Quality control for B/F examination

1) Equipment must be clean

–Slides must be clean from dust, grease, soap,
fingerprints and debris
–Lancets must be sterile& puncture should be deep
enough
–Guaze rather than cotton should be used to clean the
finger
•Cotton may leave fibers that will get in to blood films
–The finger should be cleaned thoroughly before
puncturing
 Quality control cont’d…

2) Films must be the correct density

– Thin films
• Too thick_ RBCs pile-up in layers

• Too thin_ don’t stain well

–RBCs will be distorted

–Parasites will be distorted
 Quality control cont’d…
• Thick films
– It should be possible to read small
prints through thick films
– Too thick_
• blood may flake off during staining
– Too thin
• advantage of thick film may be lost
 Quality control cont’d…
3) Films must be allowed to dry in a horizontal position for
the correct time
– If thick films are slanted, the blood may run to one
edge of the slide
– The thick area may flake off
– While drying, films must be protected from dust,
insects etc…
– Thin films must be fixed
– The methanol must be moisture free
– Never fix thick films!!!
 Quality control cont’d…
4) Stain dilutions and buffers
– The bottle of stock Giemsa stain must be
kept tightly closed and out of sunlight.
– If moisture gets in to the stain it will be
ruined
– A portion of the stock must be poured in to
a clean, dry bottle for use
– Never put a wet pipette in to the bottle of
the stock stain!!!
 Quality control cont’d…
• Diluted Giemsa is good for about 8 hours
– Prepare fresh on the day they are required
– PH of the stain: 7.2 (use buffered water)
5) The staining procedure should be followed
carefully
– Adhere to SOP
Detection of trypanosomes in blood
• The wet blood film
– Easiest and least expensive
– Least sensitive
 Method
– Collect a drop of capillary blood on to a slide
– Add an equal drop of saline and mix it
– Cover the preparation with coverslip
– Examine systematically at 10x objective
• The first sign of the presence of live
trypanosmes or microfilariae is rapid mov’t
among RBCs
• Scan the entire preparation systematically
• Trypanosomes are refractile and difficult to
see
– Reduce the light
Microfilariae (10x)
Tryps(40x)
Detection of trypanosomes in blood

Capillary Tube Centrifugation (CTC)

• It is rapid and recommended for detecting motile
trypanosomes in blood.
• It also enables the packed cell volume (PCV) to be
measured to check whether the patient is anemic
 CTC con’d…
Method
1) Fill two heparinized capillary tubes with
capillary blood to about 10 mm from the top
– EDTA anticoagulated blood can also be used
2) Seal each capillary tube by rotating it in a
suitable sealant
3) Centrifuge the capillaries in a Microhaematocrit
centrifuge (12 000 g for 5min)
 CTC con’d…
4) Wipe clean the area of each capillary where it
will be viewed
5)Mount the two capillaries on a slide, supported
on two strips of plasticine or Blutak
– Using a cloth or tissue, gently press to
embed the capillaries in the plasticine.
6) Fill the space between the two capillaries with
clean water and cover with a cover glass.
7) Examine immediately the plasma just above
the Buffy coat layer for motile trypanosomes.
– Use a 20 objective or if unavailable use a 10
objective
Microfilariae concentration
techniques
Species
Wuchereria bancrofti
Collection time

Periodic, nocturnal 22.00–04.00 h

Peak 24.00 h
Brugia malayi
Periodic, nocturnal 22.00–04.00 h
Peak 24.00 h
Brugia timori
Nocturnal 22.00–04.00 h
Peak 24.00 h
Loa loa
Diurnal 10.00–15.00 h
Peak 13.00 h
Microfilariae concentration
techniques
a) Microhaematocrit tube technique
• Capillary blood is collected in two heparanized
capillary tubes.
• The blood is centrifuged in a microhaematocrit
centrifuge and the buffy coat examined
microscopically for motile microfilariae

18
 Microfilariae concentration cont’d…
b) Membrane filtration technique
– 10 ml of venous blood is collected into sodium
citrate anticoagulant
– the blood is then passed through a polycarbonate
membrane filter of 3µm or 5µm porosity.
– The microfilariae are retained and the membrane and
examined microscopically.
– This is the most sensitive method of detecting small
numbers of microfilariae

19
 Microfilariae concentration
cont’d…
c) Lyzed capillary blood technique
• Required

– Saponin-saline solution
– Methylene blue
– Field’s stain A ( to stain the nuclei and show
whether the microfilariae are sheathed)
• Method
– Collect 100µl (0.1 ml) of capillary blood and
dispense it into a conical tube containing 1 ml of
saponin-saline lyzing solution
– Mix the blood gently and leave it for about 2
minutes
– Centrifuge for 5 minutes at slow to medium
speed, (300–500 g)
– Using a pipette, remove and discard the
supernatant fluid.
– Transfer all the sediment to a slide, add a small
drop of methylene blue or Field’s stain A, and
cover with a cover glass.
Lyzed capillary con’d
– Examine the entire preparation
microscopically for motile microfilariae using
the 10 objective or preferably examine by
darkfield microscopy.
– Count the number of microfilariae in the
entire preparation.
– Multiply the number counted by 10 to give an
approximate number of microfilariae per ml
of blood (mf/ml).