Examination of blood

Prepared by: Mathew T. Moses

RHSI 2015

General concept of blood

• Blood consists of two parts:• Cellular part which consists of three types of

cells: RBCs, WBCs and platelets. (45%)• Fluid part which consists of plasma and serum.

(55%)• many diseases of the body are recognized by:1- changes in the appearance and numbers of bloodcells;2- changes in the composition of plasma; and3- identification of infectious organisms in cells ofplasma.

Cont. …

• The average adult has a blood volume of roughly 5 liters.

Cells: One microliter of blood contains:

• 4.7 to 6.1 million (male),

4.2 to 5.4 million (female) erythrocytes/RBCs.

• 4,000–11,000 leucocytes/WBCs

• 200,000–500,000 thrombocytes/platelets

Plasma

• The blood plasma volume totals of 2.7–3.0 liters in an average human.

Important components of plasma include:• Serum albumin• Blood-clotting factors (to facilitate coagulation)• Immunoglobulins (antibodies)• lipoprotein particles• Various other proteins• Various electrolytes (mainly sodium and chloride)Serum: refers to plasma from which the clotting proteins have been removed. Most of the proteins remaining are albumin and immunoglobulins.

Functions of blood• Supply of oxygen to tissues• Supply of nutrients such as glucose, amino acids, and fatty

acids• Removal of waste such as carbon dioxide, urea, and lactic acid• Immunological functions, including circulation of white blood

cells, and detection of foreign material by antibodies• Coagulation, the response to a broken blood vessel, the

conversion of blood from a liquid to a semi-solid gel to stopbleeding.

• Messenger functions, including the transport of hormonesand the signaling of tissue damage

• Regulation of body pH• Regulation of core body temperature• Hydraulic functions

Blood collection

Capillary blood:

• Principle: collect capillary blood from the sideof the finger in adults and children.

• Avoid the tip of the finger as it is very sensitive

• In Infants, collect capillary blood from the sideof the heel.

• Transfer the capillary blood directly onto cleanglass slides or into 20ul glass pipettes.

Requirements

• Sterile lancet

• Dry cotton wool

• Alcohol swab

• Clean glass slides of 20ul pipettes.

Procedures

1. Clean the skin thoroughly at the selected siteusing alcohol swab, allow the skin to dry.

2. Prick the skin firmly so that blood flows freelywithout excessive squeezing.

3. Wipe off the first drop with a plug of dry cottonwool. The first drop is contaminated with dirtand tissue fluid.

4. Squeeze the skin gently and collect the nextdrop on to a slide or into a pipette. With smallchildren it may be easier to fill the pipette froma large drop on a slide.

Cont. …

• Apply pressure to the finger prick with a plug of dry cotton wool.

• Immediately place the lancet in safety box.

• Place the cotton wool in the bucket marked ‘INCENIRATION’

Venous blood

• Principle: collect venous blood from a vein inthe crease of the elbow, with the arm out-stretched.

• In adults, if the is not possible; use a vein inthe forearm or back of the hand.

• In babies and small infants, collect blood froma vein in the back of the hand or ask theclinician to take a sample from scalp vein.

Requirements

• Dry cotton wool

• Alcohol swab

• Tourniquet or blood pressure cuff

• Sterilized dry syringe and needle (G 21 or G 23)

• EDTA or plain blood container as required

Procedures 1. Allow the patient to sit comfortably with one arm

resting on the bench or table. Fit the needle on to thesyringe.

2. Apply the tourniquet to the upper arm. Ask thepatient to open and close the fist several times.

3. Disinfect the skin over the vein using alcohol swab,allow the skin to dry.

4. With the bevel of the needle facing up, carefullyinsert the needle into the vein. Slowly draw out theplunger until the required amount of blood fills thesyringe. Release the tourniquet or blood pressure cuff,then withdraw the the needle from the vein andimmediately apply pressure on the puncture site withplug of dry cotton wool.

Cont. …

5. Detach the needle from the syringe andtransfer the blood sample into the correctspecimen bottle. Label each bottle with thepatient’s laboratory number using a greasepencil.

6. Immediately replace the needle on to thesyringe and place it in safety box.

7. Place used cotton wool in the bucket marked‘INCENIRATION’.

Preparation of blood films

• Use capillary or anti-coagulated venous blood.

• Do not use clotted blood for making bloodfilms because blood cells and parasites aretrapped in the clot.

• Do not use blood anti-coagulated withheparin for preparing thick and thin bloodfilms because heparin cause:

• Blue-background staining, clumping of WBCsand platelets, and poor staining of white cells.

Wet preparation

Principle: a wet preparation of blood is a drop offresh blood placed on a glass slide and coveredwith a coverslip. The coverslip spreads the bloodcells evenly.

Value:

• Detection of large motile parasite in bloodsuch as: microfilaria of Wuchereria bancrofti,Loa loa and Mansonella perstans

• Sickle cell screening.

Procedures:

• Clean a glass slide with gauze or dry cotton wool.• Collect a sample of capillary or anti-coagulated

venous blood.• When using capillary blood, carefully touch the

drop of blood on to the center of the slide,making sure the slide does not touch the kin.

• When using anti-coagulated venous blood, mixwell and place a small drop of blood on to thecenter of the slide using a clean Pasteur pipetteand rubber teat. Label the slide with the patient’slab No. using a grease pencil.

Cont. …

• For detection of motile parasites, put oncoverslip and examine without delay after youmix the drop of blood with normal saline.

• Report your finding.

Thick blood film

• Is a small drop of fresh blood which is spread in a circle on a glass slide and allow to dry. It consists of many layers of blood cells on top of each other. Thick blood film is used to detect:

• Plasmodium species (malaria parasites)

• Borrelia species

• Trypanosomes

• Microfilariae

Procedure

• Clean a glass slide with gauze or dry cottonwool.

• Collect a sample of capillary by touching thedrop of blood on to the center of the slidemaking sure the slide does not touch the skin,or anti-coagulated venous blood by mixingthoroughly and place a small drop of blood onto the center of the slide using a clean Pasteurpipette and rubber teat.

Cont. …

• Spread the blood in a circle approximately 1 cm indiameter using the corner of another slide. Thefilm is the correct thickness when newsprint orthe hands of a watch can be seen through it.Label the slide using a grease pencil.

• Air dry the film in a horizontal position. Do notdry the film by heating over a flame or placing ona hot subject.

• Label the slide with the patient’s lab number.• Stain using the Feld stain technique or Giemsa

stain technique.

Thin blood film

• Is a small drop of blood that is spread thinlyon a glass slide so that red cells do notoverlap.

A thin film is used for:

• Differential white cell count.

• Examination of blood cells morphology

• Clear identification of blood parasites.

Procedures

• Clean a glass slide with gauze soaked in solublemethanol. Allow to dry.

• Collect a sample of capillary or anti-coagulatedvenous blood.

• When using capillary blood, carefully touch thedrop of blood on to the slide from one end,making sure that the slide does not touch thepatient’s skin. When using anti-coagulatedvenous blood, mix thoroughly and place a smalldrop of blood on to the slide from one end, usinga clean Pasteur pipette and rubber teat. Place theslide horizontally on a flat surface.

Cont. …

• Hold a spreader (or coverslip) in the center ofthe slide at an angle of 500.

• Move the spreader back until it is in contactwith the blood, and allow the blood to spreadalong the base of the spreader.

• Now reduce the angle of the spreader toabout 300 and move the spreader steadilyacross the slide. A good thin film should end ina conical ‘tail’ and should cover 2/3 of the slide

Cont. …

• If the drop of the blood is too large, transfer asmall portion of blood by touching it with theedge of the spreader and start the film in a freshplace ahead of the large drop.

• If the blood is anemic, hold the spreader at agreater angle and move it faster. This makes aslightly thicker film.

• Dry the film quickly by waving it in the air. Slowdrying results in shrinkage of red cells. Do not drythe film by heating over a flame or placing on ahot object.

Cont. …

• Label the slide with patient’s lab number bywriting with lead pencil across the thickestpart of the film or on the frosted end of theslide.

• Fix the film by dipping for 2 seconds inabsolute methanol. Allow to dry.

• Stain using the reverse field stain technique orGiemsa stain

Quality controls on blood films

• A thick and thin blood film may be made onthe same slide.

• It useful when examining blood film formalaria parasites.

• The thick film is examined to establish thepresence of parasites, and the thin film is usedfor species identification

• Make the thick film at one end of the slide.

Cont. …

• Spread the thin film starting from the centerand moving towards the other end of theslide.

• Allow the film to dry

• Stain the thick film using the field staintechnique. Allow to dry.

• Fix the thin film in absolute methanol.

• Stain the film separately, using the reversefield stain technique or Giemsa stain.

Common problems encountered when making thin film are:

• The film is too thick and the end of the film islost. This occurs when the drop of blood is toolarge.

• The blood clots while the film is beingprepared. This occurs when capillary blood isleft too long on the slide before spreading.

• The film has an irregular tail. This occurs whena ragged spreader is used or there is unevencontact between spreader and slide

Cont. …

• The film is too thin. This occurs when the dropof the blood is too small and the film is spreadto slowly;

• The film does not adhere o the slide. Thisoccurs if the slide is greasy and and has notbeen adequately cleaned with alcohol