Category: Human Anatomy and Physiology

Aim: To find out the number of red blood cells in one cubic millimetre of blood.

Principles: The number of RBC in a known volume of diluted blood is counted and the number of
cells in one cm of undiluted blood are calculated from this.

Apparatus Required: Hemocytometer, RBC diluting fluid, compound microscope, sterile lancet,
watch glass, cotton, rectified spirit.

Uses of R.B.C. pipette:

• WBC count in leukemias
• Platelet counting

Uses of the bead in the bulb:

• For proper mixing
• To know whether the pipette is dry
• To identify the pipette
• R.B.C diluting fluid: Hayem’s fluid is the commonly used diluting fluid.

Composition:

• Sodium chloride 0.5 Gm
• Sodium sulphate 2.5 Gm
• Mercuric perchloride 0.25 Gm
• Distilled water 100 ml sodium chloride and sodium sulphate together keep the isotonicity of fluid. Sodium sulphate also prevents the clumping of red cells. Mercuric perchloride fixes the cells and acts as a preservative.
• Other diluting fluids:
  • Gower’s fluid
  • Toison’s fluid
  • Formol Citrate solution

Procedure: Clean and dry the counting chamber and put on the special coverslip provided. Focus on the high power objective and identify the RBC counting area. Clean the RBC pipette first with distilled water, then with absolute alcohol and finally with ether and keep it dry. Take a small quantity of diluting fluid in a watch glass and keep it aside. Clean the fingertip using rectified spirit and make a deep prick with a sterile lancet, so that blood comes out freely without squeezing. Wipe off the first drop which may contain tissue fluid also. Allow a good-sized blood drop to form a hanging drop and
keep the pointed tip of the pipette touching the drop. Suck in blood up to the 0.5 marks carefully, without any air bubble. Excess blood at the tip of the pipette is removed using a blotting paper or piece of cotton. Immediately, diluting fluid from the watch glass is sucked in up to the 101 marks without any air bubble by keeping the pipette in a vertical position.

Then thoroughly mix the blood and diluting fluid in the pipette by gently rolling the pipette held horizontally between the palms and keep aside. Mixing takes place only in the bulb of the pipette. The column of diluting fluid contained in the stem of the pipette does not enter into the dilution (i.e. 101-1 = 100). So that the blood sucked up to the 0.5 marks will have a dilution of 0.5 in 100 or 1 in 200. Now take out the counting chamber for charging discard the first few drops from the pipette, as the stem contains only diluting fluid. Bring one small drop of diluted blood at the tip of the pipette, to the edge of the coverslip on the counting chamber at an angle of about 450 The fluid enters by capillary action under the coverslip and fills the counting chamber. Both areas are filled.

Focus the RBC counting area under high power. Keep the counting chamber undisturbed for about 3 minutes for the cells to settle down in the counting area, and start counting. At least 5 squares, each having 16 smallest squares (preferably 4 corners and 1 central) should be counted to obtain a satisfactory average and a better dispersal value. While counting each small square, cells touching the top and left margin of each square should be omitted and cells touching the bottom and right margin of each square should be counted. Draw a chart of the counting squares in the record and enter the number of cells in each square and when counted.

Calculations: Let the number of cells counted in (5×16) 80 smallest squares be “N”

The number of cells in 1 smallest square is N/80

Side of 1 square = 1/20mm

Side of 1 square = 1/20mm

The volume of diluted blood in 1 square=1/400×1/10=1/4000mm3

Number of cells in 1/4000mm3 diluted blood = N

80 Number of cells in 1 mm3 of diluted blood N 80×1/4000= Nx4000 80

The dilution factor is 1 in 200

(Total diluted volume in the bulb of the pipette is 100 parts, out of which 0.5 is blood. So dilution is 0.5 in 100 i.e.1 in 200)

So numberof cells in 1 mm3 of undiluted blood =Nx 4000×200 = Nxl0000 80

Precautions:

• Counting chamber and pipette should be clean and dry.
• Fingertip and pricking lancet should be sterile.
• Blood should freely come out without squeezing.
• Be careful to prevent clotting of blood inside the pipette.
• While filling the pipette and charging the counting chamber, no air bubble should enter.
• Blood should be taken only up to the 0.5 marks and diluting fluid sucked only up to 101 mark.
• Blood should be properly mixed with the diluting fluid.
• Discard first few drops before charging because it will not contain RBC5.
• While charging the counting chamber, over filling and spilling should be avoided.
• Cells should be settled down and more or less evenly distributed before counting.
• Don’t keep the microscope in tilted position.
• Count from Left to Right and avoid counting of the same cell.

Aim: To determine the clotting time of a subject.

Requirements: Fine capillary glass tubes of about 10 mm length, cotton, rectified spirit, lancet, stopwatch.

Procedure:

• Capillary tube method: (Wright’s method): Under sterile precautions make a sufficiently deep prick in the finger tip. Note the time when bleeding starts (start the stop watch). Touch the blood drop at the finger tip using one end of the capillary tube kept tilted downwards. The tube gets easily filled by capillary action. After about two minutes start snapping off small lengths of the tube, at intervals of 15 seconds, each time noting whether the fibrin thread is formed between the snapped ends. Note the time (stop the stop watch) when the fibrin thread is first seen.
• Discussion: Clotting time is the interval between the moment when bleeding starts and the moment when the fibrin thread is first seen. The normal value is 3to 10 minutes. Bleeding time and clotting time are not the same. Bleeding time depends on the integrity of platelets and vessel walls, whereas clotting time depends on the availability of coagulation factors. In coagulation disorders like haemophilia, clotting time is prolonged but bleeding time remains normal. Clotting time is also prolonged in conditions like vitamin K deficiency, liver diseases,disseminated intravascular coagulation, overdosage of anticoagulants etc.
• Modified Lee and White method: Under aseptic precaution, venepuncture is done and one ml. of blood is collected in each 3 small test tubes. Note the time when blood is taken. Keep the test tube in a water bath maintained at 37°c. Tilt the tubes every 30 seconds and see whether the blood is flowing. Repeat this till the tube can be inverted without the blood flowing out. Nore the time. The average value of the results in the 3 test tubes gives the clotting time.

Normal value is2 to 7 minutes

Aim: To determine the bleeding time of a patient.

Theory: The time required for complete stopping of blood flow from the punctured blood vessels is called the bleeding time. Normally it is 1-3 minutes for a normal human’s blood. Normal clotting time and bleeding time values differ because bleeding time is the time for stopping bleeding by the formation of fibrin network on the surface of punctured skin; that is it is the surface phenomenon. But the clotting time is the time for clotting the whole blood, collected in the capillary tube; therefore it is a volume phenomenon. For this reason, clotting time is more than the bleeding time, when determining by conventional methods.

Clinical significance: It plays a significant role in:
i) to study haemorrhagic disorders.
ii) to study the coagulation defects
iii)to have an idea about the platelets count of the patient. Bleeding time is prolonged in few disorders like vascular lesions, platelet defect, severe liver disease, uremia and anti-coagulant drug administration.

Requirements: Sterilized needle, filter paper, cotton, spirit, and stopwatch.

Procedure (Duke’s method): The finger of a subject is sterilized with spirit and pricked with a sterilized needle. Time of pricking is noted. Take the stain of the punctured point on a filter paper for 30 seconds and keep taking the stain of blood in 20-second intervals until the bleeding stops. The time of no stain has come is noted properly; it is the bleeding time of the subject.

Precaution: Following precautions should be enforced
i) Needle should be sterilized.
ii) A fain stain of blood should not be avoided.
iii) Time should be noted properly.

Aim: To determine the haemoglobin content in 20µl of the blood sample.

Principle: A hemoprotein composed of globin and heme that gives red blood cells their characteristic colour; functions primarily to transport oxygen from the lungs to the body tissues. The red blood cells are broken down with hydrochloric acid to get the haemoglobin into a solution. The free haemoglobin is exposed for a while to form hemin crystals. The solution is diluted to compare with a standard colour.

Requirements: Hemometer, Single mark pipette, Distilled water, Needle, Spirit, Cotton, HCl.

Procedure:

1. Take 1/10 HCl in the Hb tube upto the lowest mark ‘2’.
2. Prick the finger with needle and collect 20µl of blood sample with single mark pipette
3. Place the Hb tube on the working table for five minutes for the formation of hemin crystals.
4. Place the Hb tube in the compater/hemometer and add drop by drop of distilled water into it until the colour of the solution in the Hb tube coincides with the glass plates of the compater.
5. If the colour coincides with the glass plates of the compater, observe the reading in the Hb tube. The percentage of Hb can be calculated from the reading.

Evaluation: Hb content in grams X 100 / 14.5

• Normal Values:
  • Male: 14 to 18grams
  • Females: 13 to 14grams
  • Children: 10 to 13grams

Result: The hemoglobin content present in 20µl of blood sample is _____________________.