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TABLE OF CONTENTS

SNo. PRACTICAL

1 Hazards in laboratory & safety precautions.

2 Microscopic Examination of cells

3 Determination of Blood group

4 Recording of blood pressure using sphygmomanometer

5 Determination of Bleeding time

6 Determination of Clotting time

7 Estimation of total number of White blood cells (WBC)

8 Estimation of total number of Red blood cells (RBC)

9 Estimation of total number of Platelets

10 Methods of hemoglobin estimation.

11 Preparation of Haemin crystals from human blood

12 Pulmonary function testing

13 Recording of Electrocardiogram

14 Record and correlate ECG and heart sound

EXPERIMENT NO. 1

DATE :-_____________________

Hazards in Laboratory and Safety Precautions 1- Introduction: Laboratory is probably the most hazardous place in any medical set up. Laboratory workers

are exposed to a variety of hazards and hence must observe certain precautions to protect

themselves from these as much as possible.

Laboratory hazards mainly arise from:

a) Infected specimens.

b) Dangerous chemicals.

c) Faulty equipment.

2- Hazards from infected specimens: Almost all specimens in laboratory are from patients. Many of them suffer from known or

unknown infectious diseases and tests are required either to diagnose these or to follow the

course of the disease. The specimens of blood, urine, sputum, stool or aspiration fluids may

all be infected. Infections, which can be transmitted through these, may be parasitic, fungal,

bacterial or viral.

Most dangerous transmitted infections include:

A- Blood:

1- AIDS.

2- Hepatitis.

3- Hemorrhagic fever.

B- Stool:

1- Typhoid bacilli (salmonella).

2- Dysentery organisms (shigella).

3- Cholera.

4- Parasitic ova and cyst.

C- Sputum:

1- Tuberculosis.

2- Other respiratory tract infections.

3- Hazards from chemicals: A large number of chemicals are used in various laboratory tests. These include:

a- Corrosives as strong acids and alkalies.

b- Poisons as potassium cyanide, benzene compounds, mercury and toxic gases.

c- Carcinogens like benzidine, orthotoludine etc.

d- Explosives and inflammable chemicals like perchloric acid, ether etc.

With the exception of last group, which may destroy whole of the working area, others are

dangerous for working staff mainly. These may cause injury to the laboratory workers by the

following ways:

i- By direct contact with skin, mucous membranes or inadvertent swallowing while

pipetting.

ii- By inhalation of vapors or fine powder.

iii- By absorption from lungs or GIT after aspiration or inhalation.

4- Hazards from faulty apparatus: Almost all apparatus used in laboratory is capable of giving rise to some kind of injury to the

person using it.

a- Glass apparatus may break while in use causing cuts and may even soil the cut area with

its contents which may be dangerous to body tissues.

b- Gas equipment may cause explosion, fire or gas poisoning.

c- Centrifuges can cause mechanical injury when they are unbalanced or improperly closed.

d- Electrical apparatus is the most common source of electric shock or even fire in

laboratory. This may give rise to electric shock when insulation inside the equipment gets

defective. As apparatus itself is usually made of metal, current may spread into it. In some

cases switch is defective or its plug is broken and while connecting equipment to main supply

or switching on, one may receive electric shock.

5- Safety precautions: 1- Specimen containers should be such that spilling does not occur. Care should be taken in

removing and replacing caps of containers.

2- Strict aseptic precautions should be observed when collecting blood or other specimens. It

is better to wear the gloves.

3- Soiling of request forms and other papers must be prevented.

4- If a patient is known to be suffering from hepatitis or AIDS or other dangerous infectious

diseases, the specimen containers must carry a red label. This will alert all persons handling

this specimen.

5- Bench workers must wear laboratory coats and gloves. In some cases use of masks is

recommended.

6- Washing hands with soap and water should be a frequent practice particularly when

leaving the work area.

7- Eating and drinking inside the laboratory should be strictly prohibited. Foods and drinks

should not be placed in laboratory refrigerators.

8- Use of carpets and cushions should be discouraged. These are extremely difficult to clean

or disinfect in case of spills. A smooth, polished floor is easiest to clean.

9- Mouth pipetting should be avoided as for as possible. Instead use rubber teats. However if

it is to be done, it is better to plug upper end loosely with cotton wool. All care must be taken

to ensure that fluid is not sucked into the mouth.

10- Use of disposable laboratory ware is ideal but may not be possible. All disposable ware

should be collected in thick bags for later and proper disposal.

11- If re-useable glassware is used, it should be put in a strong disinfectant solution. At the

end of the day transfer these to a strong detergent. Next day place these in boiling water for

10 to 20 minutes, cool and then wash with tap water.

12- All potentially infected material like stool, urine, bacterial cultures etc. must be

incinerated after disinfections.

13- In case of spilling or breakage in centrifuge or on bench or floor, first collect all glass

pieces with forceps and then wash the area with strong disinfectant followed by washing with

strong detergent. Then wash with water and wipe to dry.

14- All reagent containers must be of proper size and shape and properly stoppered. These

shall never be left open. Containers must be clearly labeled. If these contain dangerous

chemicals or infective material, a danger sign or poison label must be pasted on the container.

15- If fuming chemicals are to be used it is advisable to use these in a cabinet or hood made

for such purpose.

16- Infected material should preferably be dealt with in a laminar flow cabinet.

17- Saline eye wash bottles must be supplied so that eyes can be immediately washed in case

of any contact with the eyes.

18- Electric and gas points should be checked from time to time for any leakage.

19- All water baths and centrifuges should be cleaned periodically.

20- Before leaving the work area laboratory coats and gloves should be removed and hands

should be washed.

6- Common disinfectants: a- FORMALINE 5 – 10 % solution for cleaning floors, surfaces and containers.

b- HYPOCHLORITE with 10 % available chlorine e.g. Clorox. This is used for overnight

soaking of contaminated laboratory glassware, cleaning surfaces, floors and equipment.

c- GLUTARALDEHYDE 2 % is good for cleaning of infected metallic objects.

d- PHENOLS as Lysol, sudol etc. for soaking of infected material.

7- Detergents: A number of detergents are available in the market. Any one can be used.

EXPERIMENT NO. 2

DATE :-_____________________

Microscopic Examination of Cells

Objectives:

Use of microscope; The metric system (estimating the size of microscopic

objects; interconversaion of different metric units).

Examination of live cells and identification of various cellular organelles.

Different between mitosis and meiosis.

Different between primary tissues and draw a typical organ showing these.

Background:

Modern definitions of units used in metric system for length, mass, volume and

temperature (general conference on wrights and measurements; international system of

units, SI).

Meter (m): unit of length; equal to 1, 650, 763, 73 wavelengths in a vaccume of the

orange- red line of the spectrum of krypton-86.

Gram (g): unit of mass; based on the mass of 1 cubic centimeter (cm3) of water at the

temperature of its maximum density.

Liter: unit of volume: equal to 1 cubic decimeter (dm3)or 0.001 cubic meter (m3).

Celius ©: temperature scale in which 0 and 100 are the freezing and boiling points of

water respectively; equivalent to the centigrade scale;

Conversion between different orders of magnitude in the metric system: based on

powers of 10.(table).

A Visual Field and estimation of microscopic size:

Objective 10 and ocular lens 10; visual field: 100 (diameter is apprix. 1600 um)

Procedure:

Use a microscopic slide having pattern of small dots and thin lines on it;

1. Using X10 object lens estimate:

a) The diameter if one dot: -------------- um

b) The distance between the edges of two adjacent dots: -------------------um

2. Using the X45 objective lens estimate:

a) The width of one line: -----------------um

b) The distance between edges of two adjacent lines: ----------------um

B Microscopic examination of cheek cells:

Membrane lining the cheeks are several layer thick: Surface cells (continuously lost &

replaced through mitosis in deeper layers in the outer layer of epithelial issue are alive

(cells of similar position in skin are dead) and can be viewed under the microscope (most

cells need staining; positively charged methylene blue combines with negatively charges

in the chromosomes to stain the nucleus blue and cytoplasm with a lower concentration

of negatively charged organic molecules appears almost clear).

Procedure:

1. Rub the inside of one cheek using cotton tip of an applicator stick and press it

against a clean glass slide rotating it while maintaining pressure and push the

cheek smear across the slide for about ½ inch

2. Observe unstained cells under X100 and X450 power.

3. Stain the slide (a drop of mthylene blue), place a cover slip, view at 100X and

450X power

EXPERIMENT NO. 3

DATE :-_____________________

Aim To find out your own blood group.

Requirement: Three glass slides, Anti sera A, Antisera B, Antisera D with separate droppers and Lancet.

Principle: The principle of the experiment is to find out the blood group by mixing the blood with the Antiserum and observe the agglutination by antigen antibody reaction.

Theoretical Explanation: At least 100 antigen can be recognized and more than 15 systems of well define antigens have been known, out of many antigen type A and type B are important which constitute OAB system. People are Rh +ve or Rh –ve In OAB system either both agglutinogens are present on the surface of RBC ,one of them present or both of them are absent on the surface of RBC .On this basis the population may be divided in to four blood group i.e .O,A,B,AB These are developed genetically according to genotypes. Blood group with their genotypes and Agglutinogen+agglutinins:

Genotype Agglutinogens Agglutinins Blood group %

OO - Anti A& Anti B O 47%

OA or AA A Anti B A 41%

OB or BB B Anti A B 9%

AB AB - AB 3%

The agglutinins are globulin which develops spontaneously due to A and B antigens which enter the body in food, bacteria and in other ways. Most naturally occurring antibodies to ABO antigen are Ig M class Rh antibodies are Ig G. Blood group antigens are present in surface of RBCs.

Procedure: 1. Take 3 dry glass slides and put them on the table. 2. Mark the slides Antisera A, Antisera B and Antisera D. 3. Put one drop of each antisera, using a separate dropper. 4. Prick the finger. 5. Add one drop of blood to each slide and mix well with the help of mixer. 6. Observe each slide for agglutination in bright light. 7. Assess the observation ,find out the blood group

The reagents used for ABO blood group determination are anti-A and anti –B. Both are Ig M antibodies. These are commercially available standardized antibodies.

Clinical Significance: When blood transfusion is necessary, the blood typing of the recipient and the donor should be done very carefully, to avoid hazards of blood transfusion.

The mismatched blood transfusion result in to hemolytic jaundice and transfusion reaction associated with hemolysis, haemoglobinurea and severe type of anemia. Erythroblastosis Fetalis is a hemolytic disease of new born characterized by progressive agglutination and subsequent phagocytosiss of RBC .If father is Rh+ve, mother is Rh –ve and child has inherited Rh +ve antigen from father. The mother develops Anti Rh antibodies that diffuse through placenta in to the fetus to cause RBC agglutination. Prevalence of disease Rh –ve mother having her first Rh +ve child does not does not develop this disease but 3% of second and10% of third baby develop erythroblastosis fetalis and they are born with severe anemia in which many blast forms appear in circulation. The incidence rises progressively with subsequent pregnancies. Hemolytic transfusion reactions occur mainly due to ABO and Rh system.

Observation chart: Antisera Antisera A &1 drop

of blood Antisera B & 1dop of blood

Antisera D& 1 drop of blood

Agglutination

Agglutination = +ve No agglutination= -ve

Result: My blood group is =__________ The Rh +ve person has Rh antigen but no antibodies. Rh –ve person neither Rh antigens nor antibodies. Rh antibodies do never develop spontaneously until and unless Rh –ve person is exposed to Rh antigen. ABO and Rh blood group are of main importance in routine use. Some other blood group include MNS ,P, Kell ,Duffy ,Kids, Lewis etc and a very important Rh group.

Precautions: 1. Slides should be washed and dried carefully. 2. Separate dropper should be used for each antisera. 3. The blood drop should not be touched to antisera. 4. A separate mixer should be used for mixing.

EXPERIMENT NO. 4

DATE :-_____________________

Aim:-

To record blood pressure of a resting subject by Sphygmomanometer.

REQUIRMENT:

Stethoscope and Sphygmomanometer,

PROCEDURE:

1. Inflate the cuff until the pressure reaches approximately 180 mmHg.

2. Slowly reduce the pressure in the cuff (approximately 1 to 2 mmHg

per second) while listening through the stethoscope for Korotkoff

sounds.

3. The systolic pressure is the pressure at which sharp, tapping sounds

are first heard.

4. Continue slowly reducing cuff pressure (at 1 to 2 mmHg per second).

The diastolic pressure is defined as the pressure at which the sounds

disappear.

5. Completely deflate the cuff once diastolic pressure is determined. Do

not leave the cuff partially inflated for a long time.

6. For each subject, record four measurements of the blood pressure.

Allow one to two minutes between measurements for recovery.

7. Repeat the procedure using other students until you feel confident in

measuring blood pressure.

RESULT:

Blood Pressure of the Subject is found to be______________.

EXPERIMENT NO. 5

DATE :-_____________________

Aim: To determine your own clotting time. OBJECTIVE: The student should be able to:

Find out the clotting time.

Know the causes which may lead to prolonged and defective clotting time.

Have knowledge bout anticoagulants used in the laboratory.

Understand the: 1) Hemophilia 2) Thrombosis 3) Thromboembolic condition and their hazards 4) Differentiate between bleeding time and clotting time. REQUIREMENT: Capillary tube, Stop watch and pricking needle. PRINCIPLE: Principle of the experiment is to observe the time when the insoluble monomer fibrin clot is formed. THEORETICAL EXPLANATION: When ever there is cut or injury the blood comes in contact with rough surface and the procoagulants present in blood are activated and convert the plasma protein Prothombin into Thrombin which converts the soluble plasma protein Fibrinogen into an insoluble protein Fibrin, jelly like semisolid substance called clot This is a loose type of plug and may be dislodged by the pressure of the blood stream. The blood clot then contracts and firmly plugs the rent in the vessel. This retraction of blood clot is the next step of haemostasis. Clotting time is the time from the time of injury to the formation of clots i.e. Fibrin Thread. Normal clotting time is 2-8 min. PROCEDURE: 1. Prick the ring finger with disposable blood lancet after sterilization of the finger by sprit swab and start the stop watch. Discard the first drop of blood. 2. Gently press the finger and allow the blood to come out freely & touch one end of capillary tube with the blood drop by holding the capillary tube horizontally and allow the tube to fill by capillarity. Capillary should be placed in the middle of blood

drop. 3. Fill the capillary tube with blood which appears in the tube by capillary action. 4. Start breaking a small piece of tube at distance of 1 c.m, separate the broken piece very gently and observe for the formation of fibrin thread. 5. Repeat the process after every 30 sec observing each time for the appearance of fibrin thread. 6. End point is reached when fibrin thread appears .stop the stop watch. 7. Note the clotting time on the observation chart and draw the figure of tube. CLINICAL SIGNIFICANCE: Excessive bleeding tendencies are related to the clotting factor defect and the deficiency and the defect of platelets .clotting time is increased due to decrease in clotting factor, factor viii deficiency causes haemophilia etc

The hemorrhagic diseases due to selective deficiencies of other clotting factors are:

Afibrinogenemia is the lack of factor 1.(fibrinogen)it may be due to complicated pregnancies ,congenital lack of fibrinogen also reported.

Fibrinogen is formed in the liver therefore it is reduced in lever disorder.

Hypo prothrombinemia (bleeding tendency in liver disease caused by lack of factor2). It occurs due to decreased hepatic synthesis of prothrombin usually secondary to vit. K deficiency.

Parahemophillia is caused by deficiency of factor v labile factor which is congenital.

Disorder caused by deficiency of factor VII(Proconvertin).

Hemophilia (classical) caused by factor VIII deficiency (antihaemophilic factor) is relatively common which may be treated by repeated transfusion of blood or factor VIII rich preparation of plasma. It is an inherited sex link recessive disorder.

Haemophilia B (Christmas)is also a congenital disease caused by deficiency of factor IX.

The congenital bleeding tendencies are also caused due to deficiency X, XI and XII.

In obstructive jaundice, absorption of vit. K along with other fat soluble vitamins is depressed. The resulting clotting factor deficiencies cause bleeding tendency.

The Thromboembolic condition is an intravascular clotting Thrombosis may occur due to deposition of cholesterol (Arteriosclerosis) i.e. formation of plaques.

The Thrombus may get detached and lead to embolism which may be pulmonary embolism, coronary embolism which may lead to ischemia and heart attack. The emboli may block the arteries in brain, kidneys or else where.

PRECAUTIONS: i) No air bubble should interrupt the column of blood in capillary tube ii) The blood should be immediately filled in the tube to avoid clotting of blood

before filling of tube. iii) The stop watch should be started immediately as the prick is made. iv) A small piece of tube (about 1cm) should be first broken & then be separated

very carefully to avoid breaking of fibrin thread. v) IMPORTANT:

Don’t use the 1st drop of blood because it is clotted unusually fast.

Note the time that the drop of blood to be used appears at the site of puncture .this is used as the starting time.

The tube should be kept at body temperature as cold retard coagulation of blood (if the outside temperature is different then the tube can be placed in a water container at 37c.

FIGURE

OBSERVATION:

RESULT: My clotting time is:

EXPERIMENT NO. 6

DATE :-_____________________

Aim: To determine your own bleeding times by Dukes method. OBJECTIVE: The student should be able to:

Learn the normal bleeding time and causes of prolonged bleeding time.

Know the process of haemostasis.

Recall the functions of platelets.

Know the bleeding disorders. REQUIREMENT: Filter paper, stop watch and all the material for pricking the finger. PRINCIPLE OF EXPERIMENT: Principle of this experiment is to note the time when bleeding stops due to formation of platelet plug. The bleeding time is taken for a small sharp incision to stop bleeding. THEORETICAL EXPLANATION: When ever there is a trauma, wound, cut or injury bleeding takes place. To prevent the excessive loss of blood, nature has provided the mechanism of Hemostasis that occurs in different steps. The first step is local myogenic effect which is prolonged by nervous control. The second step is the formation of platelet plug As the vessel traumatized, the near by passing platelets come and adhere to the rough surface (collagen fibers) and secrete ADPs and thromboxane A2 which make these platelets sticky for other passing platelets i.e platelet plug is formed which plugs the rent in the vessel. The sharply cut vessel bleeds, more than crushed vessel. The more a vessel is traumatized more is the degree of spasm. Platelet plug is unstable and temporary .proper hemostasis occurs due to addition of clotting factors leading to formation of fibrin. PROCEDURE:

1. After all usual precautionary measures, prick the finger with presterilized disposable blood lancet about 3mm deep and start the stop watch.

2. Wipe off the blood from the site.

3. Apply a gentle pressure and touch the site of prick with filter paper so that the spot of blood appears on the filter paper.

4. Repeat the procedure after every 30 sec by touching the site of prick with filter paper and wiping it off every time till there is no spot of blood on filter paper.Stop the stop watch.

5. Circle the spots of blood with pencil and note the time of each spot.

6. Note the bleeding time and paste the filter paper on your journal.

7. Bleeding time also calculated by dividing total no. of drops on the filter paper by two.

Bleeding time = No. of drops on the filter paper/ 2

CLINICAL SIGNIFICANCE: There are various conditions in which the bleeding time is prolonged. 1) Thrombocytopenic purpura is the most t important. 2) Non Thrombocytopenic purpura are also there due to:

Vitamin C deficiency (scurvy).

Infection leading to consumption of clotting factors & deficiency of platelet.

Allergic reactions.

Autoimmune reactions. 3) Idiopathic thrombocytopenia 4) Aspirin inhibit platelet aggregation inhibiting the formation of prostaglandin and

thromboxane. It rarely causes bleeding tendency because it does not inhibit aggregation to sufficient degree.

Thrombocytopenic purpura result when total platelet count falls below 50,000/cu.mm of blood .The bleeding time is the time from prick Normal bleeding time by duke’s method is 2 to 6 min. However it depends, on various factors .e.g.

Depth of wound, cut or injury.

Degree of hyperemia in the finger

Total platelet count in blood.

Elasticity and the condition of skin. Several substances are secreted at the site of pricks e.g Serotonin, thromboxane A2 ,5 hydroxy tryptophanamine(5HT) ,histamine

Thrombocytopenic purpura: Bleeding disorder which is caused by lack of platelets when their count is decrease below 50.000/cu.mm .In this the bleeding time is prolonged but the clotting time remains \normal. The blood oozes out of the blood vessels and clot forming blue patches called purpura Subcutaneously or with in the tissues Normally the blood vessels are damaged several times a day and repaired at the same Time by platelet plugs.

PRECAUTIONS: a) No need to discard first drop of blood b) The prick should be 3mm deep. c) Do not squeeze the finger, apply only a gentle pressure. d) Start the stop watch as you prick the finger.

OBSERVATION

RESULT: My bleeding time is: sec.

EXPERIMENT NO. 7

DATE :-_____________________

Objective: To count the total number of WBCs/Cu.mm3 of your own blood. Requirement: Improved Neubaur’s. Haemocytometer, Microscope, Cover slip, Truk’s fluid and the material for pricking the finger. Principle: The principle of this experiment is to dilute the blood 20 times and to calculate the number of WBC in the area in Haemocytometer meant for WBC count after multiplying by diluting factor i-e 1:20 Theory: The white blood cells (WBCs) or leucocytes are one of the three specialized cells. WBCs involve in inflammatory processes and invading organisms. The WBCs must be present in sufficient numbers to carry out these functions properly but not in an abnormal excess. Composition Truk’s Fluid. Glacial Acidic Acid (hemolytic agent) ----------02ml 1%Aqueous gentian violet solution (dye)-------50ul Distal water to make solution ------------------ 100 ml Normal WBC Count: 4000 – 11000 / cmm. Procedure:

1. Wash and dry the counting chamber & WBC pipette and focus the ruled area in low power (10x) under the microscope and put the cover slip.

2. Prick the finger by taking all the precautionary measures as directed previously.

3. Discard first drop of blood, wipe it off, allow the skin to dry and take the blood in WBC pipette having white bead, up to the mark 0.5 by holding the pipette horizontally and touching the tip of the pipette to the drop of the blood. The blood enters the pipette by capillary action, if not, apply a gentle suction.

4. Wipe the blood from the tip of the pipette and suck the Truk’s fluid up to the mark 11. This makes the dilution 1:20.

5. Mix the solution by folding the rubber tube and rolling the pipette in between the palms of hands. Don’t shake the pipette as it will break the Cell.

6. Discard 2 to 3 drops from the pipette and allow the next drop to appear at the tip of the pipette.

7. Touch the drop at the edge of cover slip and groove to charge to counting chamber.

8. Wait for few min so that the WBC settles down. 9. Observe the slide and count the number of WBC in four squares, 4 of

each corner, i-e square no. 1, 3, 7 & 9. Used for WBC count and fill the observation chart.

Precautions:

1. Discard first drop of blood. 2. While taking the drop of blood the skin should be dry. 3. Hold the pipette horizontally so that the blood appears in pipette by

capillary action 4. The tip of the pipette should be properly cleaned after taking the

blood. 5. No air bubble should interrupt the column of blood. 6. Mix the blood with Truk’s fluid immediately to avoid clotting of

blood in pipette 7. Don’t shake the pipette as it will break cells. 8. After mixing discard 2 to 3 drops from the pipette so that diluents in

the stem is discarded.

OBSERVATION CHART: Square 1. Square 3.

Square 7. Square 9.

No. of WBC in square 1. ______ No. of WBC in square 3. ______ No. of WBC in square 7. ______ No. of WBC in square 9. ______ Total No. of WBCs in Four squares: ______

No. of WBCs in one large square : ___________

Calculation: Area of one large square = 1mm2 Depth of chamber = 1/10 mm Volume = Area x Depth Area of one large square = 1/10 mm2

No. of WBCs in one large square: ___________

Total no. of WBCs in 1 cmm of diluted blood: ______ x 10

Total no. of WBCs in 1 cmm of undiluted blood: ______ x 10 x 20 = _______ /cmm

RESULT

WBC count of my blood is =___________mm3

EXPERIMENT NO. 8

DATE :-_____________________

Object: To count the total number of Platelets/Cu.mm3 of your own blood. Requirement: Improved Neubaur’s. Haemocytometer, Microscope, Cover slip, Platelet fluid(Rees Ecker) and the material for pricking the finger. Principle: The principle of this experiment is to dilute the blood 200 times and to calculate the number of Platelets in the area in Haemocytometer meant for Platelet count after multiplying by diluting factor i-e 1:200. Theory: The platelets are smallest of the blood cells. They are the granulated bodies 2-4 microns in diameter. The megakaryocytes, giant cells in bone marrow, form platelet by pinching of bits of cytoplasm and extruding them into the circulation. Platelets are important in the formation of haemostatic plug and their deficiency (thrombocytopenia) causes a bleeding disorder. Platelets are also important because they produce many important substances like serotonin (5 HT) Composition of Platelet Fluid: Sodium citrate (anti-coagulant) --------------- 3.8 gm Formaldehyde (antiseptic) ------------------- 1ml Brilliant crystal blue (dye for platelets) ------- 0.05 gm Distal water -------------------------------------- 100ml Normal Count of Platelets: 150,000 – 4,00,000 /cmm Procedure:

10. Wash and dry the counting chamber & RBC pipette and focus the ruled area in low power (10x) under the microscope and put the cover slip.


12. Discard first drop of blood, wipe it off, allow the skin to dry and take the blood in RBC pipette having red bead, up to the mark 0.5 by holding the pipette horizontally and touching the tip of the pipette to the drop of the blood. The blood enters the pipette by capillary action, if not, apply a gentle suction.

13. Wipe the blood from the tip of the pipette and suck the Platelet fluid up to the mark 101. This makes the dilution 1:200.

14. Mix the solution by folding the rubber tube and rolling the pipette in between the palms of hands. Don’t shake the pipette as it will break the Cells.



17. Wait for few min so that the Platelets settle down. 18. Observe the slide and count the number of Platelets in five squares, 4 of

each corner and one in the center, i-e square no. 1,5,13,21&25. used for RBC count and fill the observation chart.

Precautions:



blood. 13. No air bubble should interrupt the column of blood. 14. Mix the blood with Platelet fluid immediately to avoid clotting of


the stem is discarded.

OBSERVATION CHART: Square 1. Square 5.

Square 13. Square 21.

Square 25.

No. of Platelets in square 1. ______ No. of Platelets in square 5. ______ No. of Platelets in square 13. ______ No. of Platelets in square 21. ______ No. of Platelets in square 25. ______ Total No. of Platelets in Five squares: ______

Calculation: Area of one square = 1mm2 Depth of chamber = 1/10 mm Volume = Area x Depth Area of one square = 1/25 mm3 Area of one smallest square = 1/25 x 1/16 = 1/400mm2 Volume of one smallest square = 1/400 x 1/10 = 1/4000mm3 Volume of 80 smallest square = 1/4000 x 80 = 80/4000mm3 In 80/4000 m.m3of diluted blood no. Platelets = -------- x 10/1

In 1 mm3 of diluted blood no. of Platelets = --------- x 4000/80 In 1mm3 of undiluted blood no. of Platelets = -------- x 4000/80 x 200/1 RESULT Platelet count of my blood is =___________mm3

EXPERIMENT NO. 9

DATE :-_____________________

Object: To count the total number of RBCs/Cu.mm3 of your own blood. Requirement: Improved Neubaur’s. Haemocytometer, Microscope, Cover slip, Hymen’s fluid and the material for pricking the finger. Principle: The principle of this experiment is to dilute the blood 200 times and to calculate the number of RBC in the area in Haemocytometer meant for RBC count after multiplying by diluting factor i-e 1:200. Theory: Blood is a circulating connective tissue consists of plasma and formed elements, which includes RBCs, WBCs and Platelets. RBC is nonnucleated biconcave disc, having a diameter of 7.8u, 2.5u at thickest point and 1u in the center. RBC has a life span of 120 days. They are filled with Haemoglobin and their function is to transport oxygen from lungs to tissues and Co2 from tissues to lung from where it is expired. Composition Hymen’s Fluid.

1- Nacl --------------- 1.0 gram (it provides the isotonicity) 2- NaSO4 ----------- 5.0 grams (act as an anticoagulants) 3- HgCl2 ------------ 0.5 gram (act as an antibacterial agent/preservative) 4- D/W ------------- 200 ml (act as medium)

Normal RBC Count: Male ---------------------- 5.2 million/Cu.m.m Female ------------------- 4.7 million/CU.m.m Infants ------------------- 5.6 million/CU.m.m Pregnancy --------------- 4.1 million/CU.m.m Procedure:

19. Wash and dry the counting chamber & RBC pipette and focus the ruled area in low power (10x) under the microscope and put the cover slip.


21. Discard first drop of blood, wipe it off, allow the skin to dry and take the blood in RBC pipette having red bead, up to the mark 0.5 by holding the pipette horizontally and touching the tip of the pipette to the drop of the blood. The blood enters the pipette by capillary action, if not, apply a gentle suction.

22. Wipe the blood from the tip of the pipette and suck the Hymen’s fluid up to the mark 101. This makes the dilution 1:200.

23. Mix the solution by folding the rubber tube and rolling the pipette in between the palms of hands. Don’t shake the pipette as it will break the RBC.



26. Wait for few min so that the RBC settles down. 27. Observe the slide and count the number of RBC in five squares, 4 of

each corner and one in the center, i-e square no. 1,5,13,21&25. used for RBC count and fill the observation chart

Precautions:



blood. 21. No air bubble should interrupt the column of blood. 22. Mix the blood with Hymen’s fluid immediately to avoid clotting of


the stem is discarded. OBSERVATION CHART: Square 1. Square 5.

Square 13. Square 21.

Square 25.

No. of RBC in square 1. ______ No. of RBC in square 5. ______ No. of RBC in square 13. ______ No. of RBC in square 21. ______ No. of RBC in square 25. ______ Total No. of RBCs in Five squares: ______

Calculation: Area of one square = 1mm2 Depth of chamber = 1/10 mm Volume = Area x Depth Area of one square = 1/25 mm3 Area of one smallest square = 1/25 x 1/16 = 1/400mm2 Volume of one smallest square = 1/400 x 1/10 = 1/4000mm3 Volume of 80 smallest square = 1/4000 x 80 = 80/4000mm3 In 80/4000 m.m3of diluted blood no. RBCs = -------- x 10/1

In 1 mm3 of diluted blood no. of RBCs = --------- x 4000/80 In 1mm3 of undiluted blood no. of RBCs = -------- x 4000/80 x 200/1 RESULT RBC count of my blood is =___________mm3

EXPERIMENT NO. 10

DATE :-_____________________

OBJECT:

Determine the hemoglobin concentration in gm per 100 ml by method.

REQUIREMENTS : Sahli Haemometer, Haemometer diluting tube, Hb pipette(20ul), test tube and rack,

sprit swab, Blood lancet.

THEORY

Determination of hemoglobin by sahli haemometer method is one of the most accurate

method available at present. The blood obtained for testing is first diluted in Hcl solution,

which causes lysis of erythrocytes, liberating hemoglobin.

Hcl solution contains ferricyanide and potassium cyanide. Ferricyanide converts the

ferrous(Fe++) molecule of hemoglobin to the ferric (Fe+++) stat resulting in the

formation of met hemoglobin.

This compound now reacts with cyanide to form a stable colored pigment,

cyanmethemoglobin. The color comparing with standard tube.

PROCEDURE:

1.Fill the graduated measuring tube up to the bottom graduation line(mark2) with

n/10 hydrochloric acid.

2. Clean thoroughly finger tip or lobe of the ear with ether or alcohol, wipe the first

one drops of blood and let a good size drop of blood to from which is free flowing.

3. Dip the tip of clean and dry pipette in the edge of blood, suck 20ul blood into the

capillary pipette precisely up to the mark. wipe the pipette point and blow the blood

into the measuring tube and mix in Hcl with glass stirrer.

4. Add distilled water by means of the water pipette and mix with the glass stirrer

until the co lour of the solution matches the co lour of the standard test rods.

5. Read the result by diffused day-light exactly tree minute safter adding the blood to

the distilled water.

PRECAUTIONS:-

i) Prick the finger with all antiseptic measures. ii) No air bubble should interrupt the column of blood in pipette iii) The blood should be immediately filled in the measuring tube to avoid clotting of

blood before filling of tube. iv) Add distal water drop by drop carefully. v) Match the colour of measuring tube with the standard tubes in day light or in

front of tube light carefully.

OBSERVATIONS:

Name Observed

Hb gm/100ml

Normal Hb

Gm/100ml

Interpretation

Male 14-18

Female 12-16

RESULT:

My Hb is …………………% and …………………….G %.

EXPERIMENT NO. 11

DATE :-_____________________

OBJECT: To prepare Haemin/ Teachmans crystals from human blood. OBJECTIVE: The student should be able to: 1) Prepare the crystals from dried blood on some cloth, paper, file, floor etc 2) Recognize the human blood. 3) Know the medico legal value of this experiment. 4) Differentiate the human blood from animal blood from human blood or some other stain. REQUIREMENT: Two glass slides, 2cover slips, Bunsen burner, solution of glacial acetic acid mixed with sodium chloride solution and the material for pricking the finger. PRINCIPLE OF EXPERIMENT: The principle is to prepre the hydrochloride of haemoglobin (haemin’s crystals by heating it with glacial acetic acid and sodium chloride solution and observe the shape of crystal. THEORETICAL EXPLANATION: Heamin’s crystals are derived from Hb and are hydrochloride of haematin which is a ferric (Fe++) compound and obtained by gently heating hemoglobin with glacial acetic acid and sodium chloride solution. Under microscope they appear as rhomboid shape and reddish brown in color.

Hb + O2 Oxy.Hb

Oxy.Hb +CH3COOH +NaCl CH3COOHNa+ HCl + HCl of Hb This practical has a great medico legal importance and is performed to distinguish between human blood stain and some other spots. Human blood may be detected from suspected blood stain as the crystals of this shape are the only formed form of human blood. PROCEDURE: There are two methods to obtain Heamin’s crystals.

FRESH BLOOD METHOD OR DIRECT METHOD:

1. Sterilize the finger with spirit swab and prick it with prestirilized blood lancet. 2. Take two slides and put one drop of blood in center of each slide which has

been cleaned properly. 3. Keep one slide on the table; leave the blood to dry NaCl proceeds with the

other slide. 4. Put a drop of solution of with glacial acetic acid and sodium chloride

solution on the fresh blood. 5. Put cover slip and heat the slide very gently on the flame in a wave like

manner .Avoid continuous heating so as to prevent blood from burning.

6. Heat the slide till the fumes (bubbles) appear around the edges of the cover slip.

7. Stop heating and wait till the slides cool down. 8. Observe the slide under microscope first in low power 10x then in high

power 45x

DRY BLOOD METHOD OR INDIRECT METHOD: 1. Take the slide which was previously left on the table and the blood was

allowed to clot. 2. Scrape the dry blood, mash it in powder form and collect it in the center of

slide. 3. Put a drop of glacial acetic acid and sodium chloride solution and put cover

slip on it. 4. Heat the slide till the fumes (bubbles) appear around the edges of the cover

slip. 5. Wait till the slides cool down. Observe the slide under microscope .The

crystal formed by this method relatively small in size than obtained by fresh blood method.

MEDICOLEGL SIGNIFICANCE: This experiment has no clinical value but has great medico legal importance because Heamin’s crystals are formed only by human blood not from animal blood or any other stain .In animal blood the shape of crystal are entirely different . PRECAUTIONS:

1. Avoid continuous heating of slide on the flame as over heating will burn the blood.

2. Heat the slide till the fumes (bubbles) appear around the edges of the cover slip to void under heating.

3. If the crystals are not formed raises the cover slip put one drop of glacial acetic acid and sodium chloride solution and repeat the procedure.

FIGURE

HAEMIN CRYSTALS

EXPERIMENT NO. 12

DATE :-_____________________

Pulmonary Function Testing

I. Purpose of Pulmonary Function Testing

Pulmonary Function Testing has been a major step forward in assessing the functional

status of the lungs as it relates to :

1. How much air volume can be moved in and out of the lungs

2. How fast the air in the lungs can be moved in and out

3. How stiff are the lungs and chest wall - a question about compliance

4. The diffusion characteristics of the membrane through which the gas moves

(determined by special tests)

5. How the lungs respond to chest physical therapy procedures

Pulmonary Function Tests are used for the following reasons :

1. Screening for the presence of obstructive and restrictive diseases

2. Evaluating the patient prior to surgery - this is especially true of patients who :

a. are older than 60-65 years of age

b. are known to have pulmonary disease

c. are obese (as in pathologically obese)

d. have a history of smoking, cough or wheezing

e. will be under anesthesia for a lengthy period of time

f. are undergoing an abdominal or a thoracic operation

Note : A vital capacity is an important preoperative assessment tool. Significant

reductions in vital capacity (less than 20 cc/Kg of ideal body weight) indicates

that the patient is at a higher risk for postoperative respiratory complications. This

is because vital capacity reflects the patient's ability to take a deep breath, to

cough, and to clear the airways of excess secretions.

3. Evaluating the patient's condition for weaning from a ventilator. If the patient on a

ventilator can demonstrate a vital capacity (VC) of 10 - 15 ml/Kg of body weight,

it is generally thought that there is enough ventilatory reserve to permit (try)

weaning and extubation.

4. Documenting the progression of pulmonary disease - restrictive or obstructive

5. Documenting the effectiveness of therapeutic intervention

II. Equipment

The primary instrument used in pulmonary function testing is the spirometer. It is

designed to measure changes in volume and can only measure lung volume

compartments that exchange gas with the atmosphere. Spirometers with electronic signal

outputs (pneumotachs) also measure flow (volume per unit of time). A device is usually

always attached to the spirometer which measures the movement of gas in and out of the

chest and is referred to as a spirograph. Sometimes the spirograph is replaced by a printer

like the unit used in this laboratory. The resulting tracing is called a spirogram. Many

computerized systems have complex spirographs or printouts that show the predicted

values next to the observed values (the values actually measured). The unit will have in

memory all of the prediction tables for males and females across all age groups. In

sophisticated spirometers, there maybe special tables of normal values programmed into

the machine for selection when Blacks, children or other groups are being tested who

may vary from the normal PFT tables established for caucasian adults.

III. "Normal Values"

Over the last several decades much research has been undertaken to determine what are

the normal values for lung volumes and lung capacities. This has made spirometry very

useful since now we know that we can compare the patient's PFT results with those

measured on thousands and thousands of "normal" adults. By having tables of normal

values, it is then easy to compare the severity of the disease process or the rate of

recovery taking place in the patient's lungs. There are a few variables such as age, gender

and body size which have an impact on the lung function of one individual compared to

another.

Age : As a person ages, the natural elasticity of the lungs decreases. This

translates into smaller and smaller lung volumes and capacities as we age. When

determining whether or not your patient has normal PFT findings, it would be

important to compare the patient with the PFT results of a normal person of the

same age and gender.

Gender : Usually the lung volumes and capacities of males are larger than the

lung volumes and capacities of females. Even when males and females are

matched for height and weight, males have larger lungs than females. Because of

this gender-dependent lung size difference, different normal tables must be used

for males and females.

Body Height & Size : Body size has a tremendous effect on PFT values. A small

man will have a smaller PFT result than a man of the same age who is much

larger. Normal tables account for this variable by giving predicted PFT data for

males or females of a certain age and height. Sometimes as people age they begin

to increase their body mass by increasing their body fat to lean body mass ratio. If

they become too obese, the abdominal mass prevents the diaphragm from

descending as far as it could and the PFT results will demonstrate a smaller

measured PFT outcome then expected - i.e. the observed (measured) values are

actually smaller than the predicted values (predicted values from the normal

tables).

Race : Race affects PFT values. Blacks, Hispanics and Native Americans have

different PFT results compared to Caucasians. Therefore, a clinician must use a

race appropriate table to compare the patient's measured pulmonary function

against the results of the normal table written for that patient's racial group. Other

factors such as environmental factors and altitude may have an affect on PFT

results but the degree of effect on PFT is not clearly understood at this time.

IV. Terminology and Definitions

FVC - Forced Vital Capacity - after the patient has taken in the deepest possible breath,

this is the volume of air which can be forcibly and maximally exhaled out of the lungs

until no more can be expired. FVC is usually expressed in units called liters. This PFT

value is critically important in the diagnosis of obstructive and restrictive diseases.

FEV1 - Forced Expiratory Volume in One Second - this is the volume of air which can be

forcibly exhaled from the lungs in the first second of a forced expiratory manuever. It is

expressed as liters. This PFT value is critically important in the diagnosis of obstructive

and restrictive diseases.

FEV1/FVC - FEV1 Percent (FEV1%) - This number is the ratio of FEV1 to FVC - it

indicates what percentage of the total FVC was expelled from the lungs during the first

second of forced exhalation - this number is called FEV1%, %FEV1 or FEV1/FVC ratio.

This PFT value is critically important in the diagnosis of obstructive and restrictive

diseases.

FEV3 - Forced Expiratory Volume in Three Seconds - this is the volume of air which can

be forcibly exhaled in three seconds - measured in Liters - this volume usually is fairly

close to the FVC since, in the normal individual, most of the air in the lungs can be

forcibly exhaled in three seconds.

FEV3/FVC - FEV3% - This number is the ratio of FEV3 to the FVC - it indicates what

percentage of the total FVC was expelled during the first three seconds of forced

exhalation. This is called %FEV3 or FEV3%.

PEFR - Peak Expiratory Flow Rate - this is maximum flow rate achieved by the patient

during the forced vital capacity maneuver beginning after full inspiration and starting and

ending with maximal expiration - it can either be measured in L/sec or L/min - this is a

useful measure to see if the treatment is improving obstructive diseases like

bronchoconstriction secondary to asthma.

FEF - Forced Expiratory Flow - Forced expiratory Flow is a measure of how much air

can be expired from the lungs. It is a flow rate measurement. It is measured as

liters/second or liters/minute. The FVC expiratory curve is divided into quartiles and

therefore there is a FEF that exists for each quartile. The quartiles are expressed as

FEF25%, FEF50%, and FEF75% of FVC.

FEF25% - This measurement describes the amount of air that was forcibly expelled in the

first 25% of the total forced vital capacity test.

FEF50% - This measurement describes the amount of air expelled from the lungs during

the first half (50%) of the forced vital capacity test. This test is useful when looking for

obstructive disease. The amount of air that will have been expired in an obstucted patient

is smaller than that measured in a normal patient.

FEF25%-75% - This measurement describes the amount of air expelled from the lungs

during the middle half of the forced vital capacity test. Many physicians like to look at

this value because it is an indicator of obstructive disease.

MVV - Maximal Voluntary Ventilation - this value is determined by having the patient

breathe in and out as rapidly and fully as possible for 12 -15 seconds - the total volume of

air moved during the test can be expressed as L/sec or L/min - this test parameter reflects

the status of the respiratory muscles, compliance of the thorax-lung complex, and airway

resistance. Surgeons like this test value because it is a quick and easy way to assess the

strength of the patient's pulmonary musculature prior to surgery - a poor performance on

this test suggests that the patient may have pulmonary problems postoperatively due to

muscle weakness. MVV can therefore be viewed as a measure of respiratory muscle

strength. One major cautionary note is that this test is effort dependant and therefore can

be a poor predictor of true pulmonary strength and compliance.

V. What Can A PFT Be Used For ?

Pulmonary function abnormalities can be grouped into two main categories : obstructive

and restrictive defects. This grouping of defects is based on the fact that the routine

spirogram measures two basic components - air flow and volume of air out of the lungs.

Generally the idea is that if flow is impeded, the defect is obstructive and if volume is

reduced, a restrictive defect may be the reason for the pulmonary disorder.

Obstructed Airflow

The patency (dilatation or openness) is estimated by measuring the flow of air as the

patient exhales as hard and as fast as possible. Flow through the tubular passageways of

the lung can be reduced for a number of reasons:

narrowing of the airways due to bronchial smooth muscle contraction as is the

case in asthma

narrowing of the airways due to inflammation and swelling of bronchial mucosa

and the hypertrophy and hyperplasia of bronchial glands as is the case in

bronchitis

material inside the bronchial passageways physically obstructing the flow of air as

is the case in excessive mucus plugging, inhalation of foreign objects or the

presence of pushing and invasive tumors

destruction of lung tissue with the loss of elasticity and hence the loss of the

external support of the airways as is the case in emphysema

external compression of the airways by tumors and trauma

Restricted Airflow

"Restriction" in lung disorders always means a decrease in lung volumes. This term can

be applied with confidence to patients whose total lung capacity has been measured and

found to be significantly reduced. Total lung capacity is the volume of air in the lungs

when the patient has taken a full inspiration. You cannot measure TLC by spirometry

because air remains in the lungs at the end of a maximal exhalation - i.e. the residual

volume or RV. The TLC is therefore the summation of FVC + RV. There are a variety of

restrictive disorders which are as follow :

A. Intrinsic Restrictive Lung Disorders

1. Sarcoidosis

2. Tuberculosis

3. Pnuemonectomy (loss of lung)

4. Pneumonia

B. Extrinsic Restrictive Lung Disorders

1. Scoliosis, Kyphosis

2. Ankylosing Spondylitis

3. Pleural Effusion (fluid in the pleural cavity)

4. Pregnancy

5. Gross Obesity

6. Tumors

7. Ascites

8. Pain on inspiration - pleurisy, rib fractures

C. Neuromuscular Restrictive Lung Disorders

1. Generalized Weakness - malnutrition

2. Paralysis of the diaphragm

3. Myasthenia Gravis - lack of acetylcholine or too much cholinesterase at the

myoneural junction in which the nerve impulses fail to induce normal muscular

contraction. These patients suffer from fatigability and muscular weakness.

4. Muscular Dystrophy

5. Poliomyelitis

6. Amyotrophic Lateral Sclerosis - Lou Gerig's Disease

VI. Criterion for Obstructive and Restrictive Disease

Forced Vital Capacity : Forced Vital Capacity (FVC) is the amount of air that can be

maximally and forcibly expelled from the lungs after a maximal inhalation. If the patient

has an obstructive disease, the amount of air in the lungs will not be readily exhaled

because of physical obstruction and airway collapse during exhalation (loss of elastic

recoil of the lungs). FVC is, therefore, an important PFT value to look at when evaluating

the presence of obstructive pathology. In obstructive diseases, the lung's air volume will

be more slowly expelled and will be a smaller volume over the time course of the FVC

test than would be expected in a normal, healthy individual.

In patients with restrictive lung disease, the FVC will be smaller because the amount of

air that can be forcefully inhaled or exhaled from the lungs is smaller to start with

because of disease. This may be due to the fact that thoracic cage does not have the

ability to expand very much. FVC will therefore be smaller due to mechanical

limitations. However, since FVC will be smaller in obstructive disorders and in restrictive

disorders (usually no one worries about the FVC unless it is 80% - 85% of predicted

volumes), FVC alone cannot be used to diagnose obstructive and restrictive disorders all

by itself. If the patient demonstrates a reduced FVC, the patient may repeat the test after

inhaling a bronchodilator. The bronchodilator dilates the bronchial passages and reduces

airflow obstruction. The post-bronchodilator test often shows an improved FVC - often

times a 10% - 15% improvement. This simple clinical test strongly suggests that the FVC

was low due to obstructive phenomenon. If the FVC did not change, it suggests the FVC

was possibly low due to restrictive pathologies.

Another strategy that can help you decide if the low FVC is due to obstructive or

restrictive processes is to have the patient perform a Slow Vital Capacity (SVC) Test.

This test is performed by having the patient slowly and completely blow out all of the air

from their lungs. The SVC test eliminates the strong bronchoconstriction that usually

accompanies a strong forced exhalatory effort. Hence, the vital capacity of the patient

may well be much larger after a SVC test because there is little or no airway collapse

during a controlled and slow exhalatory effort. If the vital capacity improves after a SVC

test, then it can be assumed that the original small FVC was caused by airway collapse

and does not indicate the presence of restrictive disease. If the vital capacity does not

improve either with the inhalation of a bronchodilator or does not improve with the

administration of a SVC test, then restrictive pathologies must be considered as a possible

cause for the small vital capacity results.

Forced Expiratory Volume in One Second : Forced Expiratory Volume in One Second

(FEV1) is the amount of air that is forcefully exhaled in the first second of the FVC test.

In general, it is common in healthy individuals to be able to expell 75% - 80 % of their

vital capacity in the first second of the FVC test. Hence, FEV1 is a pulmonary function

value that is highly diagnostic of obstructive disease - i.e. - if an individual's FEV1 is low

compared to the predicted FEV1 in the normal population, the individual may have an

obstructive lung disease. FEV1 is also expressed as a ratio or a percentage of the FVC

and is written as %FEV1 or as FEV1/FVC. If the individual being tested displays a low

FEV1 and the FEV1% is low, then the clinician should suspect the presence of

obstructive pathologies.

In patients with restrictive lung disease, the FEV1 will be lower than predicted normal

values and so will the FVC. Since both of these values may equally be effected in

restrictive disease, the %FEV1 may well be calculated to be between 85% - 100% of

normal. Hence, in restrictive disease look closely at %FEV1 when FEV1 and FVC are

low and if the %FEV1 is 85% or greater, then you should suspect the patient has a

restrictive pathology.

Forced Expiratory Flow 25 % to 75 % : Forced Expiratory Flow 25% - 75% (FEF25%-

75%) is a measure of the flow rate in liters per second of the middle half of a FVC test.

This test is a sensitive test for the presence of obstructive airway disease. The value of

looking at the middle half becomes clear when you realize that the first quarter of the

FVC test is in part effected by the patient's effort in overcoming the inertial forces which

resist thoracic wall expansion. Additionally, the expiratory effort in the last quarter of a

FVC test is polluted by the patient's diminishing physical effort, the instigation of

bronchospasm during forced expiration and the breathlessness associated with the

terminal completion of a FVC test. Hence, the FEF25%-75% (middle 50% of a PFT) is

the most representative of true expiratory patency and is therefore a very sensitive test for

the presence of obstructive disease.

Peak Expiratory Flow Rate : Peak Expiratory Flow Rate (PEFR) is a measure of the

highest expiratory flow rate during the PFT test. It is measured in liters of air expired per

second or liters of air expired per minute. Since it is a measure of the peak or maximum

flow of expired air, it becomes a sensitive test for the presence of obstructive disease.

Patients with a low PEFR would have to be further evaluated for obstructive pathologies.

VII. How Do You Tell If The Patient Is Normal or Has Mild, Moderate or Severe

Pulmonary Disease ?

There are a number of systems which physicians use to determine the severity of disease.

Here is just one way that is very commonly used :

Normal PFT Outcomes - > 85 % of predicted values

Mild Disease - > 65 % but < 85 % of predicted values

Moderate Disease - > 50 % but < 65 % of predicted values

Severe Disease - < 50 % of predicted values

In most good spirometers on the market today, there is a set of normal tables (sometimes

multiple sets of tables) which can be chosen as you perform the PFT. Also, there are

interpretive microchips in the PFT machines which will tell you what the diagnosis is for

a particular patient. These two features make it easy for the clinician to immediately see

what the predicted values (normal table values) are for a specific patient and whether or

not the PFT has a normal observed outcome. The PFT data are examined by the

computerized spirometer and a diagnosis of obstructive or restrictive disease is made.

VIII. The PFT Before and After Aerosol Bronchodilators

Patients are almost always tested twice - once before bronchodilators are given and once

after one is administered. This is a nice way to evaluate the amount of

bronchoconstriction that was present and how responsive the patient was to a

bronchodilator medication. This assesses the degree of reversibility of the airway

obstruction. The drug that is nearly always used is a Beta-2 selective sympathomimetic

because it is a drug that causes bronchodilation but which does not stimulate the heart to

any great degree. After the drug has been administered, the PFT is repeated. If two out of

three measurements (FVC, FEV1 and FEF25% - 75%) improve, then it can be said that

the patient has a reversible airway obstruction that is responsive to medication. The

amount of improvement is variable between clinics but some standards are presented

below:

1. FVC : an increase of 10% or more

2. FEV1 : an increase of 200 ml or 15% of the baseline FEV1

3. FEF25%-75% : an increase of 20% or more

Pulmonary Function Tests - A Systematic Way To Interpretation

There is a systematic way to read the PFT and be able to evaluate it for the presence of

obstructive or restrictive disease. The following steps will be helpful.

1. Step 1. Look at the forced vital capacity (FVC) to see if it is within normal limits.

2. Step 2. Look at the forced expiratory volume in one second (FEV1) and determine

if it is within normal limits.

3. Step 3. If both FVC and FEV1 are normal, then you do not have to go any further

- the patient has a normal PFT test.

4. Step 4. If FVC and/or FEV1 are low, then the presence of disease is highly likely.

5. Step 5. If Step 4 indicates that there is disese then you need to go to the

%predicted for FEV1/FVC. If the %predicted for FEV1/FVC is 88%-90% or

higher, then the patient has a restricted lung disease. If the %predicted for

FEV1/FVC is 69% or lower, then the patient has an obstructed lung disease.

Examples:

Case # 1.

Predicted Values Measured Values % Predicted

FVC 6.00 liters 4.00 liters 67 %

FEV1 5.00 liters 2.00 liters 40 %

FEV1/FVC 83 % 50 % 60 %

Decision : This person is obstructed

Case # 2.




FEV1/FVC 84 % 79 % 94 %

Decision : This person is restricted

Case # 3.




FEV1/FVC 82 % 77 % 94 %

Decision : This person is normal. This person is normal because the FVC and FEV1 are

normal. It is irrelevant that the %Predicted for FEV1/FVC is 94% when FVC & FEV1

are normal. The % predicted values for FEV1/FVC are only relevant when the FVC and

the FEV1 are abnormal.

Case # 4.




FEV1/FVC 78 % 88 % 115 %

Decision : Case 5.




FEV1/FVC 78 % 39 % 50 %

Decision : Case 6.




FEV1/FVC 76 % 76 % 100 %

Decision :

EXPERIMENT NO. 13

DATE :-_____________________

OBJECT:-

To record and examine the major components of the Electrocardiogram (ECG).

EQUIPMENT SETUP:-

1. Make sure the PowerLab is connected and turned on.

2. Connect the push-button switch to Input 1 on the PowerLab.

3. Remove any watches and/or jewelry from your wrists and ankles.

4. Connect the electrode lead wires to Earth, NEG, and POS on the Bio Amp cable.

5. Plug the Bio Amp cable into the Bio Amp input.

Standard Connection

Attach the positive electrode to the left wrist, the negative to the right wrist, and the

ground to the right leg.

1. Using a pen, mark each point where electrodes will be placed. Clean the skin with

alcohol swabs and lightly abrade the area with abrasive gel or a pad. This reduces

the electrical resistance of the outer layer of skin and ensures good electrical

contact.

2. If you are using the Reusable Clamp Electrodes, apply a small amount of

electrode cream to the electrodes before attaching. Electrode cream is not

necessary if you are using disposable electrodes which have electrode gel on them

already.

3. If, after looking at the signal during the first exercise, you find that this does not

produce a good signal, try the alternative method.

PROCEDURE:-

1. The subject should relax and sit as still as possible to minimize signal artifacts due

to movements.

2. Type the subject's name into the Comment panel.

3. Click Start, then add the comment.

Click Autoscale as required to ensure that you can see all the data as it is being

recorded.

4. If the ECG cannot be seen, check that all three electrodes are correctly attached. If

the signal is noisy and indistinct, make sure that the subject is relaxed; consider

using the alternative attachment positions.

5. Click Stop.

6. Click Start again. While recording, ask the subject to open and close his or her

hands, and then move both arms across the chest.

The trace moves all over the place, and the ECG becomes distorted. This shows you

why it is necessary for subjects to keep still and stay relaxed while their ECG is being

recorded.

ANALYSIS:-

1. Scroll through your data and observe the regularly occurring ECG cycles.

2. In a representative cycle, measure the amplitudes and durations of the P wave,

QRS complex and T wave.

3. To measure the amplitudes, place the Marker on the baseline immediately before

the P wave. Then move the Waveform Cursor to the peak of a wave. Click to

place the number in the Value panel.

4. Drag the number from the Value panel into the appropriate column of the upper

table.

5. To measure the durations, leave the Marker at the start of the wave or complex

and position the Waveform Cursor at the end of the wave or complex.

6. Click to place the number in the Value panel and then drag the number from the

Value panel into the appropriate column of the table.

7. Now investigate how the heart rate may vary from beat to beat. To do this, set the

horizontal compression to 10:1. Measure the time interval (in seconds) between

three pairs of adjacent R waves using the Marker and Waveform Cursor.

8. Record your results in the lower table. For each interval, the heart rate is shown in

column 3 of the table, calculated using the equation HR = 60 ÷ t , where HR =

Heart Rate (beats/min) and, t = time interval (seconds).

EXPERIMENT NO. 14

DATE :-_____________________

OBJECT:-

To record and correlate the ECG and heart sounds (with a cardio microphone) in a

resting volunteer.

EQUIPMENT SETUP:-

6. Make sure the Power Lab is connected and turned on.

7. Connect the push-button switch to Input 1 on the Power Lab.

8. Remove any watches and/or jewelry from your wrists and ankles.

9. Connect the electrode lead wires to Earth, NEG, and POS on the Bio Amp cable.

10. Plug the Bio Amp cable into the Bio Amp input.

11. Microphone placed over the chest wall to record the heart sounds which can then

be displayed graphically in real time.

PROCEDURE:-

1. Plug the cardio microphone into the pod port for Input 1.

2. Place the cardio microphone on the left side of your chest. Then hold it firmly in

place either by a strap running around the chest or by placing a heavy book or

similar object on top of it. (This requires that you lie down).

It is essential that the microphone is not held onto the chest-wall by hand, as the

inevitable movement of the hand introduces considerable noise into the recording.

3. Click Start to record the ECG and cardio microphone signals. You should try

placing the microphone in different positions to get the best possible signal.

4. After about 15 seconds, click Stop.

ANALYSIS:-

1. To make it easier to compare the recordings in the two channels, the LabTutor

panel is set up so that the recordings are overlaid.

2. Clicking the buttons towards the top right of the panel allows you to select which

of the two channels is 'active' in the panel.

3. Note the relationship between the R-wave and the first sound. Using the Marker

and Waveform Cursor, follow the instructions below to measure the time between

the peak of the R wave and the beginning of the first heart sound.

1. Select the ECG channel as active.

2. Place the Marker on the R wave.

3. Select the PCG channel as active.

4. Use the waveform cursor and select the beginning of the first heart sound.

Insert this time into the table.

4. Note the relationship between the T-wave and the second sound. Now measure

the time between the peak of the T wave and the beginning of the second heart

sound by repeating steps 1 to 5 above.

References 1- Practical Haematology by Sir John Dacie, S.M. Lewis.

2- Clinical Haematology, Theory and Procedures by Mary Louise Thrgeon.

3- Clinical Haematology and Fundamentals of Haemostasis by Denise M. Haemening.

4- Diagnostic Haematology by Bernadette F. Rodak.

5- Clinical Diagnosis and Management by Laboratory Methods by John Bernard Henry MD.

6- Manual of Laboratory Medicine by Armed Forces Institute of Pathology, Pakistan.

7- Technical Manual of American Association of Blood Bank (AABB)

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