5.  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

7.  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.

10.  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.

22.  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.

24.  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.

26.  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.

29. Potassium EDTA (K2
or K3 EDTA): For tests
requiring EDTA
plasma, separate
plasma appropriately.
Use for CBC and blood
cultures
Ethylenediamine tetraacetic acid (EDTA)

30.  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

31.  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

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

33.  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

34.  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.

36.  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.

37.  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

38.  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.

39.  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.

42. * All Vacutainer should be single use only and
disposed of with the needle after use.

43. 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.

44.  F. Improperly collected (diluted) capillary
specimen.
 G. Leaking tube.
 H. Delay in transport.
 I. Collection of specimen in wrong tube.

46.  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.

47.  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.

49. Agents which prevent the coagulation of blood are
called as anticoagulants.

50.  DIFFERENT TYPES OF ANTICOAGULANTS :
 EDTA (Ethylenediaminetetra Acetic acid)
 Trisodium Citrate
 Heparin
 Double oxalate
 Sodium fluoride

51.  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.

52.  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.

53.  NEUTRAL EDTA :
 pH – 7.0
 COMPOSITION :
 EDTA, dipotassium salt -44.5 g
disodium salt – 41.0 g
 1mmol NaOH-75 ml
 Water – 1 litre.

54.  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.

55.  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.

56.  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.

57.  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.

58.  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.


59.  ADVANTAGES :
 Trisodium citrate is used for coagulation studies
 CPD,ACD,CPDA & the related solutions are used in
blood banking and blood transfusion purposes.

62.  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.

63.  SODIUM FLUORIDE :
 It prevents glycolysis
 It is the ideal additive used for blood sugar
estimation.

64.  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.

66.  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.

68.  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.

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

72.  EDTA- potassium salt.

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

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

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

80.  Plasma  Serum
1. Plasma is obtained by 1. Serum is obtained when
centrifugation of the blood undergoes clotting.
anticoagulant added blood
2. 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.