Second Hematopoietic Lab

WBCs & Differential Count

Clotting & Bleeding Time

Dr. Waleed R. Ezzat

Practical Objectives:

1. Learn how to preparing & staining a blood film

2. Be familiar with examination of blood film slides & identification of WBC type

3. Determine the percent of each type of WBC

4. Use the capillary method to determine the clotting time

5. Determine the bleeding time by using the filter paper

Differential WBC Count

Aim:

To determine the percentage of each type of leukocytes in the peripheral blood. Certain percentages can change during certain diseases so this test is valuable for the diagnosis of these diseases.

Principle:

The test depends on making a blood film from the peripheral blood and staining it with Diff Quick stain using manual method. Different types of WBCs are differentiated by their morphological and staining characteristics.

Material:

Lancets Clean slides

Microscope Diff Quick Stain

Procedure:I. Preparation of blood smear: Is made on an

ordinary slide, the steps are;

1. Slides must be spotlessly clean. Hold them always by the edges. Never put your fingers on the surface of the slide. Prepare two clean slides.

2. Obtain a drop of blood from the tip of the middle finger and place it at the end of one slide. Put the slide on the surface of your bench. Use the second slide as a spreader by placing its smooth edge at an angle of 45º to the first slide.

3. Move the spreading slide backward until it touches the drop of the blood as in the diagram. The drop of the blood will immediately spread across the edge of the spreading slide.

4. With the spreading slide at the angle of 45º, push it slowly forward and steadily across the horizontally placed first slide.

Procedure (cont.):I. Preparation of blood smear: Is made on an ordinary slide, the steps are;

5. The thickness of the film may be regulated by increasing or decreasing the angle between the slides or by varying the speed of spreading. A quick spreading gives a thick film; the wider the angle the thicker the film.

6. Allow the blood smear to dry in air at room temperature; it must not be heated.

7. A satisfactory film is very evenly distributed and yellowish-orange in colour; the red cells should be close together but not overlapping each other. Check that by looking at the film under the microscope.

8. The “zone of morphology” should be at least 2 cm in length. The smear should occupy the central area of the slide.

Procedure (cont.):II. Staining of blood smear: Do not stain a film until you have made a

satisfactory one. The stain used is the Diff-Quick stain. This stain contains;

▪ Diff-Quick fixative reagent (Triarylmethane dye, Methanol)

▪ Diff-Quick solution 1 (Xanthene dye (Eosin Y) in phosphate buffer)

▪ Diff-Quick solution 2 (Thiazine dye in methylene blue and azure A phosphate buffer)

The steps of staining are;

1. Air dry the smear

2. Dip slide for 30 seconds into Fixative. Allow excess to drain after each

dip.

3. Dip slide for 30 seconds into Stain 1. Allow excess to drain after each dip.

4. Dip slide for 30 seconds into Stain 2. Allow excess to drain after each dip.

5. Rinse slide in distilled water or Weise's buffer, pH 7.2 to remove excess

stain.

6. Allow to dry in air or rapidly dehydrate in absolute alcohol.

7. Examine at low power to identify structures and then under oil immersion.

Procedure (cont.):III. Examination of blood smear:

▪ The monolayer is the area where the cells are examined and a 100 cell

differential leukocyte counts performed.

▪ The monolayer section is usually a distance of one 10x field behind the

feathered edge of the smear and is the optimal area for examination of cells.

White blood cells are uniformly distributed in this section.

▪ The student should scan the smear at low magnification first to locate the

optimal area for differential leukocyte count.

▪ Once the optimal area has been located, switch to a higher magnification (50

or 100x oil immersion objective) and begin your differential count and morphology assessment.

▪ Begin the count by moving back and forth across the smear in a pattern that

avoids covering the same territory. This can be done by moving in a zigzag

fashion across the slide (as in the diagram).

▪ Identify each leukocyte that is encountered

until 100 cells have been counted and

sorted by type, giving you a percentage of

each cell type or a relative differential

leukocyte count.

The Granulocytes (70%-6.300/cu mm)

1. Neutrophils: (50%-70%)

- They are highly phagocytic (often called macrophages).

- Average sized (10-12μ in diameter) with abundant cytoplasm.

- Small pink granules in the cytoplasm.

- Multilobulated purple nucleus (three lobes or more).

- Increase in No. in acute pyogenic infection.

The Granulocytes (70%-6.300/cu mm)

2. Eosinophils: (1%-4%)

- They are slightly phagocytic and increase in number after allergic reaction (such as asthma, parasitic infestation, and Hay fever). They phagocytize and destroy the antigen-antibody complex

- Large sized cells with abundant cytoplasm (13μ in diameter)

- Large red-orange cytoplasmic granules, blue-purple nucleus

- Mutilobulated nucleus (2 or 3 lobules)

The Granulocytes (70%-6.300/cu mm)

3. Basophils: (0%-1%)

- They are not phagocytic and their function is to liberate histamine and heparin during allergic reactions.

- Average sized cells (7μ in diameter).

- Scanty or abundant cytoplasm.

- Large deep blue cytoplasmic granules, blue-black nucleus.

- Lobulated nucleus (2 or 3 lobules).

- They liberate heparin into the blood, a substance that can prevent blood coagulation.

The Agranulocytes (30%-2.700/cu mm)

1. Lymphocytes: (20%-40%). They are of two types; small and large.

- They are not phagocytic, specialized in production of immunity such as the synthesis of antibodies

- Variable size with no cytoplasmic granules, light blue cytoplasm

- The scanty cytoplasm is pushed to the periphery of the cell by the large deep blue or purple nucleus

- 7μ in diameter (small) to 10μ in diameter (large)

- Small lymphocytes have very large spherical nucleus, large lymphocytes have large oval indented nucleus

- They perform a major role in immune system and increase in number during chronic and viral infections

The Agranulocytes (30%-2.700/cu mm)

2. Monocytes: (2%-8%).

- They are phagocytic and help in tissue repair after injury.

- They are the scavenger cells and they are transferred into tissue macrophages to engulf the dead tissues and bacteria.

- The Largest WBC with irregular blue or purple nucleus (Kidney shaped nucleus).

- Abundant blue-gray cytoplasm (nongranular).

- They are part of the innate immune system and increase in No. in chronic inflammation.

Bleeding Time Test

(Duke Method)

Definition:

A bleeding time test determines how quickly the bleeding stopes. The test involves making small punctures in the skin. The test is a basic assessment of the platelets function (i.e. platelet plug formation).

Indication: The bleeding time test is a common test to screen patients having bleeding tendency (i.e. prolonged bleeding times). The bleeding time is used to measure the primary phase of hemostasis.

Procedure:

• Clean the puncture site with an spirit to minimize the risk of infection.

• Make small cuts with the lancet on the tip of the middle finger (or the earlobe). The puncture should be 3-4 mm deep to cause slight bleeding.

• Start the stopwatch immediately, swab the cut with filter paper every 30 seconds until no more blood is absorbed (i.e. no more blood spots on the filter paper).

• Record the time it takes for the bleeding to stop.

Bleeding Time Test

(Duke Method) – Cont.

Interpretation: Normal bleeding time is between 2-5 minutes. Prolonged

bleeding time could indicate low platelet count or the platelets are

dysfunctional, and further testing will be required. Abnormal results

could indicate one of the following conditions;

• Thrombocytopenia

• Medications (Example aspirin and other cyclooxygenase inhibitors,

& antibiotics such as penicillin and cephalosporins)

• Uremia (uremic platelets synthesize less thromboxane A2, and the

blood vessels in patients with uremia produce greater quantities of

platelet-inhibitory prostaglandin)

• Liver failure

• Leukemias

• Von Willebrand’s disease

Clotting Time Test

(Capillary Tube Method or Wright’s Method)

Definition:

• Is the time required for a fresh sample of blood to form a clot in vitro. The basis for the test is that whole blood will form a solid clot when exposed to a foreign surface such as a glass tube.

• Clotting time test is a rough measure of all intrinsic clotting factors in the absence of tissue factors.

• Variations are wide and the test sensitivity is limited. The more recent sensitive test for the intrinsic pathway is the partial thromboplastin time (PTT). Normal value of clotting time is 3-8 minutes.

Indication:

• Clotting time is used as a screening test to measure all stages in the intrinsic coagulation system and to monitor heparin therapy.

• This test is sensitive only in extreme factor deficiencies (such as in hemophilia). Therefore, it is of limited use in today’s medical practice and its use is confined to demonstration.

Clotting Time Test

(Capillary Tube Method or Wright’s Method) Cont.

Material Required:

• Sterile disposable pricking needle or lancet

• Stop-watch

• Dry glass capillary tube (narrow diameter top 2 mm, minimum 10 cm long)

• Cotton Swab of absorbent cotton

• Spirit wetted, cotton swab

• 70% v/v ethyl alcohol

Procedure:

1. Apply alcohol 70% v/v to clean the fingertip with cotton swab. Allow it to dry naturally.

2. Prick the finger. Remove the first drop.

3. Dip one end of the capillary into blood drop gently without pressure (better to use more than one capillary tube).

4. The timer is started when the first blood start to enter the first capillary tube.

5. Allow to fill the capillary with blood by lowering the end of fitted capillary. (Do not suck the blood).

6. After every 30 seconds, using stop-watch, break a small piece of capillary.

7. Repeat breaking at regular time intervals, till fibrin thread appears at the broken end of capillary tube. Do not pull away the cut pieces long apart and bristly.

8. Record time interval between pricking finger and first appearance of fibrin thread between the broken ends of capillary tube. That is clotting time of blood.

9. Don't forget to dispose of the broken capillary tube in the SHARPS CONTAINER.

Clotting Time Test

(Capillary Tube Method or Wright’s Method) Cont.

Interpretation: Conditions accompanied by increased Clotting Time are;

• Factors V, VII, VIII, IX, XI, XII Deficiencies

• Hemorrhagic disease of Newborn

• Vitamin K deficiency

• Heparin Therapy

• Presence of Circulating antibodies (inhibitors)

• Anemia and leukemia

• Afibrinogenemia and Pneumonia