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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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