Collection and Preservation of

Blood
Hematological Examination

 Capillary blood
 Venous blood
 Arterial blood
Capillary or Skin puncture

 Capillary blood : Blood from capillaries + venules + arterioles +

tissue fluid
 Used in children and infants , if small amount of blood is required.
1 Cell counts
2 Blood smear
3 Hb estimation
4 Microhematocrit
5 Blood grouping
Sites

 Adults: ring or middle finger- distal digit

and Ear lobule
 Infants: heel and great toe
Procedure

clean the site with 70% ethanol

Take a sterile disposable lancet

Puncture sufficiently deep to allow free flow of blood

First drop is wiped away with sterile dry cotton

precautions

 Donot collect from cold or cyanosed skin

 Because it will cause false increase in Hb and RBC and WBC
counts
Disadvantages

 HB, HCT and RBC count are higher than venous blood
 Platelet count lower
Venous Blood

 Prefered when multiple tests and larger quantity of blood required

 Anticoagulated blood required
 Sites:
1. Adults: Antecubital vein
2. Children: External jugular vein
Indications

 ESR, PCV estimation

 Blood constituents: sugars, urea etc….
 Bacteriological and serological tests
 Blood grouping and cross matching
procedure

Rubber tourniquet is applied to upper arm- 18x3/4 inch in

adults and 12x1/8inch in children

not too tight or not more than 2 min in place

patient is asked to make a fist so that veins become more

prominent and palpable
Cont…..

puncture site cleaned with 70% ethanol and allowed to dry

selected vein is anchored by compressing and pulling the soft tissue

below the puncture site

venepuncture is done with bevel of the needle up and along the

direction of vein
Cont…..

blood is withdrawn slowly and tourniquet should be released as

soon as blood begins to flow into the syringe

when required amount of blood is withdrawn patient is asked to open

fist, needle is withdrawn and sterile gauze is pressed over puncture site
 Needle is detached from syringe and required amount of blood is
carefully delivered into the tube containing appropriate
anticoagulant
 Check whether pt is feeling faint and bleeding has stopped
 Dispose the needle and syringe
 Container labelled
Precautions

 Never collect from IV line or arm used for IV line, sclerosed vein
,area with hematoma
 Tourniquet should not be too tight and not more than 2 min :
hemoconcentration
 It should be released before removing the needle : to prevent
hematoma formation
 Puncture site should be dry before procedure
Cont…

 To avoid hemolysis:
1. Blood is withdrawn gradually
2. Small bore needle should not be used
3. Needle is detached from syringe before dispensing blood into
container
 All samples are considered infectious and proper precautions taken
Complications

 Failure to obtain blood

 Occurrence of hematoma, thrombosis, thrombophlebitis, abscess,
bleeding
 Transmission of infections
 Anticoagulated blood should be tested within 1-2 hrs of collection
 If not possible, refrigerated at 4-6 c for max 24hrs
Arterial Blood

 Preferred when it is impossible to collect from veins

 Sites: brachial and radial artery
 Uses:
1. ABG
2. Subacute bacterial endocarditis: arterial positive for culture and
venous negative
Anticoagulants

 Calcium chelators:
1. Oxalates: potassium, ammonium, double oxalates
2. EDTA
3. Citrates: trisodium citrate , ACD ,CPA
 Heparin
Calcium chelators

 Calcium ions are essential for many steps in coagulation

 Ca. chelators act by binding to calcium, and form insoluble calcium
oxalate precipitate
Oxalates

 2mg per ml of blood

 Potassium oxalate:
1. uses: for chemical analysis
2. Disadvantages: shrinkage of RBC
3. Not recommended: PCV ,ESR, to study cell morphology
 Ammonium oxalate:
1. Disadvantages: swelling of RBC
2. Not recommended: PCV, ESR, Blood smears
Double /Balanced oxalate or Wintrobe’s
mixture

 3 parts of ammonium oxalate and 2 parts of potassium oxalate

 Advantage: decrease swelling effect of ammonium and shrinkage
effect of potassium oxalate
 Uses: HB, PCV ,ESR by wintrobe’s method, RBC AND WBC
counts
 Not recommended: WBC morphology- insoluble ca. oxalate ppt are
phagocytosed by neutrophils
EDTA

 Sodium EDTA : Versene

 Potassium EDTA: Sequestrene
 Conc: 1-2mg per ml of blood
 Most powerful ca. chelator
uses

 Gives best preservation of cell morphology

 Preferred for cell counts
 Smears can be made upto 3 hrs of sample collection
 Platelet clumping is inhibited
Disadvantages

 Expensive than oxalates

 Smears fail to demonstrate basophilic stippling of red cells
Effects of Excess EDTA

 RBC and leukocytes: shrinkage and degenerative

changes
 Hematocrit: decrease
 MCH: increase
 Platelet: swelling and disintegration gives artificially high
counts
 >2mg/ml of blood: decrease PCV and increase MCHC
Trisodium citrate/ Dacie’s solution

 Uses:
1. Westergren’s method for ESR estimation: 1:4
2. Coagulation studies: 1:9
3. Platelet studies
 ACD – Acid citrate Dextrose
 CPD: Citrate Phosphate Dextrose
 CPD-A: Adenine
Heparin
uses

 Osmotic fragility test

 Immunophenotyping
Disadvantages

 Expensive
 Highly acidic: bluish color to background when stained with
romanowsky stains
 Causes platelet and leukocyte clumping
 Inhibits enzyme activity so not used for PCR