EXPERIMENT NO: 1

HEMATOLOGICAL TESTS

RBC count:
Actual count of red corpuscles in a given amount of blood (cubic millimetre
or liter).
After puberty females have slightly lower counts (and Hgb), partly
because of menstrual blood loss and because of higher androgen (an
erythropoetic stimulant) concentrations in men.
Male: 4.6 to 6.2 X106 cells /mm3
Female: 4.2 to 5.4 X106 cells /mm3
White Blood Cell count: number of leukocytes in a given amount of blood.
NORMAL VALUE: 5000 to 10,000 cells /cu mm of blood

Haemoglobin (Hb): Hgb amount in he given volume (100ml or 1 liter) of

whole blood. Provides a direct indication of the oxygen-transport capacity of
the blood.
Male: 14 to 18g/dl
Female: 12 to 16g/dl
Hematocrit (Hct) / packed cell volume (PCV):
It is the percentage volume of blood that is composed of erythrocytes.
Hct is usually about 3 times the value of Hgb.
Normal Value:
Male: 42 to 52 %
Female: 37 to 47%
Mean cell volume (MCV): Is an estimate of the average volume of RBCs.
Derived by dividing Hct by RBC count. Abnormally large cells have an
increased MCV and are called macrocytic.
Normal Value:
-- 15
Male: 80 to 96 fl (femtolitres – 10 )
Female: 82 to 98 fl
Mean cell hemoglobin (MCH): It is the percent volume of Hb per RBC.
Derived by dividing Hb by RBC count.

1
 –12
Normal Value: 27 to 33 pg /cell [picograms = 10 ]
A low MCH corresponds with hypo chromic (pale) RBCs - as seen in
iron deficiency anaemia.
Mean cell haemoglobin concentration (MCHC): Is derived by dividing Hb
by Hct. Hct is usually about 3 times the value of Hgb.
Normal Value: 31 to 35 g/dl
Iron deficiency is the only anaemia in which the MCHC is low.
Reticulocytes: Gives indirect measurement of RBC production.
Normal Value: 0.5 to 2.5 % of RBCs
Indicates bone marrow suppression in which the percentage of
circulating is almost zero.
Erythrocyte sedimentation rate (ESR): Anemia, pregnancy and various
inflammatory diseases (including infections) can elevate ESR. Rate at which
the erythrocytes settle down.
Normal Value:
Male: 1 to 15 mm / hour
Females: 1 to 20 mm / hour

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 EXPERIMENT NO: 2

LIVER FUNCTION TESTS

Liver function tests (LFT) are a helpful screening tool, which are an effective
modality to detect hepatic dysfunction.
USES
The various uses of Liver function tests include:
Screening: They are a non-invasive yet sensitive screening modality for liver
dysfunction.
Pattern of disease: They are helpful to recognize the pattern of liver
disease. Like being helpful in differentiating between acute viral hepatitis
and various cholestatic disorders and chronic liver disease.
Assess severity: They are helpful to assess the severity and predict the
outcome of certain diseases like primary biliary cirrhosis.
Follow up: They are helpful in the follow up of certain liver diseases and
also helpful in evaluating response to therapy like autoimmune hepatitis.
CLASSIFICATION OF LIVER FUNCTION TESTS
A. Tests of the liver’s capacity to transport organic anions and to
metabolize drugs- Serum bilirubin, urine bilirubin, urobilinogen.

B. Tests that detect injury to hepatocytes (serum enzyme tests) –

Aminotransferases, alkaline phosphatase, glutamyl transpeptidase, 5
nucleotidase, leucineaminopeptidase.

C. Tests of the Liver’s biosynthetic capacity- Serum proteins, albumin,

prealbumin, serum ceruloplasmin, procollagen III peptide, prothrombin
time.

A. Tests of the liver’s capacity to transport organic anions and to

metabolize drugs
1. SERUM BILIRUBIN
Bilirubin is an endogenous anion derived from haemoglobin degradation
from the RBC. The classification of bilirubin into direct and indirect
bilirubin is based on the original van der Bergh method of measuring

3
bilirubin. When the liver function tests are abnormal and the serum
bilirubin levels more than 17μmol/L suggest underlying liver disease.
TYPES OF BILIRUBIN
i. Total bilirubin: This is measured as the amount, which reacts in 30
minutes after addition of alcohol. Normal range is 0.2-0.9 mg/dl (2-
15μmol/L). It is slightly higher by 3-4 μmol/L in males as compared to
females. It is this factor, which helps to diagnose Gilbert syndrome in
Males easily.
ii. Direct Bilirubin:
This is the water-soluble fraction. This is measured by the reaction with
diazotized sulfanilic acid in 1 minute and this gives estimation of conjugated
bilirubin.
Normal range 0.3mg/dl (5.1μmol/ L)
iii. Indirect bilirubin:
This fraction is calculated by the difference of the total and direct bilirubin
and is a measure of unconjugated fraction of bilirubin. The diazo method of
bilirubin estimation is not very
Accurate especially in detecting low levels of bilirubin. Direct bilirubin over
estimates bilirubin esters at low bilirubin levels and under estimates them
at high concentration. Thus slight elevation of unconjugated bilirubin not
detected, which is of value in detecting conditions like Gilbert syndrome.
a. Diagnostic value of bilirubin levels:
Bilirubin in body is a careful balance between production and removal of the
pigment in body. Hyperbilirubinemia seen in acute viral hepatitis is directly
proportional to the degree of
Histological injury of hepatocytes and the longer course of the disease.
Hyperbilirubinemia: It results from overproduction /impaired uptake,
conjugation or excretion / regurgitation of unconjugated or conjugated
bilirubin from hepatocytes
to bile ducts
Increased unconjugated bilirubin: This results from
overproduction/impaired uptake, conjugation.
Increased conjugated bilirubin: Impaired intrahepatic excretion /
regurgitation of unconjugated or conjugated bilirubin from hepatocytes of
bile ducts. Serum bilirubin could be lowered by drugs like salicylates,
sulphonamides, free fatty acids which displace bilirubin from its attachment
to plasma albumin.
Hyperbilirubinemia and Haemolysis

4
Bilirubin itself is not soluble in water and is bound to albumin and thus
does not appear in urine. Haemolysis with overproduction of bilirubin and
concomitant reduced GFR cause decreased excretion and can lead to high
bilirubin levels. Bilirubin levels in excess of 25 mg/dl may be seen in
haemolyses in association with liver disease.
URINE BILIRUBIN
The presence of urine bilirubin indicates hepatobiliary disease.
Unconjugated bilirubin is tightly bound to albumin and not filtered by the
glomerulus and thus not present in urine. Measurable amounts of
conjugated bilirubin in serum are found only in hepatobiliary
Disease. Because the renal threshold for conjugated bilirubin is low and the
laboratory methods can detect low levels of bilirubin in urine so conjugated
bilirubin may be found in
Urine when the serum bilirubin levels are normal. This is the case in early
acute viral hepatitis.
UROBILINOGEN
An increase in the urobilinogen in urine is a sensitive indicator of
hepatocellular dysfunction. It is a good indication of alcoholic liver damage,
well compensated cirrhosis or malignant disease of the liver. In viral
hepatitis it appears early in urine. It is markedly increased
in haemolysis. In cholestatic jaundice urobilinogen disappears from urine. It
may be intermittently present in case of gallstones.
B. Tests that detect injury to hepatocytes serum enzyme tests) : The
liver contains thousands of enzymes and these enzymes have no function
and behave as serum proteins.
A. ENZYMES THAT DETECT HEPATOCELLULAR NECROSIS –
AMINOTRANSFERASES
The aminotransferases are the most frequently utilized and specific
indicators of hepatocellular necrosis. These enzymes- aspartate
aminotransferase (AST, formerly serum glutamate oxaloacetic transaminase-
SGOT) and alanine amino transferase (ALT, formerly serum glutamic
pyruvate transaminase-SGPT) catalyze the transfer of the amino acids of
aspartate and alanine respectively to the keto group of ketoglutaric acid.

5
ALT is primarily localized to the liver but the AST is present in a wide variety
of tissues like the heart, skeletal muscle, kidney, brain and liver.
MILD, MODERATE AND SEVERE ELEVATIONS OF
AMINOTRANSFERASES:
1. Severe ( > 20 times, 1000 U/L) : The AST and ALT levels are increased
to some extent in almost all liver diseases. The highest elevations occur in
severe viral hepatitis, drug or toxin induced hepatic necrosis and circulatory
shock. Although enzyme levels may reflect the extent of hepatocellular
necrosis they do not correlate with eventual outcome.
2. Moderate (3-20 times): The AST and ALT are moderately elevated in
acute hepatitis, neonatal hepatitis, chronic hepatitis, autoimmune hepatitis,
drug induced hepatitis, alcoholic hepatitis and acute biliary tract
obstructions. The ALT is usually more frequently increased as compared to
AST except in chronic liver disease. In uncomplicated acute viral hepatitis,
the very high initial levels approach normal levels within 5 weeks of onset of
illness and normal levels are obtained in 8 weeks in 75% of cases.
3. Mild (1-3 times): These elevations are usually seen in sepsis induced
neonatal hepatitis, extrahepatic biliary atresia (EHBA), fatty liver, cirrhosis,
non alcoholic steato hepatitis(NASH), drug toxicity, myositis and even after
vigorous exercise.

ALKALINE PHOSPHATASE
Elevated serum levels of intestinal alkaline phosphatase have been found in
patients with cirrhosis, particularly those with blood group type O, and may
be associated specifically with intrahepatic disease as opposed to
extrahepatic obstruction. Hepatic and bony metastasis can also cause
elevated levels of alkaline phosphatase. Other diseases like infiltrative liver
diseases, abscesses, granulomatous liver disease and amyloidosis may also
cause a rise in alkaline phosphatase. Mildly elevated levels of alkaline
phosphatase may be seen in cirrhosis and hepatitis of congestive cardiac
failure. Low levels of alkaline phosphatase occur in hypothyroidism,
pernicious anemia, zinc deficiency and congenital hypophosphatasia.
GLUTAMYL TRANSPEPTIDASE

6
Glutamyl transpeptidase (GGT) is a membrane bound glycoprotein which
catalyses the transfer of glutamyl group to other peptides, amino acids and
water.
Normal Range is 0-30IU/L. In acute viral hepatitis the levels of glutamyl
transpeptidase may reach its peak in the second or third week of illness and
in some patients they remain elevated for 6 weeks. Other conditions causing
elevated levels of glutamyl transpeptidase include uncomplicated diabetes
mellitus, acute pancreatitis and myocardial infarction.
OTHER ENZYMES THAT DETECT CHOLESTASIS These are the other
enzymes that are not routinely estimated to detect cholestasis.
5 Nucleotidase
Leucine amino peptidase
C. Tests of the Liver’s biosynthetic capacity.
1. SERUM PROTEINS: The liver is the major source for most of the serum
proteins. The parenchyma cells are responsible for synthesis of albumin,
fibrinogen and other coagulation factors and most of a and b globulins.
Albumin is quantitatively the most important protein in plasma synthesized
by the liver and is a useful indicator of hepatic function. The serum levels
are typically depressed in patients with cirrhosis and ascites.
Normal serum values range from 3.5g/dl to 4.5 g/dl.
The average adult has approximately 300 to 500 g of albumin.
Hypoalbuminemia is not specific for liver disease and may occur in protein
malnutrition, nephritic syndrome and chronic protein losing enteropathies.
2. PREALBUMIN
The serum prealbumin level is 0.2- 0.3 g/L. These levels fall in liver disease
presumably due to reduced synthesis. Because of its short half life, changes
may precede alteration in serum albumin. Determination of prealbumin has
been considered particularly useful in drug-induced hepatotoxicity.
3. SERUM CERULOPLASMIN
Normal plasma levels are 0.2-0.4g/L. It is synthesized in the liver and is an
acute phase protein. The plasma concentration rise in infections,
rheumatoid arthiritis, pregnancy, non Wilson liver disease and obstructive
jaundice. This is an important diagnostic marker in Wilson disease, in which

7
the plasma level is usually low. Low levels may also be seen in neonates,
kwashiorkor, marasmus and copper deficiency.
PROCOLLAGEN III PEPTIDE
The serum concentration of this peptide appears to increase not only with
hepatic fibrosis but also with inflammation and necrosis. Serial
measurement of procollagen III may be helpful in the follow up of chronic
liver disease.
5. ANTITRYPSIN
Antitrypsin is a glycoprotein synthesized by the liver and is an inhibitor of
serine proteinases, especially elastase. Its normal concentration is 1-
1.6g/L. It is an acute phase protein, serum levels increase with
inflammatory disorders, pregnancy and after oral contraceptive pills (OCP).
Liver disease is usually seen with deficiency of antitrypsin, an inherited
disorder.
FETO PROTEIN
This protein, the principal one in foetal plasma in early gestation is
subsequently present at very low levels.(<25g/L) It is increased in
hepatocellular carcinoma (HCC)and more than 90% of such patients have
raised levels. Raised values are also found in other liver diseases
like chronic hepatitis, in regeneration phase of acute hepatitis and in
hepatic metastasis. This is also raised in adenomas. Feto protein elevation is
less frequent when HCC arises in non cirrhotic liver. Serial determination is
of value in cirrhotic patients and rise in the values should raise the
suspicion of HCC.
PROTHROMBIN TIME (PT)
Clotting is the end result of a complex series of enzymatic reactions that
involve at least 13 factors. The standard method to assess is the one stage
prothrombin time of quick, which evaluate the extrinsic coagulation
pathway. The results of this test may be expressed in sec or as a ratio of the
plasma prothrombin time to control plasma time. Normal control usually is
in the range of 9-11 seconds. A prolongation of more than 2 seconds is
considered abnormal.

8
 EXPERIMENT NO: 3
RENAL FUNCTION TESTS
Renal Function Tests
To Identity renal dysfunction
To Diagnose the renal disease
To monitor the progression of renal disease
To monitor the response to treatment
Classification of Renal Function Test
I. Urine Analysis
Physical Examination
Chemical Examination
Microscope Examination
II. Blood Analysis
Estimation of Plasma proteins
Estimation of NPNI.
Urine Analysis
Physical Examination
Urinary Output Volume: 1,500 ml / day
(a) Polyuria 2,500 ml /
day
Diabetes Mellitus
Diabetes insipidus
Chronic glomerulonephritis
(b) Oliguria 500ml1day
Fever
Diarrhea
Acute renal failure
(c) Anuria Complete cessation of urine
Acute tubular Necrosis
Bilateral renal stones
Prostate enlargement
Colour: Urine is transparent, pale yellow (or) amber in colour.

9
(vi). Odour: Fresh urine is normally aromatic. Foul smell indicated bacterial
Infection.
Chemical examination.

Glucose: Normal urine contains small amounts of glucose, which

cannot be detected by routine test. Presence of detectable amounts of
glucose in urine is called glycosuria.
Protein: The glomerular basement membrane does not usually
allow passage of albumin and large proteins. Increased
amounts of protein in urine are called proteinuria.
Proteinuria –Indication of leaky glomerulli. Most common type of
proteinuria is due to albumin.

Estimation of Non –Protein Nitrogenous Substances

Normal Value: 28 to 40 mg%
Components:
(i) Urea: 20 to 40 mg%
(ii) Uric acid: 2 to 4 mg %
Creatinine: 0.6 to 1.2 mg%
Normal blood levels of these substances are ↑sed in case
of impairment of renal function. An Increase of these end
products in blood -Azotaemia. Tests for Glomerular
Function Renal Clearance Tests A renal clearance test is
employed to assess the rate of glomerular filtration.
Renal clearance is defined as the volume of plasma from
which the substance is completely cleared by the kidneys
per minute. (Ml/min)
Clearance =U x V/P
Where, U -Concentration of substance in urine (mg/dl)
V -Volume of urine excreted (ml/min)
P -Concentration of substance in plasma (mg/dl)
Creatinine Clearance Test:

10
 (i) Creatinine is an excretory product derived form creatine phosphate.
This conversion is spontaneous and non-enzymatic. Creatinine is an
ideal substance for clearance test. Since, it is already present in body
fluids, it’s plasma concentration is steady throughout the day. No need
for intravenous administration as it is produced endogenously. Freely
filtered and not reabsorbed. Creatinine clearance is defined as the
routine of plasma completely cleared of creatinine per minute. Hence,
its measurement is used to assess the renal glomerular function.
Creatinine clearance = GFR = U x
V /P
Where,
U Urine concentration of creatinine (mg/dl)
V-Volume of urine (ml/min)
P -Plasma concentration of creatinine (mg/dl)
Normal value: 90 –120 ml/min
↓sed creatinine clearance indicates ↓se GFR
Urea Clearance Test
End product of protein metabolism. Sensitive than creatinine
clearance because partially reabsorbed. Increased by dietary protein.
Urea clearance Cm= U x V /P
Normal Value: 75ml/min
Inulin Clearance Test:
Polysaccharide of fructose. Neither reabsorbed nor secreted. Ideal
substance to measure GFR
Concentration of Insulin: Volume of urine excreted
GFR = U x V /P
Normal Value: 120ml/min.
IV. Test To Measure Renal Plasma Flow
Para –Aminohippurate Test: PAH: Filtered and secreted. Not
reabsorbed
PAH clearance is defined as the amount of plasma passed
through kidneys.

11
Known amount of PAH is injected into the body.
Concentration of PAH in plasma and urine volume of urine
Excreted.
RPF = U x V /P
Normal Value: 700ml/min

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 EXPERIMENT NO: 4

THYROID FUNCTION TEST:

Thyroid hormones:
Tetra iodothyronine / thyroxine (T4)
Tri iodothyronine (T3)

Binds to:
Thyroxin binding globulin (TBG)
Thyroxin binding pre albumin (TBPA)
Albumin
Apolipoproteins

THYROID DISORDERS:
1. Hypothyroidism
 Primary
 Secondary
 Tertiary
2. Hyperthyroidism
Symptoms of hypothyroidism
 Lethargy
 Constipation
 Dry and coarse skin and hair
 Facial puffiness
 Cold intolerance
 Decreased sweating
 Impaired memory
 Confusion
 Dementia
 Low speech and motor activity
 Anemia
Causes of hypothyroidism

13
Symptoms of hyperthyroidism:
 Nervousness
 Fatigue
 Weight loss
 Heat intolerance
 Increased sweating
 Tachycardia
 Atrial fibrillation
 Warm and moist skin
Causes of hyperthyroidism
a) Overproduction of thyroid hormones
a) Grave’s disease
b) TSH secreting pituitary adenomas
c) Multi nodular goiter
b) Leaking thyroid hormone due to thyroid destruction
a) Lymphocytic thyroiditis
b) Sub acute thyroiditis
c) Radiation
c) Drugs
a) Thyroid replacement drugs
b) Amiodarone
c) Iodinated radio contrast agents
Tests specific to thyroid status:
 Measure the concentration of products secreted by the thyroid gland
 Evaluate the integrity of the hypothalamic–pituitary- thyroid axis
 Assess inherent thyroid gland function
 Detect antibodies to thyroid tissue
Measure the concentration of products secreted by the thyroid gland
 Free T4
 Total serum T4
 Serum T3 resin uptake
 Free T4 index
 Total serum T3

14
 Free T4:
Reference range: 0.8 - 2.7 nanogram/dl
Measures unbound fraction of T4. Increase DED free T4 and
TSH of less than 0.01 milliunits/L is suggestive of non-pituitary
hyperthyroidism
 Total serum T4:
Reference range: 4 - 12 microgram/dl
Measures both bound and free T4
Increased total serum T4 – hyperthyroidism/ increase
concentration of thyroid binding proteins.
Decreased total serum T4 – hypothyroidism/decrease
concentration of thyroid binding proteins/ non thyroid illness [DM,
liver disease, renal failure, prolonged infection and CV diseases]
Serum T3 resin uptake (thyroid hormone binding ratio):
 Reference range: 25 – 35%
Indirectly estimates the number of binding sites on thyroid
binding proteins occupied by T3. The T3 resin uptake is high when
thyroid –binding protein is low and vice versa.
Increase in T3 resin uptake - consistent with hyperthyroidism
Decrease in T3 resin uptake - consistent with hypothyroidism
Free thyroxine (T4) index:
Reference range: 1.2 – 4.2
Free T4 index = Total serum T4 (mg/dl) X T3 resin uptake (%)
The index is high in hyperthyroidism.
The index is low in hypothyroidism.
Total serum T3:
Reference range: 78 - 195 nanogram/dl
Used to detect T3 toxicosis (increase T3 and normal T4)
TSH:
Ref range: 0.3 - 5 microunits /ml or milliunits /L
Symptomatic primary hypothyroidism: >20 mu/L
Mild symptomatic hypothyroidism: 10 to 20 mu/L
Primary hyperthyroidism: <0.05 mu/L

15
 TRH:
Regulates the TSH secretion from pituitary
TRH test measure the ability of TRH to stimulate the pituitary
to secrete

TSH

TSH rise of 5 micro units/ml over baseline - euthyroid state

A significant increase rules out – hyperthyroidism

 Increased radioactive iodine uptake noted in:

 Thyrotoxicosis
 Iodine deficiency
 Post thyroiditis
 Withdrawal rebound after thyroid hormone/ anti thyroid drug therapy
 Decreased radioactive iodine uptake noted in:
 Acute thyroiditis
 Euthyroid patients
 Patients on exogenous thyroid hormone therapy
 Patients taking anti-thyroid drugs
 hypothyroidism

16
 EXPERIMENT NO: 5

CARDIAC FUNCTION TESTS:

The goals of testing for heart disease are:

 To distinguish between symptoms that are heart-related and those
that are due to another condition.
 To determine whether the disorder is acute or chronic
 To determine the severity and extent of the disease.
 When someone presents to the emergency room, the person is
evaluated with a variety of laboratory blood tests and other tests, such
as imaging procedures.
 These are used to determine the cause of the pain and the severity of
the condition.

 CARDIAC BIOMARKERS:
 Cardiac biomarkers are substances that are released into the blood
when the heart is damaged or stressed.
 Measurement of these biomarkers is used to diagnose and monitor.
 Cardiac biomarker tests are used to help and detect the presence of
ACS and cardiac ischemia and to evaluate their severity as soon as
possible so that appropriate therapy can be initiated.
 The current biomarker test of choice for detecting heart damage is
troponin.
 Other cardiac biomarkers are less specific for the heart.

Laboratory Tests:
 Current cardiac biomarker tests used to help diagnose, evaluate,
and monitor individuals suspected of having acute coronary syndrome
(ACS) include:
 Troponin I or T
 CK
 CK-MB.
 Other biomarker tests that may be used:
 Myoglobin
 BNP (or NT-proBNP) — although usually used to recognize heart
failure, an increased level in people with ACS indicates an increased
risk of recurrent events.
Non-laboratory Tests:
These tests allows to look at the size, shape, and function of the heart
as it is beating. They can be used to detect changes to the rhythm of
the heart as well as to detect and evaluate damaged tissues and
blocked arteries.
 EKG (ECG, electrocardiogram).
 Nuclear scan.
 Coronary angiography (or arteriography)
 Echocardiogram.
 Stress testing.
 Chest X-ray.

17
 Cardiac catheterization.
 Cardiac Stress Testing.
 Nuclear imaging.
 Myocardial perfusion imaging.
 Computed tomography
 Troponin:
 Protein found in skeletal and contractual fibers of the heart (cardiac
muscle)
 Troponin I and T are cardiac specific
 Normal: 0-0.3 ng/ml
 Timing
 Earliest rise:3-4 hrs
 Peak: 10-24 hrs
 Return to Normal: 1-3 wks
 Elevated Troponin: Patients with elevated Troponin I levels, normal
CK-MB and no ST elevation have an increased risk of death.
 Useful marker for post-op cardiac surgery patient
 Troponin has three subunits, TnC, TnT, and TnI
 Troponin-C binds to calcium ions to produce a conformational change
TnI.
 Troponin-T binds to tropomyosin, interlocking them to form a
troponin-tropomyosin complex
 Troponin-I binds to actin in thin myofilaments to hold the troponin-
tropomyosin complex.

CREATINE KINASE:
 Creatine kinase (CK/CPK) is an enzyme expressed in a number of
tissues.
 Function: it catalyses the conversion of creatine to phosphocreatine
degrading ATP to ADP.
 The CK enzyme consists of two subunits, B (brain type) or M (muscle
type)
 three different isoenzymes: CK-MM, CK-BB and CK-MB
 CK-BB occurs mainly in tissues, rarely of any significance in the
bloodstream
 Skeletal muscle expresses CK-MM (98%) and low levels of CK-MB (1%)
 The myocardium has CK-MM at 70% and CK-MB at ~30%

 MYOGLOBIN:
 Myoglobin may be ordered as a cardiac biomarker, along
with troponin, to help diagnose or rule out a heart attack.
 Levels of myoglobin start to rise within 2-3 hours of a heart attack or
other muscle injury, reach their highest levels within 8-12 hours, and
generally fall back to normal within one day.
 An increase in myoglobin is detectable sooner than troponin, but it is
not as specific for heart damage and it will not stay elevated as long as
troponin.

18
 Sometimes, a urine test is ordered to evaluate myoglobin
concentrations in those who have had extensive damage to their
skeletal muscles (rhabdomyolysis).

 ELECTROCARDIOGRAPHY:
 Electrocardiography is a recording of electrical activity of the heart on
an electrocardiogram.
 Transmission, magnitude, and duration of the various electrical
impulses of the heart.
 Electrocardiograph – machine that measures and displays impulses of
the heart


 Electrocardiogram(ECG) is a tool that is used to measure and record
the electrical activity of the heart.
 An Electrocardiogram is necessary to establish many heart related
defects like
 atrial enlargement,
 ventricular hypertrophy(enlargement of the ventricles),
 arrhythmias(abnormal electrical activity of the heart) and conditions
that may lead to sudden death.
 The term Electrocardiogram was coined by Willem Einthoven in the
year 1893.
 PROCEDURE:
 The test is simple, painless and risk free.
 The patient is asked to lie down on an examination table.
 10 electrodes are attached to the body (arms, legs and chest).
 With the help of the electrodes, electrical signals generated by the
heart are transmitted to the ECG machine.
 A graph is produced by the machine. The electrodes are removed after
the graph of the cardiac electrical signals is obtained.
 Electrical rhythm as an impulse is emitted from the sinoatrial node
(heart’s natural pacemaker).
 The impulse then reaches the atrioventricular(AV) node which is
between the upper and lower chambers of the heart.

19
 The impulse is reorganized before being transmitted to the ventricle
along special bundle of electrical conducting tissue.
 Once the impulse reaches the ventricles, it causes the chambers to
contract in a consistent manner.
 If the impulse send is delayed or send through a wrong route, the
heartbeat may be irregular. These are detected on the ECG.
 An electrocardiogram (EKG, ECG) translates the heart's electrical
activity into line tracings on paper. The spikes and dips in the line
tracings are called waves.
 The P wave is a record of the electrical activity through the upper
heart chambers (atria).
 The QRS complex is a record of the movement of electrical impulses
through the lower heart chambers (ventricles).
 The ST segment shows when the ventricle is contracting but no
electricity is flowing through it. The ST segment usually appears as a
straight, level line between the QRS complex and the T wave.
 The T wave shows when the lower heart chambers are resetting
electrically and preparing for their next muscle contraction.


 Types of Electrocardiographs.
 Standard machine – 12 lead that records 12 different views at once
 Placed across the chest

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 EXPERIMENT NO: 6

ELECTROLYTES

 These are also known as blood electrolytes.

 They are basically Na, K, Cl, and HCO3 - and present in our body are
minerals like Mg, Ca and trace elements like Cu, Zn, Cr etc.
 Electrolytes are present in the human body and the balance of the
electrolytes in our bodies is essential for normal function of our cells
and our organs.
 These electrolytes help in the conduction of the cells.

 SODIUM
 Normal range: 136-145 mEq/L or mmol/L.
 Na is the most abundant cation in the extracellular fluid.
 The principle role of Na is the regulation of serum osmolality as well
as fluid and acid-base balance.
 The kidneys are the primary organs responsible for controlling body
Na and water.
 Increase in Na conc. (hypernatremia) may indicate impaired Na
excretion or dehydration. A decrease reflects over hydration, abnormal
Na loss, or decreased Na intake.
 Kidney, heart, or pulmonary disease may have difficulty with Na and
water balance, Na conc is often used as an indicator of fluid status.
 Control of Na by the body is accomplished mainly through the
hormones anti diuretic hormone (ADH) and aldosterone.
 Hyponatremia is caused by depletion of Na (Na-wasting renal disease,
replacement with non saline solution, GI losses, renal losses, loss of
Na through the skin) or dilution of serum Na (cirrhosis, CHF, renal
failure, excess water intake).
 Hypernatremia usually results from a loss of free water (diabetes
insipidus, fluid loss through skin, renal, GI), or hypotonic fluid or
through excessive Na intake.

 POTASSIUM
 Normal range: 3.5-5.0 mEq/L or mmol/L.
 Potassium is the most abundant intracellular cation.
 Major role is regulation of muscle and nerve excitability. Other roles
include control of intracellular volume, protein synthesis, enzymatic
reaction, and carbohydrate metabolism.
 The serum K concentration is buffered by the body and may be
normal despite total body K loss. K depletion caused by shift of
intracellular K to extracellular fluid to maintain K concentration.

21
  K is regulated by kidneys, aldosterone, arterial pH, insulin, K intake,
Na delivery to the distal tubule.
 Prolonged deprivation of K results in hypokalemia which may be due
to vomiting, diarrhea, laxative abuse, excess use of diuretics.
 Hyperkalemia results from decreased renal elimination, excessive
intake, from cellular breakdown and due to metabolic acidosis.
 CHLORIDE
 Normal range: 96-106 mEq/L or mmol/L.
 Most abundant extracellular anion and its intracellular concentration
is low.
 It is influenced by the extracellular fluid balance and acid-base
balance.
 Its absorption is coupled with bicarbonate secretion.
 Hypochloremia is caused due to metabolic alkalosis or acidosis caused
by organic or other acids, CRF, fasting, prolonged diarrhea or
vomiting, and diuretic therapy.
 Hyperchloremia can be due to Na water retention, ARF, dehydration,
excess Cl administration.
 BICARBONATE OR CO2 CONTENT:
 Normal range: 22-28 mEq/L or mmol/L.
 The CO2 content is the sum of bicarbonate concentration and the
concentration of CO2 dissolved in the serum.
 The HCO3 /CO2 system is the buffering system to maintain the pH
within the physiological limits.
 They maintain the acid base balance.
 Hypobicarbonatemia is caused by metabolic acidosis,
hyperventilation, and severe diarrhea.
 Hyperbicarbonatemia is caused by alkalosis, hypoventilation,
pulmonary disease and persistent vomiting.

 SERUM CALCIUM:

Calcium plays a key role in neuromuscular excitability, muscle

contraction, regulation of endocrine functions, blood coagulation, and bone
and tooth metabolism.
 Normal serum 9.0-10.5 mg/dL (includes ionized calcium and calcium
bound to protein, primarily albumin, and ions)
 Ionized calcium: 4.5-5.6 mg/dL
 Normal levels maintained by hormonal regulation using skeletal
reserves
 Ionized calcium is more accurate, especially in pt with
hypoalbuminemia; evaluate before repleting Ca+
 Hypocalcemia (serum calcium <9.0 mg/dL; ionized Ca+ <4.5 mg/dL)
 Hypocalcaemia (serum calcium >10.5 mg/dL; ionized Ca+ >5.6
mg/dL)

22
 
 SERUM PHOSPHORUS :
Phosphate is a major intracellular anion. It is important for the intracellular
metabolism of proteins, fats and carbohydrates. Phosphate is important in
maintenance of the phospholipids cellular membrane and the production of
ATP, the chemical compound that provides energy to the cell.
 normal 3.0-4.5 mg/dL
 Hypophosphatemia (<3.0 mg/dL)
 Impaired absorption (diarrhea, Vitamin D deficiency, impaired
metabolism)
 Medications: phosphate binding antacids, sucralfate, insulin, steroids)
 Alcoholism, especially during withdrawal
 Intracellular shifts in alkalosis, anabolism, neoplasm’s

 Increased losses: hyperparathyroidism, renal tubular defects, DKA

recovery, hypomagnesaemia, diuretic phase of ATN
 Hyperphosphatemia (>4.5 mg/dL)
 Decreased renal excretion: acute or chronic renal failure (GFR<20-25
mL/min); hypoparathyroidism
 Increased cellular release: tissue necrosis, tumor lysis syndrome
 Increased exogenous phosphorus load or absorption, phosphorus
containing laxatives or enemas, vitamin D excess
 Acidosis

MAGNESIUM:

Normal Range: 1.5-2.2 mEq/L

Magnesium has a diverse role in the body, especially in neuromuscular
function and enzymatic action. About 50% of the total body’s magnesium is
in the bone, 45% in the
 Intracellular fluid and about 5% in the extracellular fluid.
 Hypomagnesaemia <1.3 mEq/L
 Decreased absorption: prolonged diarrhea, intestinal or biliary fistula,
intestinal resection or bypass, steatorrhea, ulcerative colitis; upper GI
fluid loss, gastric suctioning, vomiting
 Renal losses: osmotic diuresis, DM with glucosuria, correction of DKA,
renal disease with magnesium wasting, hypophosphatemia,
hypercalcemia, hyperthyroidism
 Alcoholism
 Inadequate intake: malnutrition
 Medications
 Intracellular shift: acute pancreatitis
 Reseeding syndrome
 Hypomagnesaemia (>2.1 mEq/L)
 Acute or chronic renal failure

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 EXPERIMENT NO: 7

MICROBIOLOGICAL CULTURE SENSITIVITY TESTS

SENSITIVITY TESTING
It is the degree of activity of the selected antimicrobial agent against the
infecting bacterial strains.
 Usually almost all the bacteria in infectious disease are drug resistant.
 Hence sensitivity test is performed to select the correct antimicrobial
drug of choice.
 It may also help to identify the pathogen.
LIMITATIONS

o It helps us to measure only the antimicrobial activity against bacteria

under laboratory conditions and not in the patients.
The patients’ clinical condition, type and site of infection, drug
hypersensitivity, ADME, characters of the patients are not taken in to
consideration in sensitivity testing techniques
 Sensitivity testing can be performed by
 Diffusion technique and
 Dilution technique
1. AGAR DIFFUSION SENSITIVITY TEST
 A disc of blotting paper is impregnated with a known volume and
appropriate concentration of antimicrobial placed on a plate of
sensitivity agar inoculated with test organism.
 The antimicrobial diffuses from the disc in to the medium. After 24
hours, the culture is examined for areas of growth around the disc.
 Growth for sensitive strains is inhibited for a distance while for
resistant strains it grows up to the edge of the disc.
 The zone of inhibition caused by the antimicrobial is compared with
the control.
 The volume, moisture content, PH, constituent of agar medium,
concentration, storage and application of dose influence the diffusion
technique.
 Agar diffusion sensitivity tests are carried out either by Kirby-Bawer
(KB) method, ICS method or by Stocks method.
 Modified KB method is recommended by the National Committee for
clinical Laboratory Standards (NCCLS) and the WHO.

DILUTION SENSITIVITY TESTS.

 Dilution sensitivity tests usually measures the minimum inhibitory
concentration (MIC) or minimum bactericidal concentration (MBC)
required to kill the bacteria.
 Here dilutions of antimicrobials are added to the broth or agar.
 A standardized inoculums of test organism is added.
 After overnight the lowest antimicrobial required to prevent visible
growth is taken in to consideration.
 Dilution technique needs
Careful standardization

24
 Broth and agar medium
Antimicrobial solution
Incubation time and
Dilution time
General requirements for sensitivity testing.
1. Sensitivity testing agar.

Mueller Hinton agar (MHA)

Composition
 Meat infusion 2.0 g/l
 Casein hydrolysate 17.5 g/l
 Starch 1.5 g/l
Agar-agar 13.0 g/l
2. ANTIMICROBIAL DISC
 This disc should be refrigerated at a temperature instructed by the
manufacturer.
 This should not be used after expiry date.
 The working stock disc should be warmed to room temperature, avoid
keeping in direct sunlight.

ANTIMICROBIAL RESISTANCE
 Antimicrobial resistance can arise in bacteria in several ways.
 Microbes acquire resistance after a change in their DNA.
 Such changes may occur by
 Genetic mutation i.e. by alteration in the structure of their own
DNA.
 Genetic exchange i.e. by acquisition of extra- chromosomal DNA
from other bacteria.
 Genetic exchange is the most common cause of serious clinical drug
resistance because it can produce resistance to multiple drugs.
 In genetic exchange, the resistance genes are transferred from one
bacterial species to another by means of discrete, movable, extra
chromosomal DNA elements called transposons.
 Transfer of transposons between bacteria can occur by
 Conjugation ie directs physical mating between bacteria.
 Transduction i.e. through the agency of bacteriophages.
Transposition i.e. by means of plasmids which are transferable, extra
chromosomal DNA molecule.
DRUG RESISTANCE
 It refers to unresponsiveness of a micro-organism to an antimicrobial
agent.
 They are of 3 types:
i. Natural resistance
ii. Acquired resistance
iii. Cross resistance.
NATURAL RESISTANCE
 Some microbes have always been resistant to certain AMA.

25
 They lack the metabolic process or the target site which is affected by
the particular drug.
 eg. gram negative bacilli are normally unaffected by Pencillin G
 M.tuberculosis is insensitive to tetracyclines
ACQUIRED RESISTANCE
• It is the development of resistance by an organism (which was
sensitive before) due to the use of an AMA over a period of time.
CROSS RESISTANCE
• Cross-resistance is the tolerance to a usually toxic substance as a
result of exposure to a similarly acting substance.
• It is a phenomenon affecting e.g. pesticides and antibiotics an
example

26
 EXPERIMENT NO: 8

PULMONARY FUNCTION TESTS

Anatomy and Physiology of Lungs
• Left and right lungs are in the pleural cavity of the thorax.
• Right lung has three lobes, but the left has only two lobes; space is
thus provided for the heart.
• Lungs are connected to pharynx by trachea.
• Trachea splits into left and right main stem bronchi that deliver
inspired air to respective lungs.
• Main bronchi split into smaller bronchi, bronchioles, terminal
bronchioles and finally alveoli.
• In alveoli, the lungs exchange carbon dioxide for oxygen across a thin
membrane separating capillary blood from inspired air.
• Thoracic cavity is separated from abdominal cavity by diaphragm-a
thin sheet of dome shaped muscles-contracts and relax during
breathing.
• Lungs are contained within the rib cage but rest on the diaphragm.
• Between the ribs are two sets of intercostals muscles-attach to each
upper and lower rib.
• During inhalation, the intercostals muscles and diaphragm contracts,
thereby enlarging the thoracic cavity.
• This generates a negative intrathoracic pressure, allowing air to rush
in through nose and mouth down into pharynx, trachea and lungs.
Purpose of lungs is to take oxygen from the atmosphere and exchange it for
CO2 in blood
Pulmonary Function Tests (PFTs) are useful
- In diagnosing respiratory disorders.
- To monitor therapy for patients with respiratory diseases.

PFTs measures:
Lung volumes
Lung flows
Diffusion capacity
Airway reactivity
Compliance
Resistance and conductance
Respiratory functions are assessed by:

Lung volume tests

Lung flow tests
Lung volume tests
a) Expiratory reserve volume (ERV)

27
 b) Slow vital capacity (SVC)
c) Residual volume (RV)
d) Functional residual capacity (FRC)
e) Inspiratory capacity (IC)
f) Inspiratory reserve volume (IRV)
g) Tidal volume (TV)
h) Total lung capacity (TLC)
Lung flow tests
a) Forced expiratory volume (FEV)
b) Peak expiratory flow rate (PEFR)
c) Forced expiratory flow (FEF)
Lung volume tests
• Lung volume tests indicate the amount of gas contained in the lungs
at various stages of inflation.

Tidal volume:
Amount of air inhaled or exhaled at rest
Reference range: 500 to 750 ml
It is infrequently used as a measure of respiratory disease
Inspiratory capacity (IC):
The volume measured from the point of the TV where
inhalation normally begins to maximal inspiration is known as IC.
Reference range: 500 ml + 3.1 L = 3.6 L
Inspiratory reserve volume:
Amount of air that is inhaled with maximal inhalation after
the normal inhalation. or The volume measured from the “top” of the TV, (ie.
the initial point of normal exhalation) to maximal inspiration is known as
the IRV.
Reference range: 3.1 L
Expiratory reserve volume:
Amount of air that is exhaled with maximal expiration after
the normal exhalation. Or during exhalation, the volume from the “bottom”
of the TV (ie. Initial point of normal inhalation) to maximal expiration is
referred to as ERV.
Reference range: 1. 2 L
Slow vital capacity:
When the full inhalation-exhalation procedures is repeated
slowly – instead of forcefully and rapidly – it is known as SVC.This value is
the maximum amount of air exhaled after a full and complete inhalation.In
patients with normal airway function, SVC and FVC are usually similar
(hence shown as Vital Capacity). In patients with diseases, such as COPD
during the initial stages of disease, the FVC decreases before the SVC.

Residual volume: Amount of air that is left in the lungs after full
exhalationReference range: 1. 2 litre.RV is unmeasurable by spirometry but
measurable by body plethysmography. Without RV lungs would collapse like
deflated balloons.In asthma, RV increases due to obstruction.

28
Functional residual capacity: It is the volume of the gas remaining in the
lungs at the end of the TV. It is the sum of the ERV and RV [2.4L].An
increased FRC usually represents hyperinflation of the lungs and indicates
airway obstruction.
FRC decreases in diseases that affect many alveoli (pneumonia) and by
restrictive changes (fibrotic pulmonary tissue changes)

Functional Residual Volume (2.4 L) + Tidal volume (0.5 L) + Inspiratory

Reserve Volume (3.1L)
Total lung capacity: It is the total amount of gas contained in the lungs It
is the sum of RV and vital capacity.Reference range: 6 L.

Forced expiratory volume: Changes in FVC measurement from baseline

reflect the degree of current airway obstruction.

The normal value for FEV1 is 0.75 – 5.5 L & this wide variation is due to
physical variables among patients. Usually, patients value is described
either as a percentage of a predicted value or as a standard deviation (SD)
from the mean of a physically matched population of the same age. A value
of >80% of predicted value or within + 2 standard deviation (SD) is
considered normal. In both obstructive and restrictive diseases, the FEV
usually shows a reduction in flow
The magnitude of change reflects the severity of disease as:
Mild : 61 – 80% of predicted
Moderate: 41 – 60% of predicted
Severe: < 40% of predicted
The ratio of FEV1 to the FVC is another way to estimate the presence and
amount of obstruction in the airways. This ratio indicates the amount of air
mobilized in 1 sec as a percentage of the total amount of mobilized air. In
healthy individuals, the normal value is FVC 0.5 = 50%; FVC1 = 80%; FVC3 =
98%In patients with restrictive disease it is usually normal because both
FVC and FEV1 similarly reduced.In patients with obstructive disease, this
ratio will decrease.
Peak expiratory flow rate:
It is the measure of maximum airflow rate. Occurs within the
first millisecond of expiratory flow.Measured by using peak flow meter

Forced expiratory flow:

FEF measures airflow rate during forced expiration. While
FEV measures the volume of air during expiration, FEF measures the rate of
air movement. The FEF from 25 to 75% of vital capacity is known as FEF 25-
75.
Peak expiratory flow rate:
Range is 400 – 800 L/minute and in women 200-
600L/minute. Values of 50 – 100L / minute indicate severe acute
obstruction. PEFR reduced in obstructive disease but normal in restrictive
diseases.

29
 Forced expiratory flow:
FEF measures airflow rate during forced expiration. While
FEV measures the volume of air during expiration, FEF measures the rate of
air movement. The FEF from 25 to 75% of vital capacity is known as FEF 25-
75.

Diffusion capacity tests

Useful in assessing interstitial lung disease.Measures per minute transfer of
gas usually CO from alveoli to the blood Can be measured as the single
breath-test. Used for assessing pulmonary fibrotic changes. The mean value
for CO is 25 – 30ml/minute/mmHg
Diffusion capacity decreases in diseases that causes alveolar fibrotic
changes
The mean value for CO is 25 – 30ml/minute/mmHg. Diffusion capacity
decreases in diseases that causes alveolar fibrotic changes .

LUNG VOLUMES & CAPACITIES:

Tidal Volume (VT):The volume of air entering the nose or mouth per breath
(500 ml).
Residual Volume (RV): The volume of air left in the lungs after a maximal
forced expiration (1.5L).
Expiratory Reserve Volume (ERV): The volume of air that is expelled from
the lung during a maximal forced expiration that starts at the end of
normal tidal expiration (1.5L).
Inspiratory Reserve Volume (IRV): The volume of air that is inhaled into
the lung during a maximal forced inspiration starting at the end of a
normal tidal inspiration (2.5L).
Functional Residual Capacity (FRC): the volume of air remaining in the
lungs at the end of a normal tidal expiration (3 L).
Inspiratory Capacity (IC): The volume of air that is inhaled into the lung
during a maximal forced inspiration effort that begins at the end of a
normal tidal expiration (VT+IRV=3L).
Vital Capacity (VC): The volume of air that is expelled from the lung during
a maximal forced expiration effort starting after a maximal forced
inspiration (4.5L).
Total Lung Capacity (TLC): The volume of air that is inhaled into the lung
after a maximal inspiration effort (5-6 L)

Diffusion Capacity:

Estimates the transfer of oxygen in the alveolar air to

the red blood cell.
Factors that influence the diffusion:
1 Area of the alveolar-capillary membrane
(A) ,Thickness of the membrane (T),Driving pressure and
Hemoglobin

- Increased:

30
 • Pulmonary hemorrhage

• Polycythemia

• Early CHF

• Asthma

• Exercise

• Obesity

• Left to right shunt

Spirometry: Measures the lung volume change during forced breathing

maneuvers:
Forced vital capacity (FVC)
Forced expiratory volume in the first second (FEV-1)
Spirometry Obstruction Restriction
FEV-1 Decreased Decreased
(--) (-)
FVC Decreased Decreased
(-) (-)
FEV-1/

31