SYLLABUS BASED NOTES FOR THE POST OF JUNIOR LABORATORY ASSISTANT IN MEDICAL EDUCATION PART 1 BLOOD AND PHLEBOTOMY (Module 2)

 MODULE II- 13 Marks 

BLOOD AND PHLEBOTOMY - 10 Marks

• Methods of blood collection – 

o Capillary Puncture

o Arterial puncture and venous puncture

o Vacutainer – colour codes for vials

• Sites of blood collection: Capillary and Venous.

✅ Anticoagulants used – advantages and disadvantages

• Identification of blood cells: Leishman's staining procedure & identification

• Blood cell types: RBC, WBC, Platelets (Identifying characters and Normal Range )

ANTICOAGULANTS

INTRODUCTION 

• Anticoagulants are chemical substances which can prevent the clotting or coagulation of blood either invivo (drugs) or invitro. 

• Coagulation will instantaneously occur once a blood vessel has been injured. 

•Blood begins to solidify to prevent excessive blood loss and to prevent invasive substances from entering the blood stream

CALCIUM CHELATION 

• Calcium is required for the blood to clot, so when it is removed blood will not clot 

• Binding calcium as an insoluble salt which precipitates or converting it to a soluble non ionized form

EDTA 

• Ethylene Di Amine Tetra Acetic Acid. 

• Anticoagulant of choice in hematology.

• Most powerful Calcium Chelating agent 

• Concentration used: 1-2mg/ml of blood.

• Prepare 2g/100ml 0.1 ml solution in clean dry containers. 

• Function: prevents coagulation by binding calcium and converting it to a non- ionized soluble complex.(calcium chelation)

Different forms of EDTA 

1. Di-sodium EDTA salt (Versene) 

2. Di-potassium EDTA salt (Sequestrene)→ recommended by ICSH (International Society for Standardisation in Hematology) 

3. Committee for Clinical Laboratory Standards) Tri-Potassium EDTA → liquid form recommended by NCCLS (National 

4. Di-Lithium EDTA → same sample can be used for chemical investigation 

5. Tri sodium EDTA → not recommended because of its high pH

ADVANTAGES 

• Best preservation of cell morphology (smear can be made up to 3 hours after collecting the blood) and prevents clumping of platelets. 

DISADVANTAGES

 • Pseudo-thrombocytopenia due to EDTA dependent platelet aggregation. 

• More expensive than oxalates, but outweigherd by the advantages Not suitable for coagulation studies

USES 

Cell counts→ RBC count, total leucocyte count, eosinophil count, platelet count

 • Hemoglobin estimation

 • HbF estimation 

• Hb Electrophoresis

OXALATES 

Combines with calcium to form insoluble calcium oxalate which is precipitated. 

POTASSIUM OXALATE

 • Concentration used: 2 mg/ml of blood. 

• Used in chemical analysis 

• PCV, ESR & Blood Smear Preparation → not used → Shrinkage of RBC 

AMMONIUM OXALATE 

• Concentration used: 2 mg/ml of blood. 

• PCV, ESR & Blood Smear Preparation → not used → Swelling of RBC

DOUBLE OXALATE/WINTROBE'S MIXTURE /BALANCED OXALATE 

To balance the swelling effect of ammonium oxalate and shrinking effect of potassium 

oxalate Ammonium Oxalate: Potassium Oxalate = 3:2 (1.2gm :0.8gm-100ml DW)→ 20 mg/ml → 0.1 ml to clean dry containers 0.1 ml 2 mg of oxalate 

• Concentration used: 2 mg/ml of blood

 • Used for Hb, PCV, ESR 

• Heller and Paul's mixture

CITRATE

 • Not used for blood cell counts, Hb estimation as it causes dilution of blood →wet anticoagulant. →Binds with calcium to form non-ionised soluble calcium citrate 

a. TRISODIUM CITRATE 

3.8% (3.8gm/dl) Solution → ESR by Westergren's Method → 

sodium citrate: blood =1:4 (0.4ml citrate: 1.6ml blood) 3.2% solution →→ Coagulation studies→ 

sodium citrate: blood = 1:9 (0.5ml: 4.5ml) 

b. ACD SOLUTION (CPD,CPD-A) 

Used in blood banking. 

Citric Acid, Tri-sodium Citrate and Dextrose

HEPARIN

 • Natural biological anticoagulant.

 • Normally present in the body in small quantitites

 • Lithium or sodium salts

 • Antithrombin III in action → inhibits the formation of thrombin from Prothrombin.

 • Concentration → 0.1-0.2 mg/ml of blood. 

•Instantaneous anti-coaglulation. → best anticoagulant for opern heart surgery

DISADVANTAGES 

• Expensive

• Not used for cell counts → clumping of platelets and leukocytes 

• Not used for smear preparations → imparts bluish discoloration to the background of peripheral smears. (acidic nature) 

• Prevents coagulation only for a limited period of time until heparin is metabolized/neutralized.

 USES 

• Osmotic fragility test 

• Red cell enzyme studies → G6PD and PK deficiency Blood gas analysis 

•Electrolyte estimation 

• karyotyping

Sodium Fluoride 

• It complexes with calcium to form calcium fluoride. 

• Required in large quantities.

• Used in blood sugar estimation → 

potassium oxalate : Sodium fluoride =3:1 (FLOURIDE-OXALATE MIXTURE)

• Enzyme poison → prevents glycolysis by inhibiting enolase 

• Concentration used → 2 mg/ml of blood