Notes On Blood Banking

Note 1
Donor selection or deferral
Introduction :
The source of blood for transfusion are blood donors.
Blood donation :
Blood donors can be :
1.
Voluntary non-remunerated donors : They donate blood without any pressure or for monetary benefit, for
an unknown patient.
Advantages :
a)
Belong to low risk category.
b) Donors directory can be maintained & they can called at time of need.
2.
Replacement or relative donors : Relatives / friends of the patient replace the number of units of blood
used by him.
Drawbacks :
A paid donor can come in disguise of relative / friend of the patient.

They often insist on transfusing their blood to their patient (called directed donation), which should not be
advocated.
3.
Professional paid donors

They receive direct or indirect monetary benefit for blood they donate.
Disadvantage :
a)
May not reveal their illness
b) May donate more frequently
Message :
Voluntary unpaid donations should be promoted as the only source of blood.
Donor selection or deferral :
a)
It is the responsibility of the blood bank to ensure that blood donation does no harm, to either the donor
or the patient. Proper registration and selection of the healthy voluntary non-remunerated donor is the first
step in quality assurance. Donor selection includes Medical history questionnaire and Limited medical
examination.
2
Those found fit are accepted.
Unfit donors with treatable cause are temporarily deferred while those with chronic / incurable diseases are
permanently rejected.
Donor registration & Consent form :

The form includes :
1.
Personal information
2.
Medical history questionnaire
3.
Consent
4. Limited Medical Examination (to be filled by Blood Bank Staff).
Personal information :
Helps in :
- Identifying the donor,
- Registering him/her in Blood donation register if he/she is a first time donor,
- Linking him/her to existing donor records and updating information.
Personal information (biodata) includes :
Name
: _______________
Age
: _______ (you can donate in the age group : 18-65 years)
Sex
: _______________
Date of birth
: _______________
Postal address
: _______________
Phone number
: _______________
Occupation
: _______________
Medical history questionnaire :

Criteria for temporary deferral :
The donor having undergone surgical procedures or suffering from the following illnesses are deferred for the
duration mentioned :
1.
Donated blood in the past 3 months.
2.
Taken blood transfusion in the past 6 months.
3.
Suffering from cough, sore throat or common cold at the time of donation.
4.
Antibiotics or have you taken antibiotics in the past 7 days.
5.
Undergone dental work in the past 7 days.
6.
Undergone minor surgery in the past 3 months.
7.
Undergone acupuncture, ear piercing, body piercing or tattooing in the past 6 months.
8.
Undergone major surgery in the past 12 months.
9.
Anemic or taking treatment for anemia.
10.
Pregnant women
11.
Lactating women or who has a baby of less than 1 year age.
12.
Women who have undergone abortion in the past 6 months
13.
15 days after having taken killed or toxoid vaccine (for cholera, typhoid, diphtheria or tetanus).
4
14.
Taken anti-rabies vaccine in the past one year.
15.
Suffered from malaria and treated in the past 3 months (in endemic area) and 3 years (in nonendemic area).
16.
Suffered from typhoid and treated in the past 12 months.
17.
Suffered from hepatitis in the past 12 months.
18.
Women in menstrual period, 5 days before and 5 days after.
19. Donor under the effect of alcohol.

20. Donor on fast.
Criteria for rejection :
1.
Donor a patient of systemic disease of heart, circulatory system, lung, liver, kidney.
2.
Donor suffering from abnormal bleeding tendency.
3.
Donor a known case of diabetis.
4.
Donor a known case of cancer.
5.
Donor with a history of epileptic episode.
6.
Suffering from any sexually transmitted diseases like AIDS, hepatitis, syphilis .. Signs and symptoms
suggestive of AIDS are :
-
Unexplained weight loss (>10 kg within a month),

Prolonged diarrhea (for > 30 days),
Continuous low grade fever (for > 1 month),
Lymphadenopathy.
Consent :
The written consent includes following aspects :
Blood donation is totally voluntary act and no remuneration is been offered to the donor for the same.
The risks associated with the procedure are explained and acceptable to the donor.
Donors blood will be tested for Hepatitis B, Hepatitis C, Malarial parasites, HIV and Syphilis. The
information of the result will be kept confidential.
The donor has to state whether he/she wants his blood screening report.
Limited Medical examination :
1. Weight : Minimum acceptable weight :

-
for donation of 350 ml blood is 45 kg.
for donation of 450 ml blood is 55 kg.
2. Temperature : The donor should be afebrile (temperature < 37.5 C).

3. Physical examination of skin :
- Donor is deferred if there are boils on skin, purulent wounds or severe skin infections.
- Both arms are examined for signs of repeated parenteral entry (multiple needle puncture marks) and or
sclerotic veins as seen with drug abuse.
- Skin at the site of venipuncture should be free from lesions.
4. Pulse : should be between 60-100 beats / minute and regular.

5. Blood pressure :
-
Systolic : 100-180 mmHg,
Diastolic : 50-100 mmHg.
6. Hemoglobin :
Hemoglobin at blood donation camps is done by specific gravity method :
a)
Copper sulphate solution with specific gravity of 1.053, which is equivalent to 12.5 gm/dl hemoglobin, is
taken in a container.
b) Middle finger is pricked with a lancet..

c)
A drop of blood is dropped in the solution.
d) If drop floats, Hb is <12.5 gm/dl & donor is rejected.

e)
If drop sinks, Hb is > 12.5 gm/dl & donor is accepted.
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Note : 2
Phlebotomy in blood bank
__________________________________________________________________________________
Donor premises :
1. Well lighted.
2. Well ventilated.
3. Preferably air conditioned.
4. Peaceful.
5. Donor chairs with inclining head or donor beds.
Blood bank staff :
1. Well trained.
2. Friendly; helps reduce donor anxiety.
Material required :
1. Blood bags :
Volume
Number
2.
3.
4.
5.
: 100 ml (pediatric use),

350 ml,
450 ml.
: Single,
Double,
Triple,
Triple ADSOL / SAG-M,
Quadruple.
: Citrate phosphate dextrose adenine (CPDA).
Preservative solution
Sphygmomanometer.
Stethoscope.
Hand press.
Betadine- & spirit- soaked cotton wool swabs.
6. Blood bag weighing scale.

7. Pilot vacuette plain & citrate tubes.
8. Plastic clip or artery forcep.
9. Tube sealer.
10. Band aid, tape, plaster.
11. Scissors.
12. Emergency kit.
Check list :
1. Pre-labelled blood bag and pilot tubes are kept on donor chair.
2. Write the date of collection & expiry (for CPDA bag, 35 days from the date of collection) on the bag.
3. Place the bag on blood bag weighing scale kept under the donor chair.
4. Adjust weighing scale to zero.
5. Write the blood bag number on the registration form of the donor.
Procedure :
1. Select prominent vein : Inspect both antecubital fossa for prominent vein.
2. Scrubbing :
- Clean 3-4 cm area with betadine swab in concentric manner from center to periphery. Leave for 30
seconds.
- Clean again with spirit swab and let dry.
3. Make vein prominent :
- Inflate sphygmomanometer cuff to 60-70 mmHg.
4. Ask donor to close fist tightly. Local anesthesia : optional.
- 0.3 ml 1% lignocaine using 26 G needle is injected intradermally.
- Local anesthetic spray.
5. Venipuncture :
- Remove the needle cap.
- Introduce the needle (16 G) into the vein at 30 angle with the arm.
- See for free flow of blood in needle hub.
6. Hold needle in position with 2 pieces of tape on hub of needle. Ensure free flow of blood :
- Deflate sphygmomanometer cuff to 40 mmHg.
- Give donor a hand press and ask him to squeeze & release hand press.
7. Mix periodically, blood & preservative solution in bag.
8. Monitor volume of blood being collected to maintain ratio of blood to anticoagulant as 10:1.4 :
- Volume is indirectly inferred by weighing the blood bag. 1 ml of blood = 1.05 gm of blood.
- 350 ml x 1.05 = 367.50 gm ( 10%).
- 450 ml x 1.05 = 472.50 gm ( 10%) .
9. Stop collection after the required amount is collected :
- Clamp the tube with plastic clip or artery forcep.
- Deflate the cuff.
- Remove the needle.
- Place sterile swab on venipuncture site.
- Apply pressure & ask the donor to flex forearm at elbow joint, for 5 minutes. 10. Collect pilot samples
:
a) Insert the needle one plain vacuette tube & release the plastic clamp.
b) Remove the needle from vacuette, bend the tubing & insert into citrate vacuette tube.
11. Sealing :
- While the needle still in the citrate vacuette, seal the tubing about 5 cm away from the needle.
- Pull at the sealed site.
8
12. Sample for blood grouping & thick smear (for MP detection) :
- Take blood sample for the two tests through needle.
- Destroy the needle in needle cutter & discard safely.
13. Segments of tube :
- Seal the remaining tube at distances of about 5 inches.
- Do not cut the segments.
- These segments can be used for compatibility testing.
14. Storage of blood bag :
- Blood bag is refrigerated immediately at 4-6 C.
- If platelets are required, keep the bag at room temperature (20-24 C).
15. Donor care :
- Check venipuncture site for bleeding.
- Apply band aid or tape.
- Donor is asked to rest in refreshment room for 15-20 minutes.
- Coffee & glucose biscuits are offered.
Post donation advice :

1. Drink more fluids.
2. If donor feels giddiness, lie down with both legs raised.
3. Do not smoke for half an hour.
4. Do not consume alcohol before next meal.
5. Remove band aid after 5-6 hours.
6. If there is bleeding from venepuncture site, raise arm & apply pressure.
7. Avoid strenuous exercise for 24 hours.
Adverse donor reactions & remedy :

1. Haematoma :
- Stop blood collection. Deflate cuff & remove needle.
- Place 2-3 sterile gauze with firm pressure over venepuncture site for 7-10 minutes.
- Apply ice if desired.
- Apply pressure bandage for 24 hours.
- Apply thrombophobe (local anti-inflammatory) cream.
- If required donor may take anti-inflammatory analgesic orally.
- Reassure the donor.
2.
Syncope :
Symptoms : sweating, weakness, dizziness, unconsciousness.
Signs : cold skin low blood pressure, thready pulse.
Management :
Stop donation.
Raise legs.
Loosen tight clothing.
Ensure adequate air way.
Apply cold compresses to head.
Take donor to another room to prevent donor apprehension.
Reassure the donor.
3. Nausea & vomiting :

- Turn donors head to side to avoid aspiration of vomitus.
- Provide a plastic bag to vomit.
9
Give water to clean the mouth & towel to wipe.

Ask donor to breath slowly & deeply.
Apply cold compresses on head.
Reassure the donor.
4. Twitching or muscular spasm occurs due to hyperventilation which lowers CO2 :

- Ask the donor to breath in a paper bag.
- Reassure the donor.
5.
-
True convulsions are very uncommon. If they occur :

Keep tongue depressor between teeth to prevent tongue biting.
Loosen clothes.
Ensure adequate airway.
If convulsions persist, physician should be called.
Donor should be advised not to donate.
6.
-
Accidental puncture of artery is indicated by fast flowing bright red blood :

Stop collection.
Apply sterile gauze with pressure over venipuncture site for 15-20 minutes.
Raise arm above the level of heart.
Reassure the donor.
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Note : 3
Preservation & storage of blood bags
___________________________________________________________________________________________
PRESERVATION
Aims :
1. To prevent coagulation.
2. To preserve the life & function of red blood cells so as to have maximum post transfusion survival.
Solutions :
1. Acid Citrate Dextrose (ACD).
2. Citrate Phosphate Dextrose (CPD).
3. Citrate Phosphate Dextrose Adenine 1 (CPDA-1).
4. Citrate Phosphate Dextrose Adenine 2 (CPDA-2).
Functions of the components :

1. Citrate :
- Anticoagulant, acts by chelating calcium ions.
- Makes the solution alkaline.
- Other anticoagulants are not used :
a) Heparinized blood has to be used within 24 hours.
b) EDTA is ten times potent than citrate but is toxic and damages platelets.
2.
-
Dextrose :
Viability of red cells rapidly decreases in citrate : only 50% red cells survive after 7 days of storage.
Red cells survival can be improved by adding dextrose.
Red cells utilize dextrose as source of energy to produce ATP.
10
3. Citric acid :
- Prevents carmalization of dextrose during autoclaving.
- Is a weak acid. Along with citrate (alkaline), provides optimum pH.
4. Adenine :
- Enhances ATP production, thus improves red cell survival.
5. Sodium dihydrogen phosphate :
- Adjusts pH.
Shelf life :
Blood bag
Shelf life (days)
-
ACD
21
CPD
21
CPDA-1
35
CPDA-1 is the most commonly used solution.
Amount of solution :
- Ratio of blood to anticoagulant preservative solution should be 10 : 1.4.
Blood bag
(ml)
100
350
450
-
CPDA-2
42
Solution
(ml)
14
49
63
350 ml blood bag is most commonly used in blood banks, which deal with whole blood;
450 ml blood bag is commonly used in blood banks, which deal with component preparation.
Changes in stored blood :

1. RBC :
Physical change :
a) Shape of red cells change from discoid to spherical.
b) Loss of red cell membrane lipids.
c) Increased red cell rigidity.
d) Increased osmotic fragility.
Biochemical change :
a) Potassium (K+) and sodium (Na+) :
- K+ is lost from cells to plasma, while Na+ enters red cells.
b) pH :
- Due to lack of mitochondria, red blood cells produce energy by anerobic glycolysis (Embdem-Meyeroff
pathway); producing :
2,3-diphosphoglycerate
(2,3-DPG)
ATP
Lactate
11
Reduces pH of stored blood

c) 2,3-DPG :
- Level of 2,3-DPG falls with fall in pH on storage.
d) O2 affinity :
- 2,3-DPG level is inversely proportional to red cell O2 affinity.
Day : 0
Day : 35
2,3-DPG : 100%
2,3-DPG : <10.6%
Low O2 affinity
High O2 affinity
High O2 delivering capacity
Low O2 delivering capacity
50% of 2,3-DPG is regenerated within 3-8 hours after transfusion & 100% by 24 hours, thus regaining O2
delivering capacity.
CDPA bag
K+
Na+
pH
2,3-DPG
O2 affinity
Day : 0
4.2
169.0
7.6
100
Low
Day : 35
27.3
155.0
6.98
< 10.6
High
Significance : These changes get diluted & reversed when transfused to patient.
However, severely compromised patients and neonates do not tolerate these changes. Hence they should
not be given blood more than 7 days old.
2.
WBC :
Granulocytes :
Begin to lose their phagocytic & bactericidal property within 4-6 hours.
Become non-functional by 24 hours.
Retain antigenic properties, thus capable of causing blood transfusion reaction.
Lymphocytes :
- Few are viable even after 3 weeks.
3. Platelets :
- Lose their hemostatic function within 48 hours when stored at 4 C.
4. Coagulation factors :
- Heat labile coagulation factors (factors V & VIII), lose coagulatory activity by 50% in 1st 48-72 hours.
Thereafter, the loss is gradual.
STORAGE
12
Optimum storage temperature for whole blood or red blood cells :

Whole blood or red blood cells should be stored at 2-6 C :
1. To keep rate of glycolysis at lower level, so that dextrose is not used quickly.
2. To minimize proliferation of bacteria, which might have entered during venepuncture.
3. To minimize rate of diffusion of electrolytes across cell membrane.
4. Red cells are sensitive to freezing; at temperature lower than 2 C, they get hemolysed.
Blood bag refrigerators :

Should have internal fan to ensure uniform temperature throughout the cabinet.
Graphic record of temperature seen from outside. The graphs are changed weekly.
Battery operated alarm system to warn in case of temperature fluctuations or power failure.
Connected to alternative power supply.
Storage during transportation :

- Cold chain should be maintained from time of collection to transfusion :
- From camps : Blood is transported in insulated cold boxes. The cold boxes are surrounded by ice packs.
Bags should not touch the ice packs directly.
- On dispatch : Bags should be kept in insulated carrier boxes.
In the wards :
Once issued transfusion should be commenced within 30 minutes.
If not, bag should be stored in ward / OT refrigerator.
Keep on the shelf, upright or flat.
Do not keep anything around the bag to ensure free circulation of air.
Do not keep blood near freezer compartment.
Open door only when required, to avoid temperature fluctuations.
Optimum storage temperature for granulocytes & platelets :

- Room temperature (20-22 C).
- Never refrigerate, right from the time of collection till used.
Optimum storage temperature for plasma components :

- -20 C or below.
ADDITIVE SYSTEM
Additive system :
Are triple / quadruple bags with integral tubings, used for blood component preparation :
1. Primary 450 ml bag contains 63 ml of CPD solution.
2. One / two plain satellite bags.
3. Second / third satellite bag contains 100 ml additive solution.
Additive solutions :
1. CPD-SAG / CPD-SAGM system :
- Hogman (1978) introduced CPD-SAG (saline adenine glucose) system.
- Drawback : caused undesirable red cell lysis.
- Beutter (1979) added mannitol (CPD-SAGM system), which prevents lysis.
2. CPD-ADSOL system : Contains higher concentration of glucose, adenine & mannitol.
13
CPD-SAGM is the most commonly used additive system.
Shelf life :
Additive system
CPD-SAGM
CPD-ADSOL
Shelf life of packed red cells

42
49
Procedure :
1. Whole blood is taken in the primary bag containing CPD solution.
2. Blood is centrifuged & plasma removed into plain satellite bag.
3. Additive solution in the attached satellite bag is added to packed red cells.
Advantage :
1. Maximum amount of plasma can be harvested from blood.
2. Additive solution extends the red cell life to 42 days.
3. Lowers the viscosity of packed red cells, which eases transfusion.
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Note : 4
ABO blood group system
___________________________________________________________________________________________
Blood group systems :

There are 30 human blood group systems with 400 blood group antigens.
1. ABO
2. Rh
3. Lewis
4. MNS
5. P
6. Kell
7. Duffy
8. Kidd
9. Lutheran
10. II.
ABO BLOOD GROUP SYSTEM
History :
- Karl Landsteiner (1900) discovered A, B, O blood groups.
- Von Decastello & Sturli (1902) discovered AB blood group.
Principle :
- Blood grouping systems work on the principle of immunohaematology :
- Blood group antigens are present on red blood cells and blood group antibodies are contained in the
plasma.
14
The main aspect of blood grouping is, individuals with blood group antigen lack the corresponding
antibody in serum.
ABO blood group antigens :

Types :
1. A,
2. B,
3. H.
Subgroups :
Blood group
A
Subgroup
A1 (reacts with lectin Dolichos biflorus)
A2
A3
A intermediate
Ax.
Bm
Bx.
Location :
- Present on all cells of body except CNS.
- Secreted in all body fluids except CSF by secretor genes.
Biochemical nature :
- Precursor is a short chain sugar, oligosaccharide.
Development :
Precursor substance
L-fucosyl transferase,
product of H gene.
H-substance
N-acetyl
galactosaminyl
transferase.
Product of A gene.
A-Ag
O gene,
amorphous.
D-galactosyl
transferase.
No product.
Product of B gene.
H-substance (Ag)
B-Ag
H-substance :
- Not all H-substance is converted into A / B- antigen.
- Amount of H-substance on red cells in diminishing quantity :
O > A > A2B > B > A1 > A1B.
ABO blood group antibodies :

Types :
1. Anti-A
2. Anti-B
3. Anti-H
15
Time of production :
Develop by 3-4 months after birth.
Reach peak by 5-10 years.
Thereafter decline.
Naturally occurring antibodies :

- Appear without red cell antigenic stimulation.
- Produced due to red cell antigen-like properties of certain bacteria, virus and food; hence called naturally
occuring Abs.
Abs
- Are of IgM type.
- Blood group O also have IgG type antibodies.
IgM & IgG antibodies :
IgM antibodies
1.
Large molecule, hence cannot cross
placenta.
2.
Life span : 10 days.
3.
Called complete antibody because
on binding with Ag, they cause
clumping (agglutination) in saline.
4.
Activate complement.
ABO blood groups :

Antigen
A, H
B, H
A, B, H
IgG antibodies
Small molecule, hence can cross
placenta.
Life span : 60-70 days.
Called incomplete antibody because
they only coat the Ags & do not cause
agglutination in saline.
Does not activate complement.
Antibodies
Anti-B
Anti-A
Nil
Blood group
A
B
AB
Inheritance :
- ABO Genes are located on chromosome 9.
- Inheritance of genes of ABO system follows Mendelian law.
- Each individual has a pair of chromosomes, one inherited from each parent.
BOMBAY BLOOD GROUP
History :
Discovered by Bhende in Bombay in 1952.
Incidence :
In India : 1:7600.
Cause :
1. High consanguinous marriage among parents of Bombay phenotype.
16
2. Mutation in H gene on chromosome 19.
Pathophysiology :
Precursor substance
L-fucosyl transferase,
product of H gene.
H-substance
O gene, amorphous.
No product.
H-Antigen
Anti-A &-B
Blood group
Amorphous hh gene,
No product.
Precursor substance
A/B/O gene products,
cannot act on precursor substance.
Precursor substance
(Absence of A, B & H-Ags)
Presence of anti-A, -B & -H
Antigen
Antibody
Phenotype
Anti- A, B
OH
Bombay
Anti- A,B,H
Oh
Clinical significance :
Bombay blood group patient can receive blood from Bombay blood group donor only.
ABO-HEMOLYTIC DISEASE OF NEWBORN
Definition :
Hemolytic disease of newborn (HDN) is a syndrome associated with hemolysis in the foetus either in utero or
after delivery, with consequent hyperbilirubinaemia.
Aetiology :
HDN occurs due to blood group incompatibility between mother & foetus.
Classification :
1. ABO-HDN,
2. Rh-HDN,
3. HDN due to other blood group systems.
ABO-HDN :
Introduction :
- Occurs mostly in group O mothers with A or B foetus, because O group have IgG antibodies, which are
small and can cross placenta.
- Prior immunization is not essential, hence ABO-HDN can occur in any pregnancy including the 1st.
1.
2.
Clinical course :
Jaundice is generally mild & appears 24-48 hours after birth :
Red cell A & B antigens are not fully developed in infants.
Tissue A & B antigens neutralize anti-A & anti-B of mother, thus decreasing the number of antibody
molecules, which can agglutinate red cells.
17

1.
2.
Diagnosis :
Tests in mother :
ABO & Rh grouping.
IgG anti-A & anti-B titer : > 32 is significant.
1.
2.
3.
4.
Tests in infant :
ABO & Rh grouping.
DCT : negative or weakly positive.
S. bilirubin : moderately increased.
Hematological parameters :
- Spherocytes in smear.
- Reticulocytosis (5%)
- Increased osmotic fragility.
Management :
Phototherapy.
Blood transfusion if baby presents with anemia. More than 5 day old blood should not be used.
Exchange transfusion carried out :
Only if s. bilirubin > 20 mg/dl.
Done with fresh (>48 hours old) group O red cell concentrate of the infants Rh type.
___________________________________________________________________________________________
Note : 5
Rh blood group system
_____________________________________________________________________________________________________________
18
History :
Rh blood group system was discovered by Landsteiner & Wiener (1940) in rhesus monkey.
Inheritance :
Inheritance follows Mendelian law.
Genes of Rh system are located on chromosome 1.
There are 3 pairs of genes Cc, D(d - not identified), Ee.
Present as set of three, e.g. CDE, cDe; one set inherited from each parent.
Rh antigen :
Location :
-
Present on red blood cells only.
Are not secreted in body fluids.
-
Protein in nature
19
D-antigen :
-
D-Ag is highly antigenic while others (C/c/E/e) are weakly antigenic.
Antigenicity in diminishing order : D > c > E > C > e.
Individuals with presence of D-Ag are designated as Rh-positive; irrespective of C/c/E/e.
Individuals with absence of D-Ag are designated as Rh-negative.
D -antigen :
u
Is a weaker variant of D-antigen.
Are of 2 types :
1. High grade Du : can be detected by certain anti-D sera.

2. Low grade Du : do not react with anti-D sera; can only be detected by indirect Coombs test (ICT).
Significance of Du variant
In donors : Du blood is considered Rh +.

In recipients : Du recipients are given only Rh- blood.
Mothers with Du positive infants require Rh
immuno-prophylaxis.
Rh null syndrome :
-
Characterized by absence of all Rh antigens.
More prone to developing Rh antibodies.
Cause : red cell membrane abnormality resulting in reduced red cell survival.
20
Rh blood group antibodies :
Types :
-
Anti-C,
Anti-c.
Anti-D,
Anti-d.
Anti-E,
Anti-e.
21
Biochemical nature : Rh antibodies are predominantly of IgG type; are small molecules, hence can cross
placenta.
Rh groups :
-
Rh + individuals lack Rh antibodies.
Rh- individual also do not have Rh antibodies but can develop Rh antibodies on stimulation.
Development of Rh antibodies :
Rh antibodies develop when
Rh - person is transfused with Rh + cells.
Rh - mother carries Rh + fetus.
Rh antibodies develop by phasic response :

-
Primary response : Rh- individual when exposed to Rh+ cells for the 1st time, develops Abs in few weeks.
Abs circulate for years at non-detectable level. Individual is said to be sensitized.
Secondary response : Previously sensitized Rh- individual when re-transfused with Rh + blood, elicits
rapid hemolytic response.
Rh hemolytic disease of newborn (Rh-HDN) :
Pathophysiology :
Occurs when Rh- mother carries Rh + fetus
22
1st pregnancy (Rh- mother & Rh+ fetus).
Small amount of feto-maternal bleed is common in 3rd trimester.
Small amount of fetal red cells cross placenta & reach maternal circulation.
Due to elevated steroid levels in pregnancy, Rh antibody production is insignificant &

not affected.
the 1st child is
At delivery, trans-placental hemorrhage is common.
Amount of fetal blood entering maternal circulation is more (1-10ml or more).
Rh antibodies are produced in mothers blood in few weeks.
Mother becomes sensitized (primary response).
Subsequent Rh+ fetus.
Small amount of feto-maternal bleed occurs in 3rd trimester.
Even 0.1 ml blood is enough to stimulate the already sensitized mother to produce antibodies (secondary
response).
Rh antibodies being of IgG type, cross the placenta & causes hemolysis of Rh positive cells of fetus.
Factors influencing severity of HDN :

23
a)
ABO incompatibility between mother & fetus : provides protection to fetus from Rh-HDN. ABO
antibodies destroy fetal red cells.
b) Zygosity of father :Antigenicity of heterozygous father would be less than a homozygous father.
c)
History of Rh+ blood transfusion : antigen load in transfusion is much high, hence severity of HDN is
more.
d) MTP or miscarriage : can also immunize the Rh- mother.
Clinical features :
-
50% are mildly affected.
Severe disease is called Erythroblastosis (hydrops) fetalis; characterized by :
a)
anemia,
b) jaundice,
c)
anasarca,
d) hypotonia,
e)
hepatosplenomegaly,
f)
ascitis,
g) CCF.
24
Antenatal assessment :
Objective :
-
To assess well-being of the fetus.
Investigation done :
-
Maternal IgG anti-D titer.
To be repeated every month.
A titer of >1:32 or rising titer is significant; a candidate for amniocentesis.
Amniocentesis :
Objective : To assess the severity of hemolysis in fetus.
Time : Should not be done before 28 weeks of gestation. Should be repeated every 2 weeks.
Test done : Optical density (450-460 nm), which varies with period of gestation.
OD of amniotic fluid
Lower zone
Middle zone
Upper zone
Fetus is mildly
Moderately
Severely
affected
affected
affected
No treatment
Prepare for
Preterm delivery or exchange
intra-uterine exchange
transfusion (after assessing fetal lung
maturity)
Investigations on new born :

-
ABO grouping : cell typing.
25
Rh grouping including Du.
DCT : positive test indicates HDN.
ICT : to screen irregular antibodies.
Cord blood hemoglobin.
Cord blood s. Bilirubin.
Blood transfusion :
-
Indication : Infants with mild HDN who exhibit only anemia.
Red cell concentrate should be used.
Criteria for exchange transfusion
a)
Severe anemia (Hb <10 g/dL).
b) Hyperbilirubinemia (>20 mg/dL).

c)
Rate of bilirubin rises > 0.5 mg/dL despite optimal phototherapy.
Selection of blood for exchange transfusion :
Rh- blood of same ABO group as that of baby, if ABO group of baby & mother are same; or compatible
with mothers blood or O- blood.
Blood should be as fresh as possible, > 5 days old :

-
Ensures long post-transfusion survival of red cells.
To avoid high plasma potassium level.
If exchange transfusion is required for more than once, subsequent blood should be of the same group as
that of the 1st time.
___________________________________________________________________________________
26
Note : 6
Blood grouping techniques
___________________________________________________________________________________________
ABO & Rh grouping types :
1.
Cell (forward) typing.
2.
Serum (reverse) typing.
CELL (FORWARD) TYPING
Definition :
Unknown red cells (antigens) are typed against known blood group antibodies (anti-A, anti-B, anti-AB, antiD).
Reagent required :
Anti-sera : Anti-A, anti-B, anti-AB, anti-D.
Commercially available antisera :

Polyclonal antisera
Are derived from human donors.
Monoclonal antisera
Are derived from cultures of B-lymphocytes

secreting specific antibody.
Disadvantages :
- Low specificity (presence of non-specific
antibodies).
Disadvantages of polyclonal reagent are

overcome.
- Sensitivity varies from batch to batch.

- May contain viruses (HIV, HBV, .).
27
Abandoned.
Reagent of choice.
Type of Ig :
-
IgM antisera are used because it is a :
a)
Large molecule
b) MW : 9,00,000
c)
Serological behavior : complete
d) React optimally at 20 C (RT).
Color code :
-
Anti-A antisera is blue colored

Anti-B antisera is yellow colored
Anti-AB and anti-D antisera are colorless.
28
Methods :
1. Slide / tile method.
2. Tube method : Saline room temperature,
: Immediate spin.
3. Microplate method.
4. I.D. Micro-typing system (gel card).
5. Automated method.
Slide / tile method :
Material required :
1.
Glass slide / tile.
2.
Applicator stick.
3.
Pasteur pipette.
4.
Test tube stand.
5.
Test tubes.
6.
Centrifuge.
Procedure :
1.
Give 3 cell washes to test whole blood :
a)
Take 2-3 drops of test cells.
b) Add excess normal saline.

c)
Centrifuge at 1000 rpm for 1 min.

29
d) Discard supernatant.
e)
Repeat the procedure twice.
Objective : Cell washing removes plasma, hemolysed red cells, small clots; which can give false
positive results.
2. Make 30-40% test cell suspension : to 2 drops of cell button add 5 drops of NS.
Objective : gives high proportion of test cells, as it is an insensitive method.
3. Place 1 drop of anti-A, anti-B, anti-AB, anti-D on glass slides or tile.
4. Add 1 drop of test cell suspension to anti-sera.
5. Mix the cells & anti-sera with applicator stick, spreading over an area of 2 cm2.
6. Rock the slide / tile gently for 2 minutes.
7. See for agglutination or hemolysis. Agglutination is graded as follows :
4+ : complete agglutination of all cells, no free cells
3+ : majority of cells agglutinated with some free cells
2+ : many large clumps with many free cells
1+ : fine granular appearance visually and definite small clumps microscopically
Trace () : Grossly turbid; microscopic aggregates seen
Negative : a smooth suspension of cells.
30
Disadvantage :
1. Drying of reaction mixture.
2. Insensitive method : may not detect weak Ags.
Tube method :
Material required :
1.
Pasteur pipette.
2.
Test tubes.
3.
Test tube stand.
4.
Centrifuge.
Method :
1. Give 3 cell washes to test cells.
2. Prepare 2-4% test cell suspension : To 1 drop of cell button, add 19 drops of NS. Objective is to obtain
the optimal required concentration of antigens.
3. Set up 4 rows of test tube & label them.
4. Add 2 drops of anti-A, anti-B, anti-AB, anti-D in the pre-labeled test tubes.
5. Add 1 drop of 5% cell suspension to each tube & mix.
6. For Saline RT method : Incubate at RT for 60 min.
OR
For Immediate spin method : Centrifuge at 1000 rpm for 15-20 sec.
7. Look for hemolysis or agglutination.
Saline room temperature method
Immediate spin method

31
Advantage
1. Allows long incubation without drying. 1. Quick.

2. Centrifugation enhances Ag-Ab reaction,
hence weaker Ags can be detected.
Microplate method :
Material required :
1.
Microplate :
polystrene plate,
96 wells, each of 200-300 l volume,
V-, flat- / U-bottom; U-bottom prefered for blood bank.
2.
Microplate shaker.
3.
Microplate reader.
4.
Micropipette.
32
Method :
1. Give 3 cell washes to test cells.
2. Prepare 5% test cell suspension.
3. Add 1 drop of anti-A, anti-B, anti-AB, anti-D in wells.
4. Add 1 drop of 5% test cell suspension to each well.
5. Gently mix by tapping or on microplate shaker.
6. Incubate at RT for 20-30 minutes.
7. Gently mix by tapping or on microplate shaker.
8. Result can be read :
Manually :
-
Positive reaction : cells fall as button in the center of the bottom of the well.
Negative reaction : cells trail away from the center of the bottom.
Or results can be obtained by automatic microplate reader at 570 nm wavelength.
Advantage :
1.
Time saving.
2.
Cost effective.
3.
Ideal for testing large number of blood samples.
I.D. Microtyping system (Gel card) :
Principle :
-
6 microtubes in the form of cards are filled with gel impregnated with anti-A, anti-B, anti-AB, anti-D,
positive & negative control.
In positive reaction, red cells get agglutinated at top or remain suspended in gel.
33
In negative reactions, red cells pass through the gel to the bottom.
Material required :
1.
Gel cards for blood grouping
2.
Gel card racks.
3.
Gel card centrifuge.
4.
Micropipette.
Method :
1. Cells are not washed.
2. Add 1 drop of blood onto the gel tube.
3. Centrifuge the card.
4. Record findings.
34
Advantage :
1. Simple
2. Quick.
3. Test reagents are pre-labeled.
4. 1 card is used for 1 person, avoiding chances of error.
5. Controls are provided.
6. Clean work place.
Automated method :
All steps are automated; pipetting, reagent addition, incubation and washing steps.
SERUM (REVERSE) GROUPING
Definition :
Unknown serum are typed against known blood group antigens (A, B, O).
Preparation of red cells :
1.
Pooled A, B & O cells are taken.
2.
Cells are washed.
3.
2-4% cell suspension is prepared.
Group O cells are used to detect antibodies other than anti-A or anti-B.
Methods :
Any of the methods adopted for forward grouping can be used.
Ideally both cell & serum typing, should be performed by different workers & results cross-checked.
35
PROBLEM IN GROUPING OF CORD OR INFANT BLOOD
ABO antigens are not fully developed.
Cord red cells should be washed 3-4 times to minimize error due to Whartons jelly.
ABO antibodies are absent, those present are of maternal origin; thus serum grouping is not
recommended.
___________________________________________________________________________
Note : 7
Compatibility testing or Cross-matching
__________________________________________________________________________________
Selection of blood for transfusion :
Use ABO group specific blood. If not available, use alternate ABO compatible blood.
Use Rh specific blood. If not available, Rh negative blood can be used.
36
Compatibility testing (Pre-transfusion testing) :

Compatibility testing are a group of tests done to ensure that the particular unit of blood can be safely
transfused to the patient. Procedures :
1.
ABO & Rh blood grouping.
2.
Cross matching :
IgM cross matching,
IgG cross-matching.
37
Definition : Cross-match :
Cross-match test is carried out to ensure that antibodies in patients serum do not react with donor cells, when
transfused & vice versa.
Classification :
1.
Major cross match test (DC-PS) : Donors red cells (blood group antigens) are mixed with patients serum
(blood group antibodies).
2.
Minor cross match test : Patients cells are mixed with donors plasma.
Cross-match Techniques :
1.
IgM cross match :
Saline room temperature technique.
Immediate spin technique.
2.
IgG cross match :
Albumin addition technique at 37 C.
Indirect antiglobulin technique.

IgM
Structure
MW
Serological behavior
Optimal temperature
Pentamer
9,00,000
Complete antibody.
Example
Antibodies of ABO, Lewis, N, P,

Lutheran
React at 20 C.
IgG
Monomer, Y-shaped
1,50,000
Incomplete antibody
37 C
Rh, M, Kell, Duffy, Kidd
IgM CROSS MATCH
38
Blood samples :
Donors and patients blood samples in plain & citrate bulbs.
Material required :
1.
Glass test tubes
2.
Test tube stand
3.
Pasteur pipette
4.
Centrifuge
5.
Glass slide
6.
Microscope.
Reagent required :
Normal saline.
Procedure :
1. Take 4 pre-labelled test tubes, for patient & donor cells; donor & patient serum.
2. Give 3 cell washes to patient & donor red cells & make 2-4% cell suspension.
3.
Centrifuge patient & donor serum to remove red cells & fibrin clots.
4.
For Major cross-match : Take 1 drop of donors cell suspension, & add 2 drops of patients serum. Mix.
5. Minor cross-match : Take 1 drop of patients cell suspension & add 2 drops of donors serum.
6. For Saline RT method : Incubate at RT for 60 min.
OR
For Immediate spin method : Incubate at RT for 10-15 min. Centrifuge at 1000 rpm for 1 min.
7. Examine for hemolysis or agglutination & confirm agglutination under microscope.
39
IgG CROSS MATCH
If IgM cross-match is negative :
Look for IgG antibodies by either method :
1.
Albumin addition technique -or-
2.
Indirect antiglobulin technique (preferable due to high sensitivity).
Albumin addition technique :
Principle :
Red blood cells have a negative charge on cell membrane which makes them repel each other. This is
called zeta-potential. Albumin increases the dielectric constant of the medium, thus reducing the zetapotential between red cells, bringing them closer & causing agglutination.
40
Indirect antiglobulin technique :
Principle :
Anti-human globulin (AHG) is an antibody to human immunoglobulin (IgG). Addition of AHG
(Coomb's) reagent bridges the gap between IgG coated red cells & brings their agglutination.
Blood samples required :

Donors and patients blood samples in plain & citrate bulbs.
Material required :
1.
Glass test tubes
2.
Test tube stand
3.
Pasteur pipette
4.
Centrifuge
5.
Glass slide
6.
Microscope.
Reagents required :
1. Normal saline
2. 22% bovine albumin for albumin method
3. Coomb's reagent for anti-human globulin method.
41
Procedure :
1.
Wash 3 cell washes in excess of normal saline to the reaction mixture.
2.
Decant the last wash.
3.
For albumin method : Add 1 drop of 22% bovine albumin to the cell button.
4.
For AHG method : Add 1 drop of AHG reagent to the cell button.
5.
Centrifuge at 1000 rpm for 1 minute.
6.
Look for hemolysis or agglutination. Confirm agglutination under microscope.
Interpretation :
Hemolysis or agglutination :
incompatible,
cannot be transfused.
Absence of hemolysis or agglutination :
compatible,
can be transfused.
__________________________________________________________________________________
Note : 8
Antiglobulin (Coomb's) test
___________________________________________________________________________________
42
ANTIGLOBULIN (COOMBS) TEST
History :
Coombs, Mourant & Race in 1945 discovered a test to detect non-agglutinating (incomplete, IgG) antibodies.
Principle :
Red blood cells coated with incomplete antibodies (IgG) or C3 complement, can be made to agglutinate using
antibody against human immunoglobulin called antihuman globulin [(AHG) or (Coombs)] reagent.
Types of Coombs tests :
1.
Direct Coombs test
2. Indirect Coombs test

DIRECT COOMBS TEST (DCT)
Principle :
DCT detects in-vivo sensitization of red cells with incomplete antibody (IgG) or C3. These sensitized cells are
made to agglutinate using antihuman globulin.
Indications :
1.
Diagnosis of hemolytic disease of newborn.
2.
Diagnosis of autoimmune hemolytic anemia.
3.
Investigating drug induced red cell sensitization.
4.
Investigating hemolytic blood transfusion reaction.

43
Blood sample :
Blood is taken in EDTA bulb.
Reagents required :
1.
Antihuman globulin (Coombs) reagent.
2.
Normal saline.
Material required :
1.
75x12 mm glass test tubes.
2.
Pasteaur pipette.
3.
Glass beaker.
4.
Glass slide.
5.
Centrifuge.
6.
Microscope.
INDIRECT COOMBS TEST (ICT)
Principle :
Detects presence of incomplete antibody or C3 in the serum, after coating them on red cells in vitro. Red cells
are added to the serum, sensitizing the former. These sensitized red cells are agglutinated using antihuman
globulin reagent.
Indications :
1.
IgG compatibility testing.
2.
Screening of donors for presence of unexpected antibodies.

44
3.
Detection of red cell antigens using specific antibodies; e.g. testing for Du variant.
Blood sample :
To be taken in plain bulb for obtaining serum.
Material required :
1.
2.
Pasteaur pipette.
3.
Glass beaker.
4.
Glass slide.
5.
Centrifuge.
6.
Microscope.
Reagent required :
1.
2.
Normal saline.
3.
2-4% suspension of O Rh+ red cells (are preferred because of lack of ABO antigens).
Procedure :
1.
2.
3.
4.
5.
Give 3 cell washes to test cells & decant the last wash.
Prepare 2-4% test cell suspension by adding 19 drops of normal saline to 1 drop of cell button.
Take 1 drop of 2-4% cell suspension. Add 1-2 drops of AHG reagent to it & mix.
Centrifuge at 1000 rpm for 1 min.
See for agglutination or hemolysis.
45
___________________________________________________________________________________
Note : 9
Du variant
___________________________________________________________________________________
Du-antigen :
Is a weaker variant of D-antigen.

Du-antigen are of 2 types :
1. High grade Du : can be detected by certain anti-D sera.
2. Low grade Du : do not react with anti-D sera; can only be detected by indirect Coombs test (ICT).
Significance of Du variant :
In donors : Du blood is considered Rh +.

In recipients : Du recipients are given only Rh- blood.
Mothers with Du positive infants require Rh
immuno-prophylaxis.
Rh blood grouping :
Rh blood grouping
46
Rh blood grouping is done using monoclonal IgM anti-D.
Agglutination
Rh +
No agglutination
Test for Du variant by Indirect Coomb's test
Du testing :
Material required :
1.
2.
Pasteaur pipette.
3.
Glass beaker.
4.
Glass slide.
5.
Centrifuge.
6.
Microscope.
Reagents required :
1.
IgG anti-D.
2.
Anti-human globulin (Coombs reagent).
3.
Normal saline.
Procedure :
1.
2.
3.
4.
5.
Prepare 2-4% test cell suspension.

Take 1 drop of 2-4% test cell suspension.
Add 2 drops of IgG anti-D to it & mix.
Incubate at 37 C for 45-60 minutes.
Give 3-4 cell washes and discard the supernatant.
47
6. Add 1-2 drops of AHG to cell button.

7. Centrifuge at 1000 rpm for 1 min.
8. See for hemolysis or agglutination.
___________________________________________________________________________________
Note : 10
Antiglobulin test
___________________________________________________________________________________
History :
Coombs, Mourant & Race in 1945 discovered a test to detect non-agglutinating (incomplete, IgG) antibodies.
Principle :
Red blood cells coated with incomplete antibodies (IgG) or C3 complement, can be made to agglutinate using
antibody against human immunoglobulin called antihuman globulin [(AHG) or (Coombs)] reagent.
Types of Coombs tests :
1.
Direct Coombs test
2.
Indirect Coombs test
DIRECT COOMBS TEST
48
Principle :
DCT detects in-vivo sensitization of red cells with incomplete antibody (IgG) or C3. These sensitized cells are
made to agglutinate using antihuman globulin.
Indications :
1.
Diagnosis of hemolytic disease of newborn
2.
Diagnosis of autoimmune hemolytic anemia
3.
Investigating drug induced red cell sensitization
4.
Investigating hemolytic blood transfusion reaction
Blood sample :
Blood is taken in EDTA bulb.
Reagents required :
1.
Antihuman globulin (Coombs) reagent
2.
Normal saline
Material required :
1.
75x12 mm glass test tubes
2.
Pasteaur pipette
3.
Glass beaker
4.
Glass slide
5.
Centrifuge
49
6.
Microscope
Procedure :
1. Give 3 cell washes to test cells & decant the last wash.
2. Prepare 2-4% test cell suspension by adding 19 drops of NS to 1 drop of cell button.
3. To 1 drop of cell suspension, add 1-2 drops of AHG reagent.
4. Centrifuge at 1000 rpm for 1 min.
5. See for agglutination or hemolysis.
50
INDIRECT COOMBS TEST (ICT)
Principle :
Detects presence of incomplete antibody or C3 in the serum, after coating them on red cells in vitro.
Red cells are added to the serum, sensitizing the former. These sensitized red cells are agglutinated using
antihuman globulin reagent.
Indications :
1.
IgG compatibility testing.
2.
Screening of donors for presence of unexpected antibodies.
3.
Detection of red cell antigens using specific antibodies; e.g. testing for Du variant.
Blood sample :
To be taken in plain bulb for obtaining serum.
Reagent required :
1.
2.
Normal saline.
3.
2-4% suspension of O Rh+ red cells (are preferred because of lack of ABO antigens).
Procedure :
1.
2.
3.
4.
5.
6.
Take 2 drops of test serum.

Add 1 drop of O+ 2-4 % cell suspension.
Give 4 cell washes to the reaction mixture.
Add 1-2 drops of AHG to cell button.
Centrifuge at 1000 rpm for 1 minute.
See for hemolysis or agglutination.
51
___________________________________________________________________________________
Note : 11
Blood transmitted diseases
___________________________________________________________________________________
Diseases transmitted through blood :
Viral
Bacterial
Parasitic
Hepatitis B virus (HBV)
Syphilis
Malaria
Hepatitis C virus (HCV)
Brucella
Toxoplasma
Hepatitis D virus (HDV)
Microfilaria
Hepatitis G virus (HGV)

Human immunodeficiency virus (HIV)
Epstein Barr virus (EBV)
Cytomegalovirus (CMV)
Human T lymphotropic virus (HTLV)
Screening of donor blood for the following blood transmissible diseases was made mandatory by WHO :
- HBV : in 1968
- HIV : in 1985
- HCV
: in 1990.
Every unit of donated blood should be screened for transfusion-transmissible infections other than these, in
accordance with national policies and prevalence of infection in the population.
The other blood transmitted infections that we test for in our region are Trepenoma pallidium (syphilis) and
Malaria.
52
HEPATITIS B VIRUS
Causative organism :
DNA virus.
A double shelled particle, measuring
42 nm, called Dane particle :
Inner nucleocapsid core, measuring 27 nm.
Outer lipoprotein coat.
Nucleocapsid core : Contains :
DNA polymerase enzyme
Double stranded circular DNA
Core antigen (HbcAg)
e antigen (HbeAg) is not a part of virus. HbcAg is translated into HbeAg during viral replication.
Lipoprotein coat : Contains :

Surface antigen (HbsAg / Australia antigen).
53
HBV Antigens :
HbsAg :
In Acute hepatitis :
-
First detectable serological marker.
Its appearance coincides with the onset of clinical symptoms.
Remains elevated for 4-6 weeks.
Disappears with recovery of patient.
Chronic hepatitis :
-
Persists for years.
HbcAg :
-
HBcAg gets translated into HBeAg.
Hence not found in blood.
HbeAg :
In Acute hepatitis :
-
Appears almost at the same time as HBsAg.
- Is characterized by a high rate of viral replication.

In chronic hepatitis :
Simple carrier
Super carrier
1.
High viral replication.
2.
Detectable HBeAg.
1.
2.
3.
Low viral replication.

HBeAg is not detected.
Good prognosis.
54
3.
Poor prognosis.
HBV Antibodies :
HbsAb :
-
Appears sometime after the disappearance of HBsAg. The intervening period is called window period.
HBcAb & HBeAb are markers for window period.
Anti-HBs : is protective & persists for life in those who become immune.
Some lose HBsAb and become susceptible to future infection.
HbcAb :
Anti-HBc IgM :
-
Appears during acute illness
Disappear within few months
Indicates recent infection.
55
Anti-HBc IgG persists for life.
HbeAb :
Appears with the fall in HbeAg.
Incubation period :
50-150 days.
Diagnostic tests :
Tests
Sensitivity
Immunodiffusion
Low (> 100 ng/ml)
Latex agglutination
Moderate (10-100 ng/ml)
Reverse passive hemagglutination

'Sandwich' Enzyme immunoassay (EIA)
High (0.5-10 ng/ml)
Radio immunoassay (RIA)
Sandwich EIA :
Principle : Enzyme immunoassay is a solid phase sandwich enzyme immunoassay, detects presence of
HBsAg. Solid phase can be a well (microtiter plate) or a comb (immunocomb).
Using microtiter plate :

1. Microtiter well is coated with unlabeled anti-HBsAb.
56
2. Diluted test sample is added. HBsAg, if present combines with coated anti-HBsAb.
3. The reaction is washed to remove unbound HBsAg.
4. Conjugate, anti-HBsAb conjugated to horseradish peroxidase is added; forming Ab-Ag-Ab complex
(sandwich).
5. Reaction is washed to remove unbound conjugate.
6. Substrate containing chromogen tetramethylbenzidine is added. Substrate is activated by enzyme labelled
to conjugate; causing color change; which is read by ELISA reader at specific wavelength.
Using Immunocomb :
Solid phase is a comb with 12 projections (teeth) :
-
Each teeth is sensitized at 2 spots : Upper spot - internal control & Lower spot - monoclonal HBsAb.
The developing plate comprises of 6 rows (A-F) & 12 columns.

-
Rows : One entire row contains a specific reagent.
Columns : one entire column is for one tooth.
1.
2.
3.
4.
5.
6.
7.
8.
The developing plate is sealed with a plastic foil.

Perforator : Perforator is a plastic device used to break the plastic foil of the developing plate covering
each well.
Procedure :
Break the seal of row A & add 100 ul of test serum using micropipette.
Insert comb in Row A. Row A contains specimen diluent. HbsAg, if present in the test sample combines
with anti-HBS antibody coated on the tooth.
Comb is moved to row B containing wash solution.
Comb is moved to row C containing goat Ab to HbsAg; which combines with HbsAg forming Ab-Ag-Ab
(sandwich) complex.
Comb is moved to Row D containing avidin conjugated to alkaline phosphatase; which combines with the
sandwich.
Comb is moved to Row E containing wash solution; to wash off unbound alkaline phosphatase.
Comb is moved to Row F containing chromogen substrate. Bound alkaline phosphatase causes color
change.
The reaction is read visually.
HEPATITIS C VIRUS
57
Enveloped single stranded RNA virus.
Size : 50-60 nm in diameter.
HCV RNA :
Encodes 3 structural & several non-structural proteins :
Structural proteins : C (capsid) protein, E1 & E2 (envelop) protein.
Non-structural (NS) proteins : NS1, NS2, NS3, NS4A, NS4B, NS5A, NS5B glycoproteins.
Diagnosis :
Antibody detection
Detection of viral RNA
Indirect EIA
PCR
Antibody detection tests :
Detects anti-HCV to
1st generation
C, NS4
2nd generation
C & NS3
3rd generation
C, NS3, NS4, NS5
Indirect EIA :
Principle : The solid phase is coated with HCV Ag. Anti-HCV present in the sample binds to it. AntiHCV is recovered using an enzyme labeled antibody; thus forming Ag-Ab-Ab complex.
58
Solid phase can be a well (microtiter plate) or a comb (immunocomb).

1.
2.
3.
4.
Microtiter well is coated with unlabeled HCV antigen.

Diluted test sample is added. Anti-HCV, if present combines with coated HCV Ag.
The reaction is washed to remove unbound anti-HCV antibodies.
Conjugate, anti-human globulin conjugated to alkaline phosphatase is added; forming Ag-Ab-Ab complex
(indirect).
to conjugate; causing color change.
7. The reaction is read by ELISA reader at specific wavelength.
Using Immunocomb :
-
Each teeth is sensitized at 3 spots :

1. Upper spot - internal control
2. Middle spot - HCV core Ag
3. Lower spot - HCV non-structural Ags.

-

each well.
Procedure :
1. Break the seal of row A & add 100 ul sample using micropipette.
2. Insert comb in Row A. Row A contains specimen diluent. Anti-HCV if present in the test sample
combines with HCV antigen coated on the tooth.
3. Comb is moved to row B containing wash solution. The unbound antibody gets washed off.
4. Comb is moved to row C containing anti-human globulin conjugated to alkaline phosphatase; which
combines anti-HCV, thus forming Ag-Ab-Ab (indirect) complex.
5. Comb is moved to Row D containing wash solution. The unbound conjugate gets washed off.
6. Comb is moved to Row E containing wash solution.
7. Comb is moved to Row F containing chromogen substrate; the bound enzyme causes color change.
8. The reaction is read visually.
59
HUMAN IMMUNODEFICIENCY VIRUS
60
RNA retrovirus.
Size : 100-120 nm in diameter.
Divided into viral core & envelope.
Core :
2 copies of RNA.
2 copies of reverse transcriptase enzyme.
Core is surrounded by a shell of P-24 protein.
Envelope :
From inward outside :
1.
Shell of P-17 protein.
2.
Lipid membrane, which is penetrated by glycoproteins :
gp-41 & gp120 in HIV-1.
gp-36 & gp105/130 in HIV-2.
Incubation period :
2-4 weeks.
Serological markers :
P-24 is the 1st serological marker detected, after window period.

61
Anti-HIV antibodies to gp 120 develop with the fall in P-24 levels.
At the terminal stage : Anti-HIV antibodies fall while P-24 levels rise again.
Diagnosis :
Detecting antibodies
Screening
Confirmatory
tests
Indirect EIA
Detecting antigen
tests
Virus isolation
PCR
Western blot
Rapid test
Immunofluorosence
Particle agglutination
RIA
Indirect EIA :
Principle : The solid phase is coated with HIV Ag. Anti-HIV present in the sample binds to it. Anti-HIV
is recovered using an enzyme labeled antibody; thus forming Ag-Ab-Ab complex.
Solid phase can be a well (microtiter plate) or a comb (immunocomb).

1.
2.
3.
4.
Microtiter well is coated with unlabeled HIV antigen.

Diluted test sample is added. Anti-HIV antibody, if present combines with coated HIV Ag.
The reaction is washed to remove unbound anti-HIV antibodies.
Conjugate, anti-human globulin conjugated to alkaline phosphatase is added; forming Ag-Ab-Ab complex
(indirect).
to conjugate; causing color change.
7. The reaction is read by ELISA reader at specific wavelength.
62
Using Immunocomb :
-
Each teeth is sensitized at 3 spots :

1. Upper spot - internal control
2.
Middle spot - Coated with env gp 105 & 36 for HIV-2.
3.
Lower spot - Coated with env gp 120 & 41 for HIV-1

-

each well.
Procedure :
1. Break the seal of row A & add 100 ul sample using micropipette.
2. Insert comb in Row A. Row A contains specimen diluent. Anti-HIV if present in the test sample
combines with HIV antigen coated on the tooth.
3. Comb is moved to row B containing wash solution. The unbound antibody gets washed off.
4. Comb is moved to row C containing anti-human globulin conjugated to horseradish peroxidase enzyme;
which combines anti-HIV, thus forming Ag-Ab-Ab (indirect) complex.
5. Comb is moved to Row D containing wash solution. Unbound conjugate gets washed off.
6. Comb is moved to Row E containing wash solution.
7. Comb is moved to Row F containing chromogen substrate; which causes color change.
8. The reaction is read visually.
SYPHILIS
Syphilis is caused by bacteria Treponema pallidum.

Is a thin, delicate organism, not visible by light microscopy.
Varying in length from 5 to 15m.
It has tight spiral every 1.1m, along its length, appearing like a helical coil.
63
Transmission through blood transfusion :

Treponema palladium are sensitive to lower temperature & die within 72 hours, if stored at 2-6 C.
Incubation period :
9-10 weeks.
Diagnosis :
Direct demonstration
Serological tests
Dark ground microscopy
TP hemagglutination (TPHA)
Fluorescent microscopy
TP immobilization test (TPI)

TP antibody test (FTA)
ELISA
VDRL
RPR
64
Rapid plasma reagin (RPR) test :
Principle :
RPR is a macroscopic flocculation test. TP cardiolipin antigen is coated on charcoal or carbon particles.
Interpretation :
Positive result : antibodies in test sample, bind to antigen & cause flocculation.
Negative result : carbon particles will form a cell button.
MALARIA
Caused by protozoan parasites of the genus Plasmodium.

Four species of Plasmodium : Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale,
Plasmodium malaria.
Diagnosis :
Direct demonstration
1. Light microscopy
2. Fluorescent microscopy
Immunological methods
Molecular techniques
Detects Ab to plasmodium lactate 1. DNA hybridization
dehydrogenase(pLDH)
2. PCR
Light microscopy (gold standard) :

- Examination of thick & thin peripheral smears.
- 1 field of thick smear = 100 fields of thin smear; thus thick smear is a concentration method.
- Thin smear helps to identify the species.
Integrated Counseling and Testing Center (ICTC) :

- In the Donor registration form, the donor gives an informed consent for his blood being tested for
Hepatitis B, Hepatitis C, Malarial parasites, HIV and Syphilis; and that the information will be kept
confidential. The donor has to state whether he/she wants his blood screening report.
65
If the donor had asked for his serological status and the status is negative, the report is sent to him by post.
Blood banks are not authorized to disclose positive serological status of the donor to him or anyone. The
blood bank informs the Counselor at the Integrated Counseling and Testing Center (ICTC). The
Counselor then further informs and councils the donor.
___________________________________________________________________________________
Note : 12
Blood transfusion reactions
___________________________________________________________________________________
Definition :
Adverse reactions occuring due to blood transfusion are called blood transfusion reactions (BTR).
Classification :
Hemolytic
1. Intravascular
2. Extravascular
1.
2.
3.
4.
5.
6.
7.
8.
9.
Non-Hemolytic
Febrile non-hemolytic
Non-cardiogenic pulmonary edema
Graft versus host disease
Allergic
Anaphylactic
Circulatory overload
Transfusion siderosis
Septicemia
Transfusion transmitted diseases.
HEMOLYTIC BLOOD TRANSFUSION REACTION

Intravascular hemolysis
Extravascular hemolysis
Site of red cell destruction :
Within circulatory system.
By macrophages in spleen or liver.
Mechanism :
IgM antibody induced
IgG antibody induced.
Associated with :
ABO incompatibility
Rh, Kell or Duffy system

incompatibility.
Onset of Clinical effects :
Immediate, thus called Acute Delayed upto 2 weeks or more,

hemolytic transfusion reaction hence called delayed hemolytic
66
Severity of reaction :
(AHTR).
transfusion reaction (DHTR).
Severe.
Mild to moderate.
67
Causes :
Clerical error :
1.
Incorrect labeling of patients / donors samples / blood bag.
2.
Improper identification of patient while sample collection or transfusion.
Technical error :
1.
Error in blood grouping.
2.
Error in cross-matching.
3.
Failure to detect weak antibodies.
Signs & symptoms :
1.
Shock phase :
Fever, chill, burning sensation at the site of transfusion, pain in chest or lower back, hemorrhage.
2.
Post shock phase :

Hemoglobinemia, hyperbilirubinemia, hemoglobinuria, DIC.
3.
Anuric phase :
Renal failure : oliguria, anuria, uraemia.
4.
Recovery phase : 8-12 days.
Management :
1.
Stop transfusion immediately.
2.
Inform the attending physician.

68
3.
Keep intravenous lines open for suitable intravenous infusions.
INVESTIGATING A CASE OF SUSPECTED HEMOLYTIC BTR
69
Samples to be sent to blood bank :
1.
Immediate post-transfusion plain & EDTA blood sample.
2.
Implicated blood bag & transfusion set.
3.
1st specimen of patients urine following transfusion.
Investigations to rule out HBTR :
1.
Blood grouping
2.
Cross matching
3.
DCT
4.
ICT
5.
s. Bilirubin
6.
s. Haptoglobulin
7.
Urine for free hemoglobin
Blood grouping :
Re-blood grouping is done on :
a)
Pre-transfusion patient sample.
b) Post-transfusion patient sample.

c)
Donor blood.
Re-blood grouping rules out labeling / technical error.
70
Cross-matching :
Donor blood is re-cross-matched with :
a)

Re-cross-matching rules out labeling / technical error.
71
Direct Coombs test :

DCT is done on :
a)

Positive test indicates hemolytic BTR.
False negative result may occur if blood sample is drawn several hours later, after the cells have already
been destroyed.
Indirect Coombs test :

ICT is done on :
a)

c)
Donor sample.
Is the most sensitive method of cross-matching and rules out presence of irregular antibodies.
s. Bilirubin :
Observe pre- & post- transfusion patient sample plasma for yellow to brownish discoloration.
s. Bilirubin is done on :
a)
72
Increased indirect bilirubin indicates intravascular hemolysis.
s. Haptoglobulin :
s. Haptoglobulin is done on :
a)

Decrease in s. haptoglobulin is suggestive of intravascular hemolysis.
If all reports are negative possibility of hemolytic BTR is ruled out.
NON-HEMOLYTIC BLOOD TRANSFUSION REACTION (NHTR)
73
Febrile NHTR :
Most common, accounts for > 90% of BTR.
Cause : Antibodies to WBC & platelets; usually seen in multi-transfused or multiparous patient.
S/s : A self limiting reaction of fever, chills, malaise.
Management : Antipyretic therapy.
Prevention : Use of leucocyte poor red blood cells.
Non-cardiogenic pulmonary edema :
Cause : Leucocyte incompatibility resulting into formation of white cell aggregates; which get trapped in
pulmonary micro-circulation.
S/s : Acute respiratory distress, vascular collapse.
Management :
1.
stop transfusion,
2.
inform attending physician,
3.
give steroids, O2 and diuretics.
Prevention : use washed red cells for future transfusion.
Graft versus host disease (GVHD) :
Cause : Occur in immuno-compromised patients, post-chemotherapy, post-radiotherapy. Donor-functional

lymphocytes engraft and multiply in host tissue and react against the host cells.
S/s : Fever, skin rashes, diarrhoea, hepatitis, superinfection.
Prevention : Use leucocyte free blood or irradiated blood component.
74
Allergic transfusion reaction :
Cause : Reaction to allergens in donor plasma.
S/s : Itching.
Management : Antihistamine therapy.
Prevention :
1.
Antihistamine therapy 1 hour before transfusion.
2.
Use of washed red cells.
Anaphylactic transfusion reaction :
Cause : IgA deficient patients who have anti-IgA formed due to previous transfusion.
S/s : GIT upset, urticaria, flushing, respiratory distress, hypotension & shock.
Management :
1.
Stop transfusion,
2.
Inform attending physician,
3.
Give epinephrine.
Prevention : use plasma free products for future transfusion.
75
Circulatory overload :
Cause : excessive volume or high speed of transfusion in patients with compromised heart / lung
functions; especially infant, pregnant women (3rd trimester) & elderly.
S/s : oedema, respiratory distress.
Management :
1.
Slow the rate of transfusion to 1 ml/kg/hr.
2.
Diuretic.
Prevention : use packed red cells.
Transfusion siderosis (iron overload) :
Cause : occur in patients who receive repeated blood transfusions over a long period e.g. thalassemia
major.
1 unit of blood contains 200-250 mg iron, which get deposited in skin, heart, liver & pancreas; and
interfere with their functions.
Prevention :
1.
Iron chelating agents (desferrioxamine),
2.
Neocyte transfusion.
Septicemia :
Cause : Organism enter if septic precautions are not taken during time of collection or component
preparation. Causative organisms :
1.
Pseudomonas,
2.
Coliform,
3.
Acchromobacters.
76
S/s : shock
Management :
1.
Stop transfusion,
2.
Inform attending physician,
3.
Give antibiotics & steroids.
___________________________________________________________________________________________
Note : 13
Component therapy
___________________________________________________________________________________________
Principle :
On centrifugation, the components of blood get separated due to difference in their specific gravities :
Component
Specific gravity
Plasma
1.02-1.03
Platelet
1.03-1.04
RBC
1.08-1.09
Specific gravity of RBC & WBC are very similar.
Centrifugation :
Relative centrifugal force (g) used for component separation are :
- Heavy spin : 5000 x g for 5 minutes.
- Light spin : 2000 x g for 3 minutes.
Blood bags used for component separation :
1. Double/triple/quadruple CPDA blood bags :
Primary bag :
- volume : 450 ml,
- contains 63 ml CPDA solution.
Satellite bags : are other bags attached to the primary bag with integral tubings.
2. Four bags system with additive solution :
Primary bag contains 63 ml of CPD solution.
One of the satellite bag contains 100 ml additive solution comprised of Normal Saline, Adenine, Dextrose,
Mannitol.
Two such systems are available :
a) CPD-SAGM
b) CPD-Adsol, has higher proportion of constituents than SAGM.
Blood bag
Shelf life of RBC
(in days)
CPDA
35
CPD-SAGM
42
CPD-Adsol
49
77
Essentials of phlebotomy to good component preparation :

1. Aseptic precautions should be strictly followed at the site of venipuncture.
2. Blood should be collected by single puncture.
3. Flow of blood should be uninterrupted & constant.
4. Blood & anticoagulant solution should be mixed intermittently.
5. Maintain blood to anticoagulant ratio of 14ml of CPDA solution for 100 ml of blood, + or - 10 %.
6. Collection time should not be more than 10 minutes.
Instruments required :
1. Refrigerated centrifuge
2. Plasma expressor
3. Tube sealer
4. Laminar flow
5. Deep freezer (-20 C or below)
6. Water bath
7. Platelet agitator & incubator (20-22 C).
Components :
Cellular
RBC
Red cell concentrate or suspension
Leucocyte poor red cells
Irradiated red cells
WBC
Granulocyte concentrate
Platelet
Platelet rich plasma
Platelet concentrate
Plasma
Fresh frozen plasma
Cryoprecipitate
Cryo-poor plasma
Single donor plasma
RED CELL CONCENTRATE & SUSPENSION
Method of preparation :
Red cell concentrate :
1. Collect blood in 450 ml CPDA triple bag.
2. Centrifuge whole blood at heavy spin at 4 C. Red cells settle at the bottom.
3. Express 3/4th plasma in 1st satellite bag, leaving behind 50-70 ml plasma in primary bag.
4. Double seal the tube between primary & 1st satellite bag & separate the satellite bags.
5. Label primary bag as red cell concentrate.
Red cell suspension :
Collect blood in 450 ml quadruple bag with additive solution.
Centrifuge whole blood at heavy spin at 4 C. Red cells settle at bottom.
Express maximum plasma in 1st satellite bag, leaving behind 10-20 ml or less
plasma in primary bag.
4. Add 100 ml additive solution to packed red cells contained in primary bag.
5. Double seal the tubings & separate the bags.
6. Label the primary bag as red cell suspension.
1.
2.
3.
78
Comparison :
Composition
Total volume
Red cells
Plasma
PCV
Disadvantage
Whole blood
400-500 ml
120-250 ml
200-300 ml
45-55%
Volume overload
Red cell concentrate Red cell suspension

220-340 ml
280-320 ml
120-250 ml
120-250 ml
50-70 ml
10-20 ml or less
55-75%
50-70%
Viscous
Expensive
Advantages of using red cell component over whole blood :

1. Minimal expansion of blood volume.
2. Removal of plasma :
a) Reduces total amount of electrolytes :
- reduced Na+ is beneficial to cardiac patients.
- reduced K+ is beneficial to patients with renal failure.
- reduced NH3 & citrate is beneficial to patients with hepatic failure.
b) Reduces proteins, thus minimizing allergic & anaphylactic reactions.
c) Reduces anti-A & anti-B antibodies.
3. Plasma obtained can be used for preparation of PC, FFP.
Storage & shelf life :

Store at 2-6 C.
Shelf life :
- Red cell concentrate : 35 days in CPDA bags.
- Red cell suspension : 42 days with CPD-SAGM system & 49 days in CPD-Adsol system.
Indications :
1. Refractory anemia :
- aplastic anemia
- hypoplastic anemia
- anemia of renal failure
- carcinoma, leukemia, Hodgkins disease.
2. Non-refractory anemia :
- when urgent relief of anemia is required e.g. posted for surgery.
- late pregnancy.
3. Hemolytic anemia.
When to consider transfusion :

Based on the clinical status rather than hemoglobin level.
Transfusion trigger :
- Earlier : 10 gm/dl.
- Revised : 7.5 gm/dl.
Administration :
Group specific
Major cross-match only.
LEUCOCYTE POOR RED CELLS
Methods of preparation :
79
1.
2.
3.
4.
Centrifugation : inverted spin or upright spin.,

Washed red cells,
Filtration : leucocyte depletion filter & microaggregate filter,
Freezing & deglycerolization (costly & time consuming).
1.
2.
3.
4.
5.
6.
Centrifugation, upright spin :

Collect blood in 450 ml CPDA triple bag.
Centrifuge whole blood at heavy spin at 4 C. Red cells settle at the bottom.
Express 3/4th plasma in 1st satellite bag, leaving behind 50-70 ml plasma in primary bag.
Express buffy coat & top 10-20 ml of red cells in 2nd satellite bag.
Double seal the tubings & separate.
Label primary bag as leucocyte poor red cells.
Centrifugation, inverted spin :

Centrifuge in inverted position at heavy spin at 4 C.
Collect leucocyte poor packed red cells in satellite bag, leaving behind 70-90 ml of buffy coat mixed with
RBC & plasma in primary bag.
4. Label the satellite bag as leucocyte poor red cells.
1.
2.
3.
6.
7.
8.
9.
Washed red cells :

Centrifuge whole blood at heavy spin at 4 C. Red cells settle at the bottom.
Express 3/4th plasma in 1st satellite bag, leaving behind 50-70 ml plasma in primary bag.
Double seal the tube between primary & 1st satellite bag & separate the satellite bags.
Add 200 ml of normal saline to primary bag through bottle connector under laminar flow. Temporarily
seal the tubings.
Centrifuge at heavy spin at 4 C.
Discard supernatant saline into waste receptor.
Repeat steps 5,6,7 thrice.
After final wash, add 70-100 ml saline & mix.
Using leucocyte depletion filter :

Removes 95-100% leucocytes, with loss of 90% platelets & 10% RBC.
Special bags are devised with leucocyte depletion filters incorporated between primary & satellite bags.
Leucocyte depletion can be enhanced if blood is centrifuged before filtering.
1.
2.
3.
4.
5.
Using microaggregate filters :

- Microaggregate filters available in blood transfusion sets, remove 40-50% of leucocytes with loss of 40%
platelets & negligible loss of RBC.
- However post storage filtration (at the time of administration) does not remove the cytokines produced by
leucocytes during storage.
Indications of leucocyte poor red cells :

1. Patients receiving multiple transfusions.
2. Patients with history of severe febrile NHTR.
3. Reduces risk of transmission of CMV infection.

Store at 2-6 C.
80
Shelf life depends on production method :

Centrifugation : same as whole blood.
Washed red cells : 24 hours.
Leucocyte depletion filter : same as whole blood.
IRRADIATED BLOOD COMPONENT
Aims :
Inactivates lymphocytes, inhibiting lymphoblast transformation & mitotic activity; thus reducing risk of posttransfusion graft versus host disease.
Radiation dose :
1500-2500 rads abolishes lymphocytic activity with no cellular damage to RBC / platelet.
Indications :
1. Immuno-suppressed patients.
2. BM transplantation.
3. Pre-mature new born.
4. Patient undergoing chemotherapy.
5. Exchange transfusion & intrauterine transfusion.
LEUCOCYTE CONCENTRATE
Methods :
1. Centrifugation.
2. Leucopheresis.
1.
2.
3.
4.
5.
6.
7.
Centrifugation :
Collect blood in triple bag. Separation should be done within 4 hours of collection.
Centrifuge whole blood at light spin at 22 C.
Express 3/4th plasma in 1st satellite bag.
Seal tubings & separate.
Express plasma & upper 20-25 ml cell layer into 2nd satellite bag.
Double seal the tubings between primary & 2nd satellite bags & separate.
Label 2nd satellite bag as granulocyte concentrate.

Store at 20-22 C.
Shelf life : Use as early as possible and not later than 24 hours.
Indications :
1. Bone marrow showing myeloid hypoplasia.
2. Neutropenia (absolute count < 500/l).
3. Neonatal septicemia, not responding to antibiotics.
PLATELET RICH PLASMA (PRP) & PLATELET CONCENTRATE (PC)
1. Centrifugation.
81
2. Thrombocytopheresis.
1.
2.
3.
4.
5.
6.
7.
8.
Centrifugation :
Centrifuge whole blood at light spin at 22 C. Red cells settle at bottom.
Seal the tubings & separate.
Label 1st satellite bag as platelet rich plasma.
Centrifuge PRP at heavy spin at 22 C. Platelets settle at bottom.
Express supernatant platelet poor plasma in 2nd satellite bag, leaving 50 ml of plasma in 1st satellite bag.
Label 1st satellite bag as platelet concentrate.
Comparison :
Volume
PRP
150-230 ml.
PC
50-60 ml.

Store at 20-22 C with continous agitation in platelet agitator and incubator. Never refrigerate.
Shelf life : 5 days, the day of processing is considered as day 0.
Indications :
1. Thrombocytopenia due to reduced production :
- aplastic anemia,
- acute leukemia,
- chemotherapy or radiotherapy.
2. Thrombocytopenia due to increased loss :
- hemorrhage,
- GI bleeding,
- DIC,
- ITP.
3. Dilutional thrombocytopenia : massive transfusion with stored blood.
4. Functional platelet abnormality.
5. Viral disease associated with thrombocytopenia (Dengue).
When to consider transfusion :

Non-bleeding patients :
- Platelet count < 20,000/ul.
- Elected for surgical intervention, platelet count < 50,000/ul.
Bleeding patients :
- Platelet count < 50,000/ul.
Administration :
PRP should be ABO & Rh compatible.
PC that are ABO compatible should be given whenever possible.
Response to platelet transfusion :
Single unit of PRP / PC increase platelet count by 5000-10,000/cmm.
Expected increase will be less in patients with :
- DIC,
82
Sepsis,
Splenomegaly.
FRESH FROZEN PLASMA
1. Centrifugation.
2. Plasmapheresis.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Centrifugation :
Centrifuge whole blood at light spin at 22 C. Red cells settle at bottom.
Seal the tubings & separate.
Label 1st satellite bag as platelet rich plasma.
Centrifuge PRP at heavy spin at 22 C. Platelets settle at bottom.
Express supernatant platelet poor plasma in 2nd satellite bag, leaving 50 ml of plasma in 1st satellite bag.
Label 1st satellite bag as platelet concentrate.
Label 2nd satellite bag as fresh frozen plasma & rapidly freeze it by mechanical freezer at -70C, with bag
laid flat on the rack. Freezing should not take > 1 hour.

Storage : -20 C or below in deep freezer.
Shelf life : 1 year.
Specifications :
Volume : 200-300 ml.
Content :
1. Labile clotting factor (factor V &VIII) :
FFP is the only source for factor V.
2. Stable clotting factors.
3. Plasma proteins.
Thawing :
FFP is kept in a plastic bag & thawed in warm water (30-37 C) with continous massaging of bag to avoid
formation of cryoprecipitate.
On thawing FFP should be :
- transfused as soon as possible, not later than 6 hours; activity of labile clotting factors is lost rapidly.
- till then it should be stored at 2-6 C.
Administration :
- ABO & Rh compatible FFP should be given.
- Plasma should be checked for irregular antibodies (by ICT).
- Cross matching is not required.
Indications :
a) FFP is considered when PT/PTT is > 1.5 x normal :
1. Familial factor V deficiency.
83
2.
3.
4.
5.
6.
Hemophilia
von Willebrand disease.
Multiple coagulation factor deficiency : liver disease, DIC, dilutional coagulopathy.
Thrombotic thrombocytopenic purpura.
Patients on anti-coagulative therapy (warfarin), when actively bleeding or posted for surgery.
b) Source of Ig for patients with immunodeficiency syndrome.
CRYOPRECIPITATE
Definition :
Cryoprecipitate are protein precipitates rich in factor VIII, fibrinogen and von Willebrand factor, obtained
while thawing FFP.
Procedure :
1. Keep FFP in a plastic bag & thaw in water bath at 30-37 C, without agitating.
2. Cryoprecipitate will stick to the wall of bag.
3. Express supernatant plasma in satellite bag, leaving behind 15 ml of plasma.
4. Label satellite bag as cryo-poor plasma.
5. Re-suspend cryoprecipitate in the plasma.
Specifications :
- Volume : 10-20 ml.
- Contains : factor VIII, fibrinogen, von Willebrand factor.
Indications :
1. Hemophilia A.
2. von Willebrand disease.
3. Congenital or acquired fibrinogen deficiency.
Administration :
- Rh compatible; ABO compatible whenever possible.
- Cross matching not required.
- Infused at the earliest possible, not later than 6 hours.
CRYO-POOR PLASMA
Specifications :
- Volume : about 200 ml.
- Contents : stable coagulation factors, stable proteins.

- Stored at -20 C or below.
- Shelf life : 5 year.
Use :
- Plasma expander.
84
___________________________________________________________________________________________
Note : 14
Hemapheresis
___________________________________________________________________________________________
HEMAPHERESIS
Definition :
In Greek, apheresis = separation.
Hemapheresis is : removal of whole blood from donor/patient, separation into components, retaining
desired/unwanted component, and returning remaining components to donor / patient.
Indications :
To collect components for transfusion
To remove pathological component
Red cell apheresis
Therapeutic red cell apheresis
Leucopheresis
Therapeutic leucopheresis
Plateletpheresis
Therapeutic plateletpheresis
Plasmapheresis
Plasma exchange.
Neocytpheresis
Peripheral blood stem cells
85
Principle :
Hemapheresis machines use centrifugal force and difference in densities (specific gravities)
of various blood components for separation.
Types :
Intermittent flow centrifugation (IFC)
Continous flow centrifugation (CFC)
One vein procedure.
Two vein procedure.
Blood is drawn and reinfused after separating

desired component through the same vein.
Blood is collected from one vein,

processed & returned simultaneously through
another vein.
86
Advantages of CFC :
1.
Speed
2.
Small extracorpeal volume
Procedure :
1.
Whole blood is collected using a pump.
2.
Blood is mixed with CPD solution.
3.
Pumped into disposable centrifuge bowl through its inlet port.
4.
The bowl is centrifuged.
5.
Blood separates into its components, from bottom upwards : red cells, WBC, platelets and plasma.
6.
Agents can be added to improve separation, e.g. hydroxyethyl starch (HES).
7.
In IFC : The centrifuge stops. The pump gets reversed and the unwanted component is returned back to
individual.
8.
In CFC : the unwanted component returns simultaneously through the 2nd vein.
Adverse effects of hemapheresis :
1.
Citrate toxicity : numbness, tingling.
2.
Reaction to Hydroxyethyl starch (HES) : febrile allergic reaction, headache, mild hypertension, oedema of
extremities.
87
RED CELL APHERESIS
Introduction :
In red cell apheresis, whole blood from donor is removed, separated into components. The aspiration device
moves to the red cell column, retaining red blood cells in collection bag and returning remaining components
to donor.
Specifications :
Volume removed is replaced by saline solution.
Procedure takes about 35 - 45 minutes.
A single red cell apheresis donation results in collection of two units of red cells.
Re-donation : only after 112 days.
Storage & Shelf life :
Store at 2-6 C.
Shelf life : 35 days.
Indications :
1.
Refractory anemia :
aplastic anemia
hypoplastic anemia
anemia of renal failure
carcinoma, leukemia, Hodgkins disease.
88
2.
Non-refractory anemia :
when urgent relief of anemia is required e.g. posted for surgery.
late pregnancy.
Hemolytic anemia.
THERAPEUTIC RED CELL APHERESIS (RED BLOOD CELL EXCHANGE)
89
Steps involved :
removal of whole blood from patient,
separation into components,
disposing unwanted red blood cells into a waste bag,
returning remaining components to patient,
with simultaneous delivery of donor red blood cells to replenish the lost red cells.
Indications
Sickle cell disease during crisis.
LEUCOPHERESIS
Steps involved :
removal of whole blood from donor,
aspiration device moves to leucocyte column, retaining desired leucocytes in aspiration bag,
and returning remaining components to donor.
Steps to increase donor yield :

Aims : Granulocytes are distributed equally in circulation and marginal (body) pool. Intravascular survival of
granulocytes : half life 6-7 hours.
Measures :
Increasing granulocyte count by giving steroids or growth factors.
Adding Hydroxyethyl starch to the blood removed.

90
Yield :
Granulocyte concentrate : 0.6 x 109 granulocytes per unit.
Granulocyte apheresis : 1.0 x 1010 granulocytes per unit.
Specifications :
Storage : 20-22 C.
Shelf life : Use as early as possible and not later than 24 hours.
Indications :
Bone marrow showing myeloid hypoplasia.
Neutropenia (absolute count < 500/l).
Neonatal septicemia, not responding to antibiotics.
THERAPEUTIC LEUCOPHERESIS (LEUCOCYTE EXCHANGE)
91
Steps involved :
disposing unwanted leucocytes in waste bag,
and returning remaining components to patient,
with simultaneous delivery of donor leucocytes to replenish the lost WBC.
Indications :
To remove neoplastic cells in :
Prolymphoblastic leukemia,
chronic lymphosarcoma cell leukemia,
chronic myeloid leukemia.
PLATELETPHERESIS
92
Steps involved :
aspiration device moves to the platelet-plasma column, retaining plateletrich plasma in collection bag,
and returning remaining components to donor.
Frequency of donation :
Platelets are replaced by body within 48 hours.
One can undergo 2 plateletpheresis procedures in a week, with an interval of at least 48 hours between
procedures.
Comparison :
Platelet concentrate
Plateletpheresis
Volume
45-65 ml
200-300 ml
Yield
>0.5x1011 platelets/unit.
>3x1011 platelets/unit
Single unit transfusion increases Platelet count by
5000-10,000/l/unit
30-60,000/l/unit.
Advantages of using plateletpheresis over PC :
Provides optimal dose.
Reduces exposure to multiple donors.
Reduces risks of allo-immunization.
93
Specifications :
Shelf life
: 5 days
Storage
: 20-24 C with continous agitation.
Compatibility
: ABO and Rh specific
Indications :
1. Thrombocytopenia due to reduced production :
- aplastic anemia,
- acute leukemia,
- chemotherapy or radiotherapy.
2. Thrombocytopenia due to increased loss :
- hemorrhage,
- GI bleeding,
- DIC,
- ITP.
3. Dilutional thrombocytopenia : massive transfusion with stored blood.
4. Functional platelet abnormality.
5. Viral disease associated with thrombocytopenia (Dengue).
THERAPEUTIC PLATELETPHERESIS (PLATELET EXCHANGE)
94
Steps involved :
disposing unwanted platelets in waste bag,
and returning remaining components to patient.
with simultaneous delivery of donor platelets to replenish the lost platelets.
Indications :
Essential thrombocytopenia (Idiopathic thrombocytopenic purpura) : bleeding disorder with low platelet
count caused by autoimmune antibodies to patients own platelets.
PLASMAPHERESIS
95
Steps involved :
aspiration device moves to the plasma column, retaining plasma in collection bag,
and returning remaining components to donor,
volume removed is replaced by replacement fluids.
Remember :
Donor plasma protein :> 6.0 gm/dl.
Maximum amount of plasma donated at one sitting :
500 ml for donors weighing 50-65 kg.
900 ml for donors weighing > 65 kg.
Storage : -20 C or below in deep freezer.
Shelf life : 1 year.
Indications :
a) Source of coagulation factors for :
1. Familial factor V deficiency.
2. Hemophilia
3. von Willebrand disease.
4. Multiple coagulation factor deficiency : liver disease, DIC, dilutional coagulopathy.
5. Thrombotic thrombocytopenic purpura.
6. Patients on anti-coagulative therapy (warfarin), when actively bleeding or posted for surgery.
b) Source of Ig for patients with immunodeficiency syndrome.
96
PLASMA EXCHANGE
97
Steps involved :
retaining 2-4 liter of unwanted plasma,
and returning remaining components to patient.
removed volume of plasma is replaced by colloids or FFP.
Indications :
Removal of allo-antibodies in :
Hemolytic disease of new born.
Anti-Rh antibodies in pregnant women.
Myasthenia gravis (acetylcholine receptor antibodies).
Removal of immune-complexes :
Rapidly progressive glomerulonephritis.
Systemic lupus erythromatosus
Rheumatiod arthritis.
Hyperviscosity syndrome :
Multiple myeloma.
Removal of toxins :
Hepatic failure,
Renal failure.
PLEASE NOTE :
98
Refer text for detailed reading.

Diagrams shall be appreciated.
Mistakes are regretted.
99

Notes On Blood Banking

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Note 1

Donor selection or deferral

Belong to low risk category.

A paid donor can come in disguise of relative / friend of the patient.

Professional paid donors

May not reveal their illness

b) May donate more frequently

Donor selection or deferral :

Those found fit are accepted.

Donor registration & Consent form :

Medical history questionnaire

4. Limited Medical Examination (to be filled by Blood Bank Staff).

: _______ (you can donate in the age group : 18-65 years)

Medical history questionnaire :

Donated blood in the past 3 months.

Taken blood transfusion in the past 6 months.

Antibiotics or have you taken antibiotics in the past 7 days.

Undergone dental work in the past 7 days.

Undergone minor surgery in the past 3 months.

Undergone major surgery in the past 12 months.

Anemic or taking treatment for anemia.

Lactating women or who has a baby of less than 1 year age.

Women who have undergone abortion in the past 6 months

Taken anti-rabies vaccine in the past one year.

Suffered from typhoid and treated in the past 12 months.

Suffered from hepatitis in the past 12 months.

Women in menstrual period, 5 days before and 5 days after.

19. Donor under the effect of alcohol.

Donor suffering from abnormal bleeding tendency.

Donor a known case of diabetis.

Donor a known case of cancer.

Donor with a history of epileptic episode.

Unexplained weight loss (>10 kg within a month),

Limited Medical examination :

1. Weight : Minimum acceptable weight :

for donation of 350 ml blood is 45 kg.

for donation of 450 ml blood is 55 kg.

2. Temperature : The donor should be afebrile (temperature < 37.5 C).

4. Pulse : should be between 60-100 beats / minute and regular.

Systolic : 100-180 mmHg,

Diastolic : 50-100 mmHg.

b) Middle finger is pricked with a lancet..

A drop of blood is dropped in the solution.

d) If drop floats, Hb is <12.5 gm/dl & donor is rejected.

If drop sinks, Hb is > 12.5 gm/dl & donor is accepted.

: 100 ml (pediatric use),

6. Blood bag weighing scale.

Post donation advice :

Adverse donor reactions & remedy :

3. Nausea & vomiting :

Give water to clean the mouth & towel to wipe.

4. Twitching or muscular spasm occurs due to hyperventilation which lowers CO2 :

True convulsions are very uncommon. If they occur :

Accidental puncture of artery is indicated by fast flowing bright red blood :

Functions of the components :

CPDA-1 is the most commonly used solution.

Changes in stored blood :

Reduces pH of stored blood

High O2 delivering capacity

Low O2 delivering capacity

Optimum storage temperature for whole blood or red blood cells :

Blood bag refrigerators :