HAEMATOLOGY MAJOR

Determination RBC Count

A 30-year-old female presents to the family medicine clinic with complaints of persistent
fatigue, weakness, and headaches over the past few months. She reports increased hair fall
and has noted brittle nails. Menstrual history reveals heavy and prolonged menstrual
bleeding. On examination, there was paleness of palpebral conjunctiva and angular cheilitis
of oral mucosa.

a. What is the probable diagnosis? (1 mark)

b. Which major blood experiment would you like to perform to confirm your
diagnosis? (1 mark)
c. Perform the above experiment and give your report? (5 marks)
d. Mention the factors that affects the above cell count? (3marks)

a) What is the probable diagnosis?

The probable diagnosis is Anaemia.

b) Which major blood experiment would you like to perform to confirm your diagnosis?

I would you like to perform determination of Red Blood Cell count to confirm the
diagnosis.

c) Perform the above experiment and give your report?

Aim: Determination of red blood cell using haemocytometer.

Apparatus required: Improved Neubauer’s chamber, RBC pipette, RBC diluting fluid,
compound microscope, cover slips, watch glass, pricking apparatus.

Principle: Since the RBC count runs in millions the count is made possible by diluting the blood
sample before counting and subsequently multiplying the count by the dilution factor.

RBC DILUTING FLUID (HAYEM'S FLUID)

COMPOSITION: 100 ml of fluid contains:

1) SODIUM SULPHATE: 2.5 gm, to prevent aggregation of RBCs (Rouleaux formation)

2) SODIUM CHLORIDE : 0.5 gm, to maintain isotonicity of fluid
3) MERCURIC CHLORIDE: 0.25 gm, as preservative, antibacterial and anti-fungal
4) DISTILLED WATER: 100 ml as solvent
OBSERVATION:

A B

D C

Total (N) = A+B+C+D+E =

CALCULATIONS:

RBCs are counted in 80 smallest squares.

Area of the smallest square = 1/20 x 1/20

= 1/400 mm2

Depth of the chamber = 1/10 mm

Volume of 1 smallest square = 1/400 x 1/10

= 1/4000 mm3

Total volume of 80 smallest squares = 1/4000 x 80

= 1/50 mm3

Number of red cells in 1/ 50 cu.mm of diluted blood = N

Number of RBCs in I cu.mm of diluted blood = N/1/50

= Nx50

Dilution factor = 1:200

Number of red in 1 cu.mm undiluted blood = N x 50 x 200

Therefore, the total number of red cells in 1 cu.mm of undiluted blood = Nx10000

RESULT:

The RBC count of the given sample of blood is _________ million cells/cu.mm of blood.
d) Mention the factors that affects the above cell count?

VARIATIONS IN RBC COUNT

PHYSIOLOGICAL INCREASE

• High altitude-due to hypoxia

• Newborns and infants
• Excessive sweating-due to haemoconcentration

PHYSIOLOGICAL DECREASE

• Pregnancy due to haemodilution

• Children have lower values than adults
• Women have lower values

PATHOLOGICAL INCREASE

• Conditions that produce hemoconcentration (due to loss of body fluid) eg: severe
diarrhea, vomiting
• Conditions that produce chronic hypoxia eg: congenital heart disease, emphysema
• Polycythemia vera

PATHOLOGICAL DECREASE

• Different types of anaemia

• Relative decrease in RBC count occurs in different pathological conditions that produce
haemodilution eg: excess ADH secretion as occurs in pituitary tumours
Determination Total WBC Count

A 25-year-old male presents to the clinic with complaints of sore throat, fever and general
weakness for the past three days. On examination, he had elevated body temperature and
erythema and swelling of the tonsils.

a. What is the probable diagnosis? ( 1 mark)

b. Which major blood experiment would you like to perform to confirm your
diagnosis? (1 mark)
c. Perform the above experiment and give your report? (5marks)
d. Mention conditions where the above cell count is increased and decreased? (3marks)

a) What is the probable diagnosis?

The probable diagnosis is Acute Tonsillitis.

b) Which major blood experiment would you like to perform to confirm your diagnosis?

I would you like to perform determination of Total Leucocyte Count (TLC) to confirm
the diagnosis.

c) Perform the above experiment and give your report?

Aim: To determine the total leucocyte count using haemocytometer.

Apparatus required: Improved Neubauer’s chamber, WBC pipette, WBC diluting fluid,
compound microscope, cover slips, watch glass, pricking apparatus.

Principle: Since the WBC count runs in thousands the count is made possible by diluting the
blood sample before counting and subsequently multiplying the count by the dilution factor.

WBC DILUTING FLUID (TURK'S FLUID)

COMPOSITON : 100 ml of fluid contains:

1) GENTIAN VIOLET 1% : 1ml, stains the nuclei of WBCS

2) GLACIAL ACETIC ACID: 3ml. to destroy the RBCs and platelets
3) DISTILLED WATER: To make 100 ml
OBSERVATION:

A B

C D

Total (N) = A+B+C+D =

CALCULATIONS:

Area of one large square = 1x1 = 1mm2

Depth of chamber =1/10 mm

Volume of one large square = 1x 1/10 = 1/10 mm3

Volume of four large squares = 4x1/10=4/10 mm3

Number of cells in 1cu.mm of diluted blood =N x 10/4

Dilution factor = 1:20

Number of cells in 1 cu.mm of undiluted blood=N x 10/4x 20

Therefore, the number of cells in 1 cu.mm of undiluted blood=Nx50

RESULT:

The number of WBCs in the given sample of blood is _________ cells/cu.mm of blood.

d) Mention conditions where the above cell count is increased and decreased?

VARIATIONS IN WBC COUNT

PHYSIOLOGICAL INCREASE

• Newborn and infants

• Physical exercise
• After food intake
• Exposure to sun and increased environmental temperature
• Pregnancy
• Parturition
• Menstruation

PHYSIOLOGICAL DECREASE

Exposure to severe cold

PATHOLOGICAL INCREASE

• Leukemia
• Acute infections(pyogenic)
• Burns
• Acute hemorrhage
• Myocardial infarction
• Post Operative
• Inflammatory disorders

PATHOLOGICAL DECREASE

• Infections (non-pyogenic)
• Viral infections
• Drugs
 • Chemical poisoning-arsenic
• Malnutrition
• Exposure to radiations

Determination DLC

3.45-year-old female complaints of unusual tiredness for the last few months, with difficulty
keeping up with her daily activities. She experienced occasional low-grade fevers and night sweats,
with significant weight loss. Complete blood count showed significant increase in immature WBC
count (blast cells).

a. What are granulocytes and agranulocytes? Name them. (2 marks)

Granulocytes- Are cells having granules, they are Neutrophils, Eosinophils, Basophils
Agranulocytes- Are cells with no granules, they are Lymphocytes, Monocytes.

b. Prepare a blood smear, stain and give your report? (5 marks)

AIM:
To prepare a blood smear of the given sample of blood and to express the relative counts
of leucocytes in percentage.

OBJECTIVE:
To make a good smear, to identify the different types of leucocytes and to determine the
% distribution of leucocytes in peripheral blood.

APPARATUS:
Glass slides, compound microscope, pricking apparatus, cedar wood oil, Leishman’s
stain, wash bottle of distilled water, staining tray.

OBSERVATION:
RESULTS:

The percentage of leucocytes in the given sample of blood is

• Neutrophil: ____________%
• Eosinophil: ____________%
• Basophil: _____________%
• Lymphocyte: __________%
• Monocyte: ___________%

c. Write the composition of the stain used in the above experiment? (1 marks)

• EOSIN: Acidic dye that stains the basic structures like hemoglobin for RBCs and
eosinophilic granules
• METHYLENE BLUE: Basic dye that stains the acidic structures like nuclei
(DNA) and cytoplasm (RNA) of WBCs and basophilic granules.
• ACETONE-FREE METHYL ALCOHOL: As fixative. It is acetone-free because
acetone cause lysis of cells (damage cell membrane)

d. How will you differentiate between leukocytosis and leukemia? (2marks)

FEATURES LEUKOCYTOSIS LEUKEMIA

DEFINITION Increased WBC due to a Cancer of blood and bone

reactive process (e.g., marrow leading to
infection, inflammation) uncontrolled production of
premature WBCs.

WBC COUNT RANGE 12,000 to 30,000 cells/µL 50,000 to 1,00,000 cells/ µL