Blood smear examination

Preparation of blood smear There are three types of blood smears: The cover glass smear. The wedge smear . The spun smear. The are two additional types of blood smear used for specific purposes Buffy coat smear for WBCs < 1.0×109/L Thick blood smears for blood parasites .

WEDGE BLOOD SMEAR Specimen : EDTA blood within 2 to 3 hours & collected to the mark on tube. Not's : May change RBCs morphology such as crenated cells if : Excessive amount of anticoagulant to specimen Old blood: long standing. Warm environment (room temperature) may hasten changes.

Procedure placing a drop of blood from mixed sample on a clean glass slide. Spreader slide using another clean glass slide at 30- 40 degree angle. Control thickness of the smear by changing the angle of spreader slide Allow the blood film to air-dry completely before staining. (Do not blow to dry, The moisture from your breath will cause RBC artifact).

large angle low HCT small angle high HCT

CHARACTERISTICS OF A GOOD SMEAR Thick at one end, thinning out to a smooth rounded feather edge. Should occupy 2/3 of the total slide area. Should not touch any edge of the slide. Should be margin free, except for point of application.

tail body head

Common causes of a poor blood smear Drop of blood too large or too small. Spreader slide pushed across the slide in a jerky manner. Failure to keep the entire edge of the spreader slide against the slide while making the smear. Failure to keep the spreader slide at a 30° angle with the slide. Failure to push the spreader slide completely across the slide. Irregular spread with ridges and long tail: Edge of spreader dirty or chipped; dusty slide Holes in film: Slide contaminated with fat or grease Cellular degenerative changes: delay in fixing, inadequate fixing time or methanol contaminated with water.

Slide fixation and staining

Romanowsky staining Leishman's stain : a polychromatic stain Methanol : fixes cells to slide Methylene blue stains RNA, DNA Blue-grey color Eosin stains hemoglobin, eosin granules Orange-red color PH value of phosphate buffer is very important

Procedure Thin smear are air dried. Flood the smear with stain. Stain for 1-5 min. Experience will indicate the optimum time. Add an equal amount of buffer solution and mix the stain by blowing an eddy in the fluid. Leave the mixture on the slide for 10-15 min. Wash off by running water directly to the centre of the slide to prevent a residue of precipitated stain. Stand slide on end, and let dry in air.

Prolonged buffering or washing Old stain CAUSES & CORRECTION Too Acid Stain: Insufficient staining time Prolonged buffering or washing Old stain Correction: Lengthen staining time Check stain and buffer pH Shorten buffering or wash time

Alkaline pH of stain components Too Alkaline Stain: Thick blood smear Prolonged staining Insufficient washing Alkaline pH of stain components Correction : Check pH Shorten stain time Prolong buffering time

Manual differential

Principle White Blood Cells Perform the differential count. Check for even distribution and estimate the number present. Perform the differential count. Examine for morphologic abnormalities.

Check the WBC distribution over the smear. Observations Under 10X Check to see if there are good counting areas available free of ragged edges and cell clumps. Check the WBC distribution over the smear. Check that the slide is properly stained. Check for the presence of large platelets, platelet clumps, and fibrin strands.

Observing direction: Observe one field and record the number of WBC according to the different type then turn to another field in the snake-liked direction *avoid repeat or miss some cells

WBC estimation Under 40X Using the × 40 high dry with no oil. Choose a portion of the peripheral smear where there is only slight overlapping of the RBCs. Count 10 fields, take the total number of white cells and divide by 10. To do a WBC estimate by taking the average number of white cells and multiplying by 2000.

Reference values vary depending on age 14 yr Total WBC x 103 /µL 4.5-11.0 Cell Type 50-65 Neutrophils % 30-40 Lymphocyte % 0-10 Monocyte % 0-4 Eosinophil % 0-1 Basophil %

Morphology of WBC

Normal blood smear

Stab neurophile Diameter:12-16 Cytoplasm : pink Granules: primary, secondary Nucleus: dark purple blue Dense chromatin

Segmented neurophile Diameter: 12-16 Cytoplasm : pink Granules: primary, secondary Nucleus: dark purple blue Dense chromatin 2-5 lobes

SEGMENTED NEUTROPHIL

Basophils Diameter: 14-16 Cytoplasm : full of granules Granules: large refractile, orange-red Nucleus: blue Dense chromatin 2 lobes like a pair of glass

EOSINOPHIL

Eosinophils Diameter: 14-16 Cytoplasm : pink Granules: dark blue –black obscure nucleus Nucleus: blue

BASOPHIL

Lymphocyte Diameter: small 7-9 large 12- 16 Cytoplasm: medium blue Granules: small agranular large a few Nucleus: dark blue \round dense chromatin

LYMPHOCYTE

Monocytes Diameter: 14-20 Cytoplasm : grey blue Granules: dust-like lilac color granules Nucleus: blue large irregularly shaped and folded

MONOCYTE

LEFT-SHIFT AND RIGHT-SHIFT OF NEUTROPHIL: Left-shift: non-segmented neutrophil > 5% (Increased bands Means acute infection, usually bacterial). Right-shift: hypersegmented neutrophil >3% (Increased hypersegmented neutrophile )

Leukocytosis, a WBC above 10,000, is usually due to an increase in one of the five types of white blood cells Neutrophilic leukocytosis neutrophilia Lymphocytic leukocytosis lymphocytosis Eosinophilic leukocytosis eosinophilia Monocytic leukocytosis monocytosis Basophilic leukocytosis basophilia