This document discusses a differential leucocyte count (DLC) test. It provides information on the types of white blood cells (WBCs), how a blood smear is prepared and stained, and how the different WBCs are identified and counted under a microscope. A DLC involves counting 100 WBCs and recording the percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils present. Normal ranges for each cell type are provided. Eosinophilia, an elevated eosinophil count, and its physiological and pathological causes are also summarized.

1. Differential Leucocyte count (TLC)
& Eosinophilia
Dr Sandeep singh

2. OBJECTIVES
 Types of WBC and their counts
 Formation of WBC
 Morphology, life span, function.
 Applied aspects.
 To draw a peripheral blood smear, stain it
with Leishman's stain and do the
differential count( DLC) of the given
blood sample.

3. INTRODUCTION
 Crucial in the body’s defense against pathogens
Capable of phagocytosis.
 These are complete cells, with a nucleus and
organelles
 They lack Hb so they are colorless (i.e. white)
 Able to move into and out of blood vessels
• Can respond to chemicals released by damaged
tissues.

5. The method used to count 100 WBC and identify the
different leukocytes
 Granulocytes
Polymorphs / Neutrophils
Eosinophils
Basophils
 Agranulocytes
Lymphocytes
Monocytes

6. •Granules in their cytoplasm can be stained
inflammatory and allergic reactions.
• Neutrophils, Eosinophil, and Basophils

7. Agranulocytes
Lack visible cytoplasmic granules
Lymphocytes and Monocyte

9.  Normal levels =4,000 to 11,000 cells/mm3
Abnormal leukocyte levels
 Leucocytosis -Above 11,000 leukocytes/ml
Generally indicates an infection / Leukemia
• Leucopenia
Abnormally low leukocyte level
Commonly caused by certain drug

10. Polymorphs = 40 - 75 %
Lymphocytes = 20 - 40 %
Monocytes = 2 - 10 %
Eosinophils = 1 - 6 %
Basophils = 0 - 1 %
Normal value of
DLC (Adult)

11. Granulocytes
Feature
 Polymorph / Neutrophils
12 to 15 µm in diameter
Nucleus stains deep Purple and has 2 - 5 lobes
Cytoplasm contains numerous pink granules.
 Eosinophils
15-16 µm in diameter
Bilobed spectacle shaped nucleus
Cytoplasm contains large coarse red granules.
 Basophils
9-12 µm in diameter . Nucleus is kidney shaped or bilobed
Cytoplasm contains coarse dark purple granules,
which usually obscure the nucleus.

12. Agranulocytes
 Lymphocytes: Mononuclear cells, which are of two types-
small and large.
 Small lymphocytes are 9 to 12 µm in diameter (about
equal or slightly larger than RBC). It contains single
rounded nucleus occupying most of the cell leaving a thin
rim of clear blue cytoplasm.
 Monocytes
It is the largest cell of the normal blood film, 14 to 20 µm in
diameter. which is lobulated, deeply indented or horseshoe
shaped

13. Preparation of Blood Film

Differential leukocyte count is performed on stained
blood film.
 Material required
 Glass slides
 Blood sample
 Leishman's stain
 Buffer water (pH = 6.8)
 Cedar wood oil
 Microscope with oil immersion objective

14. Glass slides should be clean and dry.
 Place a small drop of blood on one end of a clean slide, in the
middle. Hold a second slide (i.e., spreader) above the first, right
angle of 30 to 450.
 Move the spreader forward rapidly, thus spreading the blood
over the slide in a thin film.
 Leave the slide to air dry. A properly made film should dry
quickly; if the film is not dried rapidly there will be rouleaux
formation of RBC and the cells will appear shrunken.
 A good blood film should not cover the entire surface of the
slide. It should be even, smooth and should be having three
parts i.e., head, body and tail.
 Dried film should be stained with Leishman's stain.

15. Leishman's staining and DLC
 Pour the Leishman's stain over the film to cover it completely. Keep it for
two minutes.
 After one minute, buffer water is added gently to the stain already on the
blood film to cover the slide completely (it is approximately double the
volume of stain).
 Allow the diluted stain to act for about 8 to 10 minutes.
 Wash the film in buffer water or distilled water. The film should be rose
pink in colour.
 Allow the film dry and focus under low power of microscope. Then put a
drop of cedar wood oil and see under oil immersion objective.
 Count the WBCs in a zig-zag fashion across the breadth of film, in the body
and tail junction area of film, until 100 leukocytes are counted.
 To record them draw a large square and divide it into 100 small squares.
Identify one cell and write in each square. Count different WBCs coming
across. The result thus obtained is directly in percentage.

16. N
L
RESULT:
Polymorph % =
Lymphocyte % =
Eosinophil % =
Monocyte % =
Basophil % =

17. Normal Values
Absolute count:
40 to 440
cells/cumm
Differential count:
1 to 6%
EOSINOPHILIA

18. Physiological:
Diurnal variations are seen.
Parasitic infestations: due to allergic reaction to parasites
Allergic disorders: Bronchial asthma, Urticaria, Hay fever, Drug
hypersensitivity.
Drugs: Chlopromazine, Penicillin, and Streptomycin.
Skin diseases: Pemphigus, Dermatitis herpetiformis, Erythema
multiforme, Psoriasis, Eczema, Exfoliative Dermatitis, and Scabies.
Pulmonary diseases: Löeffler’s syndrome, and Tropical
eosinophilia.
Haematopoietic diseases: Chronic myeloid leukaemia,
Polycythaemia vera, Hodgkin’s disease.
Miscellaneous conditions: Rheumatoid arthritis, Polyarteritis
nodosa, Systemic Lupus Erythematosus (SLE), Ulcerative colitis,
and Irradiation.
Causes of Eosinophilia

19. Eosinopenia
 Decrease in the count of eosinophils is known as
eosinopenia.
 Causes:
 Uraemia
 After ACTH therapy
 After steroid therapy
 After stress caused by burn or postoperative conditions
 Few hours after ECT (electroconvulsive therapy)
 In some stages of acute infections.