COLLECTION OF SPECIMEN AND ANTICOAGULANTS

-Dr Dhiraj B Shukla

The procedure in which an operator bleeds a specific amount of blood of the subject for a particular investigation can be termed as Collection of blood.

Common examinations done on blood are..??

1.Hematological 2.Biochemical3.Serological4.Cultural

Samples can be collected from:

1. Veins : Most commonly used.

2.Capillaries.

3.Arteries in case of arterial blood gas analysis and sometime in pediatric patients.

Venepuncture is a routine procedure. In order to do this safely, the phlebotomist must have a basic understanding of the following;

1. Anatomy and physiology2. The criteria for choosing a vein3. The device to use4. Skin preparation5. Personal safety – infection control policy

ANATOMY AND PHYSIOLOGY

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.

The Device

PHLEBOTOMY TRAY :

Sterile glovesSyringes and needles Tourniquet Specimen containers . Bulbs or Vacutainer.Request form70 % alcohol or 0.5% chlorhexidineSterile gauze swabs Adhesive dressingsSelf sealing plastic bagsRack to hold the specimen containers.

Skin Preparation

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 patientGood hand washing and drying techniques. If

hand washing facilities are unavailable, an alcohol based hand wash solution is an acceptable substitute.

Personal Safety

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.

Proper Labeling of the Specimens

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 onlyLabel each specimen with the patient’s name,

and time of specimen collectionWrite the Total number of specimens submitted

on the Test Requisition form.

Specimen Type

Lavender topPotassium EDTA (K2 or K3 EDTA): For tests requiring EDTA plasma, separate plasma appropriately.Use for CBC and blood cultures

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

Green tops

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

Green top

Lithium Heparin: Contains an inert gel for separating plasma, which acts as a barrier between cells and plasma after centrifugation.

Lt. Green top

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

Red top

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.

Red-Gray (tiger)

Procedure for Phlebotomy

Approach the patient in a confident manner and explain the procedure

Gather the necessary equipmentPosition 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 handsAssemble the deviceApply a tourniquet above the elbow,

ensuring that it does not obstruct the arterial flow

The veins may be tapped lightly

Select the veinPut gloves onAnchor 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 doesn't 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.

COMPLICATIONS

Immediate local complications : HaemoconcentrationCollapse of the veinFailure of blood to enter the syringe .Needle not in position

Immediate general complications:Syncope

Late local complications :Thrombosis of the vein ThrombophlebitisHematoma.

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.

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 mlWater – 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 gSodium dihydrogen phosphate, monohydrate ( 1.08 mmol / l ) - 0.15 gDextrose ( 11 mmol / l ) - 2 g 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

gCitric acid , monohydrate ( 17 mmol/l) - 3.27

gSodium dihydrogen phosphate, monohydrate ( 16 mmol/l) -

2.22 gDextrose ( 177mmol/l) - 31.8 gAdenine ( 2.04 mmol/l) - 0.275gWater - 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.

What gauge needle should be used for collection of venous blood?

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.

Why the finger should not be squeezed while collecting blood by finger prick method?

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.

Why initial 1-2 drops of blood are discarded in finger prick method?

Initial drops of the blood gets contaminated with the antiseptic and therefore discarded.

Which is the preferred anticoagulant for haemogram?

EDTA- potassium salt.

Why is ring finger preferred for capillary blood collection?

Ulnar side of the tip of ring finger is comparatively less innervated and therefore prick is less painful.

Why is liquid anticoagulant not used for haemogram?

Addition of liquid anticoagulant dilutes the blood altering the counts and different values, therefore ,powder form of EDTA salt is used

Why are double oxalates no longer used as anticoagulant for haemogram?

Oxalates induce morphological alterations in white and red cells and therefore smear morphology cannot be studied.

What are the differences between plasma and serum?

Plasma1. Plasma is obtained by

centrifugation of the anticoagulant added blood

2. It contains all clotting factors except calcium ions.

3. It is used for coagulation studies like PT ,APTT ,TT.

Serum 1. Serum is obtained when

blood undergoes clotting.

2. It does not contain fibrinogen, prothrombin F.V,VII,VIII,IX,X,XI and XII which have been used in clotting.

3. It is used for estimation of various biochemical parameters and serum enzymes like SGOT, SGPT, S. alk phosphatase, S.uric acid etc.

THANK YOU….