Methods of Blood Collection and Anticoagulants

METHODS OF BLOOD COLLECTION AND

ANTICOAGULANTS

MODERATOR – Dr MAHANTHACHAR

PRESENTED BY - Dr SHREYA PRABHU

1

BLOOD COLLECTION

2

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

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

BLOOD COLLECTION

SITE

VENIPUNCTURE/SPECIMEN COLLECTION

DEVICE

SITE PREPARATION

PERSONAL SAFTEY

TIME OF COLLECTION

THE TEST ORDER

STORAGE AND PRESERVATION

SPECIMEN REJECTION

5

TESTS PERFORMED ON COLLECTED BLOOD

HEMATOLOGICAL TESTS

BIOCHEMICAL TESTS

SEROLOGICAL TESTS

CULTURAL TESTS

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SITE

Blood can be collected from 3 different sources-

1. Capillary

2. Venous ( most common)

3. Arterial

7

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

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

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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

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15

SYRINGE

TOURNIQUET

BUTTERFLY NEEDLE

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

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

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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

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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

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

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

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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

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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.

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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)

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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

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COMPLICATIONS

Immediate local complications :

Haemoconcentration

Collapse of the vein

Failure of blood to enter the syringe .

Needle not in position

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Immediate general complications:

Syncope

Late local complications :

Thrombosis of the vein

Thrombophlebitis

Hematoma.

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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.

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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

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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

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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

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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

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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

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CAPILLARY BLOOD

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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

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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

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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

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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

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LANCET

SKIN PUNCTURE

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MICROTAINERS

CAPILLARY TUBES

ARTERIAL BLOOD

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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

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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

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MODIFIED ALLEN’S TEST49

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

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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

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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

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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

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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 countcount

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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

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ANTICOAGULANTS

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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

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COAGULATION PATHWAYS61

62CLOTTING 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

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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

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

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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

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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

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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

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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)

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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

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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

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Coding of vial- LAVENDER CAP

Recommended concentration-

K2-EDTA -1.5-2.0mg/ml

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CHANGES OCCURRING DUE TO PROLONGED STORAGE in EDTA

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USES

TLC

DLC

ESR (by wintrobe method)

CBC

RETICULOCYTE COUNT

PLATELET COUNT

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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

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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

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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

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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

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USES:

Hb

PCV

WBC

PT

ESR (Wintrobe Method)

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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

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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.

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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

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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.

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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

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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

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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

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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

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http://en.wikipedia.org/wiki/Basophil

http://en.wikipedia.org/wiki/Mast_cell

AMOUNT FOR

BLOOD STORAGE:5 – 10 IU /mL of blood

SAMPLE COLLECTION: 0.5- 2.0 IU/mL

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USES

Ammonia

Carboxyhemoglobin

Blood gases

Electrolytes ( doesnot affect the levels of

ions)

ESR

Methaemoglobin

Osmotic Fragility

HLA Typing

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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

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CRENATED RBC

PLATELET CLUMP

LEUCOAGGLUTINATIONCHANGES IN BLOOD SMEAR DUE TO EXCESS ANTICOAGULANT

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PLATELET SATELITISM

COLOUR CODING OF VIALS

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SODIUM CITRATEFOR COAGULATION STUDIES

K-OXALTE/SODIUM FLOURIDEGLUCOSE DETERMINATION

NO ADDITIVECOLLECTION OF SERUM

ACDPRESERVE RBC FOR BLOOD BANKING &HLA Typing

HEPARININHIBIT THROMBIN ACTIVATION

EDTAFOR ROUTINE HAEMATOLOGY

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

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, LithiumHeparin

Plasma/ chemistry Inhibhits Thrombin formation

ROYAL BLUE Sodium Heparin, K2EDTA Plasma/ chemistry, Toxicology

Heparin-InhibhitsThrombin formationEDTA-ChelatesCalcium

GRAY Sodium Fluoride/Potassium Oxalate

Plasma/ Glucose testing

Inhibits glycolysis

96STOPPER COLOR ANTICOAGULANT SPECIMEN ACTION

YELLOW Sterile containingSodium Polyanetholesulfonat

Serum/ microbiologyculture

Aids in bacterial recovery by inhibhitingcomplement, phagocytes and certain antibodies

YELLOW Acid citrate dextrose Plasma/ blood bank, HLA phenotypingpaternity 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 andGOLD

Clot activator separation gel

Serum/ chemistry Silica Clot activator

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


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98ADDITIVE TEST EFFECT

CITRATE ALT and AST Inhibits

Alkaline phosphatase Inhibits

Acid phosphatase Stimulates

Amylase Decreases

Calcium Decreases


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

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ADDITIVE TEST EFFECT

FLUORIDES Acid phosphatase Decreases

Alkaline phosphatase Decreases

Amylase Decreases

Creatine kinase Decreases

ALT and AST Decreases

Cell morphology Distorts

ANTICOAGULANTS FOR BLOOD STORAGE

ACD (Acid Citrate Dextrose)

CPD(Citrate Phosphate Dextrose)

CPDA-1(Citrate Phosphate Dextrose Adenine)

HEPARIN

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COMPOSITION( in 1L of distilled water)

CHEMICAL ACD CPD CPDA-1

TRI SODIUM

CITRATE22.0 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

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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

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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.

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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.

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• 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.

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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

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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

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Methods of Blood Collection and Anticoagulants

Healthcare

Transcript of Methods of Blood Collection and Anticoagulants