This document discusses methods of blood collection and anticoagulants. It covers precollection variables that can affect test results, various tests affected by factors like diurnal variation and posture. It then discusses blood collection sites like venous, capillary and arterial. The proper techniques, equipment and safety procedures for venipuncture and arterial blood collection are explained. Finally, it covers anticoagulants, their uses in preventing coagulation during storage and transportation of blood samples, and the coagulation pathways they act on.

1. METHODS OF BLOOD COLLECTION AND
ANTICOAGULANTS
MODERATOR – Dr MAHANTHACHAR
PRESENTED BY - Dr SHREYA PRABHU
1

2. BLOOD COLLECTION
2

3. PRECOLLECTION VARIABLES
 In preparing patient for phlebotomy,care should be taken to minimize physiologic
factors related to activities that might influence laboratory determinations.
 These include-
 Diurnal variation
 Exercise
 Diet
 Stress
 Posture
 Age
 Gender
 Personal Habits
3

4. TESTS AFFECTED BY DIURNAL VARIATION,
POSTURE, STRESS
CORTISOL Peaks at 4-6 AM, lowest at 8 PM-12 AM
ADRENOCORTICOTROPIC HORMONE Lower at night, increased with stress
PLASMA RENIN ACTIVITY Lower at night, higher standing>supine
ALDOSTERONE Lower at night
INSULIN Lower at night
GROWTH HORMONE Higher in afternoon and evening
ACID PHOSPHATASE Higher in afternoon and evening
THYROXINE Increases with excercise
PROLACTIN Higher with stress and at 4-8 AM and 8-10 PM
IRON Peaks early to late morning, decreases upto 30% at day
CALCIUM 4% decrease supine
4

5. BLOOD COLLECTION
 SITE
 VENIPUNCTURE/SPECIMEN COLLECTION
 DEVICE
 SITE PREPARATION
 PERSONAL SAFTEY
 TIME OF COLLECTION
 THE TEST ORDER
 STORAGE AND PRESERVATION
 SPECIMEN REJECTION
5

6. TESTS PERFORMED ON
COLLECTED BLOOD
 HEMATOLOGICALTESTS
 BIOCHEMICAL TESTS
 SEROLOGICALTESTS
 CULTURALTESTS
6

7. SITE
 Blood can be collected from 3 different sources-
1. Capillary
2. Venous ( most common)
3. Arterial
7

8. VENIPUNCTURE
 Venipuncture is a routine and common procedure done to collect venous blood
directly from the vein.
 Best site- Ante-cubital fossa
 In order to do this safely, the phlebotomist must have a basic understanding of the
following;
I. Anatomy
II. The criteria for choosing a vein
III. The device used
IV. Skin preparation
V. Personal safety – infection control policy
8

9. 9

10. 10

11. PHLEBOTOMY TRAY
 ITEMS TO BE INCLUDED-
a) Syringes and needles
b) Tourniquet
c) Specimen containers ( or evacuated tube system) – plain and with various anticoagulants
d) Request form
e) 70% isopropanol swabs and cellulose pads
f) Adhesive dressings
g) Self sealing plastic bags
h) Rack to hold specimen upright during process of filling
i) A puncture resistant disposal container should also be available
11

12. 12

13. 13

14.  NEEDLES should not be too fine/ too large/ too long
 Vary from large (16 G) to small (23 G)
 For adults- 19 or 21 G suitable
 For children- 23 G
 Ideally should have short shaft (15mm)
 Butterfly needles- when blood has to be collected from a very small vein
 Come in 21, 23, 25 G
14

15. 15
SYRINGE
TOURNIQUET
BUTTERFLY NEEDLE

16. 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 patient
 Good hand washing and drying techniques. If hand washing facilities are
unavailable, an alcohol based hand wash solution is an acceptable substitute
16

17. VENOUS PUNCTURE TECHNIQUE
PROCEDURE:
 Verify computer printed labels match requisitions
 Check patient identification band against labels and requisition forms
 Ask patient for his/her demographics
 Position patient properly
 Assemble equipment and supplies
 Apply tourniquet on the upper arm and ask to make a fist
 Select suitable vein for puncture
17

18.  Put on gloves
 Cleanse the site
 Anchor vein firm
 Enter skin with needle at 30 degree angle/ < to arm with bevel up of the needle
 Follow geography of vein and insert needle smooth
 Using a syringe , pull back on the barrel with a slow, even tension as blood flows
into the syringe
 Release tourniquet when blood begins to flow.
 Never withdraw needle without releasing tourniquet
18

19.  Withdraw needle and then apply pressure to the site.
 Mix and invert tubes with anticoagulants
 Check patient’s condition
 Dispose material in designated containers using universal precautions
 Label the tubes with –
a. Patient’s name
b. Identification number
c. Date and time of collection
d. Identification of person collecting specimen
 Deliver sample to appropriate lab section
19

20. 20

21. 21

22. PRECAUTION
 Area must be cleansed / sterilized properly
 Tourniquet should not be applied for long time
 Blind attempts should not be made
 Once needle withdrawn pressure should be applied and maintained for 1-2
mintues, if not can cause ECCHYMOSES
22

23. POST-PHLEBOTOMY PROCEDURE
 Check again patient’s details and make sure it corresponds to details on request
form
 Each submitted specimen must be labeled with the patient’s Demographics
(written exactly as it appears on the Test Request Form) and the tests to be
conducted.
 Patient dempographics include – Patient name, sex, age, DOB, DOA, DOE, hospital
number, room number, lab number, physician and physician’s pharmacy code
number
 Use one Test Requisition form only
23

24.  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
 Specimens should be sent in individual plastic bags/ set upright in a holder or
rack, separated from request form to prevent contamination in event of leaking
 Without separating needle from syringe place both together with swab and any
dressings in a puncture resistant container
24

25. PERSONAL SAFETY
 Protection for all personnel is paramount when handling blood products and body
fluids.
 Universal Precautions to be followed:
1) Every patient should be regarded as a potential biohazard
2) GLOVES MUST BE WORN.
3) Avoid needle stick injury –Hepatitis B and HIV viruses transmitted in blood and body
fluids
4) Dispose of sharps and or soiled equipment appropriately and safely; keep gloves on
whilst disposing of equipment, then dispose of gloves safely.
25

26. ORDER OF DRAW
1. Blood culture tubes (yellow)
2. Coagulation tube (blue)
3. Serum tubes with/without clot activator
4. Heparin tubes with/without gel (green)
5. EDTA tubes (lavender)
6. Glycolytic inhibitor tubes (gray)
26

27. SPECIMEN REJECTION
 Clotted specimen
 Severely hemolyzed specimen
 Improperly labeled or unlabeled specimen
 Specimen too old
 Failure to meet volume criteria
 Improperly collected (diluted) capillary specimen
 Leaking tube
 Delay in transport
 Collection of specimen in wrong tube
27

28. COMPLICATIONS
Immediate local complications :
 Haemoconcentration
 Collapse of the vein
 Failure of blood to enter the syringe .
 Needle not in position
28

29. Immediate general complications:
 Syncope
Late local complications :
 Thrombosis of the vein
 Thrombophlebitis
 Hematoma.
29

30. COLLECTION OF BLOOD FOR HEMATOLOGICAL EXAMINATIONS:
 Hb, RBC, WBC, DLC, Platelet count, Red Cell Indices, Peripheral Smear
COLLECTION OF BLOOD FOR BIOCHEMICAL EXAMINATIONS:
 Fasting conditions are advisable
 Venous blood to be preferred.
30

31. 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,
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
31

32. COMMON ERRORS IN SPECIMEN
COLLECTION
 Misidentification of patient
 Mislabelling of specimen
 Short draws/wrong anticoagulant/blood ratio
 Mixing problems/clots
 Wrong tubes/wrong anticoagulant
 Hemolysis/lipemia
 Hemoconcentration from prolonged tourniquet time
 Exposure to light/extreme temperature
 Improperly timed specimen/delayed delivery to laboratory
 Processing errors
32

33. CAUSES FOR MISLEADING RESULTS
RELATED TO SPECIMEN COLLECTION
 PRE-COLLECTION
 Urination within 30 min
 Food or water intake within 20 minutes
 Stress
 Drugs/ dietary supplement administration within 8 hours
33

34.  DURING COLLECTION
 Different time
 Posture, lying, standing, sitting
 Haemoconcentration from prolonged tourniquet pressure
 Excessive negative pressure when drawing blood
 Incorrect tube
 Capillary versus venous blood
34

35.  HANDLING OF SPECIMEN
 Insufficient or excess anticoagulant
 Inadequate mixing of blood with anticoagulant
 Error in patient and/or specimen identification
 Inadequate specimen storage conditions
 Delay in transit to laboratory
35

36. CAPILLARY BLOOD
36

37. CAPILLARY BLOOD
 For routine assays requiring small amount of blood (into capillary tubes coated with
Heparin or anticoagulated microcollection device)
 Obtained by SKIN PRICK/PUNCTURE METHOD
 Prick site should be free of congestion/ oedema/ cyanosed/ cold
37

38. 38

39. INDICATIONS
 IN ADULTS-
 Extreme obesity
 Severe burns
 Thrombotic tendencies
 In Geriatric patients because skin is thinner and less elastic- hematoma more likely to
occur from a venipunctutre
39

40. SKIN PUNCTURE TECHNIQUE
 Select an appropriate puncture site
 For infants <12 months- Lateral/ Medial plantar heel surface
 For infants >12 months, children, adults- Palmar surface of last digit of second/third/fourth
finger
 Warm the puncture site- arterial enriched blood
 Cleanse the site
 Make puncture with sterile lancet perpendicular to skin surface
 Discard first drop of blood by wiping it away
40

41.  Collect the specimen in suitable container by capillary action
 Apply pressure and dispose of the puncture device
 Label the specimen container with date and time of collection and patient
demographics
 Indicate in report that test results are from skin puncture
41

42. 42
LANCET
SKIN PUNCTURE

43. 43

44. 44
MICROTAINERS
CAPILLARY TUBES

45. ARTERIAL BLOOD
45

46. ARTERIAL BLOOD
 Technically more difficult to perform
 Increased pressure in arteries make it difficult to stop bleeding
 Order of preference- Radial> Brachial> Femoral arteries
 Specially required for estimation of ABG, pH, CO2 , O2
 Anticoagulant amount should be 0.05ml liquid Heparin for each millilitre of blood
46

47. 47

48.  Before blood collected from Radial artery in the
wrist, one should perform MODIFIED ALLEN TEST
to determine whether Ulnar artery can provide
collateral circulation to the hand after the Radial
artery punctured
48

49. MODIFIED ALLEN’S TEST
49

50. 50

51. 51

52. 52

53. ARTERIAL PUNCTURE PROCEDURE
 Prepare ABG syringe according to established procedures
 Needle (18-20 gauge) should pierce skin at 45-60 degrees for brachial, 90 degree
for femoral(23-25 gauge). Pulsations of blood into syringe confirm that it will fill
by arterial pressure alone
 After blood collected, place dry gauge over puncture site while quickly
withdrawing the needle and the collection device
 Compress the puncture site quickly, expel air from the syringe, activate needle
safety feature, discard into sharps container
 Mix specimen thoroughly by gently rotating/ inverting
 Place in ice water
53

54. CENTRAL VENOUS ACCESS
INDICATIONS:
 Ready access to patient’s circulation
 Eliminates multiple phlebotomies
 Useful in crtical care and surgical situations to draw blood/administer blood
products/drugs and provide parenteral nutrition
Indwelling catheters are surgically inserted into CEPHALIC VEIN/ INTERNAL JUGULAR
VEIN/ SUBCLAVIAN VEIN/ FEMORAL VEIN
54

55. ORDER OF DRAW FROM CATHETER LINES
 Draw 3-5ml in a syringe and discard
 Blood for blood culture
 Blood for anticoagulated tubes
 Blood for clot tubes
55

56. PRECAUTIONS TO PREVENT HAEMOLYSIS
 Apparatus should be clean and dry
 The bore of needles should not be too fine/small
 Blood should be withdrawn slowly without strong suction
 Needle should be removed from syringe before the blood is expressed into the
bottles
 Mix blood with anticoagulants by gentle swirling
 Blood should not be kept too long sitting at room temperature and if refrigerated
should not be frozen
56

57. STORAGE OF BLOOD BEFORE TESTS DONE
 Ideally tests should be done immediately after blood is collected-
 Within 2 hours- PT, other coagulation tests, Platelet count, blood
smearing
 Within 3 hours- ESR
 Within 24 hours- RBC count, PCV, Hb, WBC count, Reticulocyte count
count
57

58. SPECIMEN TRANSPORT
 Accounts for approximately 1/3rd TAT
 Excessive agitation avoided
 Protected from direct exposure to sunlight
 Kept at 4ºC immediately after collection and transported on ice for analysis of
unstable constitutents
 Specimen requiring refrigeration must be maintained between 2-10*C
58

59. ANTICOAGULANTS
59

60. DEFINITION
A substance that prevents coagulation or clotting of blood but doesn’t dissolve an
already formed clot.
Uses
• Storage of blood for blood transfusion or hematological testing
• Therapeutic
60

61. COAGULATION PATHWAYS
61

62. 62
CLOTTING FACTOR FUNCTION
I (fibrinogen) Forms clot (fibrin)
II (prothrombin) Its active form (IIa) activates I, V, VII,
VIII, XI, XIII, protein C, platelets
Tissue factor(formerly known as
factor III)
Co-factor of VIIa
Calcium(formerly known as factor IV) Required for coagulation factors to bind
to phospholipid
V (proaccelerin, labile factor) Co-factor of X with which it forms the
prothrombinase complex
VI Unassigned – old name of Factor Va

63. 63
VII (stable factor, proconvertin) Activates IX, X
VIII (Antihemophilic factor A) Co-factor of IX with which it
forms the tenase complex
IX (Antihemophilic factor B or
Christmas factor)
Activates X: forms tenase
complex with factor VIII
X (Stuart-Prower factor) Activates II: forms
prothrombinase complex with
factor V
XI (plasma thromboplastin
antecedent)
Activates IX
XII (Hageman factor) Activates factor XI, VII and
prekallikrein
XIII (fibrin-stabilizing factor) Crosslinks fibrin

64. STORAGE LESIONS
 A set of biochemical and biomechanical changes occur during storage leading to
decreased viability of the cells and its physiological functions
 RBC’s:
 Decreased ATP and 2,3 DPG levels, pH- acidic
 Poor functioning of Na-K pump- accumulation of K in stored blood
 Oxidative damage with lipid peroxidation
 Loss in membrane lipids affects deformability and osmotic fragility
 Morphological changes- Disc changes to echinocytes and to spherocytes
64

65.  Increased cellular rigidity d/t decrease in deformability
 Decrease in critical hemolytic volume(CHV) in parallel with membrane lipid
content
 CHV is largest volume to which RBC swells before haemolysis
 Decrease in osmotic fragility
65

66.  PLATELETS:
 Loss of discoid shape, microscopic platelet aggregate formation, fragmentation,
appearance of disintegrated balloon forms
 Blood stored for >24 hour at 2-6ºC has few viable platelets and granulocytes
 Heat labile coagulation factors V & VIII decrease on storage upto 50% in first
72hrs
 To ensure that blood retains its in vivo environment Anticoagulants are added
66

67.  Drop in pH and dextrose level causes anaerobic glycolysis in RBC to generate ATP.
 Decreased ph causes Decreased 2,3-DPG and cells ability to release oxygen to the
tissues.
 Metabolic functions slow down in cold temperature, ATP levels decreases.
 ELECTROLYTE: loss of potassium from RBC to plasma
passage of sodium from plasma to cells
plasma ammonia levels also increase
67

68. COMMONLY USED ANTICOAGULANTS
 EDTA (ETHYLENE DIAMINE TETRA ACETIC ACID)
 DOUBLE OXALATE
 SODIUM CITRATE
 SODIUM FLUORIDE
 HEPARIN
 ACD (ACID CITRATE DEXTROSE)
 CPD (CITRATE PHOSPHATE DEXTROSE)
 CPDA (CITRATE PHOSPHATE DEXTROSE ADENINE)
68

69. EDTA
 Used for routine hematological work
 MOA: chelating effect on calcium molecule in blood.
 Three types of EDTA salts used in salt or liquid form-
 Sodium- di/tri
 Potassium- di/tri
 Lithium- di
 DIPOTASSIUM SALT more soluble than DISODIUM SALT, hence preferred
 Solubility- K2> Na2> Li2
69

70.  DILITHIUM SALT is equally effective, sample of blood can be used for chemical
investigations
 TRIPOTASSIUM SALTS are available in liquid form with disadvantage of:
 Dilution
 Shrinkage of RBC
 Decrease in 2-3% PCV in 4 hours
 Gradual increase in MCV
 TRISODIUM EDTA not recommended because of high pH
70

71.  Coding of vial- LAVENDER CAP
 Recommended concentration-
 K2-EDTA -1.5-2.0mg/ml
71

72. CHANGES OCCURRING DUE TO
PROLONGED STORAGE in EDTA
72

73. USES
 TLC
 DLC
 ESR (by wintrobe method)
 CBC
 RETICULOCYTE COUNT
 PLATELET COUNT
73

74. DISADVANTAGES
 Excess EDTA irrespective of salt, affects both RED CELLS and
LEUCOCYTES- causing shrinkage and degenerative changes
 On RBC-
 Crenation, Spherocytic change
 Significant decrease in PCV
 Increase in MCHC
 On WBC-
 Leuco-agglutination
74

75.  On PLATELETS-
 Cause them to swell and disintegrate- causing artificially high count
 Responsible for activity of naturally occurring antiplatelet auto-antibody causing
satelitism
 Blood films from EDTA fails to demonstrate BASOPHILIC STIPPLING of RBC in
poisoning
 EDTA also appears to suppress platelet degranulation
 Monocyte activation measured by release of tissue factor & TNF is lowered with
EDTA than with citrate & heparin
75

76. DOUBLE OXALATE
 Acts by chelating calcium in blood
 POTASSIUM OXALATE: Used at a concentration of 2mg/ml
 Causes RBC shrinkage- 8% shrink in PCV
 AMMONIUM OXALATE: Used at a concentration of 2mg/ml
 Causes swelling of RBC
 Thus not used individually for PCV, ESR or Blood smears
76

77.  BALANCED OXALATE/ DOUBLE OXALATE/ WINTROBE’S MIXTURE:
 To balance swelling and shrinking effect of both salts, they are combined in a mixture
with ratio of 3 parts of NH4 oxalate to 2 parts of K oxalate which is used at
concentration of 2mg/ml of blood
 Prepared in a solution of 20 mg/ml- 2mg/0.1ml of solution- pipetted into containers-
incubated to evaporate the fluid- redissolves in blood
 Avoid high temperature as it inactivates anticoagulants
77

78. USES:
 Hb
 PCV
 WBC
 PT
 ESR (Wintrobe Method)
78

79. DISADVANTAGES:
 Calcium in the blood combines with oxalate to form insoluble calcium
oxalate which precipitates
 They are phagocytosed by neutrophils- distort WBC morphology- not
good for smears
 Never used in blood to be transfused- as it is toxic and calcium oxalate
precipitate may harm
79

80. TRISODIUM CITRATE
 Acts by chelating calcium in blood
 Used as 3.2% and 3.8% solution
It is used in a concentration of :-
 1 part sodium citrate to 4 parts whole blood for ESR (by westergren)
 1 part sodium citrate to 9 parts whole blood for coagulation profile.
80

81. CITRATE VIAL
Citrate is usually in BLUE VACUTAINER
TUBE. It is in liquid form in the tube and is
used for coagulation tests. It gets rid of the
calcium, but not as strongly as EDTA
81

82. USES
 PT
 APTT
 ESR(by westergren method)
DISADVANTAGES
 Alters concentration of blood as it is always used in solution form , hence not used
for routine hematology.
82

83. SODIUM FLOURIDE
 Sodium fluoride has a double action on the blood:
 It prevents clotting by chelating calcium.
 It prevents all phosphatase action, inhibit glucose oxidase activity
in enzymatic glucose reaction.
 Prevents glycolysis for 3 days
 It is used for determination of blood sugar.
 In bacterial specticemia, fluoride inhibition of glycolysis is
neither adequate nor effective in preserving glucose
concentration
83

84. Common disadvantages with calcium chelators
 They inhibit various plasma enzyme activities like:
 Amylase activity inhibited by oxalate and citrate
 LDH and Acid Phosphatase inhibited by oxalate
 Fluoride , Heparin or EDTA interfere with accurate determination of electrolytes
84

85. HEPARIN
 Lithium or Sodium salt of Heparin at a concentration of 10-20 IU/ml of blood is commonly
used for chemistry, gas analysis and emergency tests
 Lithium is recommended
 Doesnot alter the size of RBC- minimum chance of lysis after blood has been drawn– best
anticoagulant for osmotic fragility tests and suitable for phenotyping
85

86. ACTION
 Heparin is a naturally-occurring anticoagulant produced by basophils and mast
cells
 Heparin accelerates the action of Antithrombin III, neutralising Thrombin and
prevents formation of Fibrin
 Heparin acts as an anticoagulant, preventing the formation of clots and extension
of existing clots within the blood.
 Heparin does not break down clots that have already formed
86

87. AMOUNT FOR
BLOOD STORAGE:
5 – 10 IU /mL of blood
SAMPLE COLLECTION: 0.5- 2.0
IU/mL
87

88. USES
 Ammonia
 Carboxyhemoglobin
 Blood gases
 Electrolytes ( doesnot affect the levels of
ions)
 ESR
 Methaemoglobin
 Osmotic Fragility
 HLA Typing
88

89. Disadvantages
 Costly
 In Leishman stained peripheral blood film blue color is imparted to the
background due to presence of plasma protiens
 Not suitable for blood counts as it often induces platelet and leucocyte clumping
 Should not be used in study of PCR with restriction enzymes because it inhibits
enzyme activity
89

90. 90
CRENATED RBC
PLATELET CLUMP
LEUCOAGGLUTINATION
CHANGES IN BLOOD SMEAR DUE TO EXCESS ANTICOAGULANT

91. 91
PLATELET SATELITISM

92. COLOUR CODING OF VIALS
92

93. 93
SODIUM
CITRATE
FOR
COAGUL
ATION
STUDIES
K-
OXALTE/S
ODIUM
FLOURIDE
GLUCOSE
DETERMI
NATION
NO
ADDITIVE
COLLECT
ION OF
SERUM
ACD
PRESERVE RBC
FOR BLOOD
BANKING &
HLA Typing
HEPARIN
INHIBIT
THROMBIN
ACTIVATION
EDTA
FOR
ROUTINE
HAEMAT
OLOGY

94. TUBE COLOUR AND ANTICOAGULANT/ ADDITIVE 94
STOPPER COLOR ANTICOAGULANT SPECIMEN ACTION
RED (glass) None Serum/Chemistry, serology N/A
RED (plastic) Clot activator Serum/Chemistry, serology Silica clot activator
LAVENDER (glass) K3EDTA in liquid form Whole blood/ Hematology Chelates Calcium
LAVENDER K2EDTA/ spray dried Whole blood/ Hematology Chelates Calcium
PINK K2EDTA/ spray dried Whole blood/ blood bank and
molecular diagnostics
Chelates Calcium
WHITE EDTA and gel Plasma/ molecular genitics Chelates Calcium
LIGHT BLUE Sodium Citrate Plasma/ Coagulation Chelates Calcium
LIGHT BLUE Thrombin and Soya bean
trypsin inhibitor
Plasma/ Coagulation Fibrin degradation
products

95. 95STOPPER COLOR ANTICOAGULANT SPECIMEN ACTION
BLACK Sodium Citrate Plasma Chelates Calcium
LIGHT GREEN/
BLACK
Lithium Heparin and gel Plasma/ chemistry Inhibhits Thrombin
formation
GREEN Sodium Heparin, Lithium
Heparin
Plasma/ chemistry Inhibhits Thrombin
formation
ROYAL BLUE Sodium Heparin, K2EDTA Plasma/ chemistry,
Toxicology
Heparin-Inhibhits
Thrombin formation
EDTA-Chelates
Calcium
GRAY Sodium Fluoride/
Potassium Oxalate
Plasma/ Glucose
testing
Inhibits glycolysis

96. 96
STOPPER COLOR ANTICOAGULANT SPECIMEN ACTION
YELLOW Sterile containing
Sodium
Polyanetholesulfonat
Serum/ microbiology
culture
Aids in bacterial
recovery by
inhibhiting
complement,
phagocytes and
certain antibodies
YELLOW Acid citrate dextrose Plasma/ blood bank,
HLA phenotyping
paternity testing
WBC preservation
TAN (glass) Sodium heparin Plasma/ lead testing Inhibits Thrombin
formation
TAN (plastic) K2EDTA Plasma/ lead testing Chelates Calcium
YELLOW/GRAY and
ORANGE
Thrombin Serum/ chemistry Clot activator
RED/GRAY and
GOLD
Clot activator
separation gel
Serum/ chemistry Silica Clot activator

97. ANTICOAGULANT/ ADDITIVE EFFECT ON
BLOOD TESTS
ADDITIVE TEST EFFECT
EDTA Alkaline phosphatase Inhibits
Creatine kinase Inhibits
Leucine aminopeptidase Inhibits
Calcium and iron Decrease
PT and PTT Increase
Sodium and potassium Increase
Platelet aggregation Prevents
OXALATE Acid phosphatase Inhibits
Alkaline phosphatase Inhibits
Amylase Inhibits
LD Inhibits
Calcium Decrease
97

98. 98ADDITIVE TEST EFFECT
CITRATE ALT and AST Inhibits
Alkaline phosphatase Inhibits
Acid phosphatase Stimulates
Amylase Decreases
Calcium Decreases
Sodium and potassium Increase
Labile coagulation factors Preserve
HEPARIN T3 Increases
Thyroxine Increases
PT and PTT Increase
Wright’s stain Causes blue background
Lithium (LiHep tubes only) Increases
Sodium (NaHep tubes only) increases

99. 99
ADDITIVE TEST EFFECT
FLUORIDES Acid phosphatase Decreases
Alkaline phosphatase Decreases
Amylase Decreases
Creatine kinase Decreases
ALT and AST Decreases
Cell morphology Distorts

100. ANTICOAGULANTS FOR BLOOD STORAGE
 ACD (Acid Citrate Dextrose)
 CPD(Citrate Phosphate Dextrose)
 CPDA-1(Citrate Phosphate Dextrose Adenine)
 HEPARIN
100

101. COMPOSITION( in 1L of distilled water)
CHEMICAL ACD CPD CPDA-1
TRI SODIUM
CITRATE
22.0 g 26.3 g 26.3 g
CITRIC ACID 8.0 g 3.27 g 3.27 g
DEXTROSE 24.6 g 25.50 g 31.8 g
SODIUM
DIHYDROGEN
PHOSPHATE
____ 2.28 g 2.22 g
ADENINE _____ _____ 0.275 g
101

102.  15ml of ACD and 14ml of CPD & CPDA-1 is required for preservation of 100ml of
blood
 Initial pH of ACD 5.0 and of CPD & CPDA-1 is 5.6
 Also storage time at 2-6ºC for ACD & CPD is 21 days and for CPDA-1 is 35days
102

103. FUNCTION OF VARIOUS COMPONENTS
o CITRATE
Causes chelation of calcium
 SODIUM DIPHOSPHATE
Prevents fall in pH
o DEXTROSE
Addition of glucose prolongs survival of stored RBC as it is required for metabolism.
Glucose passes from plasma to RBC and is utilized for energy production.
2 PATHWAYS for energy production :
• 90% by Embedem Mayeroff pathway in which there is breakdown of glucose into
lactate through anaerobic glycolysis.
103

104. Prevents carmalization of glucose in citrate dextrose solution during autoclaving.
o ADENINE
It is added in modified CPD as it improves viability of RBC because of enhanced
enhanced ATP production.
104

105. • 10% by Pentose phosphate pathway through aerobic glycolysis.
The various intermediaries formed are necessary for maintaining
their ability to deliver oxygen to tissues through generation of
2,3-DPG.
Viability correlates with the level of ATP.
o CITRIC ACID
Fairly weak tribasic hydroxyacid
Along with tri sodium citrate which is alkaline gives an optimal pH.
105

106. ADDITIVE SYSTEM
To extend RBC storage to 42 days and to harvest maximum amount of plasma,
additive systems are now available in which storage environment of RBC is altered
by adding certain nutrients after removal of plasma
Its made by adding the following components to the CPD :-
• Sodium chloride-adjusts osmotic pressure
• Adenine – maintains high level of ATP in RBC
• Dextrose
• Mannitol- prevents disintegration of RBC
106

107. REFERENCES
1) Henry Laboratory Methods 22nd Edition
2) Clinical Laboratory Hematology 3rd Edition- McKenzie/ Williams
3) Dacie and Lewis Practical Hematology
4) Handbook of Medical Laboratory Technology- Robert H Carman
107

108. 108