5th year (Blood bank and blood transfusion).pptx

ABC
Dr. Mustafa Adel Ahmed
Lecturer of Clinical and Chemical Pathology
5th Year Corriculum
MINISTRY OF
Health & Population
BLOOD BANKIG

Red Blood Cells (45% of total
blood) -carries fresh oxygen
throughout the body
Plasma (55% of total blood)
-contains clotting factor proteins
Platelets (< 1% of total blood)
-help form a clot to stop
bleeding

Objectives
1. Historical notes.
2. Donor Selection
3. Blood grouping.
4. Cross matching.
5. Haemolytic disease of newborn.
6. Blood component.
7. Autologus blood transfusion
8. Blood complications.
9. Coomb’s test.

Keep in mind that Dracula
wasn’t published until 1897,
long after the idea of drinking
blood to sustain life apparently
already existed.
• China, 1000 BC The soul was contained in the blood.
• Egyptians bathed in blood for their health.
• Romans was drinking the blood of fallen gladiators
to gain strength and vitality and to cure epilepsy.

• The first recorded attempt of a blood
transfusion was described by the 15th-
century.
• In 1492, Blood of three boys was given
to Pope Innocent VIII, who had
fallen into a coma. Following orders
from a physician, the blood was
transferred to his mouth, as the concept
of intravenous circulation had not yet
been discovered. The three young blood
donors, all ten years old, had
undertaken this experiment after being
promised a ducat each. Unfortunately,
the Pope and all three boys died.

• Beginning with William Harvey's experiments on the
circulation of blood, research into blood transfusion began
in the 17th century,
VS

Ibn al-Nafis, the Pulmonary Circulation
Besides his famous discovery of the pulmonary circulation, he also gave an
early insight of the coronary and capillary circulations a contribution for
which he is sometimes described as "the father of circulatory physiology"
)1213 – 1288)

• 1665 The first recorded successful
blood transfusion occurs in England:
Physician Richard Lower keeps dogs
alive by transfusion of blood from
other dogs.
• 1667 Jean-Baptiste Denis in France
and Richard Lower in England
separately report successful
transfusions from lambs to humans.
• Finally, in 1668, the Royal Society &
the French government both banned
the procedure.
• The Vatican condemned these
experiments in 1670. Blood
transfusions fell into obscurity for the
next 150 years

• 1900 Karl Landsteiner, an
Austrian physician, discovers the
first three human blood groups,
A, B, and C. Blood type C was
later changed to O. His
colleagues Alfred Decastello
and Adriano Sturli add AB,
the fourth type, in 1902.
Landsteiner receives the Nobel
Prize for Medicine for this
discovery in 1930.
• By 1907, the blood of donors
and recipients was routinely
tested and matched

• Another important breakthrough came in 1937–40
when Karl Landsteiner (1868-1943), Alex Wiener,
Philip Levine, and R.E. Stetson discovered
the Rhesus blood group system, which was found to be
the cause of the majority of transfusion reactions up to
that time.
• Three years later, the introduction by J.F. Loutit and
Patrick L. Mollison of acid–citrate–dextrose(ACD)
solution, which reduced the volume of anticoagulant,
permitted transfusions of greater volumes of blood and
allowed longer-term storage.
• Carl Walter and W.P. Murphy Jr. introduced
the plastic bag for blood collection in 1950. Replacing
breakable glass bottle.

STANDARDS OF
BLOOD DONATION
SELECTIOM

Objectives
1. Historical notes.
2. Donor Selection
3. Blood grouping.
4. Haemolytic disease of newborn.
5. Blood component.
6. Autologus blood transfusion
7. Blood complications.
8. Cross matching.
9. Coomb’s test.

HUMAN BLOOD GROUP SYSTEMS
• The term human blood group systems is
defined by International Society of Blood
Transfusion
• As systems in the human species where cell-
surface antigens—in particular, those on blood
cells—are "controlled at a single gene locus or
by two or more very closely
linked homologous genes with little or no
observable recombination between them“

• They include the common ABO and Rh
(Rhesus) antigen systems, as well as many
others; thirty-five major human systems are
identified as of November 2014.
• In addition to the ABO and Rh blood group
systems, there are more than two
hundred minor blood groups that can
complicate blood transfusions.
• Other systems: MNS, P, Lutheran, Kell, Lewis, Duffy, Kidd, Diego, Yt,
XG, Scianna, Dombrock, Colton, Landsteiner-Wiener, Chido, Hh, XK, Gerbich,
Cromer, Knops, Indian, Ok, Raph, JMH, Ii, Globoside, GIL, Rh-associated
glycoprotein, Forssman, Langereis, Junior, Vel, CD59, Augustine, KANNO.
HUMAN BLOOD GROUP SYSTEMS

ABO SYSTEM
The difference in
blood types is
caused by
different antigen
found of the
surface of the red
blood cells known
as Antigen A and
Antigen B.

• Almost all normal healthy individuals above 3-6
months of age have “ naturally occurring
Abs” to the ABO Ags that they lack.
• Reach adult level at 5-10 years of age
• Level off through adult life
• Begin to decrease in later years: >65 years of age
• These Abs termed naturally occurring because
they were thought to arise without antigenic
stimulation.
ABO ANTIBODIES

• These “naturally occurring” Abs are mostly IgM
class. That means that, they are Abs capable of
agglutinating saline/ low protein suspended red
cell without enhancement and may activate
complement cascade.
• For Group A and Group B persons the
predominant antibody class is IgM
• For Group O people the dominant antibody
class is IgG (with some IgM)
• React best at (22-24oC) or below in vitro.
• Activates complement to completion at 37oC
• RBC Immune form: Predominantly IgG
ABO ANTIBODIES

ABO DONATION IMPORTANT NOTES
WE should answer these question first…
• What is the important in DONOR?
• What is the important in RECIPIENT?
• Is it different when give one unit of blood or
more?
• What the different in FFP than blood donation?
• What to do in Blood group AMBIGUITY?

• Blood group O
is universal
donor
• Blood group AB
is universal
recipient
• Group A can
donate group A
& AB and can
receive blood
from group A
and group O
• Group B can
donate group B
& AB and
receive blood
from group B
and group O

ABO INHERITANCE
• Blood group antigens are “codominant”, if the
gene is inherited, it will be expressed.
• Two genes inherited, one from each parent.
• Individual who is A or B may be homozygous
or heterozygous for the antigen.
• Heterozygous: AO or BO
• Homozygous: AA or BB.
• Phenotype is the actual expression of the
genotype, ie, group A
• Genotype are the actual inherited genes
which can only be determined by family
studies, ie, AO.

Genes at three separate loci control the
occurrence and location of A and B antigens
1. Hh genes – H and h alleles
H allele codes for a fucosyltransferase enzyme
that adds a fucose on precursor chain to form
the H antigen onto which A and B antigens are
built on red blood cells.
h allele is a silent allele (amorph)
ABO INHERITANCE

2. Se genes – Se and se alleles
Se allele codes for a fucosyltransferase enzyme
that adds fuscos onto Type 1 chains
(primarily) in secretory glands. Controls
expression of H antigens in secretions (i.e.
saliva, body fluids, etc.)
se allele is an a morph
3. ABO genes – A, B and O alleles
A and B alleles code for (glycosyltransferase) a
fucosyltransferase enzymes that add a sugar
onto H antigens to produce A and B antigens
O allele does not code a functional enzyme
ABO INHERITANCE

• The H substance must be formed for the other
sugars to be attached in response to an
inherited A and /or B genes
ABO INHERITANCE

ABO INHERITANCE (GENE+ENZY+CHO )

ABO SYSTEM
Blood
Types:
Antigen(s) on
RBC
Antibodies
produced
Can donate
blood to:
Can receive
blood from:
Genotypes:
A IAi
A A
ALL
B B IBIB
B
IAIA
IBi
AB
AB ALL
AB
AB IAIB
O
O
O
ii
O
Antigen B
Antigens A&B
Antigen A
Neither
Anti B
Anti A
NONE
Anti A&B

World Map of the A Allele

World Map of the B Allele

World Map of the O Allele

RH SYSTEM
• Discovered in 1940 after work on Rhesus monkeys
• The 2nd most important after ABO in the crossmatch test
• The Rhesus system is much more complex than the ABO
system and has become known for its role in hemolytic
anemia of newborn.
• Unlike ABO antigen, RH antigens are proteins.
• Rh antibodies do not occur naturally but
only develop due to allo-immunization
by previous transfusion or pregnancy.

RH inheritance
• RH genes are AD autosomal codominant located on
the short arm of chromosome 1.
• Rh inheritance is controlled by 3 closely linked loci on each
chromosome of a homologous pair.
• Each locus has its own set of alleles which are:
Dd , Cc , and Ee .
• The D gene is dominant to the d gene, but Cc and Ee are co-
dominant.
• Depending on the presence of D antigen, individuals are
▫ Classified as:
 Rh positive (85%).
 Rh negative (15%).

4 Different nomenclatures:
1. Fisher-Race Nomenclature
2. Weiner Nomenclature
3. Rosenfield Nomenclature
4. ISBT (Numerical) : International Society of
Blood Transfusion.
Nomenclature of the RH system
GENOTYPIC NOMECL.
GENOTYPIC NOMECL.
PHENOTYPIC NOMECL.
PHENOTYPIC NOMECL.

Wiener Theory
• Good for describing phenotype
• There is one Rh locus at which occurs one Rh gene, but
this gene has multiple alleles.
• For example, one gene R1 produces one agglutinogen
(antigen) Rh1 which is composed of three "factors"
• The three factors are analogous to C, D, and e
respectively
• The main difference between the Fisher-Race and
Wiener theories is that the:
▫ Fisher-Race theory has three closely linked loci,
▫ the Wiener theory has only one gene locus at which
multiple alleles occur.

Conversion of Wiener to Fisher-Race
• R in Wiener = D in Fisher-Race
• r is absence of D (d)
• 0 or no symbol implies c and e
• 1 or ′ implies C and e
• 2 or ″ implies c and E
• z or y implies C and E

Fisher-Race and Wiener Nomenclature
(Weiner Gene)
Antigens
Fisher-Race
R
0
D, c, e
Dce
R
1
D, C, e
DCe
R
2
D, c, E
DcE
R
z
D, C, E
DCE
r
c,e
dce
r′
C,e
dCe
r″
c,E
dcE
r
y
C,E
dCE

Rosenfield Nomenclature
Each antigen assigned a number
• Rh 1 = D
• Rh 2 = C
• Rh 3 = E
• Rh 4 = c
• Rh 5 = e
• In writing the phenotype, the prefix “Rh” is followed by
colon, then number (if negative, number is preceded by -)
• e.g. D+, C+, E-, c+, e+ is written as
Rh:1,2,-3,4,5

ISBT Nomenclature
HAS SIX DIGIT NUMBERS
First 3 numbers represent system (RH=004)
Second 3 numbers represent antigen specificity
• Rh 1 = D = 004001 = RH positive
• Rh 2 = C = 004002 = RH negative
• Rh 3 = E = 004003 = RH negative
• Rh 4 = c = 004004 = RH negative
• Rh 5 = e = 004005 = RH negative
• Rh 6 = ce = 004006 = RH negative
• Rh 7 = Ce = 004007 = RH negative

• Related to Hemolytic transfusion reactions
• Re-exposure to antigen cause rapid secondary
response
• Always check patients history for previous
transfusion or pregnancy to avoid re-exposure.
Clinical Significance of Rh antibodies

TYPING TECHNIQUES
1. Slide method
2. Tube method
3. Column agglutinating technique.
4. Microplate Method
5. Solid phase method

Cell Suspension for REVERSE TYPING
Although red cell reagents for serum grouping are available
commercially, most laboratories prepare their own A and B test red
cells from persons known to be group A and group B. Make pooled cell
suspension as follows:
• Tube A: Place 1 drop of red cells each from 3 of A group samples.
• Tube B: Place 1 drop of red cells each from 3 of B group samples.
• Add Normal saline and to suspend the cells. Centrifuge the tubes for
at least 1 minute at 1000 rpm.
• To make 5% red cell suspension, add 1 drop of RBC to 19 drops of
saline.
• Make 20% suspension for slide method. (4 to 20)
• Test the pooled cells prepared by adding the antisera (Anti-A, B) in
use. (Check step)

• Mark a clean slide into two halves, labeling the left and
right side side as A and B.
▫ Add a drop of blood to be tested on both sides.
▫ Add one drop each of A, B and RH antibodies to the
corresponding sides.
▫ Add a drop of serum to be tested on both sides.
▫ Add one drop each of A and B cells suspension (20%) to the
corresponding sides.
• Using a clean applicator stick, mix the serum and cell
suspension on both sides separately and spread into a
smooth round circle.
• Rock the slide gently for 2 minutes and look for
agglutination.
• Record the reactions and interpret the results.
ABO/RH TYPING
F
R
18 - 22o C
1- SLIDE METHOD

ABO/RH TYPING
1- SLIDE METHOD
• Very rudimentary method for determining
blood groups.
• CANNOT be used for transfusion purposes as
false positives and negatives do occur.
• A “false positive” is when agglutination occurs
not because the antigen is present, but cells
may already be clumpled.
• A “false negative” is one in which the cells are
not clumped because there are too many cells
or not enough reagent.
18 - 22o C

ABO/RH TYPING
1- SLIDE METHOD
18 - 22o C

REVERSE GROUPING INTERPRETATION

ABO/RH TYPING
2- TUBE METHOD
18 - 22o C

ABO/RH TYPING
2- TUBE METHOD
• Label two test tubes as A and B.
▫ Add one drops of serum to be tested in each tube.
▫ Add two drop each of A, B and RH antibody to the
corresponding test tubes.
▫ Add two drops of serum to be tested in each tube.
▫ Add one drop each of A and B cells suspension to the
corresponding test tubes.
• Mix well and centrifuge both tubes at 1000 rpm for 1
minute.
• Gently remove the tubes and completely resuspend cells
and examine macroscopically for agglutination and if
negative, microscopically.
• Record the reactions and interpret the results.
F
R
18 - 22o C

ABO /RH TYPING
3- Column Agglutination Technique (CAT)

ABO /RH TYPING
3- Column Agglutination Technique (CAT) 50μl
Patient
serum
50μl
Patient
serum
50μl
A cell
Susp.
50μl
B cell
Susp.
1. Inucbate at room
tempreture for 10
min.
2. Centrifuge the card
at 1000rpm for 10
min.
3. Record the reactions
and interpret the
results.

ABO /RH TYPING
4- Microplate METHOD

ABO /RH TYPING
5- Solid phase METHOD

TEMPREATURE CONCERN
• ABO Ag-Ab reactions occurs preferentially at
room temperature. Incubation at higher
temperature may cause false negative results, So
enhancement of weak reactions may be obtained
by incubation at 4O C.
• On the other hand weak RH Ag-Ab reactions
better incubated at 37O C.

CROSS MATCHING
To detect:
1. irregular antibodies;
2. errors in ABO grouping,
3. clerical errors in patient identification and result
recording.

CROSS MATCHING
1. The major crossmatch:
the patient’s serum with donor cells
 to determine whether the patient has an antibody which may
cause a hemolytic transfusion reaction or decreased cell
survival of donor cells.
 This is the most important cross-match.
2. The minor crossmatch
the patients cells with donor plasma
 to determine whether there is an antibody in the donor’s
plasma directed against an antigen on the patient’s cells.
There are two types of cross-matches:

CROSS MATCHING
1. Immediate spin Crossmatch:
2. Antihuman globulin Crossmatch:
1. TUBE METHOD.
2. CAT METHOD
3. SOLID PHASE RED CELL ADSORPTION METHOD.
3. The electronic (“computer”) Crossmatch
is an acceptable alternative to an IS crossmatch.
Classify

CROSS MATCHING
IMMEDIATE SPIN (IS) CROSSMATCHIN

CROSS MATCHING
CAT METHOD
• Result interpretations.

Cause of Hemolytic Disease
• Maternal IgG antibodies directed against an
antigen of paternal origin present on the fetal
red blood cells.
• IgG antibodies cross the placenta to coat fetal
antigens, cause decreased red blood cell survival
which can result in anemia.
• Produced in response to previous pregnancy
with antigen positive fetus OR exposure to red
blood cells, ie transfusion.

Three Classifications of HDN
1. Rh – anti-D alone or may be accompanied by
other Rh antibodies – anti-C, -c, -E or –e.
2. ABO (IgM ? IgG)
3. “Other” – unexpected immune antibodies
other than anti-D – Jk, K, Fy, S, etc.

HAEMOLYTIC DISEASE OF NEWBORN
• When an Rh D - negative
woman has a pregnancy with
an Rh D positive fetus.
• Rh D - positive fetal red cells
cross into the maternal
circulation (especially at
parturition and during the
third trimester) and sensitize
the mother to form anti-D.

• Anti - D crosses the placenta
to the fetus during the next
pregnancy with an RhD -
positive fetus, coats the fetal
red cells and results in
destruction of these cells
causing HAEMOLYTIC
anaemia and jaundice.

• The main aim of management
is to prevent anti - D antibody
formation in Rh D - negative
mothers.
• This can be achieved by the
administration of small
amounts of anti - D antibody
which ‘ mop up ’ and destroy
Rh D - positive fetal red cells
before they can sensitize the
immune system of the mother
to produce anti - D.
D: Coom‘b’s test D & InD

• Anti-D is the commonest form of severe HDN.
The disease varies from mild to severe.
• Anti-E is a mild disease
• Anti-c can range from a mild to severe disease -
is the third most common form of severe HDN
• Anti-e - rare
• Anti-C - rare
• antibody combinations (ie anti-c and anti-E
antibodies occurring together) - can be severe

ABO Hemolytic Disease
• Mother group O, baby A or B
• Group O individuals have anti-A and anti-B in
their plasma, fetal RBCs attacked by 2 antibodies
▫ Occurs in only 3%,
▫ Severe in only 1%,
▫ <1:1,000 require exchange transfusion.
• The disease is more common and more severe in
African-American infants.

“Other” Hemolytic Disease
• Uncommon, occurs in ~0.8% of pregnant
women.
• Immune alloantibodies usually due to anti-Kell,
-Kidd or -Duffy.
• Anti-K
▫ disease ranges from mild to severe
▫ over half of the cases are caused by multiple blood
transfusions
▫ is the second most common form of severe HDN
• Anti-M very rare

• Maternal antibodies destroy fetal red blood cells
▫ Results in anemia leads to baby tissue hypoxia.
• Baby's responds to the hemolysis by trying to make more
RBCs very quickly in the BM, liver and spleen.
▫ Organs enlarge - hepatosplenomegaly.
▫ New RBCs are prematurely released and are unable to
effeciently do the work of mature RBCs
• As the red blood cells break down, bilirubin is formed.
▫ Babies unable to get rid of the bilirubin leads to
(hyperbilirubinemia ) and jaundice.
• The placenta helps get rid of some of the bilirubin, but
not all.

Complications During Pregnancy
1. Severe anemia (Extramedullary
hematopoiesis ) leads to
hepatosplenomegally
When these organs and the bone marrow
cannot compensate for the fast destruction
of red blood cells, severe anemia results and
other organs are affected.
2. Hydrops Fetalis
This occurs as the baby's organs are unable
to handle the anemia. The heart begins to
fail and large amounts of fluid build up in
the baby's tissues and organs. A fetus with
hydrops is at great risk of being stillborn.
Varies from mild jaundice and anemia to hydrops fetalis (with ascites,
pleural and pericardial effusions)

Clinical Presentation
• Risks during labor and delivery include:
▫ asphyxia and splenic rupture.
• Postnatal problems include:
▫ Asphyxia
▫ Pulmonary hypertension
▫ Pallor (due to anemia)
▫ Edema (hydrops, due to low serum albumin)
▫ Respiratory distress
▫ Coagulopathies (↓ platelets & clotting factors)
▫ Jaundice
▫ Kernicterus (from hyperbilirubinemia)
▫ Hypoglycemia (due to hyperinsulinemnia from islet cell
hyperplasia)

Kernicterus
• Kernicterus (bilirubin encephalopathy) results from
high levels of indirect bilirubin (>20 mg/dL in a term
infant with HDN).
• Unbound unconjugated bilirubin crosses the
blood-brain barrier and, because it is lipid soluble,
it penetrates neuronal and glial membranes.
• Bilirubin is thought to be toxic to nerve cells

Laboratory Findings
• Vary with severity of HDN and include:
1. Anemia
2. Hyperbilirubinemia
3. Reticulocytosis (6 to 40%)
4. ↑ nucleated RBC count (>10/100
WBCs)
5. Thrombocytopenia (HSM)
6. Leukopenia (HSM)
7. Positive Direct Antiglobulin Test
8. Hypoalbuminemia
9. Rh negative blood type or ABO
incompatibility
• Smear: polychromasia, anisocytosis, no
spherocytes

Transcutaneous Monitoring
• Transcutaneous bilirubinometry
can be adopted as the first-line
screening tool for jaundice in
well, full-term babies.
• This leads to about 50%
decrease in blood testing.

Treatment of Mild HDN
• Phototherapy is the treatment of choice.
• Phototherapy process slowly
decomposes/converts bilirubin into a nontoxic
isomer, photobilirubin, which is transported
in the plasma to the liver.
• HDN is judged to be clinically significant
(phototherapy treatment) if the peak bilirubin
level reaches 12 mg/dL or more.

Intrauterine Transfusion
• Can be done as early as 17
weeks, although
preferable to wait until 20
weeks

Phototherapy
•
Lightweight, fiberoptic pad delivers up to 45 microwatts
of therapeutic light for the treatment of jaundice while
allowing the infant to be swaddled, held and cared for by
parents and hospital staff.
•
Compact unit is ideal for hospital and homecare.

Exchange Transfusion
• Full-term infants rarely require
an exchange transfusion if
intense phototherapy is
initiated in a timely manner.
• It should be considered if the
total serum bilirubin level is
approaching 20 mg/dL and
continues to rise despite intense
in-hospital phototherapy.
• The procedure carries a
mortality rate of
approximately 1%.

INDICATION OF BLOOD COMPONENT
1. Whole blood:
1. Acute massive blood loss with hypovolemic shock.
2. Exchange transfusion
2. Packed RBCs
1. Anemia due to heart failure , liver cirrhosis, bone
marrow failure ..ETC
3. Platelet concentrates: Indicated to stop thrombocytopenic
bleeding due to failure of platelet production, massive blood
transfusion, DIC.
4. Fresh frozen plasma (FFP) :
To treatment of haemostatic disorders due to deficiencies
of clotting factors ,massive blood loss, and liver diseases.
5. Cryoprecipitate:
Mainly to to ttt Factor VIII Def, DIC, Fibrinogen defect.

Autologus BLOOD Transfusion
Start with the ERA of anxiety of HIV and has three ways of
administration of an autologous transfusion:
1. Predeposit
Blood is taken from the potential recipient in the
weeks immediately prior to elective surgery.
2. Haemodilution
Blood is removed immediately prior to surgery once
the patient has been anaesthetized and then reinfused
at the end of the operation.
3. Salvage
Blood lost during the operation is collected during
heavy blood loss and then reinfused.

Acute complication of transfusion
They are the reactions that occurs during or shortly after (within 24 hours)
the transfusion.
IgA deficiency
Yersinia, Staph, Pseudomonas
ABO
Ab/patient’s. Donors multiparous

Delayed complication of transfusion
They are the reactions that occurs days, months, even years after the
transfusion.
donor T-lymphocytes proliferating and attacking the recipient’s tissues.
Prev sens/preg low Ab titre
antibodies directed against platelets in the recipient

5th year (Blood bank and blood transfusion).pptx

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