Standard Operating Proceduresgov1

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Revision Status : 0.0
Date : 15-05-2008
Page : 1 of 197

Established and to be maintained to the requirements of
National Accreditation Board for Hospitals and Healthcare Providers (NABH)
Standards on Blood Banks / Blood Centres and Transfusion Services

Number Effective date Pages Author Authorised By
SP-001
01-12-2007

4

Dr V Saraswathi
Version Review Period
No. of
Copies
Approved By Date

1.0

1 year 15-05-2008

Location Subject
Donor Room

Criteria for Donor Selection

Function Distribution

Assessing suitability of donor for
Blood donation

- Medical Officer in charge of Donor Area
- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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Date : 15-05-2008
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1.0 SCOPE & APPLICATION

This SOP describes the criteria for a donor to be accepted for blood donation, for
ensuring safety of donor as well as recipient. The purpose of donor selection is to
identify any factors that might make an individual unsuitable as a donor, either
temporarily or permanently.

2.0 RESPONSIBILITY

The Medical Officer is responsible for determining the suitability of donor for blood
donation. He/She should confirm that the criteria are fulfilled after evaluation of health
history questionnaire and medical examination including the results of pre-donation
screening tests.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks- 13th edition, 1999
pgs 90-97, 103-110.

4.0 MATERIAL REQUIRED

Donor Questionnaire
Donor Card

5.0 PROCEDURE

STANDARD OPERATING
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CRITERIA FOR SELECTION OF BLOOD DONORS

A. Accept only voluntary/replacement non-remunerated blood donors if following
criteria are fulfilled.

The interval between blood donations should be not less than three months.
The donor should be in good health, mentally alert and physically fit and should
not be a jail inmate or a person having multiple sex partners or a drug-addict.
The donors should fulfill the following requirements, namely:-

The donor age group between19 to 58 years.
The donor weight: Female 45 kg, Male 50 Kg
Temperature and pulse of the donor is normal
The systolic and diastolic blood pressures are within normal limits without
medication
Haemoglobin is 12.5 g/dl
The donor is free from acute respiratory diseases
The donor is free from any skin disease at the site of phlebotomy
The donor is free from any disease transmissible by blood transfusion, in so far as
can be determined by history and examination indicated above
The arms and forearms of the donor is free from skin punctures or scars
indicative of professional blood donors or addiction of self-injected narcotics

B. Defer the donor for the period mentioned as indicated in the following table:

Conditions Period of Deferment
Abortion 6 months
History of blood transfusion 6 months
Surgery 12 months
Typhoid Fever 12 months after recovery
History of Malaria duly treated 3 months (endemic)
3 months (non-endemic)
Tattoo 6 months
Breast Feeding 12 months after delivery
Immunization (Cholera, Typhoid, Diphtheria,
Tetanus, Plague, Gamma globulin)
15 days
Rabies Vaccination 1 year after vaccination
Hepatitis in family or close contact 12 months
Hepatitis immune globulin 12 months

C. Defer the donor permanently if suffering from any of the following diseases:

STANDARD OPERATING
PROCEDURES
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Cancer
Heart disease
Abnormal bleeding tendencies
Unexplained weight loss
Diabetes (on insulin)
Hepatitis B infection
Chronic nephritis
Signs and symptoms, suggestive of AIDS
It is important to ask donors if they have been engaged in any risk behaviour.
Allow sufficient time for discussion in the private cubicle. Try and identify
result-seeking donors and refer them to VCTC (Voluntary Counseling and
Testing Center). Reassure the donor that strict confidentially is maintained.
Liver disease
Tuberculosis
Polycythemia Vera
Asthma
Epilepsy
Leprosy
Schizophrenia
Endocrine disorders

D. Private interview:

A detailed sexual history is taken. Positive history should be recorded on confidential
notebook.

E. Informed consent:

Provide information regarding:

Need for blood
Need for voluntary donation
Regarding transfusion transmissible infections
Need for questionnaire and honest answers
Safety of blood donation
How the donated blood is processed and used
Tests carried out on donated blood

* Request the donors to sign on the donor card indicating that he is donating
voluntarily.

6.0 DOCUMENTATION

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 5 of 197


Enter all details in the donor questionnaire form/ Donor card and computer.

SP-002
01-12-2007

3

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

2 years

15-05-2008

Location Subject
Donor Room

Donor Screening

Physical Examination of the donor

- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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To perform a physical examination on the donor for confirming fulfillment of the criteria
which ensure safety of the donor as well as the recipient.

2.0 RESPONSIBILITY

It is the responsibility of the Medical Officer to perform the physical examination on the
donor.

3.0 REFERENCES

Introduction to Transfusion Medicine, Zarin Bharucha & Chauhan DM 1 edition
1990, Pg. 97-98
Technical Manual of American Association of Blood Banks, 13
th
edition 1999,
Pg. 93-95


Weighing scale
Sphygmomanometer
Clinical thermometer
CuSO
4
in Coplin's jar
Capillaries
Lancet

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Donor card

5.0 PROCEDURE

Medical Examination:

General Appearance:
Defer a donor who appears ill, under the influence of drugs/alcohol or do not
appear to be providing reliable answers to medical history.
Check and enter donor's weight. The weight should be >50 kg to collect 450 ml
and between 45 and 50 kg to collect 350ml blood.
Check if the blood pressure, pulse and temperature of the donor are within the
acceptable limits:
Systolic blood pressure not < 160 mm of Hg.
Diastolic pressure not <100 mm of Hg;
Pulse regular, between 60 and 72 beats / minute.
Oral temperature 37.5 C +/- 0.2C (98.6F =/- 0.5F).

HAEMOGLOBIN ESTIMATION:

Blood donation can be accepted only if the haemoglobin is > 12.5 g/dl. Test for
haemoglobin by CuSO4 specific gravity method (Refer SOP 003).

6.0 DOCUMENTATION

Enter all details in the Donor card and computer.

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PROCEDURES
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SP-003
01-12-2007

3

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Donor Room

Qualifying Test for Blood Donation

Method of estimation of donor s
Haemoglobin by copper sulphate
method

- Technicians in the Area
- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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To find a fit and healthy donor, assuring his or her safety. This also helps in assuring the
quality of the product.

2.0 RESPONSIBILITY

It is the responsibility of the technician working in the donor area.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks, 13th edition, 1999 Pg. 711-
712


Copper sulphate working solution with a specific gravity 1.053.
Sterile gauze/cotton, spirit and sterile disposable lancets.
Heparinised capillaries (dimensions: 75mmx1mm)
Containers with 1% sodium hypochlorite solution for disposing sharp lancets,
Capillaries and bio hazardous materials.
Coplin jar with lid.
For preparation of copper sulphate working solution refer SOP: SP 004.

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5.0 PROCEDURE

5.1 Principle:

This is a qualitative test based on specific gravity. The drop of donor's blood dropped
into copper sulphate solution becomes encased in a sac of copper proteinate, which
prevents any change in the specific gravity for about 15 seconds. If the haemoglobin is
equal to or more than 12.5 gm/dl the drop will sink within 15 seconds and the donor is
accepted.
N.B:
Do not depend on colour of tongue or conjunctiva.
Accept a donor only if haemoglobin is >12.5g/dL.

5.2 Method:

30 ml copper sulphate working solution (Sp.gr.1.053) in a clean, dry Coplin jar is
used for determining hemoglobin. The jar is kept covered with a lid when not in
use. The working solution is changed after every 25 tests.
The fingertip is cleaned thoroughly with a spirit swab and allowed to dry.
The finger is punctured firmly near the tip with a sterile disposable lancet. A
good free flow of blood is ensured. The finger is not to be squeezed repeatedly
since it may dilute the drop of blood with excess tissue fluid and give false low
results.
The first drop of blood is wiped and of the micro capillary is allowed to fill
with blood sample by capillary force, without any air bubbles.
Allow one drop of blood to fall gently from the capillary from a height of about 1
cm above the surface of the copper sulphate solution, into the Coplin jar.
The drop of blood is observed for 15 seconds.
The lancet and capillaries are disposed off in a container with 1% sodium
hypochlorite solution.

5.3 Interpretation:

If the drop of blood sinks within 15 seconds (i.e. donor's haemoglobin is more
than 12.5gm/dL), the donor is accepted for blood donation.
However, if the blood drop sinks midway (i.e. haemoglobin level is less than
12.5gms/dL), and then comes up, the donation or donor is deferred. If the drop
sinks slowly, hesitates and then goes to the bottom of the jar, confirm the
haemoglobin of this donor.

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PROCEDURES
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If the donor fails the CuSO4 test, repeat haemoglobin by Sahli's /Drabkin's /
Automated Cell Counter.
In case if the haemoglobin is lower than 12.5g/dL, prescribe haematinics and ask
the donor to come for a recheck after one month.

6.0 DOCUMENTATION

Enter the result on donor card

Location Subject
Donor Room
Haemoglobin Estimation


Quality Control Check

- Donor Area
- Medical Officer in-charge of Donor Area
- Master File

SP-004
NIL

2

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
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The specific gravity of 1.053 is equivalent to 12.5 g/dl haemoglobin. Hence CuSO
4
solution of specific gravity 1.053 is used for pre-donation haemoglobin.

2.0 RESPONSIBILITY

The technician/ laboratory assistant in the donor area.

3.0 REFERENCES

Introduction to Transfusion Medicine: Z.S Bharucha, D.M Chouhan, 1
st
Ed, 1990, page
99.

4.0 PROCEDURES

Stock solution is made as follows and kept in a jar or bottle
1. Dissolve 170gm crystalline CuSO
4
O5H
2
O in 1000 ml distilled water
(Working solution)
2. Every morning prepare fresh solution.
3. Add 51ml stock solution to 49 ml distilled water.
4. Check Specific Gravity which should be 1.053, if not, adjust it using either
stock solution or distilled water.

STANDARD OPERATING
PROCEDURES
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5.0 DOCUMENTATION

Record the volume of stock and working solution prepared on the register
(Table 1 of SP 005)

SP-005
01-12-2007

3

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Donor Room

Copper Sulphate Solution


Quality Control Check

- Donor Area
- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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Date : 15-05-2008
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Copper sulphate solution is used for screening blood donors by testing the haemoglobin
concentration before blood donation.
Copper sulphate solution is checked to ensure that a drop of blood sample of
predetermined haemoglobin value reacts as expected (sinks/floats).

2.0 RESPONSIBILITY

It is the responsibility of the Quality Control personnel to ensure testing of the reagent
before use.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks, 13 Edition, 1999, pg 56

4.0 MATERIALS

4.1 Equipment:

Urinometer

4.2 Reagents:

Copper sulphate working solution
Distilled water

STANDARD OPERATING
PROCEDURES
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EDTA blood samples of known haemoglobin concentration

4.3 Glassware:

Coplin jar
Heparinised capillaries

4.4 Miscellaneous:

Tissue paper
Copper sulphate record book
Tube racks

5.0 PROCEDURE

Check the copper sulphate solution against a light source for the presence of
precipitate/cloudiness.
Check the specific gravity of the solution using a urinometer
Copper sulphate being a coloured solution, the marking of the urinometer
corresponding to the upper meniscus of the solution should be 1.053=12.5g% of
haemoglobin
Arrange the blood samples according to haemoglobin concentration in a rack
Obtain samples of known Hb values
Transfer 30ml copper sulphate working solution in a Coplin jar
Mix the blood sample of known haemoglobin concentration by inversion
Fill heparinised capillary upto capacity with the blood sample
Allow the drop of blood to fall gently into the copper sulphate solution
Repeat the procedure for all the blood samples
Note the result
Record the results in the copper sulphate record book.

6.0 RESULTS

If the solution appears cloudy or precipitate is present, the solution is discarded.
The result of testing the solution is interpreted as follows:

S. No. Result Hb Concentration Interpretation
a. Blood Drop Floats Hb < 12.5 g % Fail (F)
b. Blood Drop Sinks Hb > 12.5 g % Pass (P)
c. Blood Drop Sinks slowly

STANDARD OPERATING
PROCEDURES
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or
Blood Drop hesitates
midway or sinks slowly
Hb < 12.5 g % Pass / Fail Reaction
(P/F)

7.0 DOCUMENTATION

The result are noted in the copper sulphate record book

SP-006
01-12-2007

2

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject
Donor Room

Preparation for Blood Collection


Solutions and methods for preparing
phlebotomy site

- For All Phlebotomists
- Master File

STANDARD OPERATING
PROCEDURES
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Cases of transmission of bacterial infection in blood are fortunately rare, but when they
do occur can be fatal. Thus careful preparation of the skin at the phlebotomy site before
venipuncture is very important.

2.0 RESPONSIBILITY

The phlebotomist collecting the blood unit from the donor is responsible for preparation
of phlebotomy site.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks, 13th edition, 1999 Pg. 713

4.0 MATERIALS

Sterilising tray
Demethylated spirit
Povidone Iodine
Cotton/gauze/swabs
Artery forceps
Tourniquet

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5.0 PROCEDURE

After selection of the vein for venepuncture, apply spirit, providone-iodine and finally
spirit swab, in this order, to the skin at the phlebotomy site. Start disinfection of the skin
of about an area of 5 cm diameter from the centre outwards in a circular motion. Scrub
the providone-iodine vigorously for at least 30 seconds or till froth forms. Do not touch
the site prepared for venipuncture. Should it be necessary, touch the skin away from the
point of needle insertion. If the puncture site is touched, repeat skin preparation
procedure as detailed earlier. Discreetly check the used swab. If it is physically soiled
/contaminated, take a new swab and repeat skin preparation procedure as detailed earlier.
Dispose off used swab(s) into a waste bin meant for bio-hazardous materials. Allow the
skin to air dry. Do not wipe the area with cotton wool, fan or blow on it.

SP-007
01-12-2007

3

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location
Subject

Donor Room

Selection of Bags

Choice of bag depending on component
to be prepared

- For use of all Phlebotomists &
Technicians
- Master File

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According to the components to be prepared from the blood unit and the weight of the
donor the blood bags are selected for blood collection.

2.0 RESPONSIBILITY

The technician or phlebotomist in the donor area coordinates with the component room
for deciding the type of blood bags to be used. The medical officer is consulted in case of
difficulty in making a decision or to optimise the availability of components.

3.0 REFERENCES

Introduction to Transfusion Medicine, Zarin Bharucha & D M Chouhan, s1t edition
1990, Pgs 116, 124

4.0 MATERIALS

Different types of blood bags in use

5.0 PROCEDURE

Select the bag as per the following chart:
Donor Components Bags
Weight
Aspirin
Intake
Required Type
Qty.(ml)
of Blood

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>55 Kg No PC + FFP + PLT
Triple or
Quadruple
450 ml
>55 Kg Yes
PC + FFP
PC + FVIIID + CRYO
Double
Triple or
Quadruple
450 ml/
450 ml
45-55 Kg -

PC + FFP

Double 350 ml

PC: Packed Cells, FFP: Fresh Frozen Plasma, PLT: Platelets, FVIIID: Factor VIII
Deficient Plasma, Cryo: Cryoprecipitate

Check the bag visually:
In case of puncture or discolouration, do not use
Check the expiry date of the bag
Use single bag when;
1. Components are not to be separated from that unit.
2. Therapeutic phlebotomy is being performed on a patient.

6.0 DOCUMENTATION

Enter the following details on donor card and register:

Type of bag
Manufacturer's name
Batch No.
Expiry date

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SP-008
01-12-2007

4

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Donor Room

Blood Collection


Assessing suitability of donor for
blood donation

- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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This describes a procedure for blood collection from the donor, using an aseptic method.
Blood is collected in a sterile closed system bag with a single venepuncture. A correct
performance of venepuncture is essential for the quality and safety of the blood donation.
Successful venepuncture results not only in safe collection of a full unit of blood suitable
for separation of components with good quality yields, but also contributes to the
comfort and satisfaction of the donors thus encouraging re-attendance.

2.0 RESPONSIBILITY

The phlebotomist or doctor is responsible for blood collection from the donor after
verifying the donor screening details, checking the unit number labels and preparing the
phlebotomy site.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks, 13 edition 1999 Pgs
98, 713-716
Introduction to Transfusion Medicine, Zarin Bharucha & D M Chouhan 1
st

edition 1990 Pg 99 100

4.0 MATERIALS
Cotton/Gauze swabs
Artery forceps
2% Xylocaine
Disposable plastic syringes (2ml)

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Disposable needles (26 gauge)
Pilot tubes: Plain and EDTA
Tourniquet
Oxygen cylinder with accessories
Rubber gloves - latex
First aid tray
Tubing stripper
Electronic tube sealer
Needle destroyer
Blood collecting bags
Discard jar with 10% sodium hypochlorite
Scissors
Hi Tech (adhesive) tapes

Blood collection monitor
Comfortable donor couch or chair

5.0 PROCEDURE

Make the donor lie down with a pillow under the head or recline in a comfortable
donor chair. Loosen tight garments.
Identify the donor by name. Enter the bag and segment numbers on the donor
card/form.
Ask the donor if he/she is in a comfortable position. Give the donor a hand roller
/ squeezer to hold.
Select a bag for blood collection (SP 007)
Clean the venepuncture site (SP 006)
Set the Blood collection monitor for the required volume of blood (350/450ml) to
be collected and place the bag on it.
Apply the tourniquet on donor arm.
Clamp the bleed line of the blood bag using plastic forceps to ensure that no air
enters the tubing or bag once the needle cover is removed.
Keep the level of the needle facing upward and the shaft at an angle of 150 to the
arm.
Once the needle is beneath the skin, release the clamp.
Insert the blood bag needle into the vein for about 1 to 1.5cms by a bold single
prick to ensure smooth flow of blood and secure on the arm with adhesive strips.
Advise the donor to gently squeeze the hand roller to improve blood flow.
If the venepuncture is unsuccessful do not make further attempt in the same arm.
Take the donor's permission for a second attempt. Use a new bag.
Once blood enters the bag tubing, press the bio mixer 'start' switch to allow the
blood to flow into the bag. After the programmed volume of blood is collected,
the bio mixer automatically clamps the tubing.

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Blood collection will be completed with in 6 to 8 min.
Clamp the bloodline at 2 sites and cut in the middle. Collect blood in the pilot
tubes from the tubing so that blood flows directly into the tubes from the donor
arm.
Release the tourniquet and remove the needle gently from the donor's vein
pressing the phlebotomy site. Fasten a Velcro cuff around the donor's arm in a
flexed position.
Seal the blood bag tubing with the tube sealer.
Burn the needle of the bag in the needle destroyer. Discard the tubing with the
burnt needle in a container of sodium hypochlorite solution.

6.0 THERAPEUTIC PHLEBOTOMY:
Therapeutic Phlebotomy that is part of treatment is performed to treat polycythemia
vera, a condition that causes an elevated red blood cell volume (hematocrit). This
phlebotomy is also prescribed for patients with disorders that increase the amount of
iron in their blood to dangerous levels leading to hemochromatosis.
PROCEDURE:
Before the phlebotomy is begun, the red cell level in patient blood will be checked
from a small sample of blood; pulse and blood pressure are also checked. During
the phlebotomy according to the instruction given by patients physician volume of
blood will be taken from patient vein. The actual process takes approximately 15-20
minutes. Collection Procedure is as usual as regular phlebotomy process. After the
procedure is completed, it will be necessary for patient to take rest for 15-20
minutes and to drink some juice or water. The entire procedure takes about one
hour. Blood collected from the patient is discarded.
The following donor consent is taken from the patient before procedure:

Donor consent: I am fully aware about my blood Donation. My treating Doctor has
explained about the procedure. The blood will be collected from me and discarded.

The following Physician consent is taken from the physician before the process:

Physician consent: As patient is suffering from polycythemia I am considering this
patient for therapeutic phlebotomy. I have explained about the procedure to the
patient. Bleed _________ ml of blood from this patient. I here by give my consent for
therapeutic phlebotomy.

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6.0 DOCUMENTATION

Make entries in the donor register/ computer.
Make an entry of the failed venepuncture, as double prick.

SP-009
01-12-2007

2

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Donor Room

Post Donation Care


Post Donation Care

- Master File

STANDARD OPERATING
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The donor needs to be observed after blood collection, in order to attend to any adverse
reactions in the immediate post-donation period.

2.0 RESPONSIBILITY

The medical officer in attendance attends to the donor.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks, 13th edition 1999
Pgs. 98-99
Introduction to Transfusion Medicine - Zarin Bharucha & D.M. Chouhan 1
st

edition 1990 Pgs. 100-101

4.0 MATERIALS

Sterile swabs
Adhesive tape
Thrombophobe ointment
Leaflet for post donation instructions

5.0 PROCEDURE

To prevent adverse reactions like giddiness ask the donor not to get up from the
chair/cot for 5 minutes even if he feels perfectly all right.
Observe for another 10 minutes in the refreshment area whilst having coffee.

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Inspect the venepuncture site before the donor leaves the donor room. Apply an
adhesive tape only after oozing stops. If there is persistent oozing at the site of
venepuncture, apply pressure with a dry, sterile cotton swab. If there is
haematoma apply Thrombophobe ointment gently over the area after 5 minutes.
Inform the donor about the expected change in skin colour. If the pain persists,
ask him/her to apply ice.
Instruct the donor to drink adequate fluid on that day and avoid strenuous
activities.

6.0 DOCUMENTATION

Give a leaflet of post donation instructions to the donor.
Record any adverse reaction on the donor card.

SP-010
01-12-2007

4

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject
Donor Room

Donor Adverse Reactions


Management of adverse reactions in a
donor

- Master File

STANDARD OPERATING
PROCEDURES
Section : I
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Any adverse reaction in the immediate post-donation period requires to be attended to.

2.0 RESPONSIBILITY

The medical officer in attendance is responsible for managing the adverse reaction in the
donor.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks 13th edition 1999
Pgs. 99-100
Introduction to Transfusion Medicine Z.S. Bharucha & D.M. Chouhan 1st edition
1990, Pgs. 101-102

4.0 MATERIALS

Following materials are required to attend to any emergency arising in the post
donation period.
i. Oral medication
Analgesic Tablets
Calcium and Vitamin C Tablets
Electrolyte replacement fluid

ii. Injection
Epinephrine (Adrenaline)
Atropine sulphate
Pheniramine maleate

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Diazepam
Glucocorticosteroid
Glucose (Dextrose 25%)
Furosemide
Metoclopromide
Prochlorperazine maleate
Sodium bicarbonate
Glucose saline (Sodium chloride and Dextrose 500 ml.)
iii. Antiseptics
Savlon
Mercurochrome
Tincture benzoine
Hydrogen peroxide
iv. Miscellaneous
Bandages/Dressings
Band-aids
Anti-histaminic cream
Heparin and Benzyl Nicotinate ointment.
Smelling salt-Spirit of Ammonia
Analgesic balm
Tongue depressor
Disposable syringes and needles 22 g
Clinical Thermometer
Oxygen cylinder
Infusion set
Paper bag

5.0 MANAGEMENT OF ADVERSE REACTIONS

5.1 Giddiness/Syncope (vasovagal syndrome):

Raise feet and lower head end.
Loosen tight clothing (belt, tie etc.)
Ensure adequate airway.
Check pulse and blood pressure.
Apply cold compresses to forehead and back.
Administer inhalation of spirit of ammonia if needed. The donor should respond
by coughing which will elevate the blood pressure.
If there is bradycardia and hypotension- Administer inj. Atropine 1 ml IM, if
bradycardia continues for more than 20 minutes.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 30 of 197


Administer IV normal saline or dextrose saline infusions if hypotension is
prolonged.

5.2 Convulsions:

Keep the head tilted to the side; prevent the tongue bite; keep the airway patent
by inserting a tongue blade or gauze between the teeth.

5.3 Vomiting:

Usually this provides relief. If the donor feels nauseous or if vomiting is severe,
inject Stemetil. Usually subsides on its own.

5.4 Tetany/ muscularspasm/t witching:

These are usually due to hyperventilation in an apprehensive donor. Ask the
donor to breath in a paper bag, which provides prompt relief. Do not give oxygen.

5.5 Haematoma:

Release the tourniquet/pressure cuff immediately. Apply pressure on the
venepuncture site and withdraw the needle from the vein. Raise the arm above
the head for a few minutes. Apply thrombophob ointment gently around the
phlebotomy site after about 5 minutes. Advise the donor to apply ice if there is
pain and inform about the expected change in skin colour.

5.6 Eczematous reactions of the skin around venepuncture site:

Apply steroid ointment.

5.7 Delayed syncope:

These may occur as late as 30 minutes to 1 hour after donation, usually after the
donor has left the blood bank. Permanently defer any donor who gives history of
such attacks more than twice.

6.0 DOCUMENTATION

Enter details of adverse reactions and the management in the donor card/form or
register.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 31 of 197


Keep a record of stocks of materials required, especially the expiry date of
medicines.

SP-011
01-12-2007

2

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Donor Room

Traceability of Blood bags

Method of accurately relating product
to donor

- Master File

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 32 of 197



To label the blood bags and pilot tubes after verification of donor details in order to
accurately relate the blood product to the donor. The unit number label is the unique
identifier for the donor and all the blood components separated from the unit collected
from the donor.

2.0 RESPONSIBILITY

It is the responsibility of the phlebotomist collecting the blood units to ensure proper
labeling and recording of the requisite details, even if the donor area attendant affixes the
labels.

3.0 MATERIALS

Sticker labels with pre printed serial number (10 Labels/Unit Number).

4.0 PROCEDURE

Give each donor a unique number and once his blood is collected, identify by that
number only.
Do not write donor's name on his/her blood bag or sample tube. This maintains
the donor's confidentiality.
Affix pre printed number labels on the primary bag on both sides, on all the
satellite bags in case of multiple bags and the three pilot tubes (2 plain and one
with CPD anticoagulant).

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 33 of 197


Verify the donor's identity by tallying with the name on the master registration
card. Affix the unit number label, which is loosely attached to the bag now to the
card.
Cross check the numbers on the bag, pilot tubes and master registration card to
ensure identity. Record the entry in the donor register using the same number.
Transcribe this number on all records hence forth for storage, testing and
distribution.
While issuing the unit, use the same number on issue record.

5.0 DOCUMENTATION

Make sure that the number is written clearly on all records and there are no
transcription errors, as this number will trace any product to the donor of the
blood and vice versa in case of requirement.

SP-012
01-12-2007

5

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Red Cell Serology Laboratory

ABO Blood Group


Cell and Serum Testing by Tube
Method

- Technician
- Master File

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 34 of 197



To determine the correct ABO group of an individual and ensure the reliability of the
result. This procedure describes the method of detection of ABO antigens on the red cell
and the reciprocal antibodies in the serum (Landsteiners Law). It provides guidance for
the use of blood grouping reagents (antisera & standard red cells) in order to detect weak
variants, acquired antigens, Bombay (O) blood group and irregular red cell antibodies.

2.0 RESPONSIBILITY

It is the responsibility of the technician/supervisor in the red cell serology laboratory to
perform the ABO grouping of donors and patients. One technician performs red cell
testing and the other serum testing. The supervisor checks the results. If a discrepancy is
encountered in cell and serum grouping, all tests should be repeated by the same
technician using anti A1and anti H lectins if required. If the discrepancy persists, the
sample should be handed over to the advanced red cell serology laboratory for further
workup. It is the responsibility of all staff performing the ABO grouping to ensure that
quality controlled reagents and proper cell concentrations are used.

3.0 REFERENCES

Technical Manual of the American Association of Blood Banks, 13 Edition,
1999, pages 150-151, 270, 277-280, 378-379, 285-286, 650-651.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 35 of 197


Introduction to Transfusion Medicine, Zarin Bharucha and D.M. Chouhan, 1
edition, 1990. Pages 43-47.
Procedures in Blood Banking and Immunohaematology - H.M. Bhatia, 1977.
Pages 13-15.

4.0 MATERIALS

Equipment:
Refrigerator to store samples and reagents at 2- 6
0
C.
Tabletop centrifuge.
Microscope.

Specimen:
Clotted and anticoagulated blood samples of donors.
Clotted blood sample of patients.
Test red cells suspended in native serum/plasma or saline.

Reagents:
Anti A, Anti-B, Anti-AB antisera.
Group A,B and O pooled cells.
0.9% saline.
Distilled water.

Glassware:
Serum tubes.
Micro tubes.
Pasteur pipettes.
Glass slides.

Miscellaneous:
Rubber teats.
Disposal box.
2 plastic beakers.
Wooden blocks to hold micro tubes.
Aluminium racks to hold sample tubes.

5.0 PROCEDURE

5.1 Principle:

ABO system is the only system in which there is a reciprocal relationship between the
antigen on the red cells and the naturally occurring antibodies in the serum. Routine

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 36 of 197


grouping of donors and patients must therefore include both RBC and serum tests, each
serving as check on the other.
The procedure is based on the principle of agglutination of antigen positive red cells in
the presence of antibody directed towards the antigen.
5.2 RBC Testing

1. Label tubes with donor/patient and test identification.
2. Prepare cell suspension for cells being tested (Refer SOP 015)
3. Place two drops of anti-A, anti-B and anti-AB reagent in the appropriately
labeled tubes.
4. Add to each tube one drop of a 2 - 5% cell suspension of the red cells to be
tested.
5. Mix the contents of the tubes gently and incubate at room temperature for 15
minutes.
6. Centrifuge at 1000 rpm for 1 minute.

(Note: Always follow manufacturer's instructions from package insert)
5.3 Serum Testing

Label tubes with donor-patient and test identification.
Add 2 drops of test serum in all tubes in the corresponding column.
Prepare cells for testing of A, B and O groups by pooling 3 samples of each
group. (Refer SOP 015)
Add 1 drop of 2% pooled A red cell suspension in tube labeled A
Add 1 drop of 2% pooled B red cell suspension in tube labeled B
Add 1 drop of 2% pooled O red cell suspension in tube labeled O
Mix the contents of the tubes gently and incubate the test for minimum 15
minutes at room temperature.
Centrifuge all tubes at 1000 rpm for 1 minute.
Gently resuspend the red cell button & examine for agglutination.

5.4 Results
Depending on presence (+) or absence (-) of agglutination, the position of the
tubes is changed.
Confirm the cell grouping results with those obtained in serum grouping and vice
versa.

5.5 Interpretation
Agglutination in any tube of RBC tests and agglutination or haemolysis in serum
test constitutes a positive test result. The expected agglutination reaction for
positive tests is 3+ to 4+.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 37 of 197


A smooth suspension of RBCs after resuspension of RBC button is a negative test
result. All negative results must be verified under microscope. Cells should be
separate without any clumping.
The interpretation of ABO group is as follows:

Cell Typing
Serum Typing
Interpretation of
ABO Group
Anti-A Anti-B Anti-
AB
AC BC OS
+ - + - + - A
- + + + - - B
+ + + - - - AB
- - - + + - O
- - - + + + O(Bombay Phenotype)

Resolve any discrepancies between cell and serum typing tests before the
patient's or donor's ABO group is interpreted.

6.0 DOCUMENTATION

Enter the results of donor grouping in the donor grouping register and computer.
Enter the results of patients grouping in the patient grouping register, blood group
requisition form, serial case number register and computer.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 38 of 197


SP-013
01-12-2007

4

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Red Cell Serology Lab

Rh D Typing


Tube Test for Rh testing

- Technician Red Cell Serology Lab
- Master File

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 39 of 197



This Standard Operating Procedure (SOP) provides the method to be followed to
determine the Rh D type of an individual and ensure the reliability of the result. This
procedure describes the method for detection of D antigen on the red Cells. It provides
guidance for the use of anti D blood grouping reagent.

2.0 RESPONSIBILITY

perform the D typing of donors and patients using one monoclonal and one biclonal
reagent. If a discrepancy is encountered between the two batches of anti D, the test
should be repeated by the same technician. If the discrepancy persists, the sample should
be handed over to the advanced red cell serology laboratory for further work up. If
results of D typing of a blood donor are negative, the technician should proceed with D
typing procedure. It is the responsibility of all staff performing the D typing to ensure
that quality controlled reagents and proper cell concentration are used.

3.0 REFERENCES

Technical Manual of the American Association of Blood Banks 13
th
Edition,
1999. Pages 150-151, 307-312, 657-658.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 40 of 197


Introduction to Transfusion Medicine; Zarin Bharucha & D.M. Chouhan, 1st
edition, 1990. Pages 47-48.
Procedures in Blood banking and Immunohaematology; H.M. Bhatia, 1977,
Page 37.

4.0 MATERIALS

4.1 Equipment:
Refrigerator to store samples and reagents at 2-6 C
Tabletop centrifuge
Microscope
Incubator/dry bath
4.2 Specimen:
Clotted or anti-coagulated blood samples of donors
Clotted blood sample of patients
Test red cells suspended in native serum/plasma or saline

4.3 Reagents:
Anti D biclonal
Anti D monoclonal (IgM/IgG blend)
0.9% saline
Distilled water
4.4 Glassware:
Serum tubes
Micro tubes
Pasteur pipettes
Glass slides

4.5 Miscellaneous:
Rubber teats
Disposal box
2 plastic beakers
Wooden block to hold micro tubes
Aluminium racks to hold serum tubes

5.0 PROCEDURE

5.1 Principle:

Testing with anti-D is necessary to determine if red blood cells possess or lack D blood
group antigen. Absence of agglutination is a negative test result, which indicates that the
D antigen is not demonstrable. Agglutination of red blood cells with an anti-D reagent is
a positive test result, which indicates the presence of the D antigen on the red blood cells.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 41 of 197


5.2 D Typing:

Label tubes with patient/unit and test identification.
Prepare cells for testing in accordance with the Preparation of Cell Suspension
(SOP 015)
Add one drop of reagent anti-D to the test tube.
Using a pipette, add one drop of the cell suspension to each test tube.
Mix well (incubation temperature and time depends on manufacturer's
instructions).
5.3 Results

Centrifuge all tubes at 1000 rpm for 1 minute (or as specified by manufacturer).
Gently resuspend the red cell button and examine for agglutination.
Grade and record test results.

5.4 Interpretation:

Agglutination of the red blood cells in the presence of reagent is a positive test
result and indicates the presence of the D antigen.
A smooth suspension of RBCs after resuspension of RBC button is a negative test
result. All negative results must be verified under microscope. Cells should
appear separate without any agglutination.
The interpretation of Rh D type is as follows:

Cell Typing Anti D
Biclonal
Cell Typing Anti D
Monoclonal
Interpretation of Rh D Type
+ + Positive
- - Negative

Proceed Du testing every negative result

N.B.:
Invalid test results may be obtained with this reagent if the blood tested is from a
person with auto antibodies or abnormal serum proteins. Concurrent testing for
the ABO blood group serves as a routine simultaneous control. The simultaneous
use of an Rh control is required only when the cells under test are found to be
reactive with anti-A, anti-B and anti-D. If the use of an additional control is
necessary, isotonic saline or 6% to 8% albumin in isotonic saline or a patient auto
control may be used. If the control gives a positive result, a valid interpretation
cannot be made.
Cord red blood cells heavily sensitised with anti-D may demonstrate a false
negative test result.

6.0 DOCUMENTATION

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 42 of 197


Enter the result of donor grouping in the donor grouping register and computer.
Enter the results of patients grouping in the patient grouping register, blood group
requisition form, serial case number register and computer.

SP-014 01-12-2007

3

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Advanced Red Cell Lab

Du Testing


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 43 of 197


Sample tested for Du testing by Tube
method

- Supervisor
- Master File

1. SCOPE OF APPLICATION

Du testing is mandatory to confirm whether patient sample is negative for Antigen-D.

2. RESPONSIBILITY

It is the responsibility of the technician of advanced red cell laboratory to do Du testing
and document the results.

3. REFERENCE

Technical manual of A A B B

4. MATERIALS REQUIRED

Anti D antisera
Normal saline

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 44 of 197


5. EQUIPMENT

Refrigerator to store samples & reagents at 2-6
o
C
Tabletop centrifuge
Incubator at 37
0
C

6. SPECIMEN

Plain sample

7. REAGENTS

Anti D biclonal, Normal saline
Anti D mono clonal AHG. Reagent

8. GLASSWARE
Test tubes
9. PRINICIPAL

All Rh ve samples should be tested. A weaker variant of D- antigen is termed Du.
Du positive red cells agglutinate with anti-D sera and react by the AHG technique.

10. METHOD

Take two drops of Anti- D polyclonal sera in a clean labeled test tube.
Place appropriate controls reagent in a second test tube
To both tubes add one drop of cells ( 2-5 % of RBC suspension )
Mix and incubate at 37 degree C for 45 minutes.
Centrifuge for 1 minute at 1000 rpm
See for agglutination. If there is no agglutination / show doubtful, wash the RBC
3-4 times with sterile normal saline. Completely decant the final wash and dry the
cell button.
Add 1-2 drops of AHG reagent & mix.
Centrifuge at 1000 rpm for 1 minute
If there is agglutination the test is given DU positive and treated as Rh positive as
a donor. As a patient he is Rh Negative
If there is NO agglutination the test is given DU negative and treated as Rh
negative.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 45 of 197


Re-examine and Re-centrifuge.
Every negative result Add 2drops of igg coated cells, centrifuge 1000rpm for 1
minute, If result is negative test is invalid total test should be repeated, test is
positive test is valid.

11. INTERPRETATION

Du-Positive -- Donor blood should not be given to Rh ve patient. Here the
donor considered as Rh +ve .
Du-Positive -- Patient should always receive Rh -ve blood.

12. DOCUMENTATION

All results should be entered in blood grouping register and computer.
All records are initialed by the technician who performed the test and by the
technician who has checked the results.

SP-015
01-12-2007 4 Dr V Saraswathi
No. of
Copies
Approved By Date
1.0

1 year

15-05-2008

Location Subject

Preparation of Red Cell Suspensions


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 46 of 197


To prepare RBC suspension of
appropriate concentration for a given
test
- Supervisor of Red Cell Serology
Laboratory
- For All Technicians in Lab
- Master File


This procedure applies to all testing that requires red cell suspension preparation.

2.0 RESPONSIBILITY

It is the responsibility of every technician performing a given test to prepare the
appropriate red cell suspension. Every morning, the shift duty technician must prepare A,
B & O red cell suspension for the day's use.

3.0 REFERENCES

Technical Manual of American Association of Blood Bank, 13 Edition,
1999,Pages 150, 311
Introduction to Transfusion Medicine; Zarin Bharucha & D.M. Chouhan, 1
st

Edition, 1990. Page 262.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 47 of 197


4.0 MATERIALS

4.1 Equipment:

Calibrated centrifuge.

4.2 Reagents:

0.9% saline.

4.3 Specimen:

Clotted or anticoagulated blood specimen of donor.
Clotted or anticoagulated blood specimen of patient.
Donor unit segment.

4.4 Glassware:

Pasteur pipettes.
Serum tubes.

4.5 Miscellaneous:

Discard box.
2 plastic beakers.
Rack to hold tubes.

5.0 PROCEDURE

5.1 Principle:

The ratio of serum to red cells may dramatically affect the sensitivity of agglutination
tests. Consistent preparation of either 2 to 5% red cell suspension is critical to any
agglutination test.

5.2 Pooled Cell Suspension:

Label tubes with A,B, and O groups.
Place 1 drop of red cells each from 3 of A group sample tubes or segment into the
A labeled tube.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 48 of 197


Place 1 drop of red cells each from 3 of B group sample tubes or segment into the
B labeled tube
Place 1 drop of red cells each from 3 of O group sample tubes or segment into the
O labeled tube
Fill the tube full with 0.9% saline to resuspend the cells.
Centrifuge the tubes for at least 2 to 3 minutes on high speed. Decant the
supernatant fluid.
Remove any debris or fibrin with the pipette. Add enough saline to produce a
cherry red colour comparable to that of the reagent red cell suspension.
If the colour is too dark, add additional isotonic saline to the tube until the
suspension colour is right.
If the colour is too light, repeat steps 6 and 7.
Test the pooled cells prepared using the antisera (anti-A, B, AB and D) in use.

5.3 Donor/Patients' sample

Proceed to use the same procedure to prepare cell suspension of particular donor or
patient sample for grouping and cross matching.

5.4 Limitations:

Hemolysis of the red blood cells from improper washing may result in false
results. Cell suspension that is too heavy or too light may produce false positive
or false negative results.
6. DOCUMENTATION

Enter the donor unit numbers from which pooled cells are prepared in the donor
register.
Record the results of testing with the antisera in use.
Enter the manufacturer's name and batch number of the antisera.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 49 of 197


SP-016
01-12-2007

5

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Reliability and Reproducibility of Blood
Group Results


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 50 of 197


Daily Quality Control of ABO & Rh
Blood Group reagents

- In-charge Red Cell Serology Laboratory
- Master File


This Standard Operating Procedure (SOP) provides the daily checks on blood group
reagents to ensure reliability and reproducibility of blood group results.

2.0 RESPONSIBILITY

It is the responsibility of the technician / supervisor in the red cell serology
laboratory to ensure that quality controlled reagents and proper cell
concentrations are used for testing for which daily quality control checks and test
controls are used with proper documentation. The reagents should be stored and
used as per manufacturer's instruction.
Any fault in the reagents should be immediately reported to the Quality
Assurance Manager.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 51 of 197


Technical Manual of the American Association of Blood Banks 13
th
edition 1999,
Page 22.
Introduction to Transfusion Medicine Zarin Bharucha and D.M. Chouhan; 1
st

Edition, 1990. Pages 225-226.

4.0 MATERIALS

4.1 Equipment:

Refrigerator to store samples and reagents at 2-6 C.
Table top Centrifuge.
Automated Cell Washer.
Microscope.

4.2 Reagents:

Anti-A, Anti-B, Anti-AB, Anti D(Monoclonal and Bioclone) Antisera.
Clotted or anticoagulated blood samples of random blood donors.
Group A,B and O pooled Cells.
0.9% saline.
4.3 Glassware:

Serum tubes.
Micro tubes.
Pasteur pipettes.
Glass slides.

4.4 Miscellaneous:

Rubber teats.
Disposal box.
2 plastic beakers.
Wooden block.
Aluminium racks.

5.0 PROCEDURE

5.1 Principle:

Test for reactivity and specificity is based on the principle of agglutination of antigen
positive red cells in the presence of antibody directed towards the antigen.

5.2 Quality Control Checks:

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 52 of 197


5.2.1 Visual Inspection:

Examine each vial carefully for precipitate, gel formation, turbidity or change in
colour.

5.2.2 Reactivity and Specificity:

Add one drop of 3 to 5% suspension of the appropriate red cells to the one-drop
of antiserum in a micro tube.
Mix well and incubate (as per manufacturer's instruction).
Note the reactions as under:

Red Cells for Testing
Positive Reactors Negative Reactors
Anti - A
Pooled A Cells
Pooled B Cells, Pooled O
Cells, O LISS cells
Anti B Pooled B Cells
Pooled A Cells, Pooled O
Cells, O LISS cells
Anti AB Pooled A cells, Pooled B
Cells
Pooled O Cells, O LISS
cells
Anti D Bioclonal RhD - Positive Cells
Any ABO Group)
RhD - Negative Cells
Any ABO Group)
Anti D Monoclonal RhD Positive
(Any ABO Group)
RhD - Negative
Any ABO Group)

5.3 Results:


Record presence or absence of precipitate, gel formation, turbidity or colour
change.


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 53 of 197


Centrifuge the Tubes (as per manufacturer's instruction).
Resuspend the red cell button and examine for agglutination / haemolysis.
Grade and record test results.

5.4 Interpretation:


The presence of precipitate, gel formation, turbidity, colour change indicates that
the reagent is contaminated and should not be used.
The absence of all the above indicates that the reagent is 'clear' and suitable for
use.


Agglutination of specific red cells is a positive reaction and indicates the
reactivity of the corresponding antibody in the reagent. The expected
agglutination reaction for positive test is +3 to +4.
The absence of agglutination / haemolysis is considered to be a negative reaction
and indicates the absence of the corresponding antibody specificity in the reagent.
Clear-cut negative reactions with the negative reactors rules out the presence of
irregular agglutinins and haemolysis in the reagent.

6.0 DOCUMENTATION

Enter the results in the Blood Group Register in the Red Cell Serology
Laboratory. Enter identification number of the individual donor cells used for
pooling and the reaction strengths.
Sign the results as the individual preparing the pooled cells and testing the
reagent.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 54 of 197


SP-017
01-12-2007

5

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject
Antibody Screen


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 55 of 197


Detection of Unexpected Blood Group
Antibodies

Laboratory
- Master File


This procedure applies to all testing that requires antibody screening, including donor
units, patient's pre-transfusion blood grouping and prenatal specimens.

2.0 RESPONSIBILITY

perform the antibody screen using proper cell concentrations. One technician performs
all tests and another checks it. If any unexpected blood group antibody is detected,
inform the staff of Advanced Red Cell Serology for further investigations.

3.0 REFERENCES

Technical Manual of the American Association of Blood Banks 13th Edition,
1999. Pages, 256-262, 383-384, 392-393, 379, 668-671, 676.
Procedures in Blood Banking & Immunohaematology H.M. Bhatia, 1977, Pages
72-75

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 56 of 197


Introduction to Transfusion Medicine Zarin Bharucha & D.M. Chouhan, 1
st

Edition 1990. Pages 51, 58-60, 69-71, 85.

4.0 MATERIALS

4.1 Equipment:

0
C.
Deep Freezer to store enzyme papine cystein in frozen state.
Automated cell washer (for patient pre-transfusion and prenatal testing).
Microscope.
Dri bath/ Incubator

4.2 Specimen:
Clotted blood sample of donors/patients.

4.2 Reagents:

Group O polled cells/Antibody-screening reagent red blood cells (two or three
cells).
Papain cystein.
22% Bovine albumin.
Antihuman globulin reagent (anti-IgG+anti-C3d)
IgG sensitised control cells.
0.9% saline
Distilled water

4.3 Glassware:

Serum tubes.
Coombs' tubes (for patient pre-transfusion & prenatal testing).
Micro tubes.
Pasteur pipettes.
Glass slides.

4.4 Miscellaneous:

Rubber teats.
Disposal box.
2 plastic beakers.
Aluminium racks to hold serum and Coombs' tubes.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 57 of 197


5.0 PROCEDURE

5.1 Principle:

The antibody screen test is used in the detection of unexpected blood group antibodies.
In this test, pooled O cells or the antibody-screening reagent red blood cells are
combined with serum under investigation. The addition of a potentiating medium
enzyme / albumin helps to promote the interaction of red cells and antibodies allowing
antibody/antigen reactions to occur. Positive reactions (haemolysis or agglutination) in
any tests indicate the presence of alloantibody or autoantibody in the serum.

5.2 Antibody Screen:

1. Label tubes with donor/patient and test identification.
2. Add two drops of test serum to each tube.
3. Add 1 drop of papain cystein to all tubes labeled 'enzyme' (if enzyme method is
being followed).*
4. To each of the tubes labeled 'saline' or 'enzyme/albumin', add 1 drop of 2%
pooled O red cell suspension (or 2% suspension of the antibody-screening
reagent red cells).
5. Add 1 drop of 22% abovine albumin to tubes labeled 'albumin' (if albumin
method is being followed).*
6. Add 1 drop of 5% pooled O red cell suspension (or 5% suspension of antibody-
screening reagent red cells) to tubes labeled 'IAT', followed by 2 drops of 22%
bovine albumin.
7. Mix the contents of the tubes gently and incubate for minimum 15 minutes.

Test
Incubation Temperature
Ideal Incubation Time
Saline Room Temperature 1 Hour
Enzyme
37
0
C 45 Minutes
Albumin
37
0
C 45 Minutes
IAT
37
0
C 1 Hour 90 minutes

* Either enzyme or albumin method may be followed for detection of incomplete
antibodies.

5.3 Results:

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 58 of 197


1. Centrifuge saline, enzyme and albumin tests at 1000rpm for 1 minute.
2. Examine for haemolysis.
3. Gently resuspend the red cell button and examine for agglutination.
4. Examine all visually negative tests microscopically.
5. Grade and record test results immediately.
6. Proceed to perform antiglobulin phase of the indirect antiglobulin test on tubes
labeled 'IAT'.
7. Wash the cells 3 times with saline. Decant completely after last wash.
(Washing can be done manually or using automated cell washer).
8. Add 2 drops antihuman globulin reagent to the dry cell button.
9. Mix well and centrifuge at 1000 rpm for 1 minute.
10. Read and record results.
11. Add drop IgG sensitised cells to all negative results. This shows a positive
agglutination.

5.4 Interpretation:
1. Hemolysis or agglutination in any test may indicate the presence of an
unexpected antibody.
2. The absence of agglutination and hemolysis in all tests is a negative test result.
3. After addition of IgG-sensitized cells to a negative test, the presence of
agglutination indicates that the AHG serum added was capable of reacting and
that the negative antiglobulin test is valid.

4. If IgG-sensitised cells added to confirm the activity of the anti-IgG show only
weak or no agglutination after centrifugation, the test is invalid and must be
repeated.

5.5 Limitations:
If tests with all reagent red cells are reactive, the possibility of spontaneous agglutination
should be considered. A control of cells washed three to four times added to two drops of
saline must be non-reactive.

6.0 DOCUMENTATION

Results of donor unit antibody screen are entered in the donor grouping register
and computer.
Results of patients antibody screen are entered in the patient grouping register,
blood group requisition form, serial case number register and computer.
All records are initialed by the technician who has performed the test and by the
technician who has checked the results.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 59 of 197


SP-018
01-12-2007

4

Dr V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject


Detection of Incompatibility Between
Patient and Donor

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 60 of 197


Saline / Albumin / Enzyme cross match

Laboratory
- Serology Lab
- Master File


This procedure is applied for compatibility testing of all patients requiring transfusion.

2.0 RESPONSIBILITY

It is the responsibility of the technician in the red cell serology laboratory to perform
cross match and document the results. If any unexpected antibody is detected, the
advanced Red Cell Serological should be informed.

3.0 REFERENCES

1999. Pages 380-381, 383-384, 256-257, 392-393, 667-668
st

Edition, 1990. Pages 82-85, 58-59.

4.0 MATERIALS

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 61 of 197


4.1 Equipment:

0
C
Deep Freezer to store enzyme papine cystein in frozen state.
Microscope.
Dri bath.

4.2 Specimen:

Clotted blood sample of donors/patients.

4.2 Reagents:
Group O polled cells/Antibody-screening reagent red blood cells (two or three
cells).
Papain cystein.
22% Bovine albumin.
Antihuman globulin reagent (anti-IgG+anti-C3d)
0.9% saline
Distilled water
4.3 Glassware:

Serum tubes.
Coombs' tubes (for patient pre-transfusion & prenatal testing).
Micro tubes.
Pasteur pipettes.
Glass slides.

4.4 Miscellaneous:

Rubber teats.
Disposal box.
2 plastic beakers.
Aluminium racks to hold serum and Coombs' tubes.

5.0 PROCEDURE

5.1 Principle:

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 62 of 197


The major cross-match is used to detect unexpected blood group antibodies in patient's
serum against antigens on donor cells. Positive reaction in any test indicates
incompatibility.

5.2 Cross-match:

Saline Cross Match:
1. Take two drops of patient serum.
2. Add two drops of donor cells(3 times saline washed cells)
3. Centrifuge 1000 rpm for 1 minute.
4. Shake gently and observe.

Albumin Cross Match
1. Take two drops of patient serum, add one drop of donor cells(3 times saline
washed cells), add one drop of albumin.
2. Incubate 37
0
C for 45 minutes, centrifuge and observe.
3. If you get negative result follow SP-019 procedure 4 to 8 steps.

Enzyme technique
1. Take two drops of patient serum, add one drop of donor cells(3 times saline
washed cells), add one drop of pap-cystine.
2. Incubate 37
0
C for 45 minutes, centrifuge and observe.
3. If you get negative result follow SP-019 procedure 4 to 8 steps.

5.3 Interpretation:

Hemolysis or agglutination indicates the presence of a serologically incompatible
cross-match. This result is interpreted as Incompatible.
Absence of agglutination and hemolysis is a negative test result and indicates a
serologically compatible crossmatch. This result is interpreted as Compatible.
If the IgG-sensitised control cells added to confirm the activity of the
polyspecific reagent show only weak or no agglutination the test is invalid and
must be repeated.

5.4 Limitations:
The saline / enzyme cross match will not:
Prevent isoimmunisation of the recipient
Ensure normal red blood cell survival
Detect some weakly reactive antibodies

6.0 DOCUMENTATION

Enter all results on the transfusion record card and OT/Ward transfusion register.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 63 of 197


Enter only the results of compatible units in the blood compatibility form. The
technician who performed the test and the one who checked the results sign all
records

SP-019
01-12-2007

4

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject


Anti Globulin Cross Match


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 64 of 197


Detection of incompatibilities caused by
Warm complete antibodies

- Master File


This procedure applies to compatibility testing of all multi-transfused patients and
transfusion recipients who currently demonstrate or have a history of clinically
significant antibodies.

2.0 RESPONSIBILITY

It is the responsibility of the technician in the cross match facility of the red cell serology
laboratory to perform the anti-globulin cross match using quality controlled reagents and
proper cell concentrations. One technician performs the tests and another checks it. If
any unexpected blood group antibody is detected, inform the staff of Advanced Red Cell
Serology to carry out further investigations.

3.0 REFERENCES

1999. Pages 380-381, 383-384, 258-262, 668-671.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 65 of 197


Procedures in Blood Banking and Immunohaematoology; H.M. Bhatia, 1977.
Pages 72-75.
st

Edition, 1990. Pages 69-71, 74-76, 85.

4.0 MATERIALS

4.1 Equipment:

Refrigerator to store samples & reagents at 2- 6 C.
Microscope.
Dry bath

4.2 Specimen:

Clotted blood sample of patient.
Segment from donor unit.
Donor red cells suspended in saline.

4.3 Reagents:

22% bovine albumin.
Antihuman globulin reagent(anti-IgG+anti-C3d).
0.9% Saline.
Distilled water.

4.4 Glassware:

Serum tubes.
Coombs' tubes.
Pasteur pipettes.
Glass slides.

4.5 Miscellaneous:
Rubber teats.
Disposal box.
2 plastic beakers.
Aluminium racks to hold serum and coombs' tubes.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 66 of 197


5.0 PROCEDURE

5.1 Principle:

The cross match through the anti-globulin phase permits detection of clinically
significant incompatibilities caused by incomplete antibodies that sensitise cells at 37
0
C,
but do not directly cause agglutination.

5.2 Anti-Globulin Cross-Match:

Take two drops of patient serum. Add one drop of 5% washed O pooled
cells.(sp-15)
Incubate at 37
0
C for minimum 45 to 60 minutes. (Follow manufacturer's
directions when using commercial reagents).
Wash the cells a minimum of 3 times with saline. Decant completely after last
wash. Add two drops of antihuman globulin reagent to the dry cell button.
Mix well and centrifuge at 1000 rpm for 1 minute.
Re-suspend and read for agglutination. Grade and record test results immediately.
To all negative antiglobulin tests add 1 drop of IgG-sensitised control cells
,centrifuge and observe, if result is negative test is invalid total test should be
repeated, If test is positive test is valid, result is negative.

5.3 Interpretation:

must be repeated.

5.4 Limitations:
The anti-globulin cross- match will not:

Detect error in Rh typing.
Prevent isoimmunisation of the recipient.
Ensure normal red blood cell survival.
Detect some weakly reactive antibodies.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 67 of 197


6.0 DOCUMENTATION

Enter all results on the transfusion record card and OT/Ward transfusion register.
Enter only the results of compatible units in the blood compatibility form. The
technician who performed the test and the one who checked the results sign all
records

SP-020
01-12-2007

4

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

TTI Screening Lab

LISS Coombs Gel Technique


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 68 of 197


Sample tested for LISS Coombs by ID
Card method

- Supervisor
- Master file

1. SCOPE & APPLICATION

LISS Coombs Gel Technique is mandatory for detection of unexpected antibodies.

2. RESPONSIBILITY

It is the responsibility of the technition in red cell serology laboratory toperform LISS
COOMBS GEL TECHNIQUE and document the results.

3. REFERENCE

Kit package insert


Refrigerator to store samples of reagents at 2-6
o
C

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 69 of 197


Incubator
Test Tubes
I.D. cards
Diamed Centrifuge

5. REAGENTS

LISS Solution
O pooled cells

6. SPECIMEN

EDTA Sample for DCT
Plain sample for ICT
Plain sample for Cross Matchings and grouping

7. PRINICIPAL

The LISS Coombs technique is used to detect unexpected antibodies, minor blood
group antibodies, in patients serum against O pooled cells. Positive test indicates
presence of antibodies and incompatibility if x matching done.

8. METHOD

Direct Coombs Test
Take clean test tube and place one ml LISS solution.
Add 25 ul of patient blood (EDTA) and MIX.
Add the mixture (LISS + 25 ul of blood) 50 micro liters into ID card
tube.
Centrifuge with (Diamed Centrifuge) at 1030 pm for 10 minutes.
See for agglutination and grade the result.

Indirect Coombs Test
Take clean test tube and place 1ml of LISS solution.
Add 10ul of O pooled packed RBC to tube
Add 50 ul of LISS RBC in to ID card tube
Add 25 ul of patient serum to tube
Incubate at 37
o
Cfor 15 minutes
Centrifuge in (DiaMed Centrifuge) at 1030 rpm for 10 minutes
See for agglutination and grade the result.
All negative tests to be confirmed with Ig G coated cells

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 70 of 197


LISS COOMBS X-MATCHING

Take clean test tube and place 1 ml of LISS solution.
Add 10 micro liters of packed donor red cells or 25 micro liters of
donor whole blood to tube.
Add 25 micro liters of patient serum to the ID gel card tube. And also
add 50 micro liters of LISS donor cells.
Incubate at 37 for 15 minutes.
Centrifuse in ( Diamed centrifuse ) at 1030 rpm for 10 minutes.

9. INTERPRETATION;-

For unexpected antibodies

If agglutination is there it is Positive for antibodies.
If no agglutination it is Negative for antibodies

For X- Matching

If cells settled down and form a clear red cell button it is -- compatible
If line is formed it is -- incompatible.

10. DOCUMENTATION

Enter results in Coombs register and computer.
All records are initialed by technician who performed the test and Medical
Officer who checked the results.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 71 of 197


SP-021
01-12-2007

3

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Advanced Serology Laboratory

Gentle Heat Elution Technique


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 72 of 197


Sample tested for X-matching in bellow
6months babies by Gentle heat elution
technique.

- Incharge of serology lab
- Master file

1. SCOPE OF APPLICATION

Gentle heat elution technique is mandatory do cross matching below 6 months babies
in the absence of mother sample.

2. RESPONSIBILITY

It is the responsibility of the of the technician of red cell serology lab to do the gentle
heat elution technique and record the results.

3. REFERENCE



STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 73 of 197


Normal saline

5. EQUIPMENT

o
C
Tabletop centrifuge
Water bath at 45 degree centigrade.

6. SPECIMEN:

Baby sample

7. REAGENTS

Normal saline.

8. GLASSWARE

Glass test tubes
9. PRINICIPAL

When neonate red cells are heated in normal saline the mother antibodies which coated
on neonate red cells will get eluted into the normal saline. This Elute can be used to do
cross matching and serum grouping.

10. METHOD

This Technique is used in the cross matching of below 6 months babies sample when
the mother sample is not available.
Take baby blood sample.
Add sterile normal saline.
Incubate at 45 degree C water bath for 10 minutes for heating.
Centrifuge at 2500 rpm / minute for
Take the supernatant in another test tube.
Use this supernatant as mother sample as it contains mother antibodies for cross
matching & serum grouping.
To do cross matching & serum grouping see SOP procedures.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 74 of 197


11. INTERPRETATION

Able to do serum grouping indicates procedure is successful.

12. DOCUMENTATION

All results should be entered in cross matching register and computer.
All records should be initialed by technician who performed the test and the
technician who checked the results.

SP-022
01-12-2007

6

Dr V Saraswathi
No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Advanced Red Cell Serology
Laboratory

Investigation of Transfusion Reaction

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 75 of 197



To identify cause of transfusion reaction

- In-Charge of Advanced Red Cell
Serology Laboratory
- Master File


This Standard Operating Procedure (SOP) provides the protocol to be followed to
identify the cause of an adverse transfusion reaction and prevent its reoccurrence.

2.0 RESPONSIBILITY

It is responsibility of the technician in the Red Cell Serology Laboratory to accept the
blood/component implicated in the transfusion reaction, which is returned from the
ward/OT. It is the duty of the same technician to ensure that there is documented
evidence of the nature of reaction either on the transfusion request form or on a separate
letter addressed to blood bank, along with the post-transfusion blood sample (both EDTA
and clotted) and urine specimen, if necessary. The direct antiglobulin test (DAT) should
be performed on the post-transfusion EDTA sample immediately on receipt before
refrigeration. The unit and samples should be preserved properly and handed over to the
advanced red cell serology technician who is responsible for detail investigation

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 76 of 197


- Introduction to Transfusion Medicine: Zarin Bharucha and D.M. Chouhan 1
st

Edition, 1990. Pages 216-219.

4.0 MATERIALS

4.1 Equipment:
Refrigerator to store samples & reagents at 2- 6
0
C.
Microscope.
Dry bath / Incubator
Deep Freezer to store enzyme papain-cystein in frozen state.

4.2 Specimen:
Blood/component bag returned room ward/OT.
Patient's pre-transfusion blood sample (clotted).
Patient's post-transfusion blood sample (EDTA and clotted).
Patient's post-transfusion urine sample.
4.3 Reagents:
ANTI-a, Anti-B, Anti-AB Antisera.
Group A,B &O pooled cells.
Papain-cystein/22% Bovine albumin.
Antihuman globulin reagent(anti-IgG anti-C3d).
IgG Sensitised Control Cells.
0.9% Saline.
Distilled water.
30g/l sulfosalicylic acid solution.
Ammonium Sulphate - NH
4
(SO
4
)
2

4.4 Glassware:
Serum tubes.
Coombs' tubes.
Pasteur pipettes.
Glass slides.
Micro tubes.
Small funnel.
20ml test tubes.
5ml pipette.

4.5 Miscellaneous:
Rubber teats.
Disposal box.
2 plastic beakers.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 77 of 197


Aluminium racks to hold serum and coombs' tubes.
Wooden block to hold micro tubes.
Whatman No.1 filter paper.
5ml plastic vial with screw cap.

5.0 PROCEDURE

5.1 Principle:

Red Cell Serological tests are based on the principle of agglutination and help to identify
haemolytic transfusion reactions caused either by ABO incompatible transfusion or
irregular red cell antibodies in patient's blood.

Leuco-agglutinations, if present are detected by agglutination of random donor
leucocytes in cases of febrile transfusion reaction. Serum bilirubins total and indirect are
raised in case of haemolysis. The sulfosalicylic acid test helps to differentiate between
haemoglobin and non-protein pigment, probably porphyrin in the urine. The ammonium
sulphate precipitation test is based on the fact that haemoglobin and myoglobin are
precipitated in urine at different degrees of ammonium sulphate saturation.

5.2 Serological Tests

Perform a direct antiglobulin test (DAT) on post-transfusion EDTA sample
before refrigeration immediately on receipt. If test is positive, perform DAT on
pre-transfusion sample to verify whether sensitisation is due to transfusion or it
pre-existed.
Repeat grouping and antibody screening of patient's pre-transfusion sample.
Repeat grouping and antibody screening of patient's post-transfusion sample.
Repeat grouping and antibody screening of donor sample.
Repeat grouping of unit from bag. In case of packed cell unit, do only cell
grouping. In case of FFP, do only serum grouping.
Repeat crossmatching of donor with patient's pre and post transfusion samples
using saline / enzyme / IAT. Use donor cells from blood bag and not the pilot
tube.

5.3 Interpretation:
must be repeated.

STANDARD OPERATING
PROCEDURES
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Issue : 1.0
Date : 15-05-2008
Page : 78 of 197


5.4 Leucocyte Antibody Test:
In case of febrile transfusion reaction and hypotension, look for leukocyte
antibodies.

5.5 Biochemical Tests:
Note colour of plasma. Plasma is pink, if haemoglobin is present and icteric if
bilirubin is present.
Separate the patient's pre and post transfusion serum and send to biochemistry
department in a 5 ml screw cap plastic vial bearing the date, patient and test
identification for estimation of serum bilirubin total, direct and indirect and
estimation of plasma hemoglobin.
Send the biochemistry request form with proper entries along with the sample.
Collect the report from biochemistry lab.

5.6 Tests on post-transfusion urine sample.

Red colour indicates haematuria or haemaglobinuria.

Add 3ml of 30g/l solution of sulfosalicylic acid to 1 ml urine. Mix well and filter.

No precipitate Precipitate Formed
Filter retains Colour

Non-protein pigment is Pigment is a protein
probably porphyrin

Add 2 8 g NH
4
(SO
4
)
2
to 5 ml urine
(=80% saturation)

Shake and mix to dissolve NH
4
(SO
4
)
2

Filter

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 79 of 197


Filter Retains Colour Filter is clear
Precipitate is coloured

Myoglobin Haemoglobin

5.5 Microbiology:
Send the donor unit for smear and culture (at 37
0
C, room temperature and 4
0
C) to
bacteriology department.
Make proper entries in the bacteriology dispatch book and bacteriology request
form and send along with the unit.
Collect the report from bacteriology lab.
If donor unit reveals bacteremia, then request the attending doctor to get the
patient's blood culture done and report the findings to the blood bank officer.

5.6 Interpretation:
Any red cell incompatibility found during the investigation explains a haemolytic
transfusion reaction.
The DAT will be positive and a mixed field reaction will be seen if in vivo
sensitisation of transfused red cell has occurred.
The DAT may be negative even in cases of haemolytic transfusion reaction, if the
cell destruction is severe.
If any antibody is detected in patient's serum, the donor cells should be positive
for the corresponding antigen.
Detection of leucoagglutination explains a febrile reaction or hypotension.
Serum bilirubin total and indirect are raised in case of haemolysis.
Haemoglobinemic and haemoglobinuric are highly suggestive of red cell
destruction, but are not necessarily caused by antigen-antibody reaction, unless
confirmed.

5.7 Limitations:
The non-serologic possibilities of haemoglobinemia and haemoglobinuria are:-

Hemolysis of blood before transfusion.
Poor technique of collecting post transfusion sample.
Myoglobinuria following major surgery.
Infusion of distilled water during prostatectomy.
Hemolysis due to artificial valve.
Patient's clinical condition; Autoimmune haemolytic anemia or paroxysmal
nocturnal hemoglobinuria.
Use of glucose or dextrose through the same line before starting blood.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 80 of 197


Addition of certain drugs to blood such as ethacrynic acid, hydrocortisone or
diphenyl hydantoin.

6.0 DOCUMENTATION

Enter the transfusion reaction in blood issue register, showing date and time of
return of the unit and nature of reaction.
Enter the DAT/IAT results in the Antiglobulin test book in the red cell serology
laboratory.
Document the results of the entire investigations in the Transfusion Reaction
work up form.
Keep record in the Transfusion Reaction Record Register in advanced red cell
serology laboratory.

SP-023
01-12-2007

6

DR V Saraswathi

No. of
Copies
Approved By Date

1.0

1 year

15-05-2008

Location Subject

Component Laboratory

Blood Component Separation

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 81 of 197



Method for separation of blood
components

- Master File


For judicious use of blood it is necessary to use the components as per the need rather
than using whole blood. From the whole blood collected in double bags, packed cells and
FFP or Packed cells platelets are separated. From triple bags packed cells, FFP and
platelets or packed cells, FVIII deficient plasma and cryoprecipitate are separated. When
the plasma frozen at 80
0
C is thawed at 4
0
C, a cryoglobulin remains as a precipitate
which is called cryoprecipitate. It contains mainly F-VIII fibrinogen, VWF, Factor XIII.

2.0 RESPONSIBILITY

It is the responsibility of the component room technician to separate components from
whole blood collected in multiple bags.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks 13th edition 1999
Pgs 26, 168-170, 172-173,177, 716-717, 723-726

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 82 of 197


Introduction to Transfusion Medicine - Zarin Bharucha & D.M. Chouhan 1
st

edition 1990 Pg 124-125

4.0 EQUIPMENT & MATERIALS

Tube sealer
Laminar flow
Refrigerated centrifuge
Plasma expresser
Electronic weighing scale
Double pan weighing balance
Cryoprecipitate thawing bath
Double bags (450ml) or triple bags with SAGM solution (450ml), Quadruple
bags 450 ml.
Manuals of all equipment for reference regarding use and maintenance of each
equipment

5.0 PROCEDURE

Preparation of packed cells and FFP using Double bag:

Keep the units vertical on the laminar flow table for 30 to 45 minutes
(Process all units within 8 hours of blood collection).

Keep the bags in the buckets and balance them. Keep the equally balanced
buckets with bags diagonally opposite in the refrigerated centrifuge ensuring that
the position of the bags in buckets is parallel to the direction of the spin.
After centrifugation, gently remove the bags from the bucket and place them on
the expresser stand under the laminar flow. Break the integral seal of the tube
connecting it to the satellite bag/s manually and express the supernatant plasma
into the satellite bag. In case of double bag, leave 50- 60ml of plasma back along
with the red cells in the primary bag and this component is Packed Red Cells
(PC).
Label the plasma in the satellite bag, as Fresh Frozen Plasma (FFP) if separated
within 8 hours of collection and stored immediately below -20
0
C.
Cut the segment of FFP.

Preparation of packed cells, platelet concentrates and FFP using triple bags with or
without additive solution:

Process the blood collected within 8 hours.
Keep the bags erect on the laminar flow for 30-45 minutes.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 83 of 197


Note the weight of the primary bag and record in the register.
Balance the bags in the buckets using dry rubber or unused bags.
Keep equally balanced buckets diagonally opposite each other in the refrigerated
centrifuge.
Position the bags in buckets parallel to the direction of the spin. Centrifuge the
bags at 2400 rpm for 10 minutes at 22
0
C.
Keep the bag on the separator on the laminar flow. Break the seal of the tubing
connecting to the satellite bag. And express the plasma into the satellite bag
leaving 50-60 ml plasma along with the red cells. If the bag with additive solution
is used, remove all plasma in satellite bag before clamping. Remove the clamp of
the bag containing additive solution and let the additive solution slowly pass into
the primary bag containing red cells.
Mix the contents thoroughly and seal the tubing and detach the bags.
Keep the primary bag containing packed cells with additive solution in quarantine
storage in the blood bank refrigerator kept in the component room.
Label the bag and take it on the inventory after the testing is over.
Spin the satellite bag containing platelet rich plasma (PRP) and connecting bag
from which additive solution was emptied, at 22
0
C in refrigerated centrifuge at
3100 rpm for 10 minutes after balancing the buckets.
Place the bag containing PRP on the expresser stand.
Express the plasma into the empty bag leaving 50-60 ml plasma along with the
platelets.
Seal the tubing and cut the tubing of the plasma bag short (1) to avoid breakage
during frozen storage.
A small segment of tube containing platelets (about 8 cms long) is prepared after
mixing of the bag contents as and when requested by quality control laboratory.
Leave the platelet concentrates on the laminar flow for 30 minutes, keeping the
label side down. Mix the contents of the bag manually before transferring the
units to quarantine storage in the incubator at 22
0
C on the lower shelf.
After the required test results are available place the platelet concentrates on the
agitator in the upper shelf for use.
Keep the plasma bag in the quarantine storage in the deep freezer kept in the
component room and transfer to deep freezer in issue area when the tests are
completed after labeling and entering in the inventory.
Standardise the speed of the centrifuge as it depends on the type of bag, the
amount of blood collected and centrifuge in use.

Preparation of Cryoprecipitate:
The basic material is platelet poor fresh frozen plasma. The plasma should be free
of red cell. Use the plasma frozen at -80
0
C preferably within a day or two of
freezing.
Keep the segment of the bags for potential cryo-preparation longer.
Fill the cryobath with double distilled water.
Maintain the temperature of water in continuous circular motion at 4
0
C.

STANDARD OPERATING
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Section : I
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Date : 15-05-2008
Page : 84 of 197


Keep the frozen plasma bags in this cryobath. When the plasma is thawed, place
the bags in centrifuge buckets and balance the buckets on weighing scale.
Keep the position of the bags in buckets parallel.
Spin the buckets at 3100rpm for 15 minutes at 4
0
C.
Under laminar flow, connect empty transfer bag to the bag containing plasma and
cryoprecipitate using sterile connecting device.
Place the plasma bag on expresser and separate plasma into the transfer bag
leaving approximately 15-25 ml as cryoprecipitate suspension in the original bag.
Seal the tubing and separate the cryoprecipitate and the cryopoor plasma bags.
Weigh the cryo and plasma bags and record.
The plasma separated is F-VIII deficient plasma. Both the bags are kept in
quarantine till the tests are completed.
Label, enter the inventory and place them in deep freezer in issue area after test
results are available.
Washed Packed Red Cell

Undertake the washing procedure only after the compatibility test is over.
Balance the blood bag in the centrifuge bucket with another empty bucket.
Spin the bag at 3500 rpm for 10 minutes at 4
0
C.
Remove the supernatant plasma completely in a transfer bag using expresser
under laminar flow.
Before washing the unit, red cell serology laboratory should perform the
compatibility tests. The washing procedure is undertaken only after the proposed
unit is found to be compatible with recipient.

The proposed blood unit is balanced in the centrifuge bucket with another empty
bucket. The buckets are centrifuged as per programme.
The bag is removed and supernatant plasma is completely removed in a transfer
bag using an expresser under laminar flow.
Connect the bag with a sterile 0.9% saline bag using a transfer set.
Record batch number and expiry dates of saline in use.
Introduce approximately 200 ml of saline into the packed cell bag and mix
thoroughly and centrifuge again.
Transfer the supernatant saline with some plasma into a transfer bag using the
expresser under laminar flow.
Disconnect the transfer bag, seal and discard.
Repeat the washing with saline twice more (total three times) exactly in the same
manner as described above. In the end keep 25-30ml saline with the red cells in
the bag.
Seal the final thrice washed red cell unit.
Weigh the bag and record details in the register.
Store the washed packed red cell unit at 1-6
0
C and use within 24 hours of
washing.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 85 of 197


Use this blood only for the patient for which requested. If not used discard after
24 hours with standard disposal protocol, after subjecting small sample for
bacteriological examination.

6.0 DOCUMENTATION

a. Enter following details in the Component Register

Date and time of separation.
Unit number.
Type of bag used, with batch number and manufacturer's name.
Weights of whole blood and different components.
Date of expiry of different components.
Type of centrifuge and speed used.
Blood group and serology code.

b. Enter in stock register of red cells, FFP and platelets after the testing is
completed and the units are labeled.
c. Incident reporting:
If there are any problems encountered during the component processing, enter in
the incident report form and inform the supervisor / medical officer in charge.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
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SP-024
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Location Subject
Blood Storage Area

Labeling of Blood Bags & Blood
Components

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 87 of 197


Ensure Safe Transfusion

- In-charge Blood Storage & Distribution
- Master File


The blood after it is collected remains in quarantine and is released for transfusion only
after all tests (grouping and for T T I) are completed. Before these blood bags are taken
on inventory for use they are labelled depending on their blood groups. The label is
required for identification and retrieval of blood units for use, disposal and follow up in
case of adverse reactions.

2.0 RESPONSIBILITY

It is the responsibility of the technician from the Red Cell Serology Laboratory to label
the blood and blood components units.

3.0 REFERENCES

Kit package insert.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 88 of 197


Technical Manual of American Association of Blood Banks 13th Edition,
1999.Pages 156-158.

4.0 MATERIALS

Preprinted adhesive labels for all components printed as per regulatory
requirement.
The labels are printed and colour coded for all components as per blood groups.
Group A have yellow labels, Group B pink labels, Group O blue labels and
Group AB have white labels. Negative labels also have the same colour labels
except the printing is in red colour.

5.0 PROCEDURE

5.1 Principle:

After collection and processing whole blood and component units remain in
quarantine storage areas.
Once all the reports of blood group and TTI testing are ready, place the bags on a
table in chronological order.
Segregate those, which are found reactive for any TTI or found unsuitable for use
and keep them in the area for disposal. Leave those found suitable for use on the
bench for labeling.
Write clearly the unit number, date of collection and expiry and the volume on
each label as per the grouping register records.
Date of collection and date of expiry is very important. The expiry date depends
on the type of bag and component. In case of a triple and quadruple bag with
additive solution, the expiry date is 42 days, and for double and single bags, it is
35 days. In case of a triple or quadruple bag if for some reason, the components
could not be separated, then label the expiry date as 21 or 35 days depending on
the anticoagulant present in the primary bag. The day of blood collection is
considered the day zero for calculating the expiry dates.
After the bags are labeled ask a second technician to double check the number
and group on the bags tallying them with the records.
Enter all labeled bags group wise in the stock book, which is also maintained
group wise. Label FFP and Cryo deficient plasma, and platelet concentrates in the
same manner. Cryoprecipitate labels do not indicate blood groups.
All plasma components have an expiry date of one year. The expiry date of
platelet concentrate is 5 days with PVC bags and 7 days if special bags are in use.

6.0 DOCUMENTATION

Enter all labeled bag numbers in the inventory of units for use.

STANDARD OPERATING
PROCEDURES
Section : I
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Date : 15-05-2008
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STANDARD OPERATING
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Section : I
Issue : 1.0
Date : 15-05-2008
Page : 90 of 197


Blood Storage Area

Preservation of Blood & Blood
Components


Optimum Storage of Blood and Blood
Components

- Master File


Blood components prepared are stored in conditions designed to preserve optimal
viability and function during the storage period. (Table 1)

2.0 RESPONSIBILITY

It is the responsibility of the technical staff from the component laboratory to keep the
units in the quarantine storage. The technologist who labels the units after the testing is
responsible to transfer the labeled units in their respective storage areas.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks 13th Edition, 1999
Pages 166-167, 182-183, 84, 86.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 91 of 197


Introduction to Transfusion Medicine, Zarin Bharucha & D.M. Chauhan, 1
ST

Edition, 1990, Pages 111-112.

4.0 MATERIALS

Storage Equipment
Blood bank Refrigerator
Deep Freezer
Platelet incubator
Platelet agitator

5.0 PROCEDURE

5.1 Principle:

All untested units should be kept in the quarantine area.
After testing is over, release the fully tested. Transfer those deemed suitable for
clinical use from quarantine area to the stock area after labeling. (Refer table No.
1).
Label those found unsuitable for use with a biohazard label and keep for disposal.
Store whole blood and Red Cell concentrates on metal rack stand in the Cold
Room (4-6
0
C). These stands have shelves. Each shelf is reserved for a particular
group having its label stuck on the outer side. Arrange the blood bags in
chronological order, group wise and according to the expiry dates in trays and
then stack the trays on the shelves. This makes it very easy for the technologists
on duty to remove the bags for issuing, whenever required.
Store blood collected in CPD-A1 and the red cells separated in a closed system
up to 35 days. Store the red cells suspended in additive solutions up to 42 days.
Use red cells prepared in open system within 24 hours of preparation.
Keep Fresh Frozen Plasma, cryoprecipitate and FVIII deficient plasma bags in
over wrap bags and then arrange in plastic trays in the Deep Freezer (-40
0
C)
immediately after separation. The shelf life of all these plasma components is 1
year. FFP once thawed and then refrozen is used only as FVIII deficient plasma.
Place Random donor platelets (RDP), Single Donor Platelets (SDP) in a platelet
incubator at 20-22
0
C on a agitator which has shelves to store them. Store the
concentrates prepared in PVC bags up to 5 days and those prepared in special
platelet bags up to 7 days.
Take due care to maintain sterility of all components by keeping all storage areas
clean.
Monitor to ensure the storage conditions to be appropriate and correct for each
product. Monitor the temperature of all storage areas with continuous graphic

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 92 of 197


recorder. Change the charts every week, and achieve them. Check the alarm
system every month.
Similarly, after labeling the plasma bags, enter the unit numbers group wise in the
stock register. Make FFP entries on the right hand page of the stock register,
whereas Factor VIII-D plasma & Cryo units on the left hand page.
Carry out physical stock taking every night and rewrite the inventory.

6.0 DOCUMENTATION

Record all blood/components released for use as well as the unsuitable units to be
discarded in the disposal register.
Table 1

Product Packed
cells (PC)
Fresh
Frozen
Plasma
(FFP)
Cryopoor
Plasma
Platelets Cryoprecipitate
Storage
Temperature

2-6
0
C

-30 to
80
0
C

-30
0
C

-22
0
C with
gentle
agitation

-30
0
C

Shelf life
from time of
collection
35 days
without
additive
solution. 42
Days with
additive
solution
SAGM

1 year

1 year

5-7 days
according to
the bag in
use

1 year

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STANDARD OPERATING
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Section : I
Issue : 1.0
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Page : 93 of 197


Blood Storage Area

Inventory of Blood Bags & Blood
Components


Availability of Blood for Transfusion

- Master File


Blood components prepared are stored in conditions designed to preserve optimal
viability and function during the storage period. (Table 1)

2.0 RESPONSIBILITY

The technician from the red cell laboratory checks the records and transfers all the units
which are serologically negative and labeled to inventory.

3.0 REFERENCES

Technical Manual of American Association of blood banks 13th Edition, 1999.
Pages 83-84, 86.

STANDARD OPERATING
PROCEDURES
Section : I
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Date : 15-05-2008
Page : 94 of 197


4.0 MATERIALS

Inventory Register
5.0 PROCEDURE

5.1 Principle:

Inventory is maintained on a day-to-day basis. After labeling the units, enter the
numbers of whole blood or packed cells numbers group wise on the right hand
page of the inventory register kept in the main red cell laboratory. In case of
packed cells units, write the alphabet PC above the unit number. PC denotes
packed cells without additive solutions. PCS denotes packed cells with additive
solution. The inventory bears columns for A group, B group, AB group, O group
as well as negative groups of these four groups.
Enter the units group wise and according to the date of collection in the inventory
register (daily stock). The technologist on night duty is responsible for physical
checking of the printed number tag with the hand written number on the label and
enters in the inventory.
After labeling the FFP, enter the donor units numbers group wise in the stock
register of FFP similar to blood units.
Enter FVIII Deficient Plasma units labeled group wise in the stock register
similar to plasma register.
Enter the labeled cryoprecipitate unit numbers in the register.
Clearly mark the inventory of bags that have less volume of blood collected or
are reserved for specific patients with specific instructions.
6.0 DOCUMENTATION

All unit numbers are entered group wise and expiry date wise in the inventory
register.

SP-027 01-12-2007

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STANDARD OPERATING
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Section : I
Issue : 1.0
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Page : 95 of 197


Blood Storage Area

Supply of Safe Blood for Transfusion


Reissue of Blood and Blood
Components

- Master File


The technologists have the duty to see that the blood is not wasted and made available to
another patient of the same group. This is achieved by first-in-first-out (FIFO) policy.

2.0 RESPONSIBILITY

It is the responsibility of the staff to see that the blood which has returned and not used is
once again cross matched and made safe for transfusion to another patient.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 96 of 197


Pages 10,186 & 491.

4.0 MATERIALS

Inventory Register
Issue Register

5.0 PROCEDURE

5.1 Principle:

When blood is released from the Blood Bank to operation theatre or ward of the
hospital or outside for transfusion, some times for some reason or the other, it
may not be required by the patient and it is returned to the blood bank reused for
another patient. Take care to see that this unit of blood is kept erect in the cold
room to look out for hemolysis. If there is no hemolysis seen after spinning or
standing, issue this unit safely to another patient.
In case of FFP, which comes to the blood bank unused, issue to another patient if
there is a demand for that particular group immediately within 6 hours of the first
issue. If no call arises, then use it later as FVIII deficient plasma.

6.0 DOCUMENTATION

Make entries of returned units against the issue in the issue register.
Re-enter the unit in the inventory before reissue.

SP-028
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STANDARD OPERATING
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Section : I
Issue : 1.0
Date : 15-05-2008
Page : 97 of 197


Blood Storage Area

Issue of Blood for Transfusion


Optimum utilization of blood

- Master File


The blood and blood components are used as per the need of the patients. These are
issued against the prescription of a medical officer after ensuring the compatibility and
testing results.

2.0 RESPONSIBILITY

It is the responsibility of the technician on shift duty in Red Cell Laboratory to issue the
blood for which requisition is received.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 98 of 197


3.0 REFERENCES


4.0 MATERIALS

Inventory Register
Issue Register
Request form
Compatibility Report

5.0 PROCEDURE

5.1 Principle:

In order to avoid outdating, implement FIFO policy.
Carry out compatibility testing
Ensure that the compatible units are tested for TTI and found suitable for use
Remove the correct unit from blood bank refrigerator and keep it in the
thermalbox for transport
Make entries in the issue register
Instruct the individual to take the unit straight to OT/Ward for transfusion.

6.0 DOCUMENTATION

Make following entries in issue register.

Name of patient
Hospital registration number
Blood group
Date and time of issue
Unit No. Issued
Blood group of unit
Component of blood
Signature of technician who issues
Signature of receiver

STANDARD OPERATING
PROCEDURES
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Date : 15-05-2008
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SP-029 01-12-2007

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STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 100 of 197


Location Subject

TTI Testing Laboratory

HbsAg Testing


Samples Tested for Hepatitis B
Surface Antigen by ELISA Method

- In-charge TTI Testing Laboratory
- Master File


HBSAg is mandatory test for blood unit screening before it is transfused. This is carried
out on all donor unit samples.

2.0 RESPONSIBILITY

It is the responsibility of technician from TTI testing lab to carryout the test and report as
required.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 101 of 197


Kit Package inserts.
Technical Manual of American Association of Blood Banks 13th edition 1999,
Pgs 152-153

4.0 MATERIALS

ELISA Reader
ELISA Washer.
Incubator.
Micropipettes and disposable tips.
Timer
Disposable container with Na Hypochlorite
Absorbent tissue
D. Water
Stop solution (1 mol / liter sulphuric acid)
Kit

5.0 PROCEDURE

5.1 Principle:

The wells of polystyrene microplate have been coated with HBS Ag. When incubated
with patient sample having Anti HBS Ag and HBS Ag. Peroxidase solution in wells
it forms HBS Ag + Anti HBS Ag + HBS Ag peroxidase complex. After washing to
remove unbound materials a peroxidase substrate is added and colour develops in
proportion to the amount of Anti HBS antibodies. The level of colour is greatest in
the presence of anti HBS and falls from its level with decreasing concentrations in
sample.
5.2 Method:
Anti Hbs Ag EIA test is carried out as per kit manufactures instructions. The
details as per the pack insert of the kit in use.
Remove reagents from fridge 30 minutes prior to testing. Mix the reagents gently
by inverting the vials without foaming.

Bring sample to room temperature
Prepare wash buffer solution.
Discard all disposable type in a contraire with hypochlorite
Arrange sample in rack so that well I A & 1B are Positive control and negative
control. IC & D keep the sample number wise. If any number missed already
done note down the number in work sheet.

STANDARD OPERATING
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Date : 15-05-2008
Page : 102 of 197


Bring kit solution to room temp
Fit the strip holder with the required number of strips.
(Including samples and controls)
Pipette 25 l specimen diluent in to all wells, i.e. sample & control wells.
Pipettee 100 l sample and /or control (NC-3, PC-1) into assigned wells.

NOTE : always pipette controls after pipette samples

Mix well (by use shaker) and Incubate at 37 C for 60 + 5 minutes
Pipette 50 l Conjugate into each well & Incubate at 37 C for 60 + 5 minutes
After incubation wash and soak the each well for six times with 300-350 l of
Phosphate buffer.

NOTE : Remove any remaining fluid on the top & bottom of the wells and strip
holder with absorbent tissue after last aspiration.

Pipette 100 l of substrate (50 l Urea peroxide + 50 l TMB) into each
Well (e.g., for one strip 500 l Urea peroxide + 500 l TMB)

Incubate the strips at Room Temperature. For 30+2 minutes in the dark.
Stop the reaction by adding 100 l of 1 M Silfric Acid to all wells.
Read the absorbance of the solution in the each well at 45 nm (single wavelength)
or 450 nm and 620 nm as reference (dual wavelength)

NOTE : Strips should be read within 15 min. after adding stop solution.

Assay validity: NCx must be <0.100 and NCx> 0.010
If the test run is valid, Calculate the cut of value : NCx +0.040
A test sample is reactive if sample absorbance is > cut off valve,
A test sample is non reactive if sample absorbance is < cut off valve.
Interpretation of results:
a. A Non-reactive result indicates that the sample tested either does not contain
HBsAg (OR) that it contains HBsAg concentrations at below the detection
limits if Hepanostika HBsAg ultra.
b. A reactive result indicates that the sample tested either contains HBsAg (OR)
that it contains a non-specifically reacting factor
c. That sample initially reactive result should be retested in duplicate.

5.3 Validation:

Check the validation of bank as well as negative and positive control absorbance
value as per pack insert of the kit.

STANDARD OPERATING
PROCEDURES
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Date : 15-05-2008
Page : 103 of 197


Examine absorbance values of the control before sample results can be
interpreted. If the run fails to meet criteria as per package insert consider the test
as invalid and repeat the whole test again
Cut off OD is automatically calculated.

6.0 INTERPRETATION

Check the printout carefully for absorbance values.
The sample below cut value are positive
The sample above cut off value are Negative
The sample positive for HBc Ig anti bodies do Anti HBS Ag
Equal to cut off are considered positive.
Sample close to cut off value 10% below cut off (Grey Zone) are repeated.

7.0 DOCUMENTATION

Take the O.D value on the back side of worksheet
Write name of the test and date on work sheet
Write name of the kit used
Write Lot No. and expiry date of kit
Write Positive sample Numbers
Initials of the technologist who performed the test and the medical officer who
supervised the test.
Reactive units marked in red
Transfer the record to master record.

SP-030 01-12-2007

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STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 104 of 197


Location Subject


Anti HIV Testing


Sample tested for Anti HIV Bodies by
ELISA Method

- Master File


Anti HIV antibodies testing is carried out on all bag samples before these are released for
transfusion. Pre-donation samples of pheresis donors are also tested.

2.0 RESPONSIBILITY

It is the responsibility of technician from TTI Testing lab; to carry out the test and report
as required.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 105 of 197


Kit package insert.
1999.Pages 152-153.

4.0 MATERIALS

Reagent kit
Micropipettes and disposable pipette tips
Timer
EIA reader
EIA Washer
Incubator 370C
Vortex Mixer
Glassware
Distilled water.

5.0 PROCEDURE

5.1 Principle:

Recombinant HIV-1 and HIV-2 antigens and antibodies are absorbed on to the wells of
Micorassayplate. The wells are coated with recombinant HIV-1 gp41 antigen,
recombinant HIV-1 group O gp 41 antigen, recombinant HIV-2 gp 36 antigen and
monoclonal anti HIV-1 p24 antibody.
Serum or plasma samples are added to these wells. If antibodies to HIV-1 and HIV-2 are
present in the sample, they will form stable complexes with the HIV-1 and HIV-2
antigen on the plate.
If p24 antigen is present in the sample it will bind with the monoclonal antibody
bound to the sold phase. A recombinant HIV-1 group-O gp41 antigen conjugate
recombinant HIV-2 gp36 antigen/ peroxidase conjugate, monoclonal anti HIV-1 p24
antibody/peroxidase conjugate, is added. If the antigen/antibody complex is present,
the peroxidase conjugate will bind to antibody, antigen and remain in the well.
Enzyme substrate is then added colour will change in wells containing antibody-
antigen complex.
An acidic stopping solution is added to each well and the colour is read on
photometer at 450nm. A reference wave length of 620 nm is recommended.

5.2 Method

The anti HIV antibody test is carried out as per the instructions given in the package
insert of the kit.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 106 of 197


Remove reagents from the fridge 30 minutes prior to testing. Mix the reagents
gently by inverting the vials without foaming.
Bring the samples to room temperature before testing.
Arrange all samples to be tested serially in ascending order in which they are to
be tested in a test tube rack.
Place the plate in front of the test tube rack
Fit the strip holder with the required number of strips
Pipette 100 l of Negative control into each well of 1A and 1B. 100 l of positive
control to 1C and 1D and pipette 100 l of sample into the remaining wells.
(Note: always pipette controls after pipette samples)
Mix well and Incubate at 37 C for 60 + 5 minutes
Before the last 10 minutes of 1
st
incubation, make a 1:51 dilution of conjugate
with conjugate diluent.
After incubation Wash and soak the each well for six times with 300-350 l of
Phosphate buffer.
Pipette 100 l of prepared diluted conjugate into each well.
Incubate the strips at 37
0
C for 30+ 1 minutes.
Before the last 5 to 10 minutes of second incubation, make a 1:101 dilution of
substrate with substrate buffer.
Aspirate the contents from each of the wells and wash each one 5 times with 300
l of diluted washing solution (300 /well/time).
(Note: Remove any remaining fluid on the top & bottom of the wells and strip
holder with absorbent tissue after last aspiration)
Pipette 100 l of prepared substrate into each well and incubate at controlled
room temperature (23+ 2
0
C) for 30 minutes. Avoid exposure to light.
Stop the reaction by adding 100 l of 1 M sulfuric acid to all wells.
Read the absorbance of the solution in the each well at 450 nm (single
wavelength) or 450 nm and 620 nm as reference (dual wavelength)
(Note: Strips should be read with 15 min. after adding stop solution.
Assay Validity: The average absorbance (NCx) of the Negative control should be
les than or equal to 0.100 and greater than -0.005.
The average absorbance (PCx) of both the Positive controls should be greater
than or equal to 1.0
Calculate the cut off value = NCx + 0.200

Printer prints out all numbers fed in their OD values. Reactive is printed across that
particular reactive sample number according to the O.D. value of the cutoff.

5.3 Validation:

Check the validity of the Blank (if used) as well as negative and positive control
absorbance value as per pack insert of the kit.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 107 of 197


Examine absorbance values of the controls before the sample results can be
interpreted. If the run fails to meet the criteria as per package insert consider the
test as invalid and repeat the whole test again.
Cut off O.D. is automatically calculated.

5.4 Interpretation:
Check the printout carefully for absorbance values:

The samples below the cut off are considered non-reactive.
Equal to cut off are considered initially reactive (I.R)
Above cut off are considered I.R.
Sample close to cut off value 10% below cut off (grey zone)
Samples with grey zone results are repeated in one well.

6.0 DOCUMENTATION

6.1 Paste the print out in the HIV register and also record the following details:

The date on which the test is run.
The name of the kit used.
Lot No and expiry date of the kit.
Initials of the Technologist who performed the test and the Supervisor who
verified the results.
The reactive units are marked in red.

6.2 Transfer the record to donor records.

SP-031 01-12-2007

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Location Subject

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 108 of 197



Anti HCV Testing


Sample tested for HCV Anti bodies by
ELISA Method

- Master File


Anti HCV is a mandatory test for blood unit screening before it is transfused. This is
carried out on all donor units samples and pre-donation samples of pheresis donors.

2.0 RESPONSIBILITY

It is the responsibility of technician from TTI Testing lab; to carry out the test and report
as required.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 109 of 197


Kit package insert.
1999.Pages 152-153.

4.0 MATERIALS

Elisa Reader
Elisa Washer
Microshaker
Incubator
Micropipettes and disposable tips
Timer
Disposable gloves
Disposal container with Na Hypochlorite
Absorbant tissue
Distilled water
1 mol / litre Sulphuric acid
Kit

5.0 PROCEDURE

5.1 Principle:

In HCV EIA, the microwell are coated with recombinant Hepatitis C Virus encoded
antigens as the solid phase. If the HCV antibody is present, it becomes bound to the solid
phase and can be detected by a complementary anti-human IgG conjugated to an enzyme
(capable of acting on a chromogenic substrate). When substrate is added to the bound
complex, the presence of antibody can be detected by development of a coloured end
product.
5.2 Method

Anti-HCV EIA test is carried out as per kit manufacturer's instructions. The details of the
kit procedure and interpretation of results are as per the pack insert of the kit in use.

Remove reagents from the fridge 30 minutes prior to testing. Mix the reagents
gently by inverting the vials without foaming.
Bring samples to room temperature before testing.
Prepare wash buffer solution as per pack insert.
Discard all disposable tips in a container with hypochlorite solution.
Arrange the samples so that well I A is blank I B, C & D as negative control and I
E + F as positive controls.
Dispense 100 ul diluent to each well except the blank well.
Add 10 ul of controls and specimens to appropriate wells.

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Mix the plate gently and cover with a seal.
Incubate at 37C for 60 minutes.
Remove seal and wash 5 times.
Invert the plate and tap on a clean paper towel to remove excess wash buffer.
Dispense 100 ul conjugate to all wells except blank.
Cover the plate with sealer and incubate at 37C for 30 minutes.
Prepare substrate 10 minutes prior to use.
Wash wells 5 times.
Dispense 100 ul of substrate to all wells including blank.
Incubate at 15-30C in dark for 30 minutes.
Dispense 100 ul of 4 N sulphuric acid to all wells.
Remove moisture from bottom of plate and read at wavelength of 490 nm.

5.3 Validation:

Cut off O.D. is automatically calculated.
Examine absorbance values of the controls before the sample result.
Check the validity of the Blank (if used) Negative and positive control
absorbance value as per pack insert of the kit.
If the run fails to meet the criteria as per package insert consider the test invalid
and repeat the whole test.

5.4 Interpretation:

Check the printout carefully for absorbance value:
The samples below the cut-off are considered non-reactive.
Equal to cut off are considered initially reactive
Above cut off are considered Initially reactive iv Retest ii and iii in duplicate. If one or
both positive record the result as positive. If both negative record as negative.
6.0 DOCUMENTATION

6.1 Paste the print out in the HCV register and also record the following details:

The date on which the test is run.
The name of the kit used.
Lot No and expiry date of the kit.
Initials of the Technologist who performed the test and the Supervisor who
verified the results.
Write the numbers of the samples to be repeated below the printout. After these
samples are repeated again then reactive or non-reactive is written across that unit
below the print out.
The reactive units are marked in red.

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6.2 Transfer the record to donor records and grouping register.

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Location Subject

TTI Screening Lab

Anti HBcIg Testing


Sample tested for HbcIg Anti bodies by
ELISA Method

- Supervisor
- Master File

1.0 SCOPE OF APPLICATION

Anti HBC Ig is mandotry test for blood unit screening before it is transfused.This is
carried out on all donor units samples.

2.0 RESPONSIBILITY

It is the responsibility of technician from TTI testing lab to carry out the test and report
as required.

3.0 REFERENCE

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 113 of 197


a. Kit package insert
b. Technical manual of A A B B

4.0 MATERIALS REQUIRED

ELISA Reader
ELISA Washer
Incubator
Micropipettes and disposable tips
Timer
Disposable container with hypochlorite solution.
Disposable gloves.
Absorbent tissue
Distilled Water
Stop solution 1mol / litre sulphuric acid
Kit

5.0 PRINICIPAL

The HBc Ag caoatd wells incubated with patient or donor serum along with conjugate (
Anti HBC + HRPO) . If anti Hbc Ig is present in patient sample it competes with
conjugate and attached to HBc Ag. Addition of substrate gives no colour to positive
sample. On addition of stop solution (2 N H
2
So
4
) it turns to colourless. If no antibodies
in Patient it turns to yellow colour.

6.0 METHOD

Anti HBc Ig EIA test is carried out as per kit manufactcures instructions. The details of
the kit procedure and interpretation of test results are as per the pack insert of the kit in
use.

Remove reagents from the fridge 30 minutes prior to testing. Mix the reagents gently by
inverting the vials without foaming.

Discard all disposable tips in a container with hypochlorite

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Arrange samples in a rack so that well I A & 1B are Positive control, IC & 1D are
negative controls and I E,F. keep the samples in numberwise. If any number missed or
already done note down the number in work sheet.
Bring kit solutions to room temp
Add 50 micro litres of sample + 50micro litres of conjugate into microwells.
Incubate at 37
0
c for 1 hour in an incubater.
Preparation of washing solutionWashing buffer : Distilled water in 1: 20.
To 50ml of buffer add 950 ml of distilled water to prepare IL of washing soloution.
Wash the wells for 6 times
Dry the wells, see no washing solution in wells or other contaminates
Add Substrate A 50micro liters.
Add Substrate B 50micro liters.
Incubation at Room temp for 30 min
Observe for Blue colour development in positive control and positive sample no colour
in Negative control and in negative samples.
Add stop solution 100 ml
Take O.D Values in ELISA reader at 450 nm/ 620nm within 15min
Cut off Control = 0.4 x negative + 0.6 x positive
For all HBc Ig positive samples do anti HBs Ag test

7.0 VALIDATION:

Check the validation of blank as well as negative and positive control absorbance value
as per pack insert of the kit.
Examine absorbance values of the control before sample results can be interpreted . If the
run fails to meet criteria as per package insert consider the test as invalid and repeat the
whole test again


8.0 INTERPRETALION;


1.The sample below cut off value are positive for Hbc Ig antibodies

2.The sample above cut off value are Negative for Hb c Ig antibodies.

3.The sample positive for HBc Ig anti bodies do Anti HBS Ag

4.Equal to cut off are considered positive

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5.Sample close to cut off value 10% below cut off ( grey Zone) are repeated.

9.0 DOCUMENTATION:


Initials of technologist who performed the test and the medical officer who
Reactive unit numbers are marked in red.
Transfer the record in to master record.

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Location Subject

TTI Screening Laboratory

Anti HbsAg Testing


Sample tested for HbsAg Anti bodies by
ELISA Method

- Supervisor
- Master File


Anti HBS Ag is mandatory test for blood unit screening before it is transfused. This is
carried out on all donor units samples.

2.0 RESPONSIBILITY

It is the responsibility of technician from TTI testing lab to carry out the test and report
as required.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 117 of 197


3.0 REFERENCE

1. Kit package insert
2. Technical manual of A A B B.


ELISA Reader
ELISA Washer.
Incubator.
Micropipetts and disposable tips.
Timer
Disposable container with Na Hypochlorite
Absorbent tissue
D. Water
Stop solution ( 1 mol / liter sulphuric acid )
Kit

5.0 PRINICIPAL

PROCEDURE
Principle:
The Principle of ELISCAN HBs Ag is based on a direct, non-competitive, solid-
phase enzyme immunoassay with horseradish peroxidase as the marker enzyme. The
assay proceeds according to the following reactions:
i. HBsAg() When present in patients serum, combines with the mouse monoclonal
anti-HBs antibodies coated on the polystyrene surface of the microstrip wells and
simultaneously binds with the horse-radish peroxidase conjugated sheep poly and
ouse monoclonal anti-HBs antibodies.
ii. After incubation, wells are washed and a colourless substrate(H
2
O
2
) chromogen
(TMB) solution is added. The enzyme action on substrate/chromogen produces a
coloured end product.
iii. The enzyme-substrate/chromogen reaction is terminated with addition of 1.6N
H
2
SO
4
. The colour intensity is directly related to the concentration of hepatitis B
surface antigen in the patient sample.
6.0 METHOD
HBsAg ELISA test is carried out as per kit manufactures instructions. The
details as per the pack insert of the kit in use.

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PROCEDURES
Section : I
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Date : 15-05-2008
Page : 118 of 197


Remove reagents from fridge 30 minutes prior to testing. Mix the reagents gently
by inverting the vials without foaming.
Discard all disposable type in a contraire with hypochlorite
Arrange sample in rack so that well I A & 1B are Negative control and Positive
control. IC & D keep the sample number wise. If any number missed already
done note down the number in work sheet.
Bring kit solution to room temp
Fit the strip holder with the required number of strips.
Pipette 100 l Controls & samples (NC-2, PC-2) into assigned wells.
Pipette 25 l prepared conjugate specimen diluent in to all wells, i.e. sample &
control wells.
Mix well (by use shaker) and Incubate at 37 C for 90 + 5 minutes
Before last 5-10 minutes mix substrate solution and substrate buffer at the
volume ratio 1:101.
After incubation wash and soak the each well for five times with 300-350 l of
washing solution.

NOTE : Remove any remaining fluid on the top & bottom of the wells and strip
holder with absorbent tissue after last aspiration.

Pipette 100 l of prepared substrate into each Well
Incubate the strips at Room Temperature. For 30+2 minutes in the dark.

Stop the reaction by adding 100 l of 1 M Sulfuric Acid to all wells.
Read the absorbance of the solution in the each well at or 450 nm and 620 nm as
reference (dual wavelength)

NOTE : Strips should be read within 15 min. after adding stop solution.

Assay validity: NCx must be <0.200 and PCx> 0.800
If the test run is valid, Calculate the cut off value : NCx +0.05
A test sample is reactive if sample absorbance is > cut off valve,
A test sample is negative if sample absorbance is < cut off valve.

Interpretation of results:
Samples with absorbance equal to or greater than the cut off value are considered
positive to HBsAg. Samples with absorbance less than the cut off value are
considered negative to HBsAg.
If the samples are considered positive, the test hold be conducted two more times.
In case that the re-tests show negative, the samples are considered negative, and

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on the other hand, if one of the retests shows positive the samples are considered
positive.
The samples considered positive should be tested again by confirmatory test for
final judgement.

Validation:
Check the validation of bank as well as negative and positive control absorbance
value as per pack insert of the kit.
Examine absorbance values of the control before sample results can be
interpreted. If the run fails to meet criteria as per package insert consider the test
as invalid and repeat the whole test again

Interpretation
The sample below cut value are positive
The sample above cut off value are Negative
The sample positive for HBcIg anti bodies do Anti HBS Ag
Equal to cut off are considered positive.
Sample close to cut off value 10% below cut off (Grey Zone) are repeated.

8.0 DOCUMENTATION:

Initials of the technologist who performed the test and the medical officer who
Reactive units marked in red

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Dr V Saraswathi

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Location Subject

VDRL Test


RPR test for VDRL

- Master File


The samples from donors are tested for Transfusion Transmitted Diseases. These tests
are mandatory.

2.0 RESPONSIBILITY

It is the responsibility of technician on sample receiving desk to ensure correct
samples received from patients. The responsibility of carrying out the test is of
the technician in the TTI Testing Laboratory.

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3.0 REFERENCES

Kit Package insert.
Technical Manual

4.0 MATERIALS

Carbon Antigen
Positive control, reactive with the regent
Negative control, non reactive with the reagent
Disposable slides with eight reaction circles
Disposable sample/ control dispensing pipettes
Mixing sticks
Rubber teat
Reagent dropper for dispensing the carbon antigen

5.0 PROCEDURE

5.1 Principle:

During the test procedure, the specimen, serum or plasma is mixed with
CARBOGEN reagent and allowed to react for eight minutes. If antilipoidal
antibodies are present in the specimen, they will react with CARBOGEN reagent
forming visible black floccules. If antilipoidal antibodies are not present in the
specimen, there will be no flocculation.

5.2 Sample Collection and Storage:

No special preparation of the patient is required prior to sample collection
by approved techniques. Hemolysed or lipemic samples are not suitable for testing.
Fresh serum or plasma should be used for testing.
Sample not tested immediately may be stored at 2-8
0
C for up to 48 hours.
Hazy samples should be centrifuged. Use the clear supernatant for testing.

5.3 Method:

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o Bring reagents and sample to room temperature. Thoroughly mix the
carbogen reagent suspension by gentle agitation before use.
o Take disposable slide with reaction circles
o Using sample dispensing pipette dispense one-drop (or) 50micro liters of
test sample, positive control and negative control on to separate reaction
circles.
o Add one drop of well-mixed carbogen reagent by using the reagent
dropper provided with the kit.
o Do not let the dropper tip touch the liquid on the slide
o Using a mixing stick mix the test specimen and the carbogen reagent
thoroughly spreading uniformly over the entire reaction circle.
o Immediately start stopwatch keep the slide on mechanical rotor at 180
rpm / min.
o Observe for flocculation macroscopically at 8 minutes.

5.4 Validation:

Check negative control and positive control before interpreting test.

5.5 Interpretation:

Large and medium black floccules against white background -- Reactive.
Small and medium black floccules against white background -- Reactive
No floccules or gray background -- non reactive

(Flocculation indicates presence of antilipoidal antibodies in test specimen).

5.6 Quantitative Method:

Using isotonic saline prepare serial dilutions of test sample positive in qualitative
method in 1:2, 1:4, 1:8, 1:16, 1:64,1.128 and so on.
Perform test as said above using dilutional specimen
Note the tire where antibodies can give floccules at highest dilution.
Use microscope to identify weakly reactive samples.

6.0 DOCUMENTATION

Prepare work sheet with IA as positive control, 1B as negative control followed
by numbers of test samples.
Write name of test & date on work sheet
Write name of kit used.
Write Lot no and expiry date.

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Write positive sample numbers.
Initials of technologist who performed the test and the medical officer who
Reactive units marked in red.

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Dr V Saraswathi

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Location Subject

Quality Control Laboratory

Optimum Quality Assurance


Optimum storage of consumables,
reagents and kits

- Master File


The quality assurance system requires that all the reagents used for various test
procedures are stored according to the manufacturer's instructions. Any lacunae in the
storage conditions, reduces the affectivity of the reagents.

2.0 RESPONSIBILITY

It is the responsibility of all the staff members of different laboratories to store all the
reagents and kits as per manufacturer's instructions.

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3.0 REFERENCES

Indian Pharmacopoeia, Volume II, Annexure 29 (A-29), 1996.
Reagent manufacturer's instructions.

4.0 MATERIALS

Domestic refrigerator
Blood bank refrigerator
Deep Freezer
A.C. Store room
Stock register or stock cards

5.0 PROCEDURE

(a) Donor Room:

Store disinfectants for preparation of phlebotomy sites at room temperature
(22
0
C-25
0
C) in the donor room.
Store blood collection bags and apheresis sets in air-conditioned room (22
0
C-
25
0
C).

(b) Red Cell Serology Laboratory (RCS):

Store ABO reagents Anti A, Anti-B, Anti AB, Anti D, bovine albumin,
antihuman globulin, pooled A,B, and O red blood cells, papain and cystein
powder in the cold room maintained at 4-6
0
C or as per manufacturer's
instructions.
Store 10ml aliquots of papain-cystein in the Deep Freezer at -70
0
C in RCS
laboratory.

(c) Transfusion Transmissible Infections Testing (TTI):

Store Kits for HBsAg, HIV, HCV, HbcIg, Anti Hbs and VDRL at 4-6
0
C in the
blood bank refrigerator in the TTI Laboratory or as per manufacturer's
instructions.
(d) Quality Control Laboratory (QC):

Store Kits for Factor VIII assay at 4-6
0
C in the QC laboratory or as per
manufacturer's instructions.
Store Copper sulphate stock and working solutions, 0.9% normal saline, and
distilled water at RT (22-25
0
C) in the QC laboratory.

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Store chemicals like copper sulphate, sodium chloride and calcium chloride
powders at RT (22-25
0
C) in the QC laboratory.

6.0 DOCUMENTATION

Maintain a stock register for all reagents.
On receipt, make entries of number of vials/kits received, name of manufacturer,
batch number and expiry date in this register.
Issue the reagents for use, only after a QC check is performed.
Enter all issue records in the stock register.
Order all reagents/kits, no sooner the critical level is reached.

N.B.:
Critical level for all reagents/kits is normally adjusted as per the requirement of reagents,
as well as the time taken by the procurement procedure to ensure that reagents are
received before the stock in use is exhausted. The new batch received should be tested
against the batch in use.

SP-036 01-12-2007

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DR V Saraswathi

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1.0

1 year 15-05-2008

Location Subject


Equipment Preventive Maintenance


Preventive Maintenance Contracts and
schedules

- In-charge QC Laboratory
- Master File


This procedure applies to all the instruments and equipments used within the blood bank.

2.0 RESPONSIBILITY

It is the responsibility of the supervisor of each laboratory to:

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Where relevant, prepare specifications and validation reports for new and modified
equipment.
Write an individual SOP for all equipment, which defines all the maintenance
requirements (eg. Routine, Preventative, Calibration etc.) regardless of whether carried
out by an external agent.
The requirements may be defined in the service contract referenced in the SOP.
Prepare the maintenance schedules for all equipment items. The schedule is to include:

Preventive.
Routine.
Extra maintenance.
Cleaning and sanitation.

3.0 REFERENCES

Technical Manual of American Association of Blood Banks 13th Edition, 1999,
Page 5.
Quality Manual, International Federation of Red Cross and Red Crescent
Societies, 1998, Pages 23-24.
The Gazette of India extra ordinary notification G.S.R. 245 (E) dated 5th April
1999, new Delhi, Part II Sec. 3 (i), Page 40.

4.0 PROCEDURE

4.1 Maintenance overview:

Identify each item of equipment in the unit that requires maintenance.
Ensure all items have an Asset Number.
Include clear outline of the relevant procedures, routine maintenance and
preventive maintenance and cleaning of equipment. Write operator instructions
for each item of equipment. Also include those responsible and names of service
personnel. Maintain a documented log of servicing for all items.
Identify the relevant procedures for equipment maintenance determine the
frequency of calibration and cleaning procedures clearly identifying the times eg.,
daily, monthly etc.
Prepare a complete equipment and instrumentation list consisting of the
following headings:
Equipment name / description.
Asset Number.
Serial Number.
Model Number.

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Operation
Operating Range.

Calibration:
Frequency.
Referenced documents.
Performed by

Performance Check:
Frequency.

Preventive maintenance:
Frequency.
Performed by.

Routine maintenance:
Frequency
Referenced documents

Cleaning:
Frequency.

Maintain a list of all equipment and instruments used in all sections / departments
in the QC lab to ensure all equipment within the department are documented.

4.2 Maintenance Schedules:

Draw up suitable schedules by maintenance type and frequency or by equipment
type. Define forward dates for the completion of maintenance and record the date
of actual performance in the schedule.

4.3 Service contracts:

Contracts need to be in place for all equipment items maintained by an external
agent.
Each service contract shall define exactly what is carried out / the frequency and
by whom it is completed.
At the completion of the service, a maintenance report is to be supplied, signed
by the contractor and the officer in charge. The report shall detail the work
carried out by the contractor.

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4.4 Repair & breakdown:

Operating instructions for each item of equipment shall identify the steps required
to be taken in the event of a fault or breakdown, and shall identify who is
responsible for organising service or replacement.
A log book of errors and corrective actions is to be maintained for all equipment
items. In the event of equipment breakdown, it is essential that it be clearly
labelled and identified as being OUT OF SERVICE.

4.5 Maintenance overdue:

The Quality Control Laboratory shall determine the suitability for ongoing use of any
equipment that has passed it due date for routine maintenance (where this routine
maintenance does not involve calibration). The laboratory must document their reasons
for continuing to use an item of equipment that is overdue for maintenance. Where
appropriate this should include explanation (and supportive evidence where available)
that product quality has not been compromised by this delay in maintenance. Where
possible, documentary evidence from the manufacturer supporting this decision should
be provided. Steps should be taken at the next instance of routine maintenance to
evaluate whether any discernible damage has been caused to the equipment by the delay
in maintenance.

6.0 DOCUMENTATION

Maintain individual files of service reports of all equipments.
Enter the details of routine as well as trouble-shooting service calls by the
manufacturer's engineer in the equipment maintenance register.
Maintain a file of all manufacturer's instructions and where required display them
close to the equipment.
Record the name, address and telephone number of the service engineer to be
contacted in case of need.

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1.0

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Location Subject


Equipment Maintenance: Calibration


Calibration

- In-charge QC Laboratory
- Master File


This procedure covers those measures taken to ensure the integrity, accuracy and
reliability of measurement data for equipment and instruments used in the collection,
testing and storage of blood products. The procedure is applicable to all equipment used
to control or evaluate suitability of starting materials, in process products and finished
products.

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2.0 RESPONSIBILITY

It is the responsibility of the supervisor of the section to which the equipment belongs
to:

Plan, schedule, organise and maintain records of the calibration programmes for
various equipment under their control.
Ensure that equipment and instruments are continuously calibrated or are
removed from use.
The Supervisor should train staff for performing calibration/performance checks.

3.0 REFERENCES

Blood Programme Quality Manual IFRCRCS Page 23.

4.0 DEFINITIONS

Calibration:
A set of operations, which establish under special conditions the relationship between
values indicated by measuring instruments and standards.

Performance checks:
The routine checking of the performance of an instrument to verify that it has remained
within specified range of accuracy and precision.

Accuracy:
The closeness of agreement between the result of a measure and the true value of
measurement. Calibration is used to determine the accuracy of an instrument.

Precision(Repeatability):
The closeness of agreement between the results of successive measurement of a defined
procedure several times under prescribed conditions.

Measurement standard:
A measuring instrument or material, which physically defines a unit of measurement or
value of a quantity. Measurement standards used for calibration should be traceable to
the SI units of standard measurements.

5.0 PROCEDURE

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5.1 Calibration Schedules:
Purchase each new piece of equipment or instrument according to specifications.
Place new equipment on an Asset register prior to use.
Ask the supplier prior to delivery or after installation to calibrate new equipment
and provide a certificate of calibration.
Maintain Calibration / Maintenance schedules for all equipment.
The schedules of calibration or performance checks should be based on:

Manufacturers recommendations.
The history of the item as per reliability.
Reference standards.
Recalibrate the measuring devices based on time intervals.

5.2 Reference standards, Traceability and Calibration Limits:
5.2.1 Reference Standards and Trace ability:

All measurement standards used to calibrate measuring devices should be traceable to a
national standard of measurement either:

Directly through purchase of pre-calibrated certified standards. These shall be
supported by calibration documents or certificate from the supplier stating the
date, accuracy (assigned value and units of measure), traceability and conditions
under which the results were obtained. These Standards shall be re-calibrated at
pre-determined intervals.
Indirectly by preparation of an internal working standard calibrated against a
certified standard. These shall be supported by internal test reports and any other
supporting documentation.
Where no recognised external standard exists an internal standard may be
prepared and calibrated provided a written procedure is prepared and a rationale
for assigning values, accuracy and units is established. These standards shall be
supported by suitable records of calibration as above.

5.2.2 Calibration Limits:

Calibration is concerned with the measurement of values and their comparison with
acceptable limits of standards resulting in adjustment or correction, if necessary.

Compare calibration results with established limits for accuracy for the measuring
device. If the device being calibrated does not fall within the limits then re-adjust and re-
calibrate until it falls within pre-established limits. If not, remove from use.

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The establishment of limits should be based on a combination of:

Those specified at the time of purchase.
Recommendations from the manufacturer.
Limits established in reference standards.
The acceptable limits required for satisfactory calibration of each instrument
should be identified or referenced in the relevant procedure.

5.3 Calibration and Performance Check Procedures:

Prepare documented procedures based on the instrument manufacturer's written
instructions and use for the calibration and performance checks for all measuring
instruments and measurement standards.
Calibration procedure should include:

A list of equipment to which the procedure is applicable.
Calibration points, environmental requirements and special conditions.
Limits for accuracy.
List and identity of traceable standards.
Sequence of calibration steps.
Instructions for recording data with reference to the relevant Standard
Form.
Performance checks procedures should follow a similar format.

5.4 Labelling:

Label all calibrated equipment with a label that has the following information:
Date of last calibration.
Signature of person who performed the calibration.
Date next calibration due.
Label the equipment that has passed its calibration due date until it is re-
calibrated.

6.0 DOCUMENTATION

Maintain complete records for the calibration and performance checks of all
equipment and instruments.
Calibration and Performance Check test records should include(where
appropriate):

Asset Register Number.
Instrument Serial Number.
Limits for calibration(refer 4.4.2).

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Date of calibration / performance check.
Due date for next calibration.
Any details of adjustment* or repair.
Results of the calibration*/performance check.
Statement of compliance, or details of non-compliance and action taken.
Signature/initials of the person performing the calibration/performance
check.

* It is important that the results of calibration before and after any adjustment are
recorded.
Maintain calibration and performance check records for five years.

7.0 CORRECTIVE ACTION

Conduct a review if any measuring device is found to be out of calibration and requires
adjustment. Take corrective action where appropriate.

If the item can be adjusted back into calibration, it may continue to be used. If the item
cannot be adjusted back into calibration, it must not be used until the situation is
corrected. Under these circumstances attach an identifying label stating that the item is
under repair and is not to be used.

The Supervisor must assess the likely impact of the inaccuracy of the affected
measurement on the quality of current product and product produced since the previous
satisfactory calibration.

Factors influencing the degree of risk include:

a) Critical nature of the measurement.
b) Sensitivity of quality control testing to the consequences of the inaccuracy.
c) History of production records and performance checks.

Additional quality control testing may be instituted to determine whether quality has
been compromised. Where it is likely that quality has been compromised this shall be
communicated to senior management and document reports.

8.0 RELOCATION OF INSTRUMENTS

Recalibrate the equipment (especially non-portable) when relocated. The manufacturer's
recommendations on the need for re-calibration shall be sought when relocating non-
portable instruments.

9.0 EXTERNAL CALIBRATION CONTRACTORS

STANDARD OPERATING
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Date : 15-05-2008
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Make an agreement with the contractors to supply written reports of calibrations, which
should include:

Use of standards and references traceable to national standards.
Certification/licensing by the equipment manufacturer, if available.
Check all certificates or reports supplied by approved external laboratories on
receipt. Certificates and reports should contain the same information as required
in 5.0 above.

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STANDARD OPERATING
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Section : I
Issue : 1.0
Date : 15-05-2008
Page : 137 of 197


Location Subject

Incident Report


Mechanism for correction and
prevention of errors and incidents

- Quality Assurance Manager
- Supervisor in charge of Donor Area
- Supervisor- Red Cell Serology Laboratory
- Supervisor- TTI Testing Laboratory
- Supervisor- Quality Control Laboratory
- Supervisor- Component Laboratory
- Master File


The procedure covers all incidents that would affect the quality of blood products &
services. The procedure applies to all incidents, adverse reactions, equipment used in
collection, testing & storage of blood products. The incident reporting process should be
clearly defined so that information is tracked and acted on and feedback provided.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 138 of 197


It is the responsibility of all the technical staff to report any incident/accident to
the section supervisor who will submit the report to the Quality Assurance
Supervisor/Manager.

The Quality Assurance Supervisor/Manager is responsible to review the
completed report and report to the Director for further investigation and
implementation of remedial measures if any.

3.0 REFERENCES

Technical Manual of American Association of Blood banks 13th Edition,
1999, Pages 3, 14-15.

4.0 DEFINITIONS

Incident Reporting:
Is a process improvement tool that is used to identify problems, analyse the cause,
develop solutions, execute the solution and track the effectiveness.

Performance checks:
The routine checking of the performance of an instrument to verify that it has remained
within specified range of accuracy and precision.

Corrective Action:
Is required for error and accident reports and is usually connected to a process
improvement activity. It is an immediate remedial action taken to correct the effect of a
defined event.

Preventive Action:
Follow up action taken to prevent a defined event from re-occurring.

Incident:
An Event that results from a deviation from a system, process or procedure that may
affect the:

Safety, purity, potency or effectiveness of the product.
Health or safety of a donor, product recipient, member of staff/public.
Trace ability of records.

This event may have been identified either prior to or after distribution of a product or
service

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Technician Reports to Section Supervisor
Section Supervisor completes report and Evaluates
Report to QA Manager

5.0 PROCEDURE

1) Document all incidents on the standard form (Incident Report Form).
2) Forward the incident summary report to the section supervisor for evaluation and
completion.
3) Initiate incident tracking.
4) Develop corrective/preventive Action in consultation with Section Supervisor,
QA Manager and the Director.
5) Forward original documents to the QA Manager within 3 working days of the
event.
6) The QA Manager reviews the report for completeness and appropriateness of
corrective action.
7) The status of an event remains active until effective action is taken and closed
out. Record the details, date of action and close out and get the reports form
signed by the Director.
8) Notify the Director immediately in case of critical incidents such as those that
could result in loss of life, product recall, failure to operate or adverse publicity
9) Provide monthly summary reports to the Director.

FLOW CHART FOR INCIDENT REPORTING PROCESS

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Date : 15-05-2008
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Initiate incident tracking
Corrective/Preventive Action
Submission of documents to QA Manager
Review by Director & QA Manager
Close out

6.0 DOCUMENTATION

Record all incidents on an incident report form. File all record forms.

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STANDARD OPERATING
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Section : I
Issue : 1.0
Date : 15-05-2008
Page : 141 of 197


Location Subject

Q C of Antisera


Q C of Reagents

- Incharge of Q C Lab
- Q C Record


It is mandatory to submit every lot of reagents for Q.C Anti A, Anti B, Anti AB, Anti
D, AntiH, Anti human globulin reagent( coombs) and other reagents.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 142 of 197


It is the responsibility of incharge technician of Q.C to collect samples, and medical
officer who check the results.

3.0 REFERENCE



Test tubes with rack
Antisera reagents
Normal saline

5.0 EQUIPMENT

Centrifuse

6.0 METHOD

Each lot of Antisera should be subjected to internal quality control.
Commercial antisera & reagents are licenced by FDA.
Every day observe the reagents for any change in colour, for turbidity , for any
contaminants and any leakage.
If any changes observed with the reagents stop using them and inform the
supplier to take back the lot and start using new lot.
Do quality control for new lot or new company.

RED CELLS FOR TESTING

ANTISERA POSITIVE REACTORS NEGATIVE REACTORS

Anti A Positive Reactorsp Negative Reactors
Pooled A Cells Pooled B Pooled O
Cells, O LISS Cells
Anti B Pooled B Cells Pooled A Pooled O
Cells,O LISS Cells

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Anti A B Pooled B Cells Pooled O Cells, O LISS
Cells
Anti D Rhd Positive Cells Rhd Negative Cells
Bio Clone (Any A B O group) (Any A B O group)
Anti D Rhd- Positive Rhd Negative
Mono Clonal (Any A B O group) (Any A B O group)

Avidity ( slide method )

Take a white coloured tile put two drops of antisera-A on it.
Add one drop of 5% A group red cell suspension and mix with a plastic or
glass rod.
Start the stop watch while adding red cell suspension.
Observe for agglutination.
Note the time where agglutination appeared.
Usual time is between 8-10 seconds.
Do the procedure for Anti-B , Anti-AB , Anti-D and others.

Specificity

Take four test tubes and add two drops of Antisera-A and add A cells to first
tube , B cells to second tube and O cells to third test tube, O LISS cells fourth
tube
Agglutination in first tube and no agglutination in other two tubes indicate that
the antisera is specific for the indicated cells.
Subject it to indirect coombs test. It should be negative. It indicate that
particular antisera do not have minor blood group antibodies.
Do the procedure for other Antisera also.
For Anti-D with O positive cells by indirect coombs test it should give
agglutination and with O negative cells it should not give any agglutination

Intensity

Strength of agglutination ( reaction ) done by slide method
Take a test tube and put two drops of Antisera-A and add one drop of 5% A
red cell suspension.
+ / - Doubtful for agglutination.
+1 Small scattered clumps
+2 Two or more equal sized clumps

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+3 One big clump with small clumps
+4 One big clump in center
Do the same procedure for other Antisera also.

Potency or Titre:--

The reciprocal of highest dilution of antibodies which give agglutination.
Take 10 test tubes in a row and label them with dilution numbers. 1:2, 1:4, 1:8,
1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024.
Put two drops of normal saline in all the tubes
Add two drops of Antisera-A in the first tube, Mix thoroughly and transfer two
drops of first dilution to second tube, continue like this up to last tube and
discard two drops from last tube.
Add one drop of 5% A red cell suspension to each tube, repeat procedure with
A
2
cells, A, B cells and A
2
Bcells.
Incubate at room temperature for 30 minutes.
Observe for agglutination and note at what dilution there is no agglutination.
Dilution where there is minimum agglutination is called N point.
Anti-A, Anti-B, Anti-AB, Titer is 1:256
Anti-D titer is 1:64
Potency in case of Anti-A sera and Anti-AB sera it should be tested with A1 cells , A2
cells , A1B cells and A2B cells.

Antisera Red cells for testing
A1 cells A2 cells A1B cells A2B cells
Antisera-A 1:256 1:128 1:64 1:32
Antisera-AB - - 1:256 1:64

7. 0 INTERPRETATION

Anti A, B, AB titers should be 1:256
Anti D titers should be 1:64

8. 0 DOCUMENTATION

All results should be entered in Q.C register and computer.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
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1.0

1 year

Location Subject

QC Laboratory

Sample Collection for Quality Control

Sample Collection for Quality Control

- Supervisor of QC Laboratory
- Master File


It is mandatory to submit every 100
th
bag of each component for quality control. Apart
from Whole blood the components included for Q.C are modified blood, packed rbc,
leucoreduced rbc, washed rbc, platelet concentrate, single donor platelets, fresh frozen
plasma, cry poor plasma and cryoprecipitate

STANDARD OPERATING
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Section : I
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Date : 15-05-2008
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2.0 RESPONSIBILITY

It is the responsibility of In-charge Technician of Q.C to collect samples, and medical
officer who check the results.

3.0 REFERENCES

1999.

4.0 MATERIALS

Test tubes with rack
Forceps

5.0 PROCEDURE

5.1 Principle:

To Carry out QC tests on all the components, the samples should be collected in a sterile
environment. It is advisable to collect under laminar flow cabinet

5.2 Method
Every 100
th
bag of component should be submitted for Q.C.
Weigh the bag with the help of weighing balance.
Maintain sterile working place while collecting the samples.
Clamp the tubing at proximal end of bag; mix the component thoroughly by
inverting the bag several times.
Cut the tube at the distal end of tubing and empty the contents within tubing
and collect the sample in a clean test tube.
Blood sample 5ml
Platelet concentrate 5ml
Fresh frozen plasma 5ml
Cryoprecipitate -- 5ml
Platelet concentrate sample should not be collected immediately after
preparation. Collect from bag, which was kept in agitator at least one day,
and do Q.C within 2 hours of sample collection.
FFP and cryo Thaw the bags at 37 degree C and collect the sample, issue
as early as possible no longer than 24 hours. Factors should be estimated
within 2 hours of collection otherwise keep sample in deep freezer.

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5.3 Interpretation:
Observe the samples grossly for any clots. Any lysis, any turbidity or
cloudiness, change in colour.

6.0 DOCUMENTATION

Technician who performed the test and the technician who checked the results
should initial all records.

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1.0

1 year 15-05-2008

Location Subject

Hemoglobin estimation


Cyanmethemoglobin method and
CuSo4 method

- Incharge of Q.C lab Q.C Record

1. 0 SCOPE & APPLICATION

th
bag of Whole blood , modified blood packed rbc ,
leucoreduced rbc , washed rbc .
All the donors should be submitted for Hb estimation.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 150 of 197


It is the responsibility of incharge technician of Q.C to do hemoglobin estimation , and
medical officer who check the results.

3.0 REFERENCE



CuSo4 powder
Drabkins solution
Distilled water
Spirit or betadin
Reagents of automated cell counter

5.0 EQUIPMENT

Colorimeter
20microleters pipette
Lancets

6.0 PRINCIPAL

To do Hemoglobin estimation there are three methods.

7.0 CuSo4 METHOD :--

Depending on the specificgravity of the CuSo4 and the Blood Hb can be
estimated.The drop of blood gets enclosed in a sac of copper proteinate, which
prevent any change in specific gravity for about 15 seconds.
If drop of blood remains at the surface or rises from the bottom of the solution,
sinks within 15 seconds Hb is >12 gm / dl.

Cyanmethhemoglobin Method:--

Dilution of blood in a solution containing potassium cyanide and potassium ferricyanide
Hb, Hi, HbCo ( but not HbS ) are all converted to HiCN.

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The absorbance of the solution is measured in photoelectric colorimeter at a wave length
of 540nm or with a green filter.

By automated cell counter :--
Uses colorimeter method.

Method :--

CuSo4 method :-

Preparation of CuSo4 solution: Weigh 8.33 gm of CuSo4 5 H2O powder with the help
of weighing balance and add 100ml of Distilled water in a measuring jar. Mix the
solution thoroughly and transfer it into a bottle with tight lid. Check the specific gravity
of solution with urinometer. It should be 1.053. Keep the reagent at room temperature.
Take the working solution in a wide mouthed jar and keep it in donor
Examination room.
Puncture Donor finger tip with a lancet after making it sterile with spirit or
betadin. Allow to form a drop and allow it to fall on CuSo4 solution surface from one
inch height very gently.
If drop sinks- Hb is > 12.5 gm / dl
If drop floats- Hb is < 12.5 gm / dl

Cyanmethemoglobin method:-

For detection of hemoglobin concentration.
Take 5ml of Drabkins solution in a test tube and add 20 micro liters of citrated
blood.
Wait for 3 minutes to allow the reaction to complete.
Switch on the colorimeter , set at 540nm wave length.
Take Drabkins solution in a flat bottomed cuvette as blank and adjust the
colorimeter reading to zero with the help of fine adjustment.
Take the test solution and record optical density.
Take the Hemoglobin value from the standard chart.
By automated cell counter:--
Feed the sample collected in EDTA vial after mixing thoroughly. Automation is more
sensitive than manual methods.
8.0 INTERPRETATION

If Hb value is >12.5 gm / dl accept the donor for donation. If Hb value is < 12.5
gm /dl do not accept the donor for donation.
The Hb value should be > 11.5gm / dl for all single bags
The Hb value should not be > 17gm / dl for packed cells, modified blood ,
leucocyte reduced rbc , washed rbc.

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PROCEDURES
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Date : 15-05-2008
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9.0 DOCUMENTATION

All results should be entered in Q.C register, Master record and computer.

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1.0

1 year

15-05-2008

Location Subject


Packed cell Volume

By Wintrobes method
By automated cell counter

- Incharge of Q.C lab Q.C Record


It is mandatory to see PCV for every 100
th
bag of Whole blood , modified blood ,
packed cells leucoreduced rbc , washed rbc .
Packed cell volume is mandatory to do on all blood samples collected for Q.C

STANDARD OPERATING
PROCEDURES
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Date : 15-05-2008
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2.0 RESPONSIBILITY

Sample collected for Q.C of blood bags.

3.0 EQUIPMENT

Refrigerator to store samples at 2-8 degree temperature.
Tabletop centrifuge
Lumbar puncture needle

4.0 SPECIMEN:

Q.C blood sample

5.0 REAGENTS:

NIL

6.0 GLASSWARE

Wintrobes tubes

7.0 PRINICIPAL

EDTA sample is filled in Wintrobes tube and centrifused at 3500 rpm / minute for 30
minutes to measure PCV

8.0 METHOD

Take the wintrobes tube , wash thouroughly and remove all the water by shaking
the tubes.
Take 2ml syringe and fix a lumbar puncture needle. Mix sample thouroughly and
draw 1ml of blood into syringe.
Push the needle upto bottom of the wintrobes tube and gradually draw the needle
upwards while releasing the blood.
Stop exactly at 0 (zero ) and keep the tubes in centrifuse in opposite direction
sockets. Centrifuse at 3500 rpm / min for 30 minutes.

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9.0 INTERPRETATION

Observe the tubes grossly
Green colour Pseudomonos infection
Yellow colourJaundice
Pink colour Hemolysis
Milky white Hyperlipidemia
If any change in colour go back to the bag and keep it in erect posture for 1-2
hours in the cold room. Again observe the bag for any change in plasma colour , if
present discard the bag.
If any clots or any colour change in red cells discard the bag.
Observe the buffy coat in wintrobes tube increase in thickness of buffy coat
>3mm suggestive of leucocytosis.
PCV should be 45-55 for whole blood
PCV should be always < 70 % for packed cells and other blood bags.

10.0 DOCUMENTATION


SP-043 01-12-2007

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1.0

1 year

15-05-2008

Location Subject

QC Laboratory

WBC Count


WBC Count by Neubar Chamber
Method

- Master File


WBC count is mandatory to do on all blood samples collected from leucoreduced RBC,
Platelet concentrate, Single donor platelets, granulocytepheresis for Q.C.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 157 of 197


It is the responsibility of Technician of Q.C to perform WBC estimation by Neubar
Method and record the results.

3.0 REFERENCES

1999.

4.0 MATERIALS

4.1 Equipment:

Refrigerator to store samples at 2-8
0
C temperature.
Modified Neubars chamber
Microscope
Capillary tube
Cover slips

4.2 Specimen:

QC blood sample

4.2 Reagents:

WBC fluid
Reagents for cell counter

4.3 Glassware:

Test Tubes

5.0 PROCEDURE

5.1 Principle:

Ammonium oxalate in WBC fluid dissolve the RBC and gentian violet gives colour to
the WBC. The standing out WBC can be counted under microscope in the Neubars
chamber.

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5.2 Method

Take WBC Fluid - 380 micro. It (1 in 20 dilution)
Blood - 20 Micro. It
Load the chamber carefully, wait and allow the fluid to settle. Do counting in 4 WBC
corners.

N X D
No. of WBC /Cumm =
V
Neubar Chamber Two graduated chambers on each side with a central square for RBC
Count & Platelet count. 4 peripheral corner squares for WBC count, etc.., Square meant
for WBC count is further divided into 16 small squares to facilitate counting of cells with
a length of 1 mm, breadth of 1mm and 0.1 mm depth created by cover slip when kept on
the Neubar chamber. When the fluid is allowed to fill the chamber with the help of
micropipette the volume of fluid is
V = 1 X 1 X 0.1 = 0.1
0
3 mm
(For WBC Count do counting all four corners)

N X D
No. of WBC /Cumm =
V
Where,
N = No. of cells counted in 4 corners
D= Dilution = 1 in 20
V= 0.1 X 4 = 0.4
0
3mm

= N X 200
= N X 50
4

5.3 INTERPRETATION

WBC count should be < 500 cells / cumm is one log reduction.
WBC count < 200 cells / cumm is two log reduction

6.0 DOCUMENTATION


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
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1.0

1 year

15-05-2008

Location Subject

QC Laboratory

Platelet Count of Platelet concentrate


Platelet Count of Platelet concentrate
by Neubar Chamber Method

- Master File


th
bag of Whole blood, modified blood packed rbc,
leucoreduced rbc, washed rbc. All the donors blood should be submitted for Hb
estimation.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 161 of 197


It is the responsibility of Technician of Q.C to perform Platelet count by Neubars
chamber method and record the results.

3.0 REFERENCES

1999.

4.0 MATERIALS

4.1 Equipment:

0
C temperature.
Modified Neubars chamber
Microscope
Capillary tube
Cover slips

4.2 Specimen:

Platelet concentrate sample

4.2 Reagents:

Normal Saline
Reagents for cell counter

4.3 Glassware:
Test Tubes

5.0 PROCEDURE

5.1 Principle:

Normal saline acts as a dilution fluid, and the platelets appear as refractile spherical
or comma shaped cells. It is possible to observe RBC and WBC along with the
platelets.

5.2 Method

STANDARD OPERATING
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Date : 15-05-2008
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5.2.1 Platelet concentrate
Measure volume of bag and deduct empty bag volume to get exact volume of
platelet concentrate. (Normal 50 70 ml).
Take normal concentrate - 380 Micro. It 1 in 20 dilution
Platelet normal saline - 20 micro. It
Put cover slip on the Neubar chamber.
Load undiluted platelet concentrate fluid in one side.
Load diluted platelet concentrate fluid in another side.
See under Microscope Low power & high power.

5.2.2 Undiluted sample
Look for any bacteria & fungi.
Observe RBC - Fragmented RBC Suggest hemolysis large number of RBC
reduce PH.
Do WBC Count Whole blood, modified blood WBC count in normal range.
(4000 11000/ cumm).
Increased WBC Count look for any abnormal cells. Increase neutrophilic
leucoytosis - septicemia.
Leucoreduced RBC WBC count should be less than 500 cells / cumm.
Platelet concentrate -- WBC count should be less than 500 cells / cumm.
Mostly we will see lymphocytes with occasional granulocytes.

5.2.3 Diluted sample

Neubar Chamber Two graduated chambers on each side with a central square for RBC
Count & Platelet count. 4 peripheral corner squares squares for WBC count, etc.., Square
meant for WBC count is further divided into 16 small squares to facilitate
Counting of cells with a length of 1 mm, breadth of 1mm and 0.1 mm depth created by
cover slip when kept on the Neubar chamber. When the fluid is allowed to fill the
chamber with the help of micropipette the volume of fluid is

V = 1 X 1 X 0.1 = 0.1
0
3 mm
(For platelet Count do counting in the central chamber, which include all four
corners and central small squares.)
Central chamber divided into 25 small squares and each small square again
divided into 16 small squares.
(Count 10 small squares (platelets brilliant and 2-4 microns size)

0.1
Each small square volume = ______
25

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0.1 1
10 small squares volume = ________ x 10 = _____
25 25

N = number of platelets counted in 10 small squares in the central chamber
D = dilutional factor ( 1 : 20 )

Take a test tube and put 380 micro liters of normal saline and add 20 micro liters
of platelet concentrate. Wait for few minutes. Keep the cover slip on the Neubars
chamber and charge the chamber with the help of capillary tube. Keep the entire
Neubar chamber in a Petri dish along with a wet cotton swab and close it with a
lid. Wait for 20 minutes and count the platelets under microscope.

Number of platelets / cumm = N X D = N X 20 = N X 20 X 25 = N x 500

V 1 / 25

Number of platelets for bag = N x 500 x 1000 / ml
= N X 500 X 1000 x volume of bag in ml.

5.3 Interpretation

Normal range = 2- 4 x 10 to the power of 10 / bag - for platelet
concentrate.
2-4 x 10
11
/bag for SDP

6.0 DOCUMENTATION


SP-045 01-12-2007

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STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 164 of 197


1.0

1 year

Location Subject

QC Laboratory

pH Measurement


PH Measurement of Platelet
Concentrate by pH meter / pH paper

- Master File


pH measurement is mandatory on every 100
th
bag of platelet concentrate, single donor
platelets, fresh frozen plasma, cryoprecipitate, normal saline used in lab, washing
solutions used at screening table and distilled water. 5ml of sample is collected for
measurement of pH.

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 165 of 197


It is the responsibility of In-charge Technician of Q.C to perform pH measurement and
record the results

3.0 REFERENCES

1999.

4.0 MATERIALS

4.1 Equipment:

0
C temperature.
PH meter
Commercially available pH strips.

4.2 Specimen:

FFP sample
Cryoprecipitate sample
Normal saline sample
Washing buffers

4.2 Reagents:
PH buffers (of 4, 7 & 10 pH)
Normal Saline

4.3 Glassware:

Test Tubes
Wide mouthed small jars for samples

5.0 PROCEDURE

5.1 Principle:
pH can be measured either with the help of pH meter which display the reading or with
the pH strips which can be compared with the standard colours.

5.2 Method
Switch on the pH meter. Keep the PH meter probe in the buffer with pH 4 value
Adjust the pH meter to 4 exactly.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 166 of 197


Again keep the probe in the buffer with PH 7 value and adjust the PH meter to 7
exactly.
Take the test solution in a wide mouthed small jar and keep the PH meter probe
in that solution.
See the result in PH meter recorder display and record the result.
With PH strips dip the strip in the test solution and compare the colour with the
given standard and record the result.

5.3 Interpretation

pH for platelet concentrate, single donor platelets, FFP, cryo and other solutions
should be in between 6 8.

6.0 DOCUMENTATION


SP-046 01-12-2007

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Dr V Saraswathi

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STANDARD OPERATING
PROCEDURES
Section : I
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1.0

1 year

Location Subject

QC Laboratory

Fibrinogen Assay

By manual method
By coagulometer

- Incharge of Q.C lab
- Q.C Record


Fibrinogen assay is mandatory to do on every 100
th
bag of fresh frozen plasma
and cryoprecipitate .

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 168 of 197


It is the responsibility of the of the technition of Q.C lab to do the Factor VIII
assay with coagulometer or by manual method.

3.0 REFERENCE

Manufacturer instructions.


Sample collected for Q.C from FFP and cryoprecipitate.

5.0 EQUIPMENT

Coagulometer
Water bath at 37 degree centigrade

6.0 SPECIMEN

FFP sample

7.0 PRINICIPAL

Fibrinogen reagent utilizes the clotting time method for determination of plasma
fibrinogen levels,wherein excess bovine thrombin is used to clot the diluted plasma.
First a standard curve is prepared by using a reference plasma of known fibrinogen
content
( calibrator normal ). When thrombin is added, the clotting time obtained is inversely
proportional to the fibrinogen content. The sample plasma , at a dilution of 1/10 is
clotted with thrombin and the resultant clotting time used to interpretate fibrinogen
levels from the standard curve.
8.0 METHOD

FIBRINOGEN ASSAY (MANUAL METHOD)

Principle Addition of thrombin acts on fibrinogen and converts into fibrin
Normal plasma levels are 200 400 mg| dl
Standard curve:-
Reagents 1. Fibroquint thrombin reagent (or)
Fibrinogen calibrator stock solution

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 169 of 197


2. Owrens buffer
3. Thrombin reagents
TEST TUBES

1 2 3 4
Owrens buffer NIL 800 l 900l 950 l
Fibrinogen stock solution 200 l 200 l 100 l 50 l
Dilution factor NIL 1 : 5 1 : 10 1 : 20
(Incubate at 37
o
C for 60
seconds)

Add thrombin reagent 100 l 100 l 100 l 100 l

Start stop watch and note the time in seconds where clot seen Plot the graph

TEST PROCEDURE WITH SAMPLE:-

Take 200 l plasma 1 : 10 dilution
800 l owrens buffer
Take 1 : 10 dilution plasma 200 l
Incubate at 37
o
C for 60 seconds
Add 100 l of Thrombin reagent
Start the stop watch and note the time when clot appeared.

FIBRINOGEN ASSEY BY COAGULOMETER

Test plasma sample within 2 hours
Dilute sample 1 : 10 with buffer
STANDARD CURVE
0
10
20
30
40
50
60
70
80
90
NIL 1:05 1:10 1:20
0 400 200 100
FIBRINOGEN CALIBRATOR DILUTION g/dl
T
I
M
E
/
S
E
C

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 170 of 197


Owrens buffer 900 l
Plasma - 100 l
Turn on wait until lead I lighting
Select FIB as active test
Check calibrator
Allow reagent to pre warm for 5 minutes

Procedure:-
Take 50 l of 1 : 10 diluted plasma
Pre warm for 1 minute
Transfer cuvette to measuring position
Press key Optic (key gets activated)
Add 25l of Thrombin
Again press Optic key
The result displayed in

9.0 INTERPRETATION

Fibrinogen concentration should be 200 to 400 mg for FFP and 150 to 250 for
cryoprecipitate.75 % of bags which submitted for Q.C should reach this criteria.

10.0 DOCUMENTATION


SP-047 01-12-2007

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Dr V saraswathi

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STANDARD OPERATING
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1.0

1 year

Location Subject

QC Laboratory

Factor VIII Assay


By manual method
By coagulometer

- Incharge of Q.C lab
- Q.C Record


Factor VIII assay is mandatory to do on every 100
th
bag of fresh frozen plasma
and cryoprecipitate .

2.0 RESPONSIBILITY

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 172 of 197


It is the responsibility of the of the technition of Q.C lab to do the Factor VIII
assay with coagulometer or by manual method.

3.0 REFERENCE



Sample collected for Q.C from FFP and cryoprecipitate.

5.0 EQUIPMENT

Coagulometer
Water bath at 37 degree centigrade

6.0 SPECIMEN

FFP sample

7.0 REAGENTS

Factor VIII deficient plasma
Standard human plasma
Imidazole buffer
Pathromptin reagent
Calcium chloride solution
Normal saline

8.0 GLASSWARE

Test tubes

9.0 PRINICIPAL

Plasma deficient in factor VIII ( factor of intrinsic pathway ) result in a prolonged
partial thromboplastin time, which can be used to quantify or confirm factor VIII

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 173 of 197


deficiency in patient or fresh frozen plasma and cryoprecipitate sample. A reference
curve obtained with dilutions of standard human plasma. When the sample is mixed with
factor VIII deficient plasma the sample will provide factor VIII. On addition of
pathromptin which contain phospholipids and a surface activator the intrinsic pathway
gets activated. On addition of Calcium chloride the calcium ions triggers the
coagulation process,the to formation of fibrin clot is measured.

10.0 METHOD ( MANUAL METHOD )

a) Standard graph:-

1. 1
st
Test Tube 200ul Standard human plasma 1 in 5 dilution
+
800ul Imidazole buffer ( 100% )

2. 2
nd
Test Tube 500 ul from 1st test tube 1 in 10
+
500 ul Imidazole buffer (50%)

3. 3
rd
Test Tube _ 200 ul from 2
nd
test tube 1 in 50
+

4. 4
th
Test Tube - 100 ul from 3
rd
test tube 1 in 500

From each dilution take 100 ul into 4 tubes
Add Factor VIII deficient plasma 100 ul
Add APTT reagent
Incubate at 37
0 C
water bath for 120

seconds
Add Cacl2 100ul
Note the time in seconds, observe clot formation

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 174 of 197


Test:
Sample _ 200 ul 1 in 5 dilution
Imidazole buffer 800 ul (100%)

Take 100 ul 1:5 diluted samples
100 ul Factor VIII deficient plasma
100 ul APTT
100 ul Cacl2
b). FACTOR VIII ASSAY BY COAGULOMETER:

Fibrin formation is the end point. Factor VIII is factor of intrinsic pathway. If PTT
prolonged it indicate disfunction in intrinsic pathway factors i.e .
Factor VIII , IX, XI, and XIII.

Sample: Anticoagulant sodium citrate in 1 in 9 ratio
Sample preparation:

Dilute sample with
Imidazole buffer 900 ul 1 in 10 dilution
Test Plasma 100 ul
Procedure:
Turn on
Select Fac as active test
Check calibration
Allow reagents to pre warm for 5 min

Take 25 ul 1:10 plasma in to a cuvette
Add 25 ul factor VII deficient plasma
Add 50 ul of APTT reagent
ncubate 5 min
Transfer cuvette into measuring positive and activate Optic key
Add 10 ul pre warmed Cacl 12 and simultaneously start Optic key again
Instrument starts reading in seconds
When it detects clot it display time and % of Factor VIII
0
20
40
60
80
100
1% 10% 50% 100%
Time/Sec

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 175 of 197


11. INTERPRETATION

Factor VIII concentration should be 80 to 140 units.75 % of bags which
submitted for Q.C should reach this criteria

12. DOCUMENTATION

All records should be initialed by technician who performed the test and
the technician who checked the results.

SP-048 01-12-2007

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Dr V Saraswathi

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Location Subject

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 176 of 197


QC Laboratory

VWF Assay by Manual Method

VVF ASSAY BY MANUAL METHOD

- Master File


VVF assay is mandatory on every 100
th
bag of fresh frozen plasma and
cryoprecipitate.

2.0 RESPONSIBILITY

It is the responsibility of In-charge Technician of Q.C to perform Fibrinogen
assay with manual method.

3.0 REFERENCES

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 177 of 197


1999.
Manufacturers Instructions

4.0 MATERIALS

4.1 Equipment:

0
C temperature.
V W F assay kit

4.2 Specimen:

FFP sample

4.2 Reagents:

VW reagent Stabilized platelets
Restocitine
EDTA
(Suspend with distilled water on automatic shaker)

4.3 Glassware:

Test Tubes

5.0 PROCEDURE

5.1 Principle:
Stabilized platelets are agglutinated in the presence of VWF antibiotic Restriction.

5.2 Method

5.2.1 Manual Method ( Agglutination method):

Specimen Collection
Mix 1 part of Sodium Citrate with 9 parts of venous blood.
Centrifuge immediately 1500 RPM/min for 10 minutes.
Remove supernatant and store at +5 - +25
o
C until required i.e not > 6 hours.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 178 of 197


1 2 3 4 5 6
Specimen - 50l 500l 500l 500l 250l
Saline 250l 950l 500l 500l 250l 250l
Dilution Blank 1 : 20 1 : 40 1 : 80 1 : 120 -
On to each field of enclosed glass pipette and 50 l plasma dilutions in success
along with blank.
Add 50 l reagent
Agitate slowly for 1 minute
Assess the agglutination against a dark background
Observe for distinct agglutination
Content of VWF in % = titer of the specimen given on vial x dilution at which
agglutination appeared
Normal reference value = 50 150%

5.3 Interpretation
40% to 75% of the original should be present in the bag.

6.0 DOCUMENTATION


SP-049 01-12-2007

4

Dr V Saraswathi

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15-05-2008

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 179 of 197


Location Subject

QC Laboratory

Bacteriological Examination of the
Bags

By Manual method and automation.

- Incharge of Q.C lab.
- Microbiology department, Q.C Record


It is mandatory to submit blood and its components for bacteriological
examination. Samples collected from blood, platelet concentrate, FFP,
Cryoprecipitate and Anticoagulant solution of the bag into aerobic and anaerobic
culture bottles.

2.0 RESPONSIBILITY

It is the responsibility of the of Q.C technician to send samples for culture to
Microbiology department and the Medical officer to check the results.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 180 of 197


3.0 REFERENCE

Practical manual of microbiology.


Samples collected for culture from blood, platelet concentrate, FFP,
cryoprecipitate and anticoagulant solution of bag.

5.0 EQUIPMENT

Laminar flow cabinet
Forceps, Scissors
Sterile aerobic and anaerobic culture bottles.

6.0 SPECIMEN

FFP sample
Blood sample
Anticoagulant solution

7.0 PRINICIPAL

BacT/ALERT 3D instrument MB/ BacT bacterial detection system utilizes a
colorimetric sensor and reflectance detector to determine the level of Co2 with in the
bottle. If the microorganisms grow in the bottle co2 is produced which will change
the colour of the sensor on the bottom of bottle while incubating sample. The
instrument monitors this and determine the growth of bacteria.
MB/BacT process bottle contain a media in combination with the MB/ BacT
reconstitution fluid which promote growth of bacteria.
If colour turns to deep yellow it is submitted for further investigations.
For identification of bacteria in the component the sample submitted for Grams
staining, culture on the blood agar and maCconkeys agar.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 181 of 197


8.0 METHOD

Prepare the laminar flow cabinet. Every 1000 th bag (or) one out of every month
collection should be sent for culture. Clamp the proximal end of the bag tube and cut
the distal end with scissors. Discard the blood in the tubing into hypochlorite
solution.
Open the lid of culture bottle and allow 2ml of blood or component carefully into
both aerobic and anaerobic culture bottles. Close the lid immediately. Collect platelet
concentrate F F P cryoprecipitate and anticoagulant solution simultaneously. The
sample directly submitted to BacT / ALLERT 3D instrument. If deep yellow colour
detected at the bottom of bottle it is positive for bacteria. If no yellow colour the
culture is negative for bacteria.

All positive samples processed further
with Grams stain for
_ Gram Positive cocci or
_ Gram Negative bacilli
A loopfull of sample is incubated in peptone water for 30min at 37
o
C incubator.
If bacteria present pepton water turns turbid.
Again from peptone water streak on the Blood Agar and MaCconkeys Agar.
After 12 hours incubation the morphology of bacterial colony is observed by the
incharge microbiologist and reported .If necessary for further identification of
bacteria sample submitted for Biochemical reactions.

9. INTERPRETATION;-

Culture reports Negative for bacteria indicate that blood bank is maintaining sterile
procedures at donor complex, component preparation and storage.
If culture positivity is there check whether donation site of the donor is sterilized
properly or not by the phlebotomist.
Check components preparation whether maintaining closed system or not and
environment is clean or not. Any leakage of blood bags should be noticed and
recorded.
Check the refrigerators and the temperature charts. All refrigerators should be
calibrated with standard thermometers. Send one more sample from another bag for
culture. If culture positive in the anticoagulant solution keep the entire lot of bags in
quarantine, inform manufacturer and get another new lot of bags.

10.0 DOCUMENTATION

All results entered in Q.C book .

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 182 of 197


SP-050 01-12-2007

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STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 183 of 197


Location Subject
Apheresis room

Plateletpheresis


By Apheresis cell seperator

- In charge of components
- Q.C Record.


It is mandatory to do plateletpheresis on a voluntary donor when there is a
request from treating clinician.

2.0 RESPONSIBILITY

It is the responsibility of Incharge Technician of component preparation who
were trained in apheresis procedures and Medical officer to select the donor for
plateletpheresis and to do the procedure.

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 184 of 197


3.0 REFERENCE



BP apparatus, Tornquit , Spirit swabs , Calcium tablets, Oral fluids.
ACD solution 500 ml bag ( see expiry date before using )
A disposable kit meant for plateletpheresis ( see the manufacturer instructions
for code of the procedure and expiry date )
Rapid kits for detection of HIV, HCV, VDRL, HBs Ag.

5.0 EQUIPMENT

Automated cell separator ( Hemonetics or Baxter company )
Tube sealer.
Automated cell counter
Platelet agitator com incubater.
PH meter and neaubars chamber for platelet count.
Binocular microscope.

6.0 SPECIMEN

Blood samples collected from the donor into EDTA vial and PLAIN vial.

7.0PRINICIPAL

Plateletpheresis is the removal of platelets from a donor with the return of the
donors RBC, WBC and plasma.
Anti coagulant is added to the whole blood as it is drawn into the bowl, chamber or
belt depending on the cell separator used. The layering of the blood components
occurs based on density the desired fraction is diverted and remaining elements
returned to the donor.

8.0 REAGENTS: -

Anticoagulant - A C D Solution
Normal Saline -

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 185 of 197


Oral fluids
Tab Calcium

9.0METHOD

Donor Selection:
Donor must meet all the criteria of routine whole blood donation
Hb% - More than 12.5 gm/dl
HCT - 40% --45%
Donor must not have taken aspirin containing medication with in last three days.
Donor platelets count should be more than 3 X 10
5
/ cumm.
Reaming cell counts should be with in normal limit
ABO / Rh (D) typing and also antibody screening before platelet pheresis.
Screening for TTD always by ELISA method or other superior methods. As
donor waiting time is more with the routine screening methods go for rapid
method for HIV, HCV, HBs Ag and VDRL .If all are negative do the platelet
pheresis.
During platelet pheresis the donor platelet count decreases by about 20 30% of
original platelet count. Within 72 hours the original count returns back.
The interval between two donations should be at least 48 hours.
If donor donates a unit of whole blood, or if it becomes impossible to return red
cells during apheresis at least eight weeks should elapse before a subsequent
platelet -pheresis
Donor should not undergo platelet pheresis more than 24 times in a year.

10. HEMONETICS SDP PPROCEDURE

(HEMONETICS MCS 3P MODEL NO. 8000 IN U.S.A)

1. Press power switch : On
2. Instruction on monitor : Open the centrifuge cover
3. Instruction on monitor : Close the centrifuge cover
5. Protocol to select : For SDP select a3p / for

6. To continue press : yes

7. Now install the kit : Bowl/ Tubes etc
8. Press prime : Prime Prime Prime
9. Disable drip monitor : No

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 186 of 197


10. Confirm : Yes
11. Press prime : Prime
12. Hemo calculator : Press Modify program
13. Enter donor details : By using yes / no buttons
Enter details like : Height / weight / hct/ pl count
14. Machine will calculate : Platelet yield / blood volume /
Number of cycles
15. If above calculation is ok : Press yes
16. If wants to change it : Press Modify program
17. Monitor display : Ready
18. Now keep donor ready : Apply Bp cuff
19. Press cuff button : On
Select vein & clean with spirit
Give prick and verify the blood flow, by releasing clamp
20. Press draw button : On
ask donor to press the ball 10 times / minutes
check the donor pressure monitor (dpm)
21. If blood is flowing slow : Reduce pump pressure
50 or 40
22. After completion of cycles machine displays message
Procedure complete
23. Clamp the SDP bag
24. Remove needle from the donor
25. Seal / cut the tube of SDP bag label it
26. Preserve the SDP bag in platelet agitator at 22
0
c

11.0 STORAGE AND ISSUE

Platelets Collected by aphaeresis should be stored 22 - 24
0
in platelet agitator cum
incubator for 5 days.
Routinely group matched aphaeresis platelets should be issued in case of non
availability of any group units are without serious problems.

12.0 QUALITY CONTROL:

At least 75 % of units tested should conation 3 X 10
11
/ bag
PH 6-8.
If more than 2 ml of red cell contamination compatibility test carried out
WBC count should be < 500 cells / cumm 1 log reduction and < 200 cells / cumm
2log reduction.
Platelet count should be 3 X 100000/ bag

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 187 of 197


Dose of platelets
P1 X BV/ F
P1 = Desired Platelet increment X 10
9

BV = Patient blood volume in liters
F= Correction factor of 0.67
(for pooling of 33% transfused platelets in spleen)
Example: If a platelet increment of 40 X 10
9
is desired for patient with blood
volume 5 liters.
40 X 10
9
X 5 300 X 10
9

Dose = 0.67

13.0 COMPLICATIONS
Citrate toxicity
Haematoma
Vasovagal reaction
Hypovolemia
Allergic reaction
Hemolysis
Depletion of clotting factors.
Circulatory of respiratory distress
T T D
Lymphocyte loss
Depletion of proteins and immunoglobulin

14.0 DOCUMENTATION

Master Record
Quality Control Record.

SP-051 01-12-2007

5

Dr V Saraswathi

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Approved By Date

1.0

1 year

15-05-2008

Location Subject

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 188 of 197


Apheresis room

Plasmapheresis


By cell separator

- In charge of components lab
- In Charge medical officer


It is mandatory to do plasmapheresis on a voluntary donor when there is a request
from treating clinician .
To do exchange plasma transfusion on a patient with high antibody titer.

2.0 RESPONSIBILITY

It is the responsibility of In charge Technician of component preparation who
plasmapheresis and to do the procedure.

8.0 REFERENCE

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 189 of 197




A disposable kit meant for plasmapheresis ( see the manufacturer instructions for
code of the procedure and expiry date )
Rapid kits for detection of HIV, HCV , VDRL , HBs Ag. ( for patient it is not
necessary )

5.0 EQUIPMENT

Tube sealer.
PH meter .

6.0 PRINICIPAL

Plasmapheresis is the removal of plasma from a donor/ patient with the return of the
donor/ patients RBC , WBC and plasma.

7.0 REAGENTS

Anticoagulant - A C D Solution
Normal Saline -
Oral fluids
Tab Calcium

8.0 METHOD

Donor Selection:

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 190 of 197


HCT - 40% --45%
Donor must not have taken aspirin containing medication with in last three days.
Blood counts should be with in normal limit
ABO / Rh (D) typing and also antibody screening before plasmapheresis.
method for HIV, HCV, HBs Ag and VDRL .If all are negative do the platelet
pheresis.
platelet -phereis

9.0 METHOD

Donor Selection:
Informed consent from donor or patient
R B C loss must not be more than 25 ml/wk
Interval between two procedures 48 hours.
Donor weighing 50 -80 kg do not collect more than 500ml or > 600 ml.
Do Hb estimation
Serum total proteins and A/G ratio PT and APTT

10.0 HEMONETICS SDP PPROCEDURE

1. Press power swtich : On
3. Instruction on monitor : Cose the centrifuge cover
5. Protocol to select : For SDPlasma select ppp
6.To continue press : yes
7. Now instal the kit : Bowl/ Tubes etc
8. press prime : Prime Prime Prime
9. disable drip monitor : No
10. Confirm : Yes
12. Hemo claculator : Press Modify program

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 191 of 197


Enter details like : Height / weight / hct/ plasma
volume
14 . Machine will calulate : Plasma yeild / blood volume /
Number of cycles
18. now keep donor ready : Apply bp cuff
50 or 40
22. After completion of cycles machine displays message procedure complete
23. Collect plasma 20ml/kg body weight.
24. If plasmapheresis is done for patient start normal saline through another
venous access depending on clinical situation run hemaccel or Fresh frozen
plasma equivalent to the volume removed.
25. Remove needle from the donor/ patient
26. Seal / cut the tube of SDPlasma bag label it with registration number.
27. Preserve the SDPlasma bag in deep freezer at - 20
0
c or discard it if collected
from patient properly.


Plasma Collected by aphaeresis should be stored at - 20 to - 40
0
c in deep freezer
for one year.
Routinely group matched aphaeresis plasma should be issued.

12.0 QUALITY CONTROL

At least 75 % of units tested should meet the desired factors.
PH 6-8

13.0 COMPLICATIONS

Citrate toxicity
Hemataoma
Vasovagal reaction

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 192 of 197


Hypovolemia
Allergic reaction
Hemolysis
Depletion of clotting factors.
T T D
Lymphocyte loss

14.0 DOCUMENTATION

Master Record

SP-052 01-12-2007

5

Dr V Saraswathi

Copies
Approved By Date

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1 year

15-05-2008

Location Subject

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 193 of 197


Apheresis room

GranulocytePheresis


By Apheresis cell seperator

- In charge of components lab
- Q C Record


It is mandatory to do Granulocytepheresis on a voluntary donor when there is a
request from treating clinician .

2.0 RESPONSIBILITY

It is the responsibility of Incharge Technician of component preparation who
granulocytepheresis and to do the procedure.

3.0 REFERENCE

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 194 of 197




A disposable kit meant for granulocytepheresis ( see the manufacturer
instructions for code of the procedure and expiry date )
Rapid kits for detection of HIV, HCV , VDRL , HBs Ag.

5.0 EQUIPMENT

Tube sealer.
PH meter

6.0 SPECIMEN

Blood samples collected from the donor into EDTA vial and PLAIN vial.

7.0 PRINICIPAL

Granulocytepheresis is the removal of Granulocytes from a donor with the return of
the donors RBC, platelets and plasma.

8.0 REAGENTS

Anticoagulant - A C D Solution- 500ml
Normal Saline -
Oral fluids

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 195 of 197


Tab Calcium

9.0 METHOD

Donor Selection:
HCT - 40% --45%
Donor must not have taken aspirin containing medication within last three days.
CBP cell counts should be within normal limit
ABO / Rh (D) typing and also antibody screening before granulocytepheresis.
method for HIV, HCV, HBs Ag and VDRL .If all are negative do the
granulocytepheresis.
platelet -pheresis
WBC count should be 4000 10000 / cumm

Granulocyte aphaeresis

Approximately 23% of neutrophulis replaced daily in a healthy. Daily 1% RBC
and 10% platelet also replaced.
Hence harvesting more cells from a pool of fewer cells is the problem.
H E S is recommended for better separation of W B C.

Give G- CSF along with corticosteroid to granulocyte gets mobilized dose-
5-10mg / kg body with given 12 hours before leucopheresis.
Dexamethasone 8 mg only 1 hour before Granulocytepheresis.

Storage:
At 2 8
0
C for 24 hours
More than 24 hours cryopreserved
Dont transfuse through filter.
Irradiation required before administration to immunodeficient recipient
Compatibility testing should be done as R B C contamination is inevitable.

Dose:

1 X 10
10
cells / square meter of body surface area / day until recovery of marrow
function. Or resolution of infection

STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 196 of 197


Unacceptable toxicity to the transfused granulocytes is the adverse effect.

10.0 HEMONETICS SDP PPROCEDURE


1. Press poweer swtich : On
2. Instruction on monitor : Oopen the centrifuge cover
3. Instruction on monitor : Oose the centrifuge cover
5. Protocol to select : For Granulocytes select PBSC/ for
Plasmaphersis select ppp
6. To continue press : yes
7. Now instal the kit : Bowl/ Tubes etc
8. press prime : Prime Prime Prime
9. disable drip monitor : No
10. Confirm : Yes
12. Hemo claculator : Press Modify program
Enter details like : Height / weight / hct/ pl count
14 . Machine will calulate : Platelet yeild / blood volume /
Number of cycles
18. now keep donor ready : Apply Bp cuff
50 or 40
22. After completion of cycles machine displays message
procedure complete
23. Clamp the granulocyte bag.
24. Remove needle from the donor
25. Seal / cut the tube of Granulocyte bag and label it
26. Preserve the Granulocyte bag in cold room at 2-8
0
c


STANDARD OPERATING
PROCEDURES
Section : I
Issue : 1.0
Date : 15-05-2008
Page : 197 of 197


Routinely group matched Granulocytephaeresis bag should be issued .

12.0 QUALITY CONTROL

Do WBC count to a sample collected from Granulocytepheresis bag by automated
cell counter.

13.0 COMPLICATIONS

Citrate toxicity
Hemataoma
Vasovagal reaction
Hypovolemia
Allergic reaction
Hemolysis
T T D
Lymphocyte loss

14.0 DOCUMENTATION

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