Experiment



Blood:

It consists of:
Formed elements (erythrocytes, leukocytes and platelets).
Plasma.
Methods of obtaining blood:
When only a few drops are required, sufficient blood may be obtained from capillary puncture.
When more than a few drops of blood are required, venepuncture is necessary.
Red Blood Cell Count
The number of red blood cells is determined by diluting a definite small amount of blood with a specific amount of diluting fluid. The number of cells in a very small known volume of diluted blood is counted and the number finally multiplied to give the number of cells in a cubic millimeter. The instrument used is called hemocytometer. The hemocytometer consists of:
1. Counting chamber:
It is a heavy glass slide, in the center of which is ruled platform. The platform is lower than the rest the slide by 0.1mm. The graduated area is ruled both horizontally and vertically (the crossed lines). The crossed lines form nine square ABCD is subdivided into 16 medium squares, each of which is subdivided into 16 small squares. The area of each small square is 1/400 square mm, and since the depth of fluid between the ruled surface and the cover glass is 1/10mm, the volume of the fluid covering one of the small squares is:
1/400 x 1/10 = 1/4000 cubic mm


Figure (1.1): Counting chamber, red and white cell pipettes.






Figure (1.2): Ruled counting area of Neubauer hemocytometer (ABCD marks the area used for counting erythrocytes)

2. Special pipette (red cell pipette):

The pipette has a narrow stem, graduated with figures indicating 0.5 and 1, which widens into a bulb containing a red glass bead, which helps in mixing the blood with the diluting fluid (Hayem's solution). The bulb narrows again, and at this point it is marked 101.
3. Special thick plane covers glass:
(Cover slip) of the standard weight and thickness.



Figure (1.3): Cell counting. Five erythrocytes are counted in this square. The shaded cells touching the bottom and right side of the square are not included for this square.

Calculation:

The 0.5 units of blood, have been diluted by 99.5 units of diluting fluid. The dilution is 1 in 200
X: the number of cells counted over 80 small squares
The volume of 80 small squares represent = 80 x 1/4000 = 1/50


80 x =


X


? 1 cu mm



? = = X x 1 x = X x 50 x 200 (diluting factor)

= X x 10000



White Blood Cell Count

Introduction and principle:
The white blood cell count denotes the number of white blood cells in 1 liter of whole blood. In a normal healthy individual WBC range 4.5 -11.0 x 103 / c. mm this count varies with age. WBC count is useful to indicate infections or may be employed to follow the progress of certain diseases.
Objectives:
To do total WBC count of a provided sample.
Methods
Manual method.
Electronic cell counting (coulter counter)

Manual White Blood Cell Count

Materials and instruments:
Whole blood using EDTA as the anticoagulant, capillary blood may be also used.
Turk's diluting fluid:
Glacial acetic acid 3 ml
to haemolyse RBC
Aqueous gention violet (1% w/v) 1ml
to color the nuclei of WBC
Distilled water 100 ml
WBC pipette (Figure 1).
Haemocytometer (Neubauer's counting chamber) with coverglass.
Microscope.
Lancet.
Alcohol 70%.



Figure (2.1) White cell Thoma pipette attached to rubber aspiration tube (or rubber sucking tube)

Procedure

Obtain a drop of blood in the same manner as in RBC count. Draw blood up to the mark 0.5 using WBC pipette.
Aspirate diluting fluid up to mark 11. The dilution is 1:20.
Remove blood from the outside of the pipette with a clean guaze.
Gently rotate the pipette horizontally with your hand to ensure a proper amount of mixing for 3 minutes.
After mixing, discard the first four drops of the mixture.
Fill the counting chamber with diluted blood by holding the pipette at 45 with the slide and allow the mixture to seep under the coverslip. The filled chamber should be allowed to stand for about 1 minute prior to counting.
Count the WBC using low power 10x objective. Scan the four large corner squares marked A, B, C and D on the counting chamber as shown in Figure (1.3) in the experiment of RBC.
Count all W.B.C. in four large corner squares and add the results together to obtain the total number of cells counted. In counting the cells that touch the outside lines of the large square, count only those that touch the left and upper outside lines, discharging those that touch the right and lower outside margin. The WBC look like black dots (Figure 2.2).



Figure (2.2) Examples of white blood cells counted (and not counted) in a representative square.

Leucocytes:

They are commonly known as white blood cells. They are larger than erythrocytes, contain a nucleus and do not contain haemoglobin. Normal leucocytes count various from 4000 to 11000 cells / Cu mm of blood.

The leucocytes are classified into granulocytes and agranulocytes.

Granulocytes:
1. Neutrophil:
They constitute 60 70% of the total leucocute count. They have a multilobed nucleus and the number of lobes usually range from 3 to 5, cytoplasm contains fine granules, which take neutral stain. Neutrophils exhibit phagocytosis and their count increases in acute infections. They form the first line of defense in the body.
Increase neutrophil count during menstruation, pregnancy and muscular exercise, in acute infections like pneumonia, appendicitis, tonsillitis.
Neutrophil count decreses in typhoid, malaria, aplastic anaemia and under the influence of various drugs.


2. Eosinophil:
It has usually a bilobed nucleus and shows the presence of relatively large granules, which take an acidophilic or orange stain. Normal eosinophil count ranges from 2 to 5%. It has been observed that in allergic conditions eosinophil count increases (eosinophilia), the decrease in eosinophil count constitutes eosinopenia and can be seen in acute pyogenic infections or steroid therapy.


3. Basophil:
The nucleus in these cells is usually bilobed and cytoplasm contains blue granules. Their cont ranges from 0 1o 1%. Basophil releases heparin, which is an anticoagulant and it also releases histamine and this occurs specially in hypersensitivity reactions such as anaphylactic shock, the basophil count increases in polycythemia and chronic myloid leukemia.

Agranulocytes:

1. Lymphocyte:
The cell has a large oval or round nucleus and there is a thin strip of clear nongranular cytoplasm between the nucleus and cell membrane. The lymphocytes are responsible for providing immunity to the body. There are two functionally distinct types namely T lymphocytes and B lymphocytes, they are involved in immune and defense mechanisms.

2. Monocyte:
They are the largest white cell (15 microns) and have a kidney shaped nucleus. The normal monocyte count is 3 to 8%. Monocytes are phagocytic in function and they are the second line of defense in the body.

The life span of white cells is very short and ranges from a few hours to 2 -3 days.

The age of a neutrophil can be determined by the number of lobes present in its nucleus. The number of lober increase as the cells grow old. The grouping of neutrophils based upon the number of lobes
N1 5 10%
N2 25 30%
N3 45 50%
N4 15 20%
N5 Less than 3%
Functions of Leucocytes:
1. Phagocytosis: It is a process by which leucocytes engulf bacteria and foreign material.
2. Antiallergic effect: histamine release during allergic conditions is inhibited by eosinophils, their count increases in allergy.
3. Heparin production: Basophils produce heparin, which prevents intravascular clotting.


Leucocytosis: This term is used to indicate an increase in the white cell count and is a common feature in most infections. A physiological increase in leucocytes is seen in:
Menstruation.
Pregnancy.
Muscular exercise.

A reduction in white cell count is known as leucopenia; this condition is seen in:

Bone marrow suppression by drugs and x ray radiation.
Pernicious anaemia.
Infections such as typhoid and malaria.

Leucocytosis is associated with the presence of premature white cell in the peripheral blood. This condition which may prove fatal is termed leukemia.




Platelet Count (Thrombocyte Count)

Introduction and principle:
Platelet count is of great importance in helping to diagnose bleeding disorder.
Platelet play important role in the stoppage of bleeding. There are two methods for counting of platelets:
Electronic (Caulter counter).
manual (microscopic).


Materials and instruments:
Diluting fluid (31.3 gm sodium citrate, 10 ml of formaldehyde 40%) added to it distilled water up to 100 ml.
Hb pipette.
Hemocytometer.
Blood.
Petri dish.
Filter paper.
Microscope.

Procedure:

Draw blood by the Hb pipette up to the mark 20l and mix it with 2 ml of diluting solution, mix for 5 minutes.
Fill the counting chamber of hemocytometer with a mixture and place it in a moist chamber for 15 minutes.
Place the counting chamber under the microscope and examine under objective lens 40 in the 25 medium size squares of middle large square.

Results:

Number of platelets (cu.mm) = N x 1000
10: correction for volume
100: correction for dilution
Number of platelets in L = N x 1000 x 106
The normal range of platelet count is 150.000 400.000 / ml


Discussion:
Decreased platelet count; the condition is called --------------------- lead to -----------------------.
The main functions of platelets are ----------------------.

Thrombocytes:

They are commonly known as platelets. They are small, biconvex nonnucleated cells, usually found in clusters in a dried film. The average size of platelet is 2.5 microns and their count range from 2-4 lacs/Cum. The life span of platelets is about 10 days.
Variations in count:
An increase in count is observed in:
Haemorrhage.
Splenectomy.
Hodgkin's disease.
A reduction in platelet count is known thrombocytopenia, it is seen in:
Splenomegaly.
Aplastic anaemia.
Acute infections.
Leukemia.
Idiopathic thrombocytopenic purpura.
Functions:
Arrest of bleeding. Platelets aggregate at site of injured vessel and form a haemostatic plug to prevent blood loss.
Coagulation. Platelets release clotting factors, phospholipids and prostaglandins which help in the clotting process.
Clot retraction. The release of thrombesthanin from the platelets helps in clot retraction.
Repair of endothelium. Platelets release PDGF (platelet derived growth factor) which help in the repair of damage capillary endothelium and other tissues.
Release of serotonin and epinephrine. These substances released from platelets produce vasoconstriction and consequently reduce the blood loss.


Purpura:
A reduction in platelet count results in purpura. It is a bleeding disorder in which haemorrhagic tendency increases and there may be subcutaneous haemorrhage.



Packed Cell Volume (PCV) or

Haematocrit (HCT) Value
Introduction and principle:
Whole blood is centrifuged for maximum R.B.Cs packing. The space occupied by the R.B.Cs is measured and expressed as a percentage of the whole blood volume.

Objectives:

To determine the volume or the amount of the R.B.Cs in 100 ml (dl) of blood.

Methods:

Microhaematocrit. It requires less blood and also less time to determine a haematcrit.
Macrohaematocrit or Wintrobe Method. This method is of little use today since it is time consuming requires large amount of blood and contains a higher degree of plasma trapping.
Electric cell counting. E.g., Coulter counter.

Microhaematocrit Method

Materials and instruments:
Microhaematocrit tube 75 mm in length and 1 mm in diameter which contain heparin and show a red ring at the end of the tube.
Microhaematocrit centrifuge capable of producing a relative centrifugal force of 10000 to 15000 gm.
Plastic seal of Bunsen burner flame to seal one end of microhaematocrit tube.
Microhaematocrit reader.


Procedure:
Blood is frawn into the tubes by capillary phenomenon by holding the tubes in a horizontal manner and allow 2/3 to 3/4 to be filled with blood. Air bubbles denote poor technique but do not affect the results of the test.
Seal the dry end of the tube by plastic seal or by heating the dry end of the tube rapidly on a fine flame of Bunsen burner combined with rotation.
The sealed tube is then placed in the radial grooves of the microhaematocrit centrifuge with the sealed end away from the center of the centrifuge for 5 min.
When looking at a centrifuged haematocrit tube, you can see three distinct layers. A top layer of clear slightly milky plasma, a thin buffy coat layer (consisting of W.B.Cs and platelets) and a dark packed R.B.C layer.
Obtain the results using the microhaematocrit tube reading device, adjust the movable line to touch of the R.B.Cs in the tube.



Estimation of Haemoglobin Concentration

Introduction:
Hb is a protein in nature composed of two portions:
Heme: the heme consists of iron and prophyrin ring.
Globin: which consists of two pairs of chains α and β. The α consists of 141 A.A while β consists of 146 A.A.

Types of Hb:

There are three types of Hb:
Hb A1: consists of 2 α and 2β.
Hb A2: consists of 2 α and 2δ.
Hb F (F present in infants): consists of 2 α and 2γ.


Normal value of Hb:
In males 14 gm/dl
In female 16 gm/dl

Materials and Methods

Manual method
Electric cell counter.

Manual method by:

Sahlis haemoglobin binometer which usually graduated 3 columns: the lateral two are scaled and contain standard acid.

Procedure:

10 ml HCl take, because it hydrolysis all types of Hb to form acid haemation.
Clean the tip of finger and then the blood is sucked by Hb pipette up to 20 mm3.
After that the colour compared in light day.
Then distilled water is added 3 4 drops each time.

Note: The important precautions should include when taken blood from finger should never squeeze.

Other methods like:

Haldanes method.
Gower's dilution and colour matching method.
Photoelectric method.


Discussion:
What are the abnormal of Hb?
Hb pathics.
That mean polypeptide chain of Hb abnormal.
2. Thalasemia disease.
That mean polypeptide chain produce normally but decrease in amount.
3. Sickle cell anaemia.
That mean changes in shape of R.B.C cells to form as sickle in shape.

What is anaemia?

Definition and types.



Figure (5.1) Haemoglobinometer.



Figure (5.2) Microhaematocrit reader.




Erythrocyte sedimentation rate (ESR)
If fluid (kept fluid by means of an anticoagulant) is allowed to stand in a narrow tube (Westergren pipette), the corpuscles settle progressively to the botton leaving clear plasma above. The rate at which the heavier elements of the blood settle toward the bottom of the tube is known as sedimentation rate.

Sedimentation of erythrocytes proceeds in 3 phases:

Formation of rouleaux (clumping of red cells together like a stack of coins).
Rapid settling.
Final packing of the red cell mass.

The rate of sedimentation varies with the speed of rouleaux formation. The most important factor controlling this process outside the body is the composition of the plasma: fibrinogen, other globulins and certain products of tissue destruction increase rouleaux formation and hence the sedimentation rate. Albumin reduces it. Usually the rate is increased in acute general infections, in the presence of malignant tumors, in inflammatory conditions, in hypothyroidism, and also in pregnancy. The test is nonspecific and is not diagnostic of any disease. It can be used in the prognosis of any disease.



Blood Indices

Introduction and principle:
The red blood cell indices are used to define the size and haemoglobin content of the red blood cell. They consist of mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration.
Objectives:
To learn how to calculate the blood indices.
Information required:
RBC count in millions/cu.mm
Hb estimation in g/dl.
PCV estimation as %.


Calculations:
Mean corpuscular volume (MCV)
The MCV indicates the average volume of single RBC in femtoloters (fl)

MCV =


= x 10

= fl

2. Mean corpuscular hemoglobin (MCH)
The MCH indicates the average weight of Hb in single RBC in picograms (Pg)

MCH =


= x 10

= Pg

3. Mean corpuscular hemoglobin concentration (MCHC)
MCHC is an expression of the average concentreation of Hb in the red blood cells. It is expresses as %.

MCH =


= x 100


 = %

or: MCHC = x 100

Results:
MCV: Normal range 80 -= 100 fL
MCH: Normal range 27 31 Pg.
MCHC: Normal range 31 36 %
Discussion:
Find the MCV, MCH and MHCH and identify the type of anaemia from the following data:
1. Hb =11.8 g/dl, PCV= 41%, RBC = 4.5 x 106/ cu.mm
2. Hb =9 g/dl, PCV= 30%, RBC = 4.5 x 106/ cu.mm
3. Hb =14 g/dl, PCV= 41%, RBC = 4.5 x 106/ cu.mm



Blood Grouping

Introduction:
Blood grouping is based on the presence of antigen (agglutinogen) on the RBC membrane. The main types of these antigens are A, B, and D. Accordingly blood groups are classified into ABO blood group and (Rh) blood group.

Objectives:
To identify the groups ABO and Rh (D) of a given blood sample


Methods:
Slide method.
Tube method.

Materials and instruments:

Anti A serum
Anti B serum
Anti D serum
These anti sera are available commercially
Slide.
Microscope.
Applicator sticks for mixing.
isotonic saline (0.9% NaCl) for dilution RBC's to prevent false agglutination due to rouleaux formation.
WBC pipette 0.5 to 11 mark.

Procedure:

Place one drop of each anti A, anti B and anti D serum on a slide.
Add to each drop one drop of whole blood or aspirate blood to the 0.5 mark of WBC pipette and dilute to the 11 mark with isotonic saline dilution 1:20.
Mix with an applicator stick.
The reaction will be visible within 20 60 seconds.
Examine for reaction with naked eye or by microscope.
The ABO grouping of the subject can then be obtained.
The Rh(D) grouping also can be obtained.
Positive (+) indicates agglutination while negative (-) indicates no agglutination.
Blood grouping tests should be performed in a warm room.
The ABO blood group and Rh(D) blood group usually are designated together.


Cross Matching Test
Introduction and principle:
This is the direct test of compatibility of donor's cells and recipient's serum or plasma.

Objectives:

To find if the blood of a donor is compatible with that of the recipient.

Methods:

Slide method.
Tube method.
Materials and instruments:
Slide.
Serum or plasma from recipient (contain antibody).
Red blood cell from donor (contain antigen).
Isotonic saline (0.9% NaCl) for dilution.
Applicator sticks.
Microscope.
WBC pipette.

Procedure:

Recipient serum or plasma. Draw 1ml of blood from recipient and allow to clot, when the clot retracts, the serum can be pipetted off.
Donor's red blood cell, prick the ear or finger of donor. One drop of blood should be placed in 1 ml of isotonic saline and mixed, Aspirate blood to the 0.5 mark of WBC pipette and dilute to 11 mark with 0.9% NaCl. Mix and blow out the saline in the stem.
Place one drop of suspension of donor's cell on a microscope slide and over it place one drop of the recipient's serum or plasma. Mix and wait for 10 minutes. Examine under microscope.
If the blood is incompatible this will be shown by an agglutination (+) or clumping or red blood cell.
If the blood is compatible this will be shown by uniform suspension with cells i.e., no addlutination (-).







Incompatible Compatible

Cross Matching Test
Ratio of different blood types
A 41%
B 9%
AB 3%
O 47%

Rh antigens Rh positive and Rh negative people

There are six common types of Rh antigens, each of which is called an Rh factor. These types are designated C, D, E, c, d, and e. A person who has a C antigen does not have the c antigen, but the person missing the C antigen always has the c antigen, the same is true for the D.d and E.e antigens. Each person has one of each of the three pairs of antigens.
The type D antigen is widely prevalent in the population and considerably more antigen than the other Rh antigens. Any one who has this type of antigen is said to be Rh positive whereas the person who does not have type D antigen is said to be Rh negative.



Blood Pressure

It may be defined as the pressure blood exerts against the vessel walls. It depends on:
Cardiac output.
Peripheral resistance.
Total blood volume.
Viscosity of the blood.
Elasticity of the arterial wall.


Systolic pressure: The maximum pressure during ventricular systole.
Diastolic pressure: The minimum pressure during ventricular diastole.

The arterial pressure is written as systolic pressure over diastolic pressure (e.g., 120/70 mm Hg).
Blood pressure is measured indirectly by means of an instrument called a Sphygmomanometer. The Sphygmomanometer consists of two main parts:
One part consists of a hand pump, which is used to measure the air pressure in an inflatable rubber bag. The bag is enclosed in a material case (the cuff) and is attached by a rubber tubing to the second part.
The second part is a mercury manometer to measure the pressure of air in the bag.



Figure (9.1) Sphygmomanometer

Palpation method:

Palpate the subject's radial artery with the tips of the index and middle fingers.
Screw down the escape valve, which is placed just above the bulb of the pump.
Pump up the arm cuff rapidly (to about 150 mm Hg) till the pulse disappears, then let out the air slowly by unscrewing the valve.
Watch the manometer and note the reading when the pulse returns, this is the systolic pressure.

Auscultatory method:

Place the arm cuff around the left arm just above the elbow.
Place the bell of the stethoscope over the brachial artery and inflate the cuff until the pressure in it is well above the expected systolic pressure in the brachial artery.
The brachial artery is occluded by the cuff, and no sound, is heard with the stethoscope.
Let the pressure down slowly. At the point at which systolic pressure in the artery just exceeds the cuff pressure, a spurt of blood passes through with each heart beat and, synchronously with each beat, a tapping sound is heard.
The cuff pressure at which the sounds are first heard is the systolic pressure.
As the cuff pressure is lowered further, the sounds become louder, then dull and muffled and finally in most individuals they disappear. These are the sounds of koratkow. These sounds are produced by turbulence in the intermittent blood flow as the artery opens and shuts with each cardiac cycle.
The cuff pressure at which the sounds disappear is the diastolic pressure.




Figure (9.2) Indirect measurement of blood pressure with Sphygmomanometer



Bleeding Time

Introduction and principle:
A standardized puncture of the ear lobe is made and the time course for bleeding to stop is recorded. Cessation of bleeding indicates the formation of haemostatic plugs which are in turn dependent on an adequate number of platelets and the ability of the platelets to adhere to the subendothelium and to form aggregates.

Objectives:

To find the time between the puncture of the skin and the stoppage of blood oozing.

Methods:

Duke test, is the easiest to perform.
Ivy test.
Template test.

Materials and instruments:

Sterile disposable lancet.
Stopwatch.
Circular test paper.
Alcohol.


Procedure:
The ear lobe is cleansed with an alcohol spong and allowed to dry.
A standardized puncture of the ear lobe is then made, using a sterile blood lancet.
The stopwatch is started at the moment of the puncture.
Using circular filter paper the blood is blotted every 30 seconds without allowing the filter paper to touch the wound.
When bleeding ceases, the stopwatch is halted and the bleeding time recorded as shown in the figure.



 Normal spots on filter paper




Results:

Normal range: 1 3 minutes, borderline times are 3 6 minutes.
Discussion:
What is the clinical significance of determining bleeding time?

Coagulation Time (clotting Time)

Introduction and principle:
The coagulation time of whole blood is the time required for a measured amount of blood to clot under certain specialized conditions.


Objectives:
To determine clotting (coagulation) time.

Method:

Lee and White method.

Materials and instruments:

Water bath, 37C.
Glass test tubes, 13x100 mm.
Stopwatch.
Plastic syringe (10 ml) and 20 gauge needle.
Fresh whole blood, 4 ml.

Procedure:

Label three test tubes with the patients name and number them 1, 2 and 3.
Withdraw 4ml of blood.
Carefully place 1 ml of the blood in test tube 3, then 1ml in test tube 2 and lastle, 1ml in the tube 1. Start the stopwatch as soon as blood is placed in tube 3.
Place the three test tubes in a 37C water bath.
At exactly 5 minutes, title test tube 1 gently to 45 angle. Repeat this procedure every 30 seconds until the test tube can completely be inverted without spilling the contents i.e., until the blood is completely clotted.
Record the time it took for the blood in test tube 1 to clot.
Thirty seconds after the blood in test tube 1 is clotted, proceed with tube 2 and repeat the preceding procedure, tilting the test tube every 30 seconds until a clot is formed. Record the results. Repeat this procedure for test tube 3.
Since agitation and handling speed up coagulation, the coagulation time of test tube 3 is handling the reported result.


Results:
Normal range 5 15 minutes.

Discussion:

How can you differentiate between purpura and haemophilia.
Test tubes are incubated in water bath at 37C. Why?

Prothrombin Time

Introduction and principle:
In the presence of calcium, thromboplastin is the cofactor of factor VII which activates the extrinsic coagulation pathway. Coagulation begins after adding thromboplastin to a normal citrated plasma which ends within few seconds with the formation of fibrin clot.

Objectives:

To estimate the length of time taken for the plasma to clot in the presence of excess thromboplastin and calcium.

Methods:

Manual method.
Automated system.

Manual Method

Material and instruments:
Thromboplastin calcium reagent which is commercially available.
Water bath 37C.
Test tube, 13x100 mm.
Stopwatch.
Sodium citrate (3.8%) as anticoagulant, 0.5 ml of sodium citrate added to 4.5 ml of whole blood.
Centrifuge.
Micropipettes, 0.1 ml, 0.2 ml.


Procedure:
Centrifuge anticoagulated blood at 1200 1500 g for 15 minutes.
Separate the plasma for patient and control RBC's and store at room temperature until ready for testing, perform the test within 4 hours of blood collection.
Prepare thromboplastin calcium reagent which is available commercially.
Pipette 0.2ml of thromboplastin calcium reagent into the duplicate test tubes. Warm the test tubes in the water bath for 10 15 minutes.
Incubate the plasma at 37C for 2 3 minutes.
Pipette 0.1 ml of patients plasma into the duplicate test tubes containing 0.2 ml of thromboplastin calcium reagent and simultaneously start the stopwatch.
Mix the contents of the tubes, remove the tubes from the water bath wipe and dry. Gently tilt the tube back and forth until a clot forms, at which point timing is stopped.
Average the two results and report the patient's results.




Special Senses

Vision:
Visual receptors are located in the retina, which forms the inner coat of the eyeball, the anterior surface of the eyeball is kept moist by the lacrimal fluid secreted from lactrimal glands.
Lacrimal gland: it secretes the lacrimal fluid, which is thin watery secretion contain chlorides and glucose.


Structure of the eyeball

Eyelids: They provide mechanical protection to the eye against injury, foreign body and excessive light. The sudden closure of the eyelids (blinking) helps the spread of lacrimal fluid in the conjuctival sac.
Eyeball: It is rounded or spherical in shape and it consists of three distinct coats:
Sclerocornea.
choroids.
Retina.
Visual pathway:






Procedure include:

1. Visual field:
When we look to an object a number of other objects in the neighbor hood, more or less distinctly are seen, the full extent of this vision is called visual field.

Objectives:

To map out the field of vision of the subject by using perimeter and detect any defects in the visual fields.

Subject and Instruments:

A perimeter.
A subject.
A perimeter chart.


Procedures:
The concavity of the arc of the perimeter is towards the face of the subject. The subject is made to rest his chin on the chin rest of the perimeter, the other eye is covered.
The eye to be examined should keep looking at the test object which is coloured, 5 mm object at the center of the metallic arc. The subject should keep focusing on the white object.
The white disc fixed to the carrier is moved along the arc and is gradually brought from the periphery to the center. The subject say's "yes" the moment he sees the object and mapping of the points is done on the chart the field. i.e., temporal, nasal, superior, inferior at interval of 15 and a total circle of 360.
marking should be done in clock wise fashion.
We find also the blind spot which is to the temporal side of the central point of vision which represent the optic nerve head area in which there are no light receptors.
We do the same procedure for mapping the visual field of the other eye.

2. Visual Acuity:

It is measured with Snellen's test type a series of letters of varying size are so constructed that the top letters are visual or visible to be seen to the normal eye at 60 m and the subsequent lines at 36, 24, 18, 12, 9, 6, respectively

Visual Acuity =


=

Note:

each eye is tested separately.
patient is normally placed at distance of 6 meter from the test types d = 6.
if only the tap letters are visible v= 6/60, read at least the seventh line
mean v = 6/6
4. if visual acuity is less 6/60 move the patient toward the Snellen's test type until he can read the top line for example about 2 meter
V = 2/60
Myopia = mean need concave lens.
Hypermetropia = mean need convex lens



3. Colour sense:
we use pseudo isochromatic plates a person with normal colour. Vision can read on number on plate while patient with defective colour vision will read a different number, on the same plate

Red green colour:

Blindness most commonly inherited as sex linked recessive. Condition occur in 8% male and 1% female.

Blue yellow colour:

Blindness in these two colours less commonly and usually defective in the number 74, 21, 42.



Pseudo isochromatic plates for testing colour blindness





If damage in optic nerve, mean total loss of vision.

If damage in optic chiasma, mean Bitemporal hemianopia.
If lesion in optic tract mean Right homonymous hemianopia.
If lesion of the lower fibers of the optic radiation in the temporal lobe lead to upper right quadrantic hemianopia
If lesion of the upper fibers of the optic radiation in the anterior part of parietal lobe lead to less commonly a lower quadration hemianopia.
If lesion in optic radiation in the posterior part of the parietal lobe lead to Right homonymous hemianopia.




Body Temperature Measurement (Clinical Thermometry)

Measurement of body temperature is a routine procedure used in all clinical work. It varies considerably under many physiological and pathological conditions. It a curate measurement helps in many ways to reach an accurate diagnosis.
Materials:
An accurate clinical thermometer of good quality.
Cotton wool and alcohol to wash the thermometer with.
Procedure:
The body temperature may be measured in the mouth axilla, groin and rectum.


Clinical Thermometer

1. Oral Temperature Measurement:
Procedure:
Clean the thermometer by rubbing its bulb with a cotton wool soaked in alcohol, holding the thermometer from the end away from the bulb with your right thumb and index figures.
Holding the thermometer from the same end, observe the level of the indicator fluid (mercury or alcohol column) If the column is not seen easily rotate the thermometer forward and backward until the column an appear clearly.
If the mercury level is 5C lower its level to below this mark. This is the best done by sharp jerky shakes of the thermometer.
Insert the bulb of the thermometer into your mouth, under the tongue. Then mouth is shut tightly and you must breath through the nose, during the whole time of the measurement. He sure that the thermometer does not slip firm under your tongue and guard against any breathing through the mouth.
Leave the thermometer in its place for 2 minutes, withdraw it and the level of the indicator fluid in the same way as in (2 record your results).
Repeat the measurement but immediately after a hot drink and again after a cold rink of water. Compare these results with these obtained. Take the measurement at intervals and record the time taken for the temperature to return to its normal level after each drink.
Repeat the measurement immediately after 1 minute heavy exercise. Take the measurement at intervals and record the time take for the temperature to return to its resting level.
Wash the thermometer and cleans it with alcohol soaked cotton wool before returning to its case or bottle.


2. Axillary and groin Temperature Measurement:

Follow the same steps 1, 2 and 3 as in oral measurement.
Dry the skin of axilla or the groin from any sweat and take aired to keep the part as free as possible for perspiration both during the measurement and for few minutes before it. The arm must be drown near the side (for the axillary measurement) and tight up towards the abdomen (for the groin measurement) for a short time before the thermometer is inserted. This is necessary to guard against the skin being chilled or exposed to air.
Insert the thermometer in the axillary flexure (or into the groin) and bring down the arm to the side of the body (or flex the thing on the abdomen) keeping the thermometer there in for 4 minutes.
Repeat the measurements but keeping the thermometer for periods of 1,3,4 and 6 minutes. Be sure to shake down the mercury to a level below 35C before each measurement.

3. Rectal Temperature Measurement:

This procedure is used mainly for children or for unconscious patients. In some clinics it is used regularly.




Reflex action in man

A reflex is an involuntary activity in an effectors organ (muscle, gland) elicited by the stimulation of a receptor organ.
There are five component parts to most reflexes:
Receptoo organ.
Afferent (sensory) neuron.
Interneuron.
Efferent (motor) neuron.
Effectors organ.


These structures form a reflex arc. You will examine a number of reflexes, which are especially significant from both physiological and clinical points of view.

Figure (13.1): Reflex arc showing the pathway of impulses and cross section of the spinal cord.
I. Cutaneous or superficial reflexes:
These consist of muscular contractions evoked by cutaneous stimulation.
1. Plantar reflex:
Scratch the lateral border of the sole of the foot using a blunted point (e.g., key), the normal adult response is a downward flexion of all toes. If the toes fan out with the big toe flexed dorsally (upward) it is referred to as Babinski reflex; this is often associated with damage to the pyramidal tract fibers.
Babinski reflex is the normal response of a child in the first year because the nerve are still undergoing myelination during this time.

2. Abdominal reflexes:

Stroke the skin of the abdominal wall. The reflex contraction of the abdominal muscles will pull the umbilicus to the side stroked.

II. Tendon or deep reflexes (stretch reflexes):

They are evoked by a stretch of the tendons of appropriate muscles, and this is done by a tap from a tendon hammer.

1. Knee jerk:

With the tendon hammer hit the patellar tendon, the extensor muscle will contract and the foot will be kicked forward. If no reflex is obtained ask the subject to hook his fingers together and to pull them apart (reinforcement maneuver).
Ankle jerk (Achilles reflex).
Biceps jerk.
Triceps jerk.






III. Eye reflexes:

1. Corneal (Blink) reflex:
Gently touch the periphery of the cornea with the corner of a clean handkerchief or a piece of a clean paper. The eyelids close immediately. If the stimulus is more intense, lacrimation will occur (lacrimation reflex). This reflex protects the cornea from injury by foreign material.

Afferent limb: Ophthalmic division of the trigeminal nerve.

Interneurons: Their cell bodies are located within the reticular formation of the brain stem.
Efferent neurons: Alpha motor neurons of the facial nucleus, which supply the orbicularis oculi muscles.
Afferent limb of lacrimation reflex is the same as for the blink reflex. Efferent limb involves the restral salivatory nucleus of the brain stem and the facial nerve fibers to the lacrimal gland.
2. Pupillary light reflex:
Swirch on the light and observe papillary constriction.
Receptor organ: Retina.
Afferent limb: Optic nerves and tracts.
Interneurons in the pretectal nucleus.
Efferent limb: Oculomator nerves.
Effector organ: Circular smooth muscles of the iris.

Fibers of both retinas project to both pretectal areas and both pretectal areas project fibers to both oculomator nuclei. Therefore, stimulation of either retina alone will induce the constriction of both pupils. The constriction of the pupil receiving the stimulus is referred to as the simple papillary light reflex, whereas the constriction of the pupil of the opposite eye is referred to as a consensual papillary light reflex.



Sensory Physiology
Information about the internal and external environment reaches the CNS via a variety of sensory receptors. These receptors are transducers that convert various forms of energy in the environment into action potentials in neurons.

Tactile sensation (touch):

This sense allows us to distinguish between hard and soft bodies and to judge their shape. Touch receptors are most numerous in the skin of the fingers and lips and relatively scarce in the skin of the trunk. There are many receptors around hair follicles.

Localization:

This tests the ability of the subject to localize the point touched when his eyes are closed. The power of localization depends on:
The position at which the nerve fibers from the tactile end-organs enter the spinal cord and on their higher connections.
Experience.
Information can be transmitted via cutanoeus endings in densely innervated zones such as the fingertips, and this is the basis of a reading method for blind persons.

Discrimination (two point discrimination):

These tests the ability to distinguish the contact of two separate points applied simultaneously to the skin. Discrimination can be made by using aesthesiometer, compass or caliper.
Discrimination magnitude varies from place to place on the body and is smallest where the touch receptors are most abundant. Find the two point discrimination threshold (in millimeter) for the tip of tongue, fingertip, palm of hand, back of hand and back of neck.

Stereognosis:

It is the ability to identify objects by handling them without looking at them. This ability depends upon relatively intact touch and pressure sensation. It also has a large cortical component.

Deep pressure touch:

With a blunted object (e.g., pencil) press on different areas of the skin.


Pain sensation:
The sense organs for pain are the naked nerve endings. Pain is tested by pinprick.

Temperature sensation:

There are two types of temperature sense organs; those responding maximally to temperatures slightly above body temperature (warmth receptors), and those responding maximally to temperatures slightly blow body temperature (cold receptors).
Prepare two test tubes, one containing water at 15C and another at 40C. Touch different areas of the skin. Notice which areas of the body are more sensitive to touch.

Joint movements and posture sensation:

Joint receptors are:
Golgi end organs in the ligaments.
The end organs of Ruffini in the capsule.
Pacinian corepuscles in the ligaments.

Change the position of the different joints passively whilst the eyes are closed. Ask the subject to describe the passive movements induced.

Vibration sensation:

When a vibrating tuning fork is applied to the skin, a buzzing or thrill is felt. The sensation is most marked over bones.
The receptors involved are the receptors for touch and pressure, Merkel's disks. Apply the base of tunning fork to the superficial bones and describe the sensation.





Hearing
Auditory acuity:
The relative acuity of hearing can be determined by the distance at which a person can hear a given intensity of sound. Auditory acuity should be performed in a quiet room.

Localization of sound:

Determination of the direction from which a sound emanates depends upon:
Detecting the differences in sound intensity in both ears.
Detecting the differences in time of arrival of the sound wave in both ears.

Tuning fork tests:

If a subject's sensitivity is low, this loss may be due to a defect in the external ear (such as wax blocking the meatus), or to some defect of the middle ear. This is called conduction deafness. There may be a loss of sensitivity in the cochlea or a more central defect, this is called nerve deafness. Conduction and nerve deafness can be distinguished by a number of tests with a tuning fork (A 512 Hz tuning fork is performed).

Rinne's test:

Place the handle of tuning fork on the mastoid process at the level of the upper portion of the ear canal. When the sound is no longer audible place the tuning fork in front of the auditory canal. In normal subject the air conducted sound is heard longer than heard by bone conduction (Rinne's test is positive). In conduction deafness Rinne's test may be negative (bone conduction is greater than air conduction).

Weber's test

The base of tuning fork is placed on the forehead in the midline or on the top of the head. If hearing is normal the sound appears to arise in the midline. If there is a damage to the cochlea or its neural connections (nerve deafness), the sound will be perceived less well on the affected side and will appear to arise on the healthy side. If there is conduction deafness, the sound is referred to the affected ear and is heard more in the affected ear.





Taste sensation
Introduction and principle:
The four primary taste sensations are sweet mainly felt by the tip of the tongue, sour sensation on the sides of the tongue, salty sensation on the dorsum of the tongue and bitter sensation on the posterior part of the tongue.

Objectives:

To examine the localities of the taste sensations.

Materials and subjects:

Volunteer from students.
Four solutions are used in this experiment:
Strong solution of sugar.
Strong solution of salt.
Weak solution of citric acid (sour).
Weak solution of quinine (bitter).

Procedure:

Ask the subject to put out his tongue and with a glass rod apply a fine drop of each solution (only one at a time) to the tip, upper surface, back and the sides of the tongue.
Ask the subject to describe what sort of taste he perceives each time. Repeat the test with all of the solutions, (bitter at the end). Put down your results and map the different parts of the tongue to all solution on a diagram.

Results:

Bitter substances are tasted on the back of the tongue, Sour along the edges. Sweet at the tip, and Salt on the dorsum anteriorly (see figure16.1).


Discussion:
Enumerate the types of the taste papillae and their taste specialization.
Mention the substances that cause the four primary taste sensations and their chemical structures.
Is it possible to perceive bitter taste on the dorsum of the tongue? How and why?



Figure (16.1): The location of different taste modalities on the surface of the tongue.









PAGE 

PAGE 54

Experiment (1)

1

4000

50

1

50

1

X x 1

50

1

1

50


Experiment (2)

Experiment (3)

Experiment (4)

Experiment (5)

dl = 100 ml

Experiment (6)

Occipital cortex

Optic chiasma

Ganglion cell

Optic nerve

Experiment (7)


Receptor

Bipolar cell

PVC (%)

RBC count x 106 cumm

Hb (gm / dl)

RBC count x 106 cumm

Hb (gm / dl)

PCV(%)

RBC

PVC

RBC


Hb

Hb

PCV

MCV

MCH

Experiment (8)

Experiment (9)

Experiment (10)

Lateral geniculate body

Optic tract

d


D

Distance at which the letter read

Distance at which they should read

Occipital cortex

Visual Pathway

Experiment (11)

Experiment (12)

Oral Temperature Measurement

Experiment (13)

Experiment (14)

Experiment (15)


Epiglottis

Root

Sour

Body

Bitter

Sour

Salt

Sweet

Experiment (16)

WBC

WBC


WBC

WBC

RBC

1 mm

One corner (1mm2)

Counted

Uncounted

Mixing chamber

Stem

Aspiration tube

(rubber sucking tube)

1


2