Blood concentration Methods Part Of peripheral Blood Film (PBF) Use to detect parasites

1. BlOOD CONCENTRATION
METHODS
CHOYTOO Shiksha

2. Contents
▫ Microfilaria count
▫ Microhaematocrit centrifugation
▫ Triple centrifugation
▫ Buffy coat concentration
▫ Knot concentration
▫ Membrane Filtration
▫ Gradient centrifugation
▫ Malarial Parasite – QBC Method
▫ References

3. Note:
• The thin and thick blood films for detection of
parasites are routinely done
• However, if the parasites are not seen in these
blood films, the concentration methods are used

4. Microfilaria count
• The microfilaria is an early stage in the life
cycle of certain parasitic nematodes.
• Microfilaria count : with the help of a
haemoglobinometer pipette, 29 mm3 of blood is
placed in a clean glass, dried as thick film,
dehaemoglobinised and stained in the usual
manner.

5. • The total number of microfilariae in the thick
smear multiplied by 50 gives the number per ml
of blood

6. Blood Concentration Method
• Concentration methods are used to detect
haemoparasites
• Some of the methods are
▫ Microhaematocrit centrifugation
▫ Triple centrifugation
▫ Buffy coat concentration
▫ Knot concentration
▫ Membrane Filtration
▫ Gradient centrifugation

7. Microhaematocrit Centrifugation
• Blood is collected in a haematocrit tube up to
2/3rd of its volume
• End of the tube is sealed
• Centrifuge at 1500g for 7 minutes
• The RBC-plasma is examine under oil-
immersion lens
• Examination of malarial parasite and
trypanosomes

9. Triple Centrifugation
• Brief description:
▫ 9ml of blood is mixed with 1ml of 6%sodium
citrate
▫ Centrifuge for 10 min at 100g
▫ The supernatant* is collected and is centrifuge
again at 700g for 10 min
▫ Sedimentation is examined under wet film or
stained smear
*
denoting the liquid lying above a solid residue after crystallization,
precipitation, centrifugation, or other process.

10. • Uses:
▫ This method is used to detect trypanosomes in
peripheral smears when they are scanty
Trypanosomes

11. Buffy Coat Concentration
• Brief description
▫ 5ml of citrated or oxalated blood is centrifuged in
a tube
▫ Buffy coat present between the plasma and packed
red cells is collected and stained

12. • Uses:
▫ For detection of
▫ Lieshmania Donovani
▫ Trypanosome

13. Knott Concentration
• Brief Description:
▫ 2ml of blood is thoroughly mixed with 10 ml of 2%
solution of formalin
▫ Mixture is allowed to stand for 10 min or longer
▫ Then centrifuged at 200g for 2min

14. • Use:
▫ It is primarily used to detect microfilariae in
blood, especially when a light infection is
suspected
▫ Disadvantage of this method is that microfilariae
are killed by the formalin and are therefore not
seen as motile organisms.

15. Membrane filtration
• 1ml of venous blood is drawn into a 10 ml of syringe
containing 0.1 ml of a 5% solution of sodium citrate
• In the same syringe, 9ml of 10% solution of Teepol
in physiological saline is drawn and shaken gently
for 1 min
• Needle is removed and attached to a Swiney filter
holder containing a 25 mm membrane filter of 5um
porosity placed over a filter paper pad of the same
size and moisten with saline

16. • With gentle and steady pressure the blood is
forced through the filter
• The filter is washed three times by passing 10 ml
of physiological saline
• Filter is removed and stained for 5 min in hot
haematoxilin
• It is then briefly “blued” in running tap water

17. • It is dried, covered with mounting medium and
coverslip, and examined under a microscope
• This technique has been proved highly efficient
in demonstrating filarial infections when
microfilaremia are of low density
• It has also been successfully used in field
surveys.

18. PARASITES

19. Gradient Centrifugation
• 5ml of Ficoll-Hypaque solution is mixed with an
equal volume of heparinised blood.
• This is centrifuged at 150g for 40 min
• Shows three layer:
▫ White cell layer (bottom)
▫ Ficoll-Hypaque layer (middle)
▫ Plasma layer (top)

21. • Uses:
▫ The middle layer is used for detection of
microfilariae

22. Malarial Parasite – QBC Method
(quantitative buffy coat method)
• new method for identifying the malarial parasite
in the peripheral blood
• involves staining of the centrifuged and
compressed red cell layer with acridine orange
and its examination under UV light source

23. • QBC test tube (hematocrit tube)- pre-coated
internally with acridine orange stain and potassium
oxalate
• is filled with 55-56 ml of blood
• centrifuged at 12,000 rpm for 5 minutes
• components of the buffy coat separate according to
their densities, forming discrete bands
• The QBC tube is placed on the tube holder and
examined using a standard white light microscope
equipped with the UV microscope adapter

24. • Fluorescing parasites are then observed at the
red blood cell/white blood cell interface.

26. References

27. THANK YOU