BlOOD CONCENTRATION

METHODS

CHOYTOO Shiksha

Contents

▫ Microfilaria count

▫ Microhaematocrit centrifugation

▫ Triple centrifugation

▫ Buffy coat concentration

▫ Knot concentration

▫ Membrane Filtration

▫ Gradient centrifugation

▫ Malarial Parasite – QBC Method

▫ References

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

Microfilaria count

• Themicrofilaria 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.

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

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

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

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.

• Uses:

▫ This method is used to detect trypanosomes in peripheral smears when they are scanty

Trypanosomes

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

• Uses:

▫ For detection of

▫ Lieshmania Donovani

▫ Trypanosome

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

• Use:

▫ It is primarily used to detect microfilariae in blood, especially when a light infection is suspected

▫ Disadvantage of this method is that microfilariaeare killed by the formalin and are therefore not seen as motile organisms.

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 Teepolin 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

• 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

• 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.

PARASITES

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)

• Uses:

▫ The middle layer is used for detection of microfilariae

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

• 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

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

References

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