hematology practical

1. Clotting time, Bleeding time &
Blood groups
Practical session briefing

2. Blood grouping
• Also known as blood typing
• PRINCIPLE
• The surfaces of red cell membrane contain a variety of genetically
determined antigens, called agglutinogens, while the plasma
contains antibodies (agglutinins)
• To determine the blood group of a person, his/her red cells are
made to react with commercially available antisera containing
known agglutinins
• The slide is then examined under the microscope to detect the
presence or absence of clumping and hemolysis (agglutination) of
red cells which occurs as a result of antigen-antibody reaction

3. • APPARATUS AND MATERIALS
• Microscope
• Dropper
• Sterile blood lancet
• Sterile cotton
• Alcohol
• Toothpicks
• Glass slides
• Normal saline
• Anti-A serum (contains monoclonal anti-A antibodies)
• Anti-B serum (contains monoclonal anti-B antibodies)
• Anti-D serum (contains monoclonal anti-D antibodies)

5. • Preparation of red cell suspension
• Add two drops of blood with 2ml of saline in a small test tube
• A suspension of red cells in saline should preferably be
prepared and used instead of adding blood drops directly from
the fingerpick to the antisera for the following reasons:
• Dilution of blood permits easy detection of agglutination and
hemolysis, if present. (Red cells in undiluted blood tend to
form large rouleaux and masses. These may be difficult to
disperse and may be mistaken for agglutination)
• Plasma factors likely to interfere with agglutination are
eliminated

6. Put one drop of anti-A serum on the left half
Put one drop each of normal saline on the right halves (C)
Add a drop each of red cell suspension to both sides
Mix the anti-sera and red cells, and saline and red
cells
Put one drop of anti-B serum on the left half
Put one drop each of normal saline on the right halves (C)
Add a drop each of red cell suspension to both sides
Mix the anti-sera and red cells, and saline and red
cells
Put one drop of anti-D serum on the left half
Put one drop each of normal saline on the right halves (C)
Add a drop each of red cell suspension to both sides
Mix the anti-sera and red cells, and saline and red
cells

7. • The red cells-saline mixture on the “control” sides of each
slide will act as a control to confirm agglutination or no
agglutination on the corresponding test side
• Wait for 8–10 minutes, then inspect the 3 antisera-red cell
mixtures (“test” mixtures) and ‘control ‘ mixtures, first with
naked eye to see whether agglutination has taken place of
not
• Then confirm under low magnification microscope,
comparing each “test mixture” with its corresponding
“control mixture”

8. • OBSERVATIONS AND RESULTS
• i. If agglutination occurs, it is usually visible to the naked
eye. The hemolysed red cells appear as isolated (separate),
dark-red masses (clumps) of different sizes and shapes.
• ii. There is brick-red coloring of the serum by the
hemoglobin released from ruptured red cells.
• iv. Under LP objective, the clumps are visible as dark masses
and the outline of the red cells cannot be seen.
• No agglutination in the ‘control’ mixtures

11. BT and CT

12. WHAT IS
THE
CLINICAL
SIGNIFIC
ANCE OF
DOING BT
AND CT?
1. History of frequent, persistent or
spontaneous bleeding.
2. Before every minor and major
surgery.(eg; tooth extraction)
3. Before taking biopsy (bone marrow,
kidney, liver etc.)
4. Before and during anticoagulant
therapy.
5. Family history of bleeding disorder.

13. BLEEDIN
G TIME
First functional platelet evaluation
test
Introduced by duke in 1900
Used to detect defects in primary
hemostasis
Used as a screening test for
vascular disorders
as well as platelet function test

14. REQUIMENTS
A) Blotting papper
B) Stop- watch
C) Sterile Lancet
D) Alcohol swabs

15. PROCEDURE
a) After following sterile precautions, prick the fingertip deep enough (2- 3 mm) to result in
bleeding. Note the time of onset of bleeding.
b) Apply a piece of filter paper to the blood-drop every 30 seconds until the bleeding stops.
Note the time when no trace of blood on the filter paper
c) Count the spots of blood on filter paper and express bleeding time in minutes and seconds.
d) The bleeding time estimated by this method of a normal subject is within the range of 2-5
minutes.
Normally, bleeding time by this methods range by 1-7minutes. A value exceeding 10 minutes is
clearly abnormal.

16. Results
6 x 30 sec = 180 sec

17. CLINICAL
SIGNIFICANCE
•Normal Bleeding Time: 1 to 7 minutes
Prolonged BT in
Thromboctytopenia
Disorders in platelet: -thrombasthenia,storage
pool disease,Bernard –Soulier syndrome
Afibrinogenemia
Severe hypofibrinogenemia
Vascular disorders
Aspirin

18. CLOTTING TIME
WHOLE BLOOD CLOTTING TIME
The time it takes for whole blood , drawn from
a vein and immediately placed in a container to
clot.
It measures all stages of intrinsic coagulation
It is not a very sensitive method
Avoid contamination with tissue fluid.

19. Aim: To determine Clotting
time by capillary method.
*The time from pricking the finger to the
appearance of the fibrin clot is the clotting
time

20. REQUIREMENT
1. Glass capillary tubes ( Non heparnised )
2. A Petri dish
3. Alcohol swabs
4. Cotton wool
5. Sterile lancet

21. GLASS CAPILLARY TUBES

22. PROCEDURE
a) Clean finger with aseptic alcohol swab.
b) Prick the finger with lancet resulting in bleeding, note the time of onset of bleeding.
c) Wipe away the first drop of blood, fill freely flowing blood is taken into a capillary tube up to about 8 cm long without air
bubble, holding the tube horizontally let it fill by capillary action.
d) Roll the capillary gently between the palms, Fill more than one tube.
e) At the end of two minutes after making the puncture, break off 0.5cm of capillary tube (blood containing end) and separate
the two halves slowly.
f) Repeat the procedure at 30 second intervals with the remaining tubes.
g) When the blood forms a continuous thread-like clot between the broken ends of the tube, the end-point has been reached,
Note the time.
h) Clotting time by this method is 3-8 minutes.

23. RESULTS….
Normal Clotting Time: 3-8
minutes

24. CLINICAL
SIGNIFICANCE
• Only severe clotting factor deficiency can be recognized.
• Prolonged CT more than 10’-pt subjected to more detailed test.
• Used to monitor heparin therapy.
• Can be due to the deficiency of plasma factors such as
Antihemophiliac globulin,plasma
thromboplastin,fibrinogen,prothrombin.
• Circulating anticoagulants