
2- T cell count with MAB (CD3, CD4, CD8 ).
3- In vitro stimulation of T lymphocytes by mitogen , or antigen and estimation of cytokine
production or blast transformation .
4-Delayed hypersensitivity skin test i e tuberculin test .

III- Phagocytic dysfunction :

1- Assessment of chemotaxis.
2- Assessment of opsonins i e immunoglobulins & complements .
3- Assessment of intracellular killing by nitroblue tetrazolium test
( NBT ) .
4- Enzyme determination ( NADPH ,NADH oxidase , myelo – peroxidase and others ) .

IV Complement deficiency :
1- Assay of complement components commonly C3 & C4 (mean 125 mg/dl & 28 mg/dl)
respectively.
2- Functional complement assay CH50 .

Immunologic Laboratory diagnosis of SLE :

 ANA .
 Anti ds DNA .
 Decrease C3 & C4 levels.
 Anti smith antibodies, SSA, SSB.
 Other autoantibodies associated with different autoimmune diseases RF,
anticytoplasmic, antiphospholipid, antiplatelet antibodies and etc.

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Laboratory diagnosis of rheumatoid arthritis:
1- RF in 80% of patients .
2- ANA in 15% of patients .
3- Complement level normal or may be low in vasculitis.
4- Cry globulin in rheumatoid vasculitis.
5- HLA DR4 genetic predisposition .

Some MHC (HLA- allele ) – associated diseases :

1- Rheumatoid arthritis DR4 and DR1.
2- Systemic lupus erythematosus DR2 .
3- Insulin dependent diabetes DR3 & DR4.
4- Graves diseases DR3.
5- Ankylosing spondylitis B27

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