BLOOD FILMS NORMAL

and ABNORMAL
Dr N.O.Ogidi
What is a Blood Film
• Blood film or peripheral blood smear is a thin layer of blood smeared
on a slide and then stained in such a way to allow the various blood
cells to be examined microscopically. A blood smear is a blood test
that gives information about the number and shape of blood cells.
The cell types are examined under a microscope to
investigate hematological problems (disorders of the blood)
Applications of Blood Film
• To detect infection or inflammation, determine the effects of possible
poisoning by chemicals, drugs, chemotherapy, radiation, etc.
• To monitor blood diseases like leukemia, anaemia or to detect allergic
and parasitic infections.
Background
• Blood film focuses on morphology of

red cells: (which carry oxygen throughout the body)

white cells: (which function as part of the body’s immune system)

Platelets: (which are important for blood clotting).

How to make a blood film
Blood Film preparation
• Wedge technique
• Coverslip technique
• Automated Slide Making
and Staining
Wedge technique
1. Easiest to master
2. Most convenient and most commonly
used technique
Material needed
3. Glass slide 3 in X 1in
4. Beveled/chamfered edges
Procedure for Blood Film Making
• Drop 2-3 mm blood at one end of the slide
Precaution: Too large drop = too thick
smear
Too small drop = too thin
smear
2. The pusher slide be held securely with the dominant hand in a 30-
45 deg angle.
- quick, swift and smooth gliding motion to the other side of the
slide creating a wedge smear
Blood Film Making
Wedge Technique
1. Push Type wedge preparation
2. Pull Type wedge preparation
Precautions:
Ensure that the whole drop of blood is picked up and spread
Too slow a slide push will accentuate poor leukocyte distribution,
larger cells are pushed at the end of the slide
Maintain an even gentle pressure on the slide
Keep the same angle all the way to the end of the smear.
Blood Film Making
Precautions:
Angle correction:
1. In case of Polycythemia: high Hct - angle should be lowered
- ensure that the smear made is not too thick
2. Too low Hct: Angle should be raised
Characteristics of a well made blood
film
• Smear is 2/3 or ¾ the entire slide
• Smear is finger shaped, very slightly rounded at the feathery edge:
widest area of examination
• Lateral edges of the smear visible
• Smear is smooth without irregularities, holes or streaks
• When held up in light: feathery edge should show rainbow
appearance
• Entire whole drop of blood is picked up and spread
Blood Film making (Coverslip
technique)
• Cover Slip Technique
rarely used
used for Bone marrow aspirate smears
Advantage: excellent leukocyte distribution
Procedure
• 22 x 27mm clean coverslip
• More routinely used for bone marrow aspirate
• Technique:
1. A drop of marrow aspirate is placed on top of 1 coverslip
2. Another coverslip is placed over the other allowing the aspirate
to spread.
3. One is pulled over the other to create 1 thin smears
Procedure
4. Mounted on a 3x1 inch glass slide
Precautions:
• Very lgiht pressure should be applied between the index finger and
the thumb
• Crush preparation technique
• Too much pressure causes rupture of the cells making morphologic
examination impossible
• Too little pressure prevents the bone spicules from spreading
satisfactorily on the slide
Automated Film Making and Staining
• Automatic Slide Making and Staining
• SYSMEX 1000i
• AUTOMATED SLIDE STAINERS
1. It takes about 5-10 minutes to stain a batch of smears
2. Slides are just automatically dipped in the stain in the buffer and a
series of rinses
• Disadvantages:
1. Staining process has begun, no STAT slides can be added in the batch
2. Aqueous solutions of stains are stable only after 3-6 hours
Staining Blood and Bone Marrow
Films
Romanowsky Stain
• Eosin + Methylene Blue = thiazine eosinate complex
• The complex will not stain any color unless a buffer is added: 0.05M
sodium phosphate (pH 6.4) and aged distilled water (pH 6.4-6.8)
• Methanol is added to fix the cells on the slide
Romanowsky Stain
• Free Methylene Blue:
- basic
- stains acidic cellular components such as RNA
• Free Eosin
- acidic
- stains basic cellular components such as Hgb and eosinophilic
granules
Problems Encountered During
Staining
• Water artifact: moth eaten RBC, heavily demarcated central pallor on
the RBC surface, crenation, refractory shiny blotches on the RBC
• What contributes to the problem:
1. humidity in the air as you air dry the slides.
2. Water absorbed from the humid air into the alcohol based stain
• Solution:
1. Drying the slide as quickly as possible.
2. Fix with pure anhydrous methanol before staining.
3. Use of 20% v/v methanol
Characteristics of a Well Stained
Film
• Macroscopically: color should be pink to purple
• Microscopically:
RCS: orange to salmon pink
WBC: nuclei is purple to blue
cytoplasm is pink to tan
granules is lilac to violet
Eosinophil: granules orange
Basophil: granules dark blue to black
Characteristics of a Well Stained
Film
• Troubleshooting:
1. RBC gray, WBC too dark Eosinophil granules are gray
Cause: stain or buffer is to alkaline
inadequate rinsing
Prolonged staining
heparinized sample
Characteristics of a Well Stained
Film
• Troubleshooting:
2. RBC too pale, WBC barely visible
Causes: Stain or buffer is too acidic
Underbuffering
Over rinsing
Blood Film Examination
Blood Film Examination
• Macroscopic
1. Overall bluer color: increased blood proteins (multiple myeloma,
rouleaux formation)
2. Grainy appearance: RBC agglutination (cold hemagglutinin
diseases)
3. Holes: increased lipid
4. Blue specks at the feathery edge: Increased WBC and Platelet
counts
Blood Film Examination
Blood Film Examination
• Microscopic:
• 10x Objective
1. Assess overall quality of the smear i.e feathery edge, quality of the color,
distributin of the cells and the lateral edges can be checked for WBC
distribution
2. Snow-plow effect: more than 4x/cells per field on the feathery edge: Reject
3. Fibrin strands: Reject
4. Rouleaux formation, large blast cell assessment
Blood Film Examination
• Microscopic:
• 40x Objective
1. Correct area where to star counting is determined
2. WBC estimate: internal quality control
Blood Film Examination
• Microscopic:
• 100x Objective; OIO
1. Highest magnification
2. WBC differential counting
3. RBC Analysis
4. Platelet Analysis
Blood Film Examination
Blood Film from a healthy adult
Blood Film Examination
Blood Film Examination
Blood Film Examination
Identifying a Leucocyte
• A leukocyte is identified from its size, its nucleus, and the cytoplasm—
its color, whether vesicles (granules) are visible or not, their color and
size if visible, and the cytoplasm/nucleus ratio.
 Identifying a Leucocyte

Lymphocytes
Monocytes
Differential Count on Blood Film
• Count each WBC seen and record on a differential cell counter until 100
WBCs have been counted. For instance if 25 of the 100 WBCs where
lymphocytes, then the percentage of lymphocytes is 25%.
• The normal range percentage of the different types of WBCs is as
follows:
Neutrophils 45-70%
Eosinophils 1-4%
Basophils 0.4%
Monocytes 2-8%
Lymphocytes 20-40%