Blood Transfusion Science

RBCs
PLT Plasma

Prepared by: Hassan Deeb Suleiman

Central Blood Bank Shifa Hospital
1
Blood Transfusion Director
Together, we can save
human lives.
It is one of the most
important things we can do.
2
 Donor Selection and Blood
Collection

Blood Centers And Transfusion Services

depend on voluntary donors to provide
the blood ,and to meet the need of the
patients they serve.
So that, the the general rule for blood
donation relies basically on :
1. Donor protection and safeness.
2. Safe blood transfused to the patient.
3
:Donor selection criteria
.Donor's age: 18-60 years .1
.Donor's weight: not less than 50 Kg .2
Interval between donations: 3 month .3
.duration
Donor's appearance: special notes should be .4
taken of plethora, poor physique,
debilitation, under nutrition, jaundice, cyanosis,
dyspnea, mental instability and intoxication from
.alcohol, drugs and narcotics
:Donor's pulse and blood pressure .5
.Pulse: 50-110 beats per minute
:Blood pressure
Not less than 100/60
.Not more than180/100
:Hemoglobin .6
.Male: Not less than 13.5 g/dl
.Female: Not less than 12 g/dl 4
 Donor Blood Testing
B lo o d C o lle c tio n
S to r a g e
T ra n s p o rt
D o c u m e n ta tio n

A B O g r o u p in g R e ty p in g a n d G r o u p in g T e s tin g : R P R , A lt,
R e v e r s e G r o u p in g C r o s s m a tc h H C V , H B S ag,
R h ty p in g R e le a s in g C o m p a tib le U n its H B C o re , H T L V I
A n tib o d y D e te c tio n F o llo w - u p H T L V III, C M V 5
6
 Patient Specimen Handling
P a tie n t
Id e n tific a tio n
D o c u m e n ta tio n
L a b e lin g

A B O g ro u p in g A n tib o d y Id e n tific a tio n Is s u in g b lo o d

R h ty p in g S e le c tin g a p p ro p ria te b lo o d D o c u m e n ta tio n
R e v e rs e ty p in g C ro s s m a tc h in g A d v e rs e R e a c tio n s
A n tib o d y D e te c tio n L a b e lin g R e c o r d K e e p in g
7
 Pre-Transfusion Patient Testing
 Patient testing depends on the type of blood
component requested and the patient’s previous
history.
 For red blood cells:
Type and Screen (ABO & Rh group, antibody
screen)
Compatibility test
 Electronic crossmatch if no antibody
present
 Serological crossmatch if antibody present
 For plasma, platelets, cryo:
ABO & Rh group
Historical ABO & Rh on file is acceptable
8
 Transfusion Medicine Requisition or
Request Form

• Identify the requisition with patient addressograph

with full name and unique identification number.
• Verify the patient identity against the clinician’s
order.
• Complete the patient transfusion history.
• Complete other questions if applicable.
• Specify blood product requested.
• Specify amount required.
• Specify when required.
• Specimens will require signature of phlebotomist
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 Requesting Blood and Blood
Components
ASSESS TRANSFUSION NEED

DEFINITE NEED FOR

EMERGENCY BLOOD BLOOD, i.e., POSSIBLE NEED FOR
STAT NOTIFY TM - ANEMIAS BLOOD
- SURGERY

REQUEST EMERGENCY BLOOD

REQUEST TYPE & SCREEN RED
- GROUP SPECIFIC – with specimen
BLOOD CELL ON REQUEST
- O NEG BLOOD – no specimen

REQUEST TYPE & SCREEN

RED BLOOD CELLS & TIME 10
REQUIRED
 Testing and Requesting Blood
Components
 Testing Performed on Donated Blood
 Pre-Transfusion Patient Testing
 Request for Blood Products
 Patient Specimen Collection
 Type & Screen Requests
 Emergency Blood Request
 Time-frame for Obtaining Blood Products 11
Specimen with Requisition

12
Blood Transfusion

Serological Techniques

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 Blood Group Antibodies

 Blood group antibodies are usually IgM or

IgG

 Reactions are visualised by agglutination

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15
 Agglutination

 IgM antibodies agglutinate red cells with

corresponding antigen in saline solution

 IgG antibodies bind to antigen but cannot

agglutinate red cells in saline solution

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Immunoglobulin M (IgM)

Pentamer with 10 antigen

combining sites

17
Immunoglobulin G (IgG)

Monomer with 2 antigen combining sites

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 Red Blood Cells

Negative surface charge due to sialic acid

residues maintains a gap between red cells
19
Therefore,
Main Factors Affecting Agglutination

 Red cell surface charge

 Ig type of antibody

20
 Factors Affecting
Antigen/Antibody Reactions

 Concentration of Ag/Ab
 Temperature
 Ionic strength of reaction medium
 Antibody avidity
 Antibody affinity

21
 Recap
 IgM antibodies can  IgG antibodies only
be visualised easily combine with the
as they agglutinate corresponding
red cells with the antigen in saline
corresponding (sensitisation) and
antigen in saline therefore need help
solution to agglutinate red
cells

22
 Techniques

 Indirect anti-human globulin (Coomb’s)

test

 Direct anti-human globulin (Coomb’s) test

 Enzymes
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24
 Anti-Human Globulin (AHG)
 IgM antibody

 Directed against human IgG molecule

 Historically made in rabbits

 Now produced in cell culture

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 Indirect Anti-Human Globulin
Test (IAGT, IAHGT)
 Uses AHG to detect antibody coating on
red cells after incubation phase

 Detect antigens on red blood cells

 Detect antibodies in plasma

 Perform crossmatches
26
Incubate Cells and Serum

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 Negative test
Positive test
No sensitisation
Sensitisation

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 Remove Excess IgG
Positive test Negative test

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 Add AHG
Positive test Negative test
Agglutination No Agglutination

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Today's , LISS gel (low ionic strength saline)
is used to detect comb's test(A.H.G.)
LISS is more sensitive and accurate than the
old tube methods.

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Direct Anti-Human Globulin Test
(DAGT, DAT, DCT)
 Uses anti-human globulin to detect
antibody coating on red cells without
incubation
 It therefore reflects in vivo state

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 Enzymes

 Papain, Ficin, Bromelin, Trypsin

 Remove sialic acid from red cell surface

 Only used for antibody investigation

 Sialic acid residue contains some antigens

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DIRECT ANTIGLOBULIN TEST
 Detects in vivo sensitization of RBCs
 Procedure (see Color Plate 1)
 Wash unknown RBCs >3 X with saline
(removes unbound globulins)
 Add AHG (binds to IgG or C3d, if any, that
is bound to RBCs; forms agglutinates)
 Centrifuge (accelerates agglutination)
 Grade and record agglutination as 0 to 4+
 Add Ab-coated RBCs (check cells) to all
negatives, spin, read and record (checks
that AHG was added and was functioning) 34
DAT: CLINICAL CONDITIONS
 Hemolytic disease of the newborn (HDN)
(maternal IgG crosses placenta and binds to
infant RBCs; may be up to a 4+ reaction)
 Hemolytic transfusion reaction (recipient Ab
coats donor RBCs; usually about a 1+
reaction)
 Autoimmune hemolytic anemia (AIHA)
(autoAbs coat own RBCs; reaction strength
variable)
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 DAT: INTERPRETATION
 Usually do initial DAT using polyspecific AHG
and then more detailed testing, if necessary,
with mono-specific AHG
 With a positive DAT, consider:
 Evidence of in vivo hemolysis?
 Recently transfused?
 Unexpected allo- or autoAbs?
 Medications?
 Positive DATs with no clinical significance may
be detected in up to 2% of population
36
 INDIRECT
ANTIGLOBULIN TEST (IDAT)
 Detection of in vitro sensitization of RBCs
 Procedure (see Color Plate 1)
 Same as DAT, except:
 Step 1 entails incubating RBCs (reagent or
unknown) with antisera (reagent or
unknown) allowing time for in vitro
attachment of Abs to RBCs

37
 IDAT: APPLICATIONS

 When the unknown is sera:

 Detection of Abs in recipient sera that may be
incompatible with donor RBC Ags (compatibility
test - in this case, the RBCs may also be
considered “unknown”)
 Detection of unexpected allo- or autoAbs in
unknown sera (antibody screen)
 Identification of unexpected allo- or autoAbs in
unknown sera (antibody identification)
 Titration of Ab in unknown sera or amniotic fluid
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COMPATIBILITY TESTING

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 PURPOSE
 Selection of the safest blood components to
infuse which meets the needs of the patient
 With acceptable donor cell survival rates
 Without destruction of recipient cells
 Potential risks must be weighed against the
benefits

40
 CONSISTS OF:
 ABO/Rh type (including Dw) and Ab screen on
donor (D)
 ABO/Rh type (Dw optional) and Ab screen on
recipient (R)
 Crossmatch (tube X-match – major side, +/-
auto control; electronic)

41
 SPECIMENS
 Patient ID (many errors occur here)
 Patient sample
 Serum preferred over plasma
(anticoagulant in plasma may inactivate C’)
 Non-hemolyzed
 Free of IV contaminants
 72 hours old (esp. important if patient
was recently transfused or pregnant)
 Keep after infusion for 7 days
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 SPECIMENS
 Donor samples
 Drawn when unit is drawn
 Labeled with unit #
 Kept after infusion for 7 days

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 PROCECURES
 Donor testing includes:
 ABO/Rh including Dw (must repeat at
transfusing site)
 Tests for transmittable infections (CMV
optional)
 Ab screen (if negative or giving packed
cells, eliminates need for minor X-match)
 All testing performed with acceptable
protocols and reagents
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 PROCEDURES
 Recipient testing (may be performed in
advance)
 Review of medical history, esp. previous
transfusions and pregnancies
 ABO/Rh (Dw optional)
 Ab screen
 Performed with accepted protocols and
reagents
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SHIFA HOSPITAL
LABORATORY
Blood units whole
blood, packed RBC
stored at 2-6°C

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47
ABO & Rh(D) Blood Groups

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The ABO System
 Discovered in 1901 by Dr. Karl
Landsteiner
 4 Main Phenotypes (A, B, AB, O)
 ABO gene located on long arm
of chromosome 9

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 The ABO Antigens
 Added stepwise to Proteins or Lipids on
Red Cells
 Substrate Molecule is H (fucose)
 A antigen is N-acetyl-galactosamine
(GalNAc)
 B antigen is Galactose (Gal)
 A and B genes code for transferase
enzymes

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Structure of A & B Antigens

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 ABO Antibodies
 A and B substances very common
 Antibodies produced to “non-self”
 Produced after first few months of life
 A & B people have mainly IgM
 O people have IgG
 May fade in old age

52
 Antigens & Antibodies

Blood Antigens on
Antibodies in Serum Genotypes
Group RBCs

A A Anti-B AA or AO

B B Anti-A BB or BO

AB A and B Neither AB

O Neither Anti-A and anti-B OO

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Inheritance of ABO Groups
Allele from Allele from Genotype of Blood types of
the mother the father offspring offspring

A A AA A

A B AB AB

A O AO A

B A AB AB

B B BB B

B O BO B

O O OO O

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 The Bombay Phenotype
 H substrate is required to attach A or B
 Rare recessive allele h
 Persons who are hh produce no H
 Genetically they can carry A or B genes
 Transferase Enzymes are produced
 Have a potent anti-H

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 Subtypes of A
 80% of A are type A1
 A1 cells carry approx 1 million antigens
per red cell
 Type A2 carry only 200,000
 Type A2 people can make anti-A1
 Other weaker subgroups exist

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 ABO Typing
 Cell Group  Reverse Group
 Test Washed Cells  Test plasma/serum
With: with:
 Monoclonal Anti-A
 Known A1 cells
 Known B cells
 Monoclonal Anti-B
 Known O cells
 Inert control
 ? Known A2 cells
 Agglutination is a  Reactions may be
positive result weaker than cell
group
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 Universal Donor and Recipient
 Universal Donor
 Group O
 Carries no A or B
antigens
 Packed and
processed units have
little antibody
 Universal Recipient
 Group AB
 Patient has no anti-A
or anti-B present
 Cannot lyse any
transfused cells
 Beware: other
 antibodies may be
present

Using the patient’s own group is the most preferrable 58

 Significance of ABO Group
 ABO mismatched transfusions:
 Rare
 May be life threatening
 Can be caused by technical or clerical error
 Intravascular haemolysis
 More severe in group O patients

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The Rh(D) Antigen
 RH is the most complex system,
with over 45 antigens
 Discovered in 1940 after work on
Rhesus monkeys
 Subsequently discovered to be
unrelated to monkeys
 RH gene located on short arm of
chromosome 1

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 Simple Genetics of Rh(D)
 86% of Caucasians are Rh(D) pos
 The antithetical antigen d has not been
found
 The d gene is recessive:
 Dd, dD, DD, persons are Rh(D) pos
 Only dd persons are Rh(D) neg

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 Distribution of Rh(D) Types
Population Rh(D) pos Rh(D) neg

Caucasian 86% 14%

African-American 95% 5%

Oriental >99% <1%

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 Significance of Rh(D)
 80% of Rh(D) neg persons exposed to Rh(D)
pos blood will develop anti-D
 Anti-D can also be stimulated by pregnancy
with an Rh(D) positive baby
 Sensitisation can be prevented by the use of anti-
D immunoglobulin, antenatally and post natally
 Rh(D) neg females of childbearing potential
should never be given Rh(D) positive blood
products
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 Inheritance
 ABO & RH genes are not linked
 ABO & Rh(D) type are inherited
independently
For example:
An A Rh(D) pos mother
and a B Rh(D) pos father
could have an O Rh(D) neg child

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Inheritance of ABO and Rh(D)
Mother Father
Group A AO Mating Group B BO
Rh(D) pos Dd Rh(D) pos Dd

Group A AO Group B BO Group O OO

Rh(D) pos Dd Rh(D) pos Dd Rh(D) neg dd

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Blood group systems other than ABO
system
 Lewis system
 Duffy system
 Kidd system
 Kell system
 Lutheran system
 I system
These system are less important than ABO &
RH(D) system, as well as do not routinely done.

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The Risks of Blood Transfusion

 Transfusion transmitted diseases

 Immunological reactions
 Non-immunological reactions
 Signs and symptoms of adverseevents
 Nursing actions

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Transfusion Transmitted Diseases
 It is impossible to be certain about the exact risk
of infection.
 HIV (AIDS)
Hepatitis B
Hepatitis C
HTLV Rare

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 Immunological Reactions
 Acute hemolytic (mostly ABO incompatibility)
 Delayed hemolytic (antibody present)
 Febrile (temperature increase seen in 1% of
transfusions)
 Transfusion Related Acute Lung Injury
(TRALI), antibody in donor plasma against
patient’s leukocytes
 Allergic (allergens in donor blood)
 Anaphylactic (possibly IgA related)
 Bacterial (contaminated blood or equipment)
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 Non-Immunologic Reactions

 Circulatory overload
 Microaggregate infusion
 Air embolism
 Hypothermia
 Citrate toxicity, Hypocalcemia
 Hyper/Hypo-kalemia
 Iron overload
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Common Signs and Symptoms of
Transfusion Reactions
Symptoms Severity
Mild if temperature rise ≤ 1°C from
 Fever baseline temperature and no other
symptoms
 Dyspnea Serious
 Bronchospasm Serious
 Rash Mild if rash is over <25% of body
 Urticaria Mild if hives over <25% of body
 Flank Pain Serious
 Hypotension Serious
 Shock Serious
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 Nurses’ Actions
STOP THE TRANSFUSION
 Keep the line open with normal saline, assess, O2
Notify the attending MD and Transfusion Medicine
 If the reaction appears to be life threatening, the
physician on call for Transfusion Medicine should
be notified immediately
 Complete Transfusion Medicine requisition section
on transfusion reaction
 Draw 7 mL (3mL pediatrics) EDTA specimen and
send to Transfusion Medicine with blood unit and
administration set
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Transfusion Complications
Positive DAT (Comb's)

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 Immediate Complications
 Within 96 hours
 Antigen-Antibody reactions
 Acute hemolysis
 need only 10 - 15 ml to start reaction
 95% due to clerical error
 two types - extravascular and
intravascular

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 Immediate Complications

 Antigen-Antibody reactions
 Extravascular hemolysis
 destroyed in the RES
 anticipate increased bilirubin
 may be undetected except by decreased hemoglobin
and hematocrit

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 Immediate Complications

 Antigen-Antibody reactions
 Intravascular hemolysis
 Severe
 Complement binding and usually ABO
 symptoms
 chills, fever, decreased blood pressure, release of
histamine, hemoglobinemia, hemoglobinuria, DIC
and oozing during surgery, renal shutdown

76
 Immediate Complications
 Antigen-Antibody reactions
 Antibodies against leukocytes, platelets
and plasma proteins
 cause febrile reactions
 classic response begins 5 minutes into
transfusion with flush, palpitation,
increased pulse, tightness in chest and
cough, increase in blood pressure,
increased fever and chills
 neutrophilic leukocytosis - peaks 3-5 hours
77
 Immediate Complications

 Antigen-Antibody reactions
 Reactions from transfused antibodies
 penicillin, foods, mild HTR
 Allergic reactions
 usuallya rash and due to proteins in
plasma, food or drugs

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 Delayed Complications
 After 96 hours
 Diseases
 hepatitis,
malaria, syphilis, brucellosis, infectious
mononucleosis, toxoplasmosis, HIV,
cytomegalovirus, Chaga’s, Babesiosis
 Delayed ag-ab
 old antibody restimulated
 Allotypic immunizations
 Hemosiderosis 79
 Prevention

 Most are self evident

 Use leukocyte poor red cells
 Careful donor screen and selection
 Careful control against mistakes

80
 Investigation
 Stop transfusion
 Collect blood sample and first urine
 Obtain blood and infusion set
 Immediately
 direct
antiglobulin on pre and post sample
 Check ABO and Rh on all samples
 Observe all specimens for visible hemolysis
 Check urine for red cells and hemoglobin
81
 Investigation
 As Soon As Possible
 Repeat antibody screen on all specimen
 Repeat compatibility on pre and post
including major and minor testing
 Chemistries on pre and post
 plasma hemoglobin - clears in 4-12 hours
 serial bilirubin - intra peaks 6-24 hours and extra
peaks 3 - 10 days
 haptoglobin

 Bacterial smear if needed

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 Investigation

 Febrile reaction
 rule out hemolytic
 may be able to demonstrate
leukoagglutinins
 use leuko poor blood

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Thank You.

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