Venepuncture is a routine procedure. In order to do this safely, the phlebotomist must have a basic understanding of the following; 1. Anatomy and physiology 2. The criteria for choosing a vein 3. The device to use 4. Skin preparation 5. Personal safety – infection control policy
The circulation is a closed sterile system and venepuncture, however quickly completed, is a breach of this system providing a method of entry for bacteria. Infection sustained at the venepuncture site can at its worst result in septicemia. The superficial veins of the upper limbs are most commonly used for venepuncture. If venepuncture is unsuccessful in these sites alternatives may be sought i.e. back of hand, but this may require a more experienced phlebotomist.
PHLEBOTOMY TRAY : Sterile gloves Syringes and needles Tourniquet Specimen containers . Bulbs or Vacutainer. Request form 70 % alcohol or 0.5% chlorhexidine Sterile gauze swabs Adhesive dressings Self sealing plastic bags Rack to hold the specimen containers.
Skin cleansing with an alcohol swab. Asepsis should be maintained. The two main sources of microbial contamination are: a) The hands of the phlebotomist b) The skin of the patient Good hand washing and drying techniques. If hand washing facilities are unavailable, an alcohol based hand wash solution is an acceptable substitute.
Protection for all personnel is paramount when handling blood products and body fluids. Universal Precautions to be followed: a) Every patient should be regarded as a potential biohazard b) Gloves MUST be worn. c) Avoid needle stick injury –Hepatitis B and HIV viruses transmitted in blood and body fluids d) Dispose of sharps and or soiled equipment appropriately and safely; keep gloves on whilst disposing of equipment, then dispose of gloves safely.
Each submitted specimen must be labeled with the patient’s name (written exactly as it appears on the Test Request Form) and the tests to be conducted. Use one Test Requisition form only Label each specimen with the patient’s name, and time of specimen collection Write the Total number of specimens submitted on the Test Requisition form.
Potassium EDTA (K2or K3 EDTA): For testsrequiring EDTAplasma, separateplasma appropriately.Use for CBC and bloodcultures Ethylenediamine tetraacetic acid (EDTA)
The interior of the tube wall is coated with sodium heparin, lithium heparin, or ammonium heparin The anticoagulant heparin activates antithrombins, thus blocking the coagulation cascade and producing a whole blood / plasma sample instead of clotted blood plus serum
Sodium Heparin: Preferred heparin tube for send out testing. It is glass and Does Not contain inert gel For plasma determinations in avian and reptiles
Lithium Heparin: Contains an inert gel for separating plasma, which acts as a barrier between cells and plasma after centrifugation.
Contains no anticoagulant.. Red No Gel tubes are available in "No Additive" clot tubes as well as "Clot Activator" tubes for serum collection. Use for serum determinations in chemistry, serology and Immunohematology (blood banking). TESTS -SEROLOGY
Contains clot activator and inert gel for separating serum, which acts as a barrier between cells and serum after centrifugation. During centrifugation the barrier gel moves upward to the serum - clot interface, where it forms a stable barrier separating the serum from fibrin and cells.
Approach the patient in a confident manner and explain the procedure Gather the necessary equipment Position the patient in a suitable place, request the patient to sit upright, although in those with a history of fainting it is best to position the patient lying on a bed or couch.
The arm should be supported, comfortable and relaxed Wash your hands Assemble the device Apply a tourniquet above the elbow, ensuring that it does not obstruct the arterial flow The veins may be tapped lightly
Select the vein Put gloves on Anchor the vein by applying manual traction to the skin just below the proposed insertion site. The tourniquet should be released once blood starts flowing into the syringe. Delay may lead to haemoconcentration as a result of stagnation.
Insert the needle through the skin at a plane parallel to the vein & care should be taken that the vein doesnt get counter punctured. The piston of the syringe should not be withdrawn fast as it may cause hemolysis. Sufficient amount of blood should be withdrawn and dispensed in appropriate bulbs. The bulbs should be shaken for a while so that the blood gets mixed with the anticoagulant properly.
* All Vacutainer should be single use only and disposed of with the needle after use.
Reasons to reject specimens for hematology are: A. Clotted specimen. B. Severely hemolyzed specimen. C. Improperly labeled or unlabeled specimen. D. Specimen too old. E. Failure to meet volume criteria.
F. Improperly collected (diluted) capillary specimen. G. Leaking tube. H. Delay in transport. I. Collection of specimen in wrong tube.
Immediate local complications : Haemoconcentration Collapse of the vein Failure of blood to enter the syringe . Needle not in position Immediate general complications: Syncope Late local complications : Thrombosis of the vein Thrombophlebitis Hematoma.
COLLECTION OF BLOOD FOR BIOCHEMICAL EXAMINATION: Fasting conditions are advisable Venous blood to be preferred. COLLECTION OF BLOOD FOR SEROLOGICAL EXAMINATIONS: 5 ml of blood is collected in a plain bulb & is allowed to clot at 37 deg for 1– 2 hours. Alternately if immediate investigations are needed, the blood is defibrinated & is centrifuged & serum is separated. E.g. ; Diagnosis of Syphilis, Enteric fever ( Widal Test ) , HIV , HBsAg determination. COLLECTION OF BLOOD FOR CULTURAL EXAMINATION: 5 - 10 cc of blood is collected in a 25 – 50 cc of Harley’s broth or Robertson’s cooked meat medium & incubated. E.g. : Bacterial endocarditis, Enteric Fever , Septicemias & pyaemias.
Agents which prevent the coagulation of blood are called as anticoagulants.
DIFFERENT TYPES OF ANTICOAGULANTS : EDTA (Ethylenediaminetetra Acetic acid) Trisodium Citrate Heparin Double oxalate Sodium fluoride
EDTA : It is the most commonly used anticoagulant in routine practice. The potassium & sodium salts of EDTA are powerful anticoagulants. Mechanism Of Action: It acts by chelating the calcium molecules in the blood. 1.2 mg of EDTA is required for each ml of blood to get the desired results. EDTA is used for mainly Blood counts.
COMPOSITION OF EDTA: Ethylenediamine tetra acetic acid,dipottasium salt – 100g. Water – 1 litre. Allow appropriate volume to dry in bottles at 20 deg to give concentration of 1.5 +/- 0.25 mg/ml of blood.
NEUTRAL EDTA : pH – 7.0 COMPOSITION : EDTA, dipotassium salt -44.5 g disodium salt – 41.0 g 1mmol NaOH-75 ml Water – 1 litre.
ADVANTAGES : It is the anticoagulant of choice in routine hematological work. Best for platelet counts. DISADVANTAGES: RBC morphology is hampered if the concentration is more than the required. Not good for coagulation studies.
Trisodium Citrate : It is used for coagulation studies. Mechanism of action : It also works on the principle of calcium chelation. 9 volumes of blood are added to 1 volume of 109 mmol/l sodium citrate for coagulation studies. For ESR estimation, 4 volumes of blood are added to 1 volume of sodium citrate solution & well mixed.
COMPOSITION OF TRISODIUM CITRATE: Dissolve 32 g in 1 litre of water. Distribute appropriate volumes into small bottles & sterilize by autoclaving at 121 deg for 15 min.
CITRATE PHOSPHATE DEXTROSE ( CPD ) : pH – 6.9 Trisodium citrate, dihydrate( 102 mmol / l) - 30 g Sodium dihydrogen phosphate, monohydrate ( 1.08 mmol / l ) - 0.15 g Dextrose ( 11 mmol / l ) -2g Water - 1 litre Sterilize the solution by autoclaving at 121 deg for 15 min After cooling to 20 deg, it should have a brown tinge & pH should be 6.9.
CITRATE PHOSPHATE DEXTROSE ADENINE : pH – 5.6 – 5.8 COMPOSITION : Trisodium citrate, dihydrate ( 89 mmol/l) – 26.30 g Citric acid , monohydrate ( 17 mmol/l) - 3.27 g Sodium dihydrogen phosphate, monohydrate ( 16 mmol/l) - 2.22 g Dextrose ( 177mmol/l) - 31.8 g Adenine ( 2.04 mmol/l) - 0.275g Water - 1 litre Sterilize the solution by autoclaving at 121 deg for 15 min For use, add 7 volumes of blood to 1 volume of solution.
ADVANTAGES : Trisodium citrate is used for coagulation studies CPD,ACD,CPDA & the related solutions are used in blood banking and blood transfusion purposes.
HEPARIN: It is used for chemistry , blood gas analysis & emergency tests. It is the best anticoagulant for osmotic fragility tests & immunophenotyping. The lithium or sodium salt of heparin at a concentration of 10- 20 IU / ml blood is used generally. It is not suitable for blood counts as it induces platelet & leucocytes clumping & gives faint blue color on PS.
SODIUM FLUORIDE : It prevents glycolysis It is the ideal additive used for blood sugar estimation.
DOUBLE OXALATE: It contains ammonium oxalate ( 6 parts) potassium oxalate ( 4 parts ) This combination minimizes the morphological changes that occur in the erythrocytes. Mechanism of action : Calcium chelation Routine hematological examinations & ESR determination.
A 20-22G needle is suitable for collection of venous blood. If thinner needle of 23 or 24G are used, there may be haemolysis of blood resulting in hemoglobinemia, rendering the specimen unfit for various hematological and biochemical investigation.
Prick should be deep enough for free flow of blood or gentle squeezing to start the flow of blood. Squeezing the finger tip hard results in tissue fluids diluting the blood , thus lowering the hematological values.
Initial drops of the blood gets contaminated with the antiseptic and therefore discarded.
Ulnar side of the tip of ring finger is comparatively less innervated and therefore prick is less painful.
Addition of liquid anticoagulant dilutes the blood altering the counts and different values, therefore ,powder form of EDTA salt is used
Oxalates induce morphological alterations in white and red cells and therefore smear morphology cannot be studied.
Plasma Serum1. Plasma is obtained by 1. Serum is obtained when centrifugation of the blood undergoes clotting. anticoagulant added blood2. It contains all clotting 2. It does not contain factors except calcium fibrinogen, prothrombin ions. F.V,VII,VIII,IX,X,XI and XII which have been used in clotting.3. It is used for coagulation 3. It is used for estimation of studies like PT ,APTT ,TT. various biochemical parameters and serum enzymes like SGOT, SGPT, S. alk phosphatase, S.uric acid etc.