IMMUNOHEMATOLOGY / BLOOD BANKING

HISTORY
First blood transfusion: 1492, ________________
Pope Innocent V11

Braxton Hicks: recommend Sodium phosphate as blood preservative

Karl Landsteiner: discover the ABO blood group system
Hustin: uses sodium citrate as an anticoagulant
Lewisohn determined the minimum amount of citrate needed for anticoagulation and demonstrated its nontoxicity in small
amounts
Rous and Turner: introduced the citrate dextrose preservative
Dr. Charles Drew: director of first American Red Cross blood bank
Loutit and Mollison: introduced the ACD preservative
Gibson: introduced Citrate Phosphate Dextrose preservative

ISBT TERMINOLOGY OF RED BLOOD CELL SURFACE ANTIGENS

ISBT SYSTEM SYSTEM CHROMOSOMAL
NUMBER NUMBER
001 tng ABO 9
002 Mens MNS 4
003 Po P 22
ni
004 Rhea Rh 1
005 Lumabas Lutheran 19
006 Kaya Kell 7
007 Lang Lewis 19
008 Di Duffy 1
009 Kita Ini) Kidd 18
010 Diego Diego 17
011 Cartwright (Yt) 7
012 Xg 2
013 Scianna 1
014 Dombrock 12
015 Colton 7
016 Landsteiner Weiner 19
017 Chido/Rodgers 6
018 H 19
019 Kx Xp
020 Gerbich 2
021 Cromer 1
022 Knops 1
023 Indian 11
024 Ok 19
025 Raph 11
026 John Milton Hagen 15
027 I 6
028 Globoside 3
029 Gill 9
030 RHAG 6
031 FORS 6p
032 JR 4q
033 LAN 2q
034 Vel 1q
035 CD59 11p
036 Augustine 6p
 BASIC GENETICS
Genetics= Study of transmission of inherited characteristics
Important in the study of antigen inheritance and inherited disorders
Normal Number of Human chromosomes: 46 (1 pair of 23 chromosomes coming from each parent)
Gene: A segment of DNA arranged along the chromosome at a specific position called locus. Gene at a specific locus that
differ in their nucleotide sequence are called alleles
Dosage effect: Presence of homozygous genotype can express itself with more antigen than the heterozygous
genotype
Genotype: Total genetic composition of an individual, representing maternally and paternally derived genes. It is the
complement of DNA that is inherited.
Phenotype: Detectable or expressed characteristics of genes
Most of the antigens in the various blood group system generally follow straightforward inheritance patterns, usually
CODOMINANT nature.
Autosomal dominant traits are routinely encountered in the blood bank, as most blood group genes are codominant and are
on autosomal chromosomes

Population genetics Concerning genetic traits in large numbers of individuals

Cellular genetics Pertains to the cellular organization of genetic material
Molecular genetics Based on the biochemistry of genes and the structures that support them
Gregor Mendel The father of genetics. The one who postulated the law of inheritance
Law of Independent or random segregation = First Law
inheritance Law of Independent assortment = second law
Law of Dominance
Law of segregation Each gene is passed on to the next generation on its own
Law of Dominance alleles of a gene, the dominant allele is always expressed
because it masks the recessive allele. Recessive traits are only seen when both alleles are recessive
Law of assortment State that genes for different traits are inherited separately from each other. This allows for all
possible combinations of genes to occur in the offspring
Autosomal refers to traits that are not carried on the sex chromosomes.
Recessive trait carried by either parent or both parents but is not generally seen at the phenotypic level unless both
parents carry the trait
Heterochromatin Stains as dark bands
Achromatin stains as light bands and consists of highly condensed regions that are usually not transcriptionally
active
Eurochromatin the swollen form of chromatin in cells, which is considered to be more active in the synthesis of RNA
for transcription
Human chromosomes Humans have a complement of 46 chromosomes arranged into23 pairs; one member of each pair is
inherited from the father and the other from the mother. Each of the members of one chromosome
pair is referred to as a chromosome homologue. Of the pairs, 22 are called autosomes; the
remaining pair represents the sex chromosomes of which males have an X and a Y and females have
two Xs
Meiosis Meiosis is the process of cell division unique to gametes (ova and sperm). In contrast to mitosis, the
process of meiosis produces four gametes with genetic variability. This results in four unique, rather
than two identical, daughter cells.
Mitosis Is the process of replication in nucleated body cells (except ova and sperm cells).
Transcription the cellular process by which one strand of duplex DNA is copied into RNA
Translation the cellular process by which RNA transcripts are turned into proteins and peptides, the functional
molecules of the cell

Interphases / Non-Mitosis stages

G0 (quiescence/resting phase) The cell is not actively in the cell cycle.
G1 (gap 1) *L
*Period of cell growth and synthesis of components necessary for replication
* During this period, the nucleolus (nucleoli) becomes visible, and the chromosomes are
extended and active metabolically. The cell synthesizes RNA and protein in preparation for
cell division
S phase *L
(DNA synthesis) *This is the time of DNA replication, during which both growth and metabolic activities are
minimal.
*The centrosome is also duplicated during the S stage
G2 (gap 2) *R
*This is the second period of growth, when the DNA can again function to its maximum in the
synthesis of RNA and proteins in preparation for mitotic division.
*The tetraploid DNA is checked for proper replication and damage
 M phase/Mitosis phase
Prophase *The chromatin becomes tightly coiled.
*Nucleolus and nuclear envelope disintegrate.
*Centrioles move to opposite poles of the cell.
Metaphase * Sister chromatids move to the equatorial plate.
Anaphase * Sister chromatids separate and move to opposite poles.
telophase * Chromosomes arrive at opposite poles.
*Nucleolus and nuclear membrane reappear.
*The chromatin pattern reappears

MUTATIONS
Definition Any change in the structure or sequence of DNA, whether it is physical or biochemical.
An organism is referred to as a mutant if its DNA sequence is different from that of the parent
organism
Point mutation The simplest type of mutation is the point mutation, in which only one nucleotide in the DNA
sequence is changed. Point mutations include r substitutions, - insertions, and r deletions.
Transition one purine is substituted for another purine, or one pyrimidine is substituted for another
pyrimidine.
Transversion A type of mutation in which purine is substituted for a pyrimidine or a pyrimidine for a purine

Missense mutation A missense mutation results in a change in a codon, which alters the amino acid in the
corresponding peptide. These changes cannot be accommodated by the peptide while still
maintaining its function.
Example alterations in the hemoglobin molecule at a single base pair, resulting in different types of
inherited anemias
Non sense mutation A very specific type of serious mutation, called a nonsense mutation, results when a point change in
one of the nucleotides of a DNA sequence causes one of the three possible stop codons
to be formed
Frameshift mutation results in a nonfunctional transferase protein that is seen phenotypically as the O blood group

Duplication Give rise to pseudogenes and other so-called junk DNA that does not code for proteins
E.g glycophorin A and B genes; the second involves the genes RHD and RHCE. The Chido and
Rodgers blood group antigens, carried on the complement components of the C4A and C4B genes
Recombination Mutations involving recombination or crossing over take place during the process of meiosis in
the formation of gametes. An example of such an event resulting in a hybrid formation is
seen in the MNSs blood group system.

Transcription Transcription is the cellular process by which DNA is copied into RNA. Although mRNA accounts
for only a small percent of the total RNA inside a eukaryotic cell, it has the extremely
form of the genetic code.

Transcription is an enzymatic process whereby genetic information in a DNA strand is copied into an mRNA
complementary strand. Eukaryotic mRNA is modified after it is made by various processing steps, such as
the removal of introns and addition of a poly-A tail to the end. These processing steps take place in
the nucleus of the cell before the mRNA is exported to the cytoplasmic ribosomes for translation.

Translation Translation is the cellular process by which RNA transcripts are turned into proteins and peptides, the
structural and functional molecules of the cell.

Translation is a complicated process and involves three major steps: initiation, elongation, and termination.

Translation takes place on the rough endoplasmic reticulum (ER) in the cytoplasm. Also called the rough
ER, it is the site of the ribosomes, which are organelles composed of proteins and ribosomal RNA (rRNA).
 ISBT 001 ABO BLOOD GROUP SYSTEM
Most important of all blood group
Most common cause of HTR and HDN
ABO Forward Typing / Front/ Direct Typing
Specimen: Patient RBC > Trypan blue > Acriflavine dye

Reagent: uses commercial antisera, Anti A (Blue color), Anti- B (Yellow color)
Use: Detection of ABO Antigens
Blood Type Antigen Reaction With Anti-A Reaction with Anti-B
A 4-1 0
B 0 4-1
AB 4-1 4-1
O O O

ABO Reverse Typing / Back / Indirect Typing /Serum Typing

Specimen: Serum/plasma
Reagent: A cells, B cells
Use: Detection of ABO antibodies

Blood Type Antigen Reaction with A cells Reaction with B cells

A O 3-1
B 3-1 O

AB O O
O 4-1 4-1

inverse reciprocal Relationship between forward and reverse typing

30 seconds Centrifugation time

Type 0 negative Universal donor of whole blood or packed RBC

Type AB positive Universal acceptor/recipient of whole blood or packed RBC

Type AB Universal donor of plasma products

Type AB Universal acceptor/recipient of plasma products

GRADING OF AGGLUTINATION
Grade Description
Cells Supernate
0 No agglutinates Dark, turbid,
homogenous
W+ Many tiny agglutinates Dark, turbid
Many free cells
May not be visible without
microscope
1+ Many small agglutinates Turbid
(25%) Many free cells
2+ Many medium-sized agglutinates Clear
(50%) Moderate number of free cells
3+ Several large agglutinates Clear
(75%) Few free cells
4+ One large, solid agglutinate Clear
(100%) No free cells
 Mixed-field agglutination may look like small to large agglutinates with unagglutinated cells. Mixed-

CAUSES: receiving non-ABO-type specific RBCs, ABO subgroups (A3), and bone marrow or hematopoietic stem cell transplantation

ABO Genotypes and Phenotypes

Phenotype Genotype
A1 A1A1 , A1O , A1A2
A2 A2O , A2A2
A1B A1B
A2B A2B
B BB, BO
O OO

ABO MATING
AxA AA x AA A (AA)
Example: both parents are blood type A AA x AO A (AA or AO)
A 0 A 0
A A
A AH AH AAA AO AAA AO
AO x AO A (AA or AO) or O (OO)
AA A HA HO O AO 00
A At

Possible outcome: Blood type A and O

BxB BB x BB B (BB)
Example: both parents are blood type B BB x BO B (BB or BO)
B O B O
B B
BB BB BBB BO BBB BO
BO x BO B (BB or BO) or O (OO)
B

p BB BB BBB BO 0 13000

Possible outcome: Blood type B and O

AB x AB AB x AB AB (AB)
Example: both parents are blood type AB A(AA)
A B
AB
B (BB)
A AA

B AB BB

Possible outcome: Blood type A, B, AB

OxO OO x OO O (OO)
Example: Both parents are blood type O
Possible outcome: Blood type O
AxB AA x BB AB
Example: Parent A Blood type A AO x BB AB , B(BO)
Parent B- Blood type B AA x BO AB , A(AO)
B O
B °
B B B B
A AB AO
A AB AO AO x BO AB, A(AO) , B(BO) , O(OO)
A AB AB
A AB AB

O BO 00
A AB AO
AB
O BO BO
A AB

Possible outcome: A, B , AB ,O

Formation of THE ABO Blood Group Antigen

Inheritance
ABO Genes Chromosome #9
A and B genes Dominant
O gene Amorph /silent
Recessive
-No antigen is produced
 GENE GLYCOSYLTRANSFERASE IMMUNODOMINANT SUGAR Antigen
H L-fucosyltransferase L-fucose H
A N-acetylgalactosyltransferase N-acetyl-D-galactosamine A
B D-Galactosyltransferase D-galactose B
AB N-acetylgalactosyltransferase N-acetyl-D-Galactosamine AB
D-Galactosyltransferase D-galactose
O -- -- Unchanged

D-
galactose

D- galactose
fucose
N acetyl D
-
-
-

glucosamine

D- galactose

Glucose

REMINDERS
Genes does not actually code for the production of antigens but rather produce specific glycosyltransferase
The H antigen is actually the precursor structure on which A and B antigens are made
The H and Se genes are not part of the ABO system; however, their inheritance does influence A and B antigen expression
Without fucose no other immunodominant sugar will be attached

0 > Az > B > AZB At > AIB

Amount of H (From greatest to least): __________________________
>

Frequencies: O>A>B>AB

COMPARISON OF TYPE 1 AND TYPE 2 CHAINS

Type 1 TYPE 2
Linkage Beta 1,3 Beta 1,4
Origin Plasma Erythrocyte precursors
Controlling genes H, A, B, Se, Lewis H, A, B

I. A-SUBGROUPS
- A1 and A2 describe by Von Dungern
ANTIGEN PRESENT Anti-A ANTI-A1 Lectin
Anti -
A plus Anti -
Ai

A1 - 80% of the A population * +

A2 20% of the A population A 1-

Note
1-8% of A2 produces anti A1, 22 -35% of A2B produces Anti-A1
The very potent gene A1 creates between 810,000 and 1,170,000 antigen sites on the adult A1 RBC, whereas inheriting
an A2 gene results in production of only 240,000 to 290,000 antigen sites on the adult A2 RBC
Source of Anti-A1 lectin: From Plant Dolichos biflorus
There are four Forms of H antigen (H1, H2, H3, and H4)
H1 AND H2 = Unbranched straight chain
H3 AND H4 = Complex branched chain
 II. OTHER SUBGROUPS OF A
PHENOTYPES DESCRIPTION

a A3 Mixed field agglutination with anti-A and or anti-AB

b Ax Weak agglutination with anti-AB only

Aend <10% red cell shows very weak mf agglutination
Am No agglutination with anti-A and anti-AB, secretors demonstrate quantities of A substance in
c
saliva
Ay No agglutination with Anti-A and anti-AB, secretors contain small amount of A substance in
saliva
Ael No agglutination with anti-A and Anti-AB, secretors contain only H substance and no
d
substance in saliva

III. B Subgroups
PHENOTYPES DESCRIPTION
B3 a Mixed field agglutination with anti-B and or anti-AB
Bx b Agglutination with Anti-AB (wk /0 with anti-B)
Bm c No agglutination with anti-B and anti-AB, secretors demonstrate quantities of B substances
in saliva
Bel No agglutination with anti-B and anti-AB, secretors contain only H substance and no B
d
substance in saliva

Bandeiraea simplicifolia Source of anti-B lectin. Used to agglutinates B cells that have been enzyme pretreated
ANTI-AB
1. used to identify weak subgroups of A and B
2. Has a higher titer compared to Anti-A or Anti-B alone

ABO HISTOBLOOD GROUP AND SOLUBLE ANTIGENS

ABO antigens are present in all tissue and organs of the body
May be expressed in secretions depending on secretor status

THE SECRETOR GENE (Se) Detection Of Secretor Status

SeSe or Sese (80%) secretor Specimen: saliva
sese (20%) non secretor Principle: Hemagglutination inhibition

Body fluids associated with ABH Substances

a, tears, urine, digestive juices, bile, milk, amniotic fluid, Pathological fluids: peritoneal, pleural, pericardial, and ovarian
cyst STUD BAM

ABH ANTIGENS ON RBC ABH ANTIGENS ON SECRETION

RBC antigens can be-
glycolipids, -
glycoproteins, or Secreted substances are r
glycoproteins.
glycosphingolipids
r Secreted substances are primarily synthesized on_
type
RBC antigens are synthesized only on/
type 2 precursor 1 precursor chains.
chains. Type 1 chain refers to a beta-1 to 3 linkage in which
Type 2 chain refers to a beta 1 to 4 linkage in which the number one carbon of the galactose is attached to
the number one carbon of the galactose is attached to the number three carbon of the N-acetylglucosamine
the number four carbon of the N-acetylglucosamine sugar of the precursor substance.
sugar of the precursor substance.
 ABO GROUP SECRETORS ABH SUBSTANCES IN SALIVA
A B H
A
↑↑ None ↑
B None ↑↑ ↑
AB TT TT ↑
O None None ↑↑
Non secretor None None None

ABO ANTIBODIES
ABO antibodies may be IgG, IgM (Predominant), and IgA (Henry ).
They are naturally occurring antibodies because they are produced without any exposure to RBCs.
It has been postulated that bacteria, pollen particles, and other substances present in nature are chemically similar to A and
B antigens. Bacteria are widespread in the environment, which constantly exposes individuals to A-like and B-like antigens.
This exposure serves as a source of stimulation of anti-A and anti-B.
ABO antibody production is initiated at birth, but titers are generally too low for detection until infants are 3 to 6 months
old.
In people whose blood type are B and A may have Anti-A and anti B correspondingly, predominantly are IgM
In patient with blood type O, Anti-A, Anti-B, Anti-AB may be seen and predominantly are IgG

BOMBAY INDIVIDUAL (Oh)

Do not inherit H gene
Inherited as an autosomal recessive trait. The underlying molecular defect is most often a mutation in the gene FUT1
(H gene), producing a silenced gene incapable of coding for the enzyme, a-2-L-fucosyltransferase (H transferase).
H null or hh genotype
Lacks: A, B, H antigen
Presence of: Anti-A, Anti-B, Anti-H
Anti-A Anti-B A cells B cells Anti-H lectin
Blood type O - - + +
Oh - - + +

÷
Source of Anti- H lectin: Ulex europeus

ACQUIRED ANTIGENS
Acquired A antigen Reported in persons of type O or B in association with severe infections caused by Proteus
mirabilis
Tn-activated erythrocytes c
Acquired B antigen Associated with conditions: EPIC
E.Coli 086 infection, Proteus vulgaris, Intestinal obstruction, Carcinoma of colon
Occurs only on Group A individual, and maybe mistyped as Group AB in forward grouping
Anti-A Anti-B A cells B Cells
Group AB + +
Acquired B + Wk+ 4-1

TECHNICAL ERRORS
- Incorrect or inadequate identification of blood specimens, test, tubes, or slides
- Cell suspension either too heavy or too light
- Clerical errors or incorrect recording of results
- A mix-up in samples
- Missed observation of hemolysis
- Failure to add reagents and/or sample
- Uncalibrated centrifuge
- Overcentrifugation or undercentrifugation
- Contaminated reagents
- Warming during centrifugation
REMEMBER
Serum and antiserum should always be added first, followed by the patient or reagent RBCs to avoid both reagent contamination
and potential omission of either patient sample or reagent.

DISCREPANCIES

Group I Causes 6
Weakly reacting or Newborns
missing antibodies Elderly patients
Patients with leukemia demonstrating hypogammaglobulinemia
-Most common Patient with immunodeficiency
-discrepancies on Patient with bone marrow transplantations
reverse typing Patient taking immunosuppressive drugs

REMEDY
Obtaining patient clinical history may immediately resolve this type of discrepancy.
An auto control and O cell control must always be tested concurrently with the reverse typing
when trying to solve discrepancy
incubating the patient serum with reagent A1 and B cells at room temperature for approximately
15 to 30 minutes or by adding one or two drops more plasma or serum to the test.
Group II Causes

:
Weakly reacting or Subgroups of A and B
missing antigens Leukemias
disease
-least common Excess BGSS
-discrepancies on Acquired B phenomenon
forward typing Antibodies to low incidence antigens
Chimerism

REMEDY
The agglutination of weakly reactive antigens with the reagent antisera can be enhanced by
incubating the test mixture at room temperature for up to 30 minutes, which will increase the
association of the antibody with the RBC antigen.
RBCs can also be pretreated with enzymes and retested with reagent antisera.
For acquired B
a.Check for secretor status only A substance is secreted in acquired B
b.Treating the RBC with acetic anhydride reacetylates the surface molecules, then markedly
decreases the reactivity of the cells tested with anti-B (note: normal B cell reactivity is not
affected by acetic anhydride)
Acquired B antigen is also not agglutinated when reacted with anti-B that has a pH
greater than 8.5 or less than 6
In cases of Antibodies to low incidence antigens, repeating the forward type, using antisera
with a different lot number will resolve the problem
Group III Causes
Protein or plasma in cord blood samples
abnormalities High level of globulin: MM, WM, plasma cell dyscracias
High level of fibrinogen
 PUP
-discrepancies on Plasma expanders such as Dextran and polyvinylpyrrolidone
both forward and Rouleaux
reverse
REMEDY

For rouleaux =
Saline replacement technique - serum is removed and replaced by an equal volume of saline
a. Rouleaux = will free the cells in the case of rouleaux formation
b. True Agglutination = clumping will still remain after the addition of saline
Group IV Causes
6
Miscellaneous Polyagglutination
problems Cold reactive autoantibodies
Unexpected ABO isoagglutinins (E.g., Anti-A1 from A2, Anti-H from A1B individual)
-discrepancies on Unexpected non-ABO alloantibodies
both forward and Circulating RBCs of more than one ABO group due to RBC transfusion or marrow/stem cell transplant
reverse

REMEDY
Cold auto antibodies = perform DAT test, then
a.
three times and retyped. If this is not successful in resolvin
RBCs can be treated with 0.01 M dithiothreitol (DTT) to disperse IgM-related agglutination. As
for the serum, the reagent RBCs and serum can be warmed to 37°C for 10 to 15 minutes,
mixed, tested, and read at 37°C
b. a cold autoabsorption (patient cells with patient serum) could be performed to remove the cold
autoantibody from the serum

Unexpected ABO isoagglutinins

a. reverse typing can be repeated using at least three examples of A1, A2, B cells; O cells;
and an
b. Dolichos biflorus to confirm the presence of subgroup
Anti -
Al lectin

Unexpected alloantibodies
a. rum
b. for antibody against Acriflavine (yellow dye used in anti-B reagents) -Washing the

Chimerism presence of two cells population in an individual. True chimerism occurs only in twins and is rarely
found
BGSS Blood group specific soluble (BGSS) substances present in the plasma, which sometimes occurs with certain
diseases, such as carcinoma of the stomach and pancreas. Excess amounts of BGSS substances will neutralize
the reagent anti-A or anti-B, leaving no unbound antibody to react with the patient cells and yields a false-
negative or weak reaction in the forward grouping
REMEDY: Washing the
This substance is a viscous mucopolysaccharide material present on cord blood cells that may cause the red
jelly
CIS AB inheritance of both AB genes from one parent carried on one chromosome and an O gene inherited from the
other parent that results in the offspring inheriting three ABO genes instead of two.
 ABO DISCREPANCIES BETWEEN FORWARD AND REVERSE TYPING (Harmening)

FORWARD REVERSE
GROUPING GROUPING
Anti Anti-B A1 B O Auto Possible Cause RESOLUTION
-A Cells cells cells Control
-Group O newborn or Check age and diagnosis

0 0 0 0 0 0 elderly patient of patient and immunoglobulin levels; if

possible incubate at RT for 30 min or at
-Patient may have
4°C for 15 min; include group O and
agammaglobulinemia/Hypo
autologous cells at 4°C
gammaglobulinemia
-patient may be taking
immunosuppressive drugs
4+ 0 1+ 4+ 0 0 -Subgroup of A: probable React patient cells with anti-A1 lectin,

A2 with Anti-A1 test serum against additional

A1, A2, and O cells; run autocontrol

4+ 4+ 2+ 2+ 2+ 2+ -(1) Rouleaux (1) Wash RBCs; use saline dilution or

-(2) Cold autoantibody saline replacement technique

(2) Perform cold panel and autoabsorb
-Cold autoantibody with
or rabbit erythrocyte stroma (RESt)
underlying cold or RT
absorb or reverse type at 37°C
reacting alloantibody
(3) Perform cold panel autoabsorb or
RESt, and run panel on absorbed
serum; select reverse cells lacking
antigen for identified alloantibody;
repeat reverse group on absorbed
serum to determine true ABO group or
at 37°C
4+ 4+ 1+ 0 0 0 -Subgroup of AB; probable Use anti-A1 lectin, test serum against

A2B with Anti-A1 additional A1, A2, and O cells

4+ 0 0 4+ 3+ 0 -A1 with potent anti-H Confirm A1 group with anti-A1 lectin;

test additional A2, O, and A1 cells and
an Oh if available
0 0 4+ 4+ 4+ 0 -Oh Bombay phenotype Test with anti-H lectin; test Oh cells if
available; send to reference laboratory
for confirmation
0 0 2+ 4+ 0 0 -Subgroup of A; probable Perform saliva studies or

Ax with Anti-A1 absorption/elution

4+ 2+ 0 4+ 0 0 -Group A with an acquired B Check history of patient for lower

antigen gastrointestinal problem or septicemia;

acidify anti-B typing reagent to pH 6.0
by adding 1 or 2 drops of 1 N HCl to
1 mL of anti-B antisera, and measure
with a pH meter (this acidified anti-B
antisera would agglutinate only true B
antigens not acquired B antigens), test
serum against autologous cells
4+ 4+ 2+ 0 2+ 0 Group AB with alloantibody Perform antibody screen and panel,
identify room temperature antibody,
repeat serum type with antigen
negative reagent cells or perform
serum type at 37°C
0 4+ 4+ 1+ 1+ 1+ Group B with cold Enzyme-treat RBCs and perform

autoantibody autoabsorption at 4°C or perform

prewarmed testing

ISBT OO4 Rh BLOOD GROUP SYSTEM

Second most important blood group system in terms of transfusion
The most important blood group system associated with HDN
Landsteiner and Wiener described an antibody made by guinea pigs and rabbits when they were transfused with rhesus
macaque monkey RBCs. The antibody agglutinated 85% of human RBCs and was named anti-Rh after the rhesus monkey.
RH genes are inherited as codominant alleles. The product of RH genes are nonglycosylated proteins. This means no
carbohydrates are attached to the protein. Rh antigens reside on transmembrane proteins and are an integral part of
the RBC membrane
Rh gene: RHD and RHCE are two closely linked genes located on chromosome 1 that control expression of Rh proteins
Rh function:rmaintain structural integrity of RBC , andr
Carbon dioxide transporters

TERMINOLOGIES OF Rh System

1.Fisher-Race (DCE)
-based on the theory that antigen of the systems were produced by three closely linked set of alleles, each gene was responsible for
producing a product
Fisher race antigens: D, C ,E ,c, e

Rh null = -
- -

f- -

Order of Immunogenicity: D>c>E>C>e

2. Wiener: Rh-Hr terminology

- Wiener believed there was one gene responsible for defining Rh that produced an agglutinogen containing a series of blood factors
R0 D
r d
C
E
Z or y CE
c
e

3. Rosenfield (Alpha numeric)

-Number is assigned to each antigen of the Rh system in order of its discover
-the nomenclature has no genetic basis
D = Rh1 C =Rh2 E= Rh3 c= Rh4 e= Rh5

4. International Society of Blood Transfusion (ISBT) : Numeric terminology

-Adopted a six-digit number for each blood group specificity
-First three number represents the system and the remaining three represents the antigenic specificity
-Establish a uniform nomenclature that is both eye and machine readable
 COMPARISON AND EQUIVALENT OF THE DIFFERENT NOMENCLATURE
ISBT FISHER-RACE WIENER ROSENFIELD
004 001 D Rh0 Rh1
004 002 C RH2
004 003 E RH3
004 004 c RH4
004 005 e RH5

FISHER- WIENER ROSENFIELD

RACE
Gene Gene Agglutinogen Short Blood Factors Phenotype
hand
Dce -Rh0 Rh0 /Ro -
RH: 1, -2, -3, 4, 5
DCe Rh1 Rh1 R1 RH: 1, 2, -3, -4, 5
DcE Rh2 Rh2 R2 RH: 1, -2, 3,4, -5
DCE RhZ Rhz RZ RH:1,2,3, -4, -5
dce rh rh r RH: -1-2-3, 4,5
dCe r1 RH: -1,2, -3, -4, 5
dcE r2 RH: -1, -2,3,4, -5
dCE rhy rhy ry RH: -1,2,3, -4, -5

Number of D antigen sites in various phenotypes Note!

Rh Phenotype Number of D antigen sites
7 R 1r Dce/doe 9,900-14,600
6 R 0r Dce/dce 12,000-20,000
5 R 2r DCE /dce 14,000-16,600
4 R1R1 Dce/ Dce 14,500-19,300
2 R1R2 Dce /Dct 23,000-31,000
3
R2R2 Dct/DCE 15,800-33,300
D-- 110,000-202,000

:
WEAKENED D ANTIGEN EXPRESSION
Genetic weak D D antigens expressed appear to be complete, but few in number
C Trans to D Positional effect or gene interaction effect
Allele carrying D is trans (opposite haplotype) to the allele carrying C
D mosaic or Partial One or more parts of the D antigen is missing. The expression can be weakened is when one or more D
D epitopes within the entire D protein is either missing or altered
Patient: Rh (+) with anti-D to the missing part
Once anti-D is identified, Rh-negative blood should be used for transfusion
Phenotype whose red blood cells possesses an extremely low number of D antigen that most reagent anti-
D are unable to detect
-due to mutation of RHD gene
-common in individuals of asian ethnicity
Test for Weak D
Antigen (Du) Note: In case of Weak D (+), Individually it must be reported as Rh(+)
But during transfusion/donation

Patient: Typed as Rh (-)

Donor: Typed as Rh (+)
Rh ANTIBODIES
They are immune antibodies
Do not bind complement
Causes extravascular hemolysis
Number one cause of HDN (previously but now replaced by ABO)
Causes delayed HTR
Rh
foreign RBCs, through either transfusion or pregnancy
HDFN caused by Rh antibodies is often severe because the Rh antigens are well developed on
fetal cells, and Rh antibodies are primarily IgG, which readily cross the placenta.
Anti-LW Originally identified as anti-Rh in early experiment involving rabbits immunized with rhesus
monkey blood
Anti LW is different from Anti-D
Anti LW agglutinates Rh+ and Rh- cells except Rh null (--/--)
Also, anti-LW more frequently appears as an autoantibody, which does not present clinical
problems
techniques can help distinguish between Anti-D and LW :
a. Treat the reagent panel cells with 0.2 m dithiothreitol (DTT) and test the patient serum against
the treated cells. LW antigens are denatured with DTT treatment while D antigens are unaffected
b. Test the patient serum against Rh-positive and Rh-negative cord blood cells. Anti-D will react
only with the Rh-positive cord cells whereas the LW antibodies will react with all cord cells
tested, regardless of Rh type

RH null syndrome Individuals lack all RH antigens on their RBCs

They are negative for the high-prevalence antigen✓ LW and for /FY5, an antigen in the Duffy
blood group system. S, s, and U antigens found on glycophorin B may also be depressed

:-.
When transfusion of individuals with Rhnull syndrome is necessary, only Rhnull blood can be given
The Rhnull phenotype is usually written in Fisher-Race as / , in Wiener as Rhnull, and in
Rosenfield nomenclature as (RH: 1, 2, 3, 4, 5).
Individuals with Rhnull syndrome demonstrate a mild compensated hemolytic anemia,
.reticulocytosis, stomatocytosis, a slight-to-moderate decrease in hemoglobin and hematocrit
levels, .
an increase in hemoglobin F, a decrease in serum haptoglobin, and .
possibly an elevated
bilirubin level.

UNUSUAL PHENOTYPES Cw , f(ce), rhi(Ce) , G , Hr0, Rh:23, Rh:30, Rh:40 , e variants, V, Vs, Rh43

The f antigen is expressed on the RBC when both c and e are present on the same
haplotype. It has been called a compound antigen
G is an antigen present on most D-positive and all C-positive RBCs.
For transfusion purposes, it is not necessary to discriminate anti-D and anti-C from anti-G,
as the patient would receive D-negative and C-negative blood regardless if the antibody is
anti-D, anti-C, or anti-G.
Rh43, also known as the Crawford antigen, is a low-prevalence antigen on a variant Rhce
protein

Saline reactive reagents
High protein Anti-D
reagents
Chemically modified Rh
typing reagents
Rh monoclonal
antibodies reagents
RH TYPING REAGENTS
Saline reactive reagents, which contain IgM immunoglobulin, were the first typing reagents available
to test for the D antigen
Saline anti-D has the advantage of being low-protein based and can be used to test cells that are
already coated with IgG antibody, as in patients who have autoantibodies binding to their RBCs
The primary disadvantages of saline typing reagents are their limited availability, cost of production,
and lengthy incubation time. Because saline anti-D is an IgM immunoglobulin, it cannot be used for
weak D typing.
High-protein anti-D reagents (poly-specific reagent) were developed that consisted primarily of IgG
anti-D. Human plasma containing high-titer D-specific antibody was used as the raw material.
Potentiators of bovine albumin and macromolecular additives, such as dextran, were added to the
source material to optimize reactivity
Advantages: reduced incubation time, ability to perform weak D and slide typing with the same
reagent
Chemically modified Rh typing reagents alter the IgG anti-D molecule by breaking the disulfide
Can be used for both slide and tube testing and do not require a separate, manufactured Rh
control as long as the samples type as A, B, or O.
When samples test AB Rh-positive or when the Rh test is performed by itself, a separate
saline control or 6% to 8% albumin control must be used to ensure the observed
reactions are true agglutination and not a result of spontaneous agglutination.
these reagents are not human-derived, they lack all potential for transmitting infectious disease.
These reagents are derived from single clones of antibody-producing cells
It is prepared by hybridoma technology

FALSE REACTIONS WITH RH TYPING REAGENTS

FALSE POSITIVE FALSE NEGATIVE
Cold agglutinins Immunoglobulin coated cells (in vivo)
Cell suspension too heavy Saline suspended cells
Test incubated too long or drying Omission of reagent
Rouleaux Resuspension too vigorous
Fibrin interference Incorrect reagent selected

i
Contaminating low incidence antibody in reagent Variant antigen
Polyagglutination Reagent deterioration
Bacterial contamination on reagent vial Centrifugation too short
Incorrect
' reagent selected Rpm too low
Over centrifugation Incorrect reagent selected
RPM too high

ISBT 007 LEWIS BLOOD GROUP SYSTEM

Produced by tissue cells (found in secretions)
Not well developed at birth
Lewis gene (Le, FUT3) codes for the production of fucosyltransferase enzyme
Unique because Lewis antigens are not intrinsic to RBCs but are type 1 glycosphingolipids that are passively absorbed on
_

the RBC membrane plasma

LEWIS ANTIGENS
Cord blood and red cells from newborn /infants phenotype as Le(a-b-)
Decrease in expression on red cells from many pregnant women
Lea and Leb are NOT antithetical
Le(a+b ) RBCs are from ABH nonsecretors and Le(a b+) RBCs are from ABH secretors. Individuals with the Le(a b )
RBC phenotype are either secretors or nonsecretors.
The Le(a b ) phenotype is found more frequently among Africans.
The Le(a+b+) phenotype is rare among whites and Africans but is more frequent among Asians
In children who inherit both Le and Se genes, the transformation can be followed from the Le(a b ) phenotype at birth to
Le(a+b ) after 10 days to Le(a+b+) and finally to Le(a b+), the true Lewis phenotype, after about 6 years.
In contrast, children who inherit Le and sese genes phenotype as Le(a b ) at birth and transform to Le(a+b ) after 10
days; the Le(a+b ) phenotype persists throughout life
Lack of expression of Lewis antigens (Lea and Leb) has been demonstrated on the RBCs of patients with cancer, alcoholic
cirrhosis, and viral and parasitic infections

LEWIS ANTIBODIES
Anti-Lea (most common), and Anti-Leb
Naturally occurring antibody
IgM in nature
Easily neutralized by plasma
Can bind complement and rarely can cause HTR if anti-Lea
The antibodies do not cause hemolytic disease of the fetus and newborn (HDFN).
Most Lewis antibodies agglutinate saline suspended RBCs, but these agglutinates are often fragile and can be easily
dispersed if the cell button is not gently resuspended after centrifugation. Lewis antibodies can bind complement, and
when fresh serum is tested, anti-Lea may cause in vitro hemolysis of incompatible RBCs, though this is more often seen
with enzyme-treated RBCs than with untreated RBCs

Note about Anti-Leb

Anti-Leb is not as common or generally as strong as anti-Lea.
It is usually an IgM agglutinin and can bind complement.
Anti- Leb is infrequently made by Le(a+b ) individuals and can be
classified into two categories: anti-LebH and anti-LebL.
Anti-LebH reacts best when both the Leb and the H antigens are present
on the RBC, such as group O and A2 cells. Anti-LebH represents an antibody to a compound antigen Anti-LebL recognizes
any Leb antigen regardless of the ABO type.

GENOTYPE SUBSTANCE(SECRETION) REDCELL PHENOTYPE ANTIBODY PRESENT

ABH lele sese none ABH (Le a-b-) Anti-Lea, Anti-Leb
ABH lele SeSe ABH ABH (Le a-b-) Anti-Lea, Anti-Leb
ABH LeLe sese Lea non secretor
-
ABH (Le a+b-) Anti-Leb
ABH LeLe SeSe ABH, Lea, Leb ABH (Le-b+) None
 Pi , P.PK P P, Pk

RBCs platelets plasma -

glycosphingolipids
cells
lymphocytes epithelial hyadatid cyst
-

glycoproteins
granulocytes fibroblasts
monocytes

* ISBT 003 P: P1PK BLOOD GROUP SYSTEM

The P blood group was introduced in 1927 by Landsteiner and Levine.
In their search for new antigens, they injected rabbits with human RBCs and produced an antibody, initially
called anti-P, that divided human RBCs into two groups: P+ and P .
The P blood group antigens, like the ABH antigens, are synthesized by sequential action of glycosyltransferases, which add
sugars to precursor substances. The precursor of P1 can also be-
glycosylated to type 2H chains, which carry ABH antigens.
P1, P, or Pk may be found on RBCs, lymphocytes, granulocytes, and monocytes; P can be found on platelets, epithelial
cells, and fibroblasts. P and Pk have also been found in plasma as glycosphingolipids and as glycoproteins in hydatid cyst
fluid. The RBC antigens of the P blood group exist as glycosphingolipids.
The P blood group antigens are resistant to treatment with ficin and papain, DTT, chloroquine, and glycine-acid EDTA.
Reactivity of the antibodies can be greatly enhanced by testing with enzyme-treated RBCs

P1 Antigen Poorly expressed at birth and may take up to 7 years to be fully expressed
It deteriorates rapidly on storage - older RBCs used as control for typing reagents can lead to false
negative result
P1 substance has been identified in Hydatid cyst fluid, Lumbricoides terrestris (common earth
worm) and Ascaris suum
P1 like antigen has been found on:
- plasma, -
droppings of pigeon and turtledoves,
- eggwhite of
turtle doves
Blacks have a stronger expression of P1 than whites
Pk The Pk antigen is a marker of apoptosis in germinal center B cells, Burkitt lymphoma, and
lymphoblastic leukemia
Anti-P Naturally occurring alloantibody in sera of all Pk individuals
It is typically weak, cold, reactive saline agglutinin
testing
Auto Anti-P specificity is found as an IgG autoantibody in patient with Paroxysmal
cold hemoglobinuria
Anti-P1 Common, naturally occurring IgM antibody in the sera of P2 individuals
Anti-P1 is usually IgM; IgG forms are rare. HDFN is not associated with anti-P1
Strong anti-P1 was observed in individuals infected with Echinoccocus granulosus
Strong antibodies to P1 have also been found in patients with fascioliasis (bovine liver fluke
disease) and in bird handlers
Anti- PP1 Pk Originally called as Anti- Tja, was first described in the serum of Mrs. Jay, a p individual with
adenocarcinoma of the stomach. T in the Tja refers to tumor
Produced by p individuals early in life without RBC sensitization and reacts with all RBCs except
those of the p phenotype
Reacts over a wide thermal range (both IgM and IgG)
Associated with spontaneous abortions in early pregnancy
Has potential to cause severe HTRs and HDFN.
Anti-Pk Isolated from some examples of Anti- PP1Pk by selective adsorption with P1 Cells
Associated with -
billiary cirrhosis and-
autoimmune hemolytic anemia

Phenotype Detectable Antigens Possible antibodies

P1 P P1 Pk None
P2 P, Pk Anti- P1
p (P null) None Anti- PP1Pk
P1 K P1, Pk Anti- P
P2 K Pk Anti P, anti- P1
Disease association
UPEC
The P system antigens also serve as receptors for P-fimbriated uropathogenic E. coli a cause of urinary tract infections. The Pk
antigen is a receptor for Shiga toxins, which cause Shigella dysentery and E. coli associated hemolytic uremic syndrome. In
addition, P is the receptor of human parvovirus B19. Pk provides some protection against HIV infection of peripheral blood
mononuclear cells

ISBT 002 MNSs BLOOD GROUP SYSTEM

Forty-nine antigens have been included in the MNS system, making it almost equal to Rh in size and complexity.

MN antigens Found on glycophorin A and they are antithetical antigens

MN antigens differ in their amino acid residue at positions 1 and 5
M has a serine and glycine Sg
N has leucine and glutamic acid 1g
Well developed at birth
They are easily destroyed by enzymes
S and s antigens Found on Glycophorin B and they are antithetical antigens
S and s are differentiated by the amino acid at position 29 on GPB.
S has methionine; s has threonine
Well developed at birth
Less easily degraded by enzymes
Anti- M May be IgG and IgM
Most examples are cold reactive saline agglutinins
Usually do not bind complement
Do not react with enzyme treated cell
pH-dependent, reacting best at pH 6.5
Others react with red cell exposed to glucose solution
Rarely cause HDN
Shows dosage effect
Anti-N Cold reactive IgM saline agglutinin
Does not bind complement
Implicated with rare case of HDN
Anti-Nf - seen in renal patient, who are dialyzed on equipment sterilized with formaldehyde
Anti- S and Anti-s antiglobulin test phase
Implicated with severe hemolytic transfusion reaction, hemoglobinuria, and HDN
Anti- U U- universal
Rare but can be formed in S-s- individuals, found on black people
Can also cause HDN
Enhanced by Enzyme treatment
En(a-) phenotype They are M-N-
Confer resistance to Plasmodium falciparum merozoite
Mk Phenotype The RBCs of these individuals typed M N S s U En(a )Wr(a b ), but they had a normal
hematologic picture.
It is the null phenotype in the MNS blood group system

Disease Associations
Glycophorin A may serve as the receptor by which certain pyelonephritogenic strains of E. coli gain entry to the urinary tract
The malaria parasite Plasmodium falciparum appears to use alternative receptors, including GPA and GPB
 I (027) BLOOD GROUP SYSTEM
I I antigen for
I and i Antigens Both I and i are high-prevalence antigens, but they are expressed in a reciprocal relationship
that is developmentally regulated.
At birth, infant red cells are rich in i ; I is almost undetectable
During the first 18 months of life, the quantity of i slowly decreases as I increases until adult
proportions are reached; adult red cells are rich in I and have only trace amount of i antigen
There is no true I or i phenotype.
I and i are no antithetical antigens. Rather, they are branched and linear carbohydrate
structures, respectively, that are formed by the action of glycosyl transferases
I and i antigens are found on the membranes of leukocytes and platelets in addition to RBCs
I and i have also been found in the plasma and serum of adults and newborns and in saliva,
human milk, amniotic fluid, urine, and ovarian cyst fluid
Treatment of RBCs with ficin and papain enhances reactivity of the I and i antigens with their
respective antibodies. They are resistant to treatment with DTT and glycine-acid EDTA.
Rare i adult or I Negative Phenotype Individuals who do not change their i status after birth
Anti-I Common antibody that can be benign or pathologic
Demonstrates strong reactions with adult cells and weak reactions with cord cells
Not associated with HDN because the antigen is poorly expressed on infant red cells
Incubating tests in the cold enhances anti-I reactivity and helps confirm its identity; albumin
and enzyme methods also enhance anti-I reactivity

Benign Anti-I Found in the serum of many normal healthy individuals

Not associated with an vivo red cell destruction
Weak, naturally occurring, saline-reactive IgM agglutination
Usually reacts only at

Pathologic Anti-I or Potent IgM agglutinins with higher titers and broader thermal range of activity, reacting up to
Autoanti-I 30° or 32°C
phenomenon) or
intravascular hemolysis
Production of autoanti-I may be stimulated by microorganisms carrying I-like antigen on their
surface
Patients with Mycoplasma pnuemoniae often develop strong cold agglutinins with I
specificity as a cross-reactive response to Mycoplasma antigen
Listeria monocytogenes organism from a patient with cold autoimmune hemolytic anemia has
been reported to absorb anti-I and stimulate its production in rabbits
It is associated with cold agglutinin disease

Anti-i Autoanti-i is not seen as a common antibody in healthy individuals.

Reacts best with saline-suspended cells at 4°
Most autoanti-i are IgM
IgG anti-i has also been described and has been associated with HDN
Potent examples are associated with: -
INFECTIOUS MONONUCLEOSIS, alcoholic
- cirrhosis,
myeloid
- leukemia and-
reticuloses
Anti-IT Naturally occurring antibody among Melanesians, coastal residents of Papua new Guinea,
Yayoma, and Venezuela
T= transition Examples of IgM and IgG anti-IT reacting preferentially at 37°C have also been found in
patients with warm autoimmune hemolytic anemia, with a special association with
lymphoma
I and i antigens reactivity
Phenotype Anti-I Anti-i Anti-IT
Adult I RBC Strong -/Weak -/Weak
Cord RBC -/Weak Strong Strong
Adult i RBC -/Weak Strong -/Weakest

Disease Associations
Anti-I associated with cold agglutinin disease and M. pneumoniae, and anti-i is associated with infectious mononucleosis.
Conditions associated with increased i antigen on RBCs include those with shortened marrow maturation time or
dyserythropoiesis: acute leukemia, hypoplastic anemia, megaloblastic anemia, sideroblastic anemia, thalassemia, sickle cell
disease, paroxysmal nocturnal hemoglobinuria (PNH), and chronic hemolytic anemia
Chronic dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with a positive acidified serum test
(HEMPAS) is associated with much greater i activity
In Asians, the i adult phenotype has been associated with congenital cataracts

ISBT (008) DUFFY BLOOD GROUP SYSTEM

The Duffy blood group system was named for Mr. Duffy, a multiply transfused hemophiliac who in 1950 was found to
have the first described example of anti-Fya.
Fya and Fyb Identified on fetal red cells as early as 6 weeks gestational age and are well developed at birth
The antigens have not been found on platelets, lymphocytes, monocytes, or granulocytes, but
they have been identified in other body tissues, including brain, colon, endothelium, lung,
spleen, thyroid, thymus, and kidney cells
Destroyed by common proteolytic enzymes such as ficin, papain, bromelin, and chymotrypsin,
and by ZZAP (which contains either papain or ficin in addition to DTT)
They are not affected by DTT alone, AET, or glycine-acid EDTA treatment
Fyx An inherited weak form of Fyb that reacts with some examples of anti-Fyb
Fy6 Is an important for invasion for P.vivax and P.knowlesi
Anti-Fya and anti-Fyb Usually, IgG and react best at the antiglobulin phase
Activity is enhanced in a low ionic strength solution
Do not react with enzyme treated cells
Anti-Fya and anti-Fyb have been associated with acute and delayed HTRs
Associated with hemolytic transfusion reactions, not often severe
Anti-Fya is the most common antibody found in Duffy BGS
Some examples of anti-Fya and anti-Fyb show dosage effect
Anti- Fya is more common than anti-Fyb
Fy3 antigen and Anti Found in the serum of an Fy(a b )
fy3 Unlike Fya and Fyb, the Fy3 antigen is not destroyed by enzymes.
Fy5 antigen and In 1973, Colledge and coworkers42 discovered anti-Fy5 in the serum of an Fy(a b ) black
antibody child who later died of leukemia.
Initially it was thought to be a second example of anti-Fy3, because it reacted with all Fy(a+)
or Fy(b+) RBCs but not with Fy(a b ) cells.
The antibody differed in that it reacted with the cells from an Fy(a b )Fy: 3 white female, but
it did not react with Fy(a+) or Fy(b+) Rhnull RBCs and reacted only weakly with Fy(a+) or
Fy(b+) D RBCs
The molecular structure of Fy5 is not known, but it appears to be the result of interaction
between the Rh complex and the Duffy glycoprotein. People who are Fy(a b ) or Rhnull do
not make Fy5 antigen and are at risk of making the antibody, although few do
Like Fy3, Fy5 is not destroyed by enzymes.
 Phenotype Whites (%) American Blacks (%) Chinese (%)
Fy (a+b-) 17 9 90.8
Fy (a+b+) 49 1 8.9
Fy(a-b+) 34 22 0.3
Fy(a-b-) Very rare 68 0

Fy(a-b-) resist infection in vitro by the monkey malaria organism Plasmodium knowlesi and Plasmodium vivax
has been linked to lower neutrophil counts, susceptibility to infection, renal disease, and reduced graft survival
following renal transplantation
Commonly found in black population

The Duffy glycoprotein is a member of the superfamily of chemokine receptors and is known as the atypical chemokine receptor 1
(ACKR1, previously known as DARC). Thus, in addition to being a receptor for the parasite P. vivax, the Duffy glycoprotein binds a
variety of proinflammatory cytokines.

ISBT 009 KIDD BLOOD GROUP SYSTEM

-Found on Mrs.Kidd whose infant has HDN

JKa and JKb Commonly found on RBCs of most individuals

Detected on fetal red cells as early as 11 weeks for JKa, and 7 weeks for Jkb
Not altered by enzymes

Anti Jka and Anti- Kidd antibodies have a notorious reputation in the blood bank.
Jkb They demonstrate dosage, are often weak, and are found in combination with other antibodies, all
of which make them difficult to detect.
They are Immune antibodies made in response to pregnancy or transfusion
Anti-Jka is more frequently encountered than anti-Jkb, but neither antibody is common.
Antibody reactivity can also be enhanced by using LISS or PEG (to promote IgG attachment), by
using four drops of serum instead of two in a saline tube method (to increase the antibody-to-
antigen ratio), or by using enzymes such as ficin or papain.
Many examples of the Kidd antibodies bind complement.
The titer of anti-Jka or anti-Jkb quickly declines in vivo.
A strong antibody identified following a transfusion reaction may be undetectable in a few weeks
or months. The decline in antibody reactivity and the difficulty in detecting Kidd antibodies are
reasons why they are a common cause of HTRs, especially of the delayed type
Most Common cause of DHTR (delayed hemolytic transfusion reaction)
Contrary to their hemolytic reputation in transfusion, most Kidd antibodies are only rarely
associated with severe cases of HDFN

Alloanti-Jk3 An IgG antiglobulin reactive antibody that looks like an inseperable anti-JkaJkb. The individual
making the antibody will type Jk(a-b-)

Drug Induced Associated with KIDD BGS on patient receiving ALDOMET/METHYLDOPA and another was
Autoantibody chlorpropamide-dependent
 Phenotype Whites (%) Blacks (%) Asians (%)

Jk (a+b-) 23 57 23
Jk (a+b+) 49 34 50
Jk (a-b+) 23 9 27
Jk (a-b-) Exceedingly rare Exceedingly rare <0.1-0.9

Note about Jk (a-b-)

Jk(a-b-) or the null phenotype- lack Jka , Jkb, and common Jk3 antigen, although very rare, this phenotype is
most abundant among Polynesian, and is identified in Filipinos, Indonesian, Chinese, and Japanese. It also resists
lysis to 2M urea
With Jk(a+) or Jk(b+) RBCs, lysis in 2M urea occurs within 1 minute; with Jk(a b ) cells, lysis is delayed by 30 minutes

ISBT 006 KELL BLOOD GROUP SYSTEM

KELL ANTIGENS
Excluding ABO, K is rated second only to D in terms of immunogenicity
The Kell blood group system consists of 36 high-prevalence and low-prevalence antigens; it was the first blood group
system discovered after the introduction of antiglobulin testing.
The K antigen can be detected on fetal RBCs as early as 10 weeks and is well developed at birth.
Kell blood group antigens are found only on RBCs. They have not been found on platelets or on WBCs.
The Kell glycoprotein is covalently linked with another protein, called Xk, by a single disulfide
bond. Kell antigen expression is dependent upon the presence of the Xk protein
The absence of Xk results in McLeod syndrome. It is also associated with CGD (Chronic granulomatous disease)
The prevalence of K antigen is low, and the chance of receiving a K+ unit is small
-Depressed Kell antigens are seen on RBCs with the rare Gerbich-negative phenotypes Ge: 2, 3, 4 and Ge: 2, 3, 4.
-Patients with autoimmune hemolytic anemia, in which the autoantibody is directed against a Kell antigen, may have depressed
expression of that antigen.
-K (Kell), k (Cellano), Kpa(Penny), Kpb(Rautenberg), Jsa(Sutter), and Jsb(Matthews)

Phenotypes Whites Black

K-k+ 91.0 96.5
K+k+ 8.8 3.5
K+k- 0.2 <0.1
Kp (a+b-) <0.1 0
Kp(a+b+) 2.3 Rare
Kp (a-b+) 97.7 100
Js (a+b-) 0 1
Js (a+b+) Rare 19
Js (a-b+) 100 80

KELL ANTIBODIES
Anti- K
Outside of the ABO and Rh antibodies, anti-K is the most common antibody seen in the blood bank
Anti-K was identified in 1946 in the serum of Mrs. Kelleher.
Usually an IgG antibody reactive in the antiglobulin phase
Naturally occurring IgM examples of anti-K are rare and have been associated with bacterial infections
Implicated in severe hemolytic transfusion reaction
Associated with severe hemolytic disease of newborn
Antibodies to Kpa ,Jsa ,and other low frequency Kell antigens
Antibodies are rare because so few people are exposed to the antigen
Most often detected through unexpected incompatible cross-matches or cases of HDN

Antibodies to k, Kpb , Jsb ,and other High frequency Kell antigen

Antibodies to high frequency Kell antigens are rare, because so few people lack the antigen

OTHERS
Kx antigen Kx is present on all RBCs except those of the rare McLeod phenotype. Ko and Kmod phenotype RBCs have
increased Kx antigen
K0 phenotype Ko RBCs lack expression of all Kell antigens.
Anti -Ku Anti-Ku has caused both HDFN
and HTRs

McLeod Rare phenotype with decreased Kell system antigen expression and abnormal RBC morphology,
Phenotype which has been associated with X-linked CGD, which is rare disorder affecting only males
McLeod phenotype RBCs lack Kx and the Kell system high prevalence antigen, Km, and have
marked depression of all other Kell antigens
Individuals develop a slow, progressive form of muscular dystrophy
Findings: hemolytic anemia, reticulocytosis, bilirubinemia, splenomegaly, and acanthocytosis

ISBT 005 LUTHERAN BLOOD GROUP SYSTEM (LU)

Lutheran BGS (Named from the donor Lutheran), when an anti-Lua was discovered in serum of a patient with SLE following
transfusion.
In 1956 Cutbush and Chanarin described anti-Lub, which defined the antithetical partner to Lua.
Blood bankers seldom deal with the serology of the Lutheran blood group system.
The antigens are either high prevalence, so only a few people lack the antigen and can make an alloantibody, or very low
prevalence, so that only a few people are ever exposed
Antigens have not been detected on platelets, lymphocytes, monocytes, or granulocytes
Lutheran glycoproteins is widely distributed in tissue: brain, lung, pancreas, placenta, skeletal muscle, and hepatocytes
Lu and Lub
a
Although the antigens have been detected on fetal RBCs as early as 10 to 12 weeks of gestation,
they are poorly developed
HDFN is rare and only mild at birth
Lutheran glycoprotein on placental tissue may result in adsorption of maternal antibodies to
Lutheran antigens, thus decreasing the likelihood of HDFN
Anti-Lua Most are naturally occurring saline agglutinins (IgM)
Experienced technologists recognize Lutheran antibodies by their characteristic loose, mixed-field
reactivity in a test tube
Anti-Lub Most are IgG although IgM and IgA antibodies have been noted
Implicated with shortened survival of transfused cells and post transfusion jaundice
Anti Lu3 Anti-Lu3 is a rare antibody that reacts with all RBCs except Lu(a b ) RBCs
The antibody looks like inseparable anti- Luab and recognizes a common antigen, Lu3, that is present
whenever Lua or Lub is present
 Prevalence of most Common Lutheran phenotypes
Phenotype Most population (%)
Lu (a+b ) 0.15
Lu (a+b+) 7.5
Lu (a b+) 92.35
Lu (a b ) Very rare

THE Lu (a-b-) Phenotypes

Mode of inheritance Lu Antigens Other RBC antigens affected Presence of Anti-Lu3
Dominant Type Lu(a b ) Extremely weak Reduced P1, i, Inb, AnWj, No
MER2, CD44
Recessive Type Lu(a b ) None Not affected Yes
Recessive X-Linked Inhibitor Type Extremely weak Not affected No

Lu10 and Lu15 are obsolete Lutheran antigens

MINOR BLOOD GROUPS

ISBT 010 DIEGO Antigens: Dia /Dib and Wra Wrb , Wda, Rba, WARR
Dia antigen has served as a useful tool in anthropologic studies of Mongolian
ancestry
Dia antigen is rare in all population except those of Mongolian ancestry, asian people, and
native south American Indians
Antigens are located on the anion exchange molecule (AE-1)
Associated with hereditary spherocytosis, Acanthocytosis, and southeast asian
ovalocytosis
ISBT 011 Antigens: Yta high incidence antigen, Ytb low incidence antigen
CARTWRIGHT Yt antigens have been located on erythrocyte acetylcholinesterase, which is an enzyme
involved in neurotransmission

Antibodies: Both anti-Yta and Anti-Ytb are IgG antibodies reactive in IAT and are considered to be
clinically important
ISBT 012 XG Antigen: Xga
Gene that encodes for the Xg allele is located on the short arm of the X chromosome
Xga antigen is carried by a protein with cell adhesion properties that has been demonstrated
to have homology with the CD 99 molecule
Difference in the frequency of the Xga antigen is noted between sexes
89% of the female population expresses Xga
66% of male population expresses Xga
Antibodies: Anti-Xga are predominantly IgG
ISBT 013 SCIANNA Antigens: Sc1, Sc2 and Sc3 antigens
The very rare Sc: 1, 2, 3 phenotype is the Scianna null type.
The SC gene is located on chromosome 1 at 1p34. The product of the gene is a protein called
erythroid membraneassociated protein (ERMAP), which is an RBC adhesion protein.
The prevalence of Sc2 in Northern Europeans is 1% but is higher in the Mennonite
population.
ISBT 014 DOMBROCK Antigens: Doa , Dob , Gregory (Gya) , Holley (Hy) , Joseph (Joa)
Antibodies:
Anti-Doa and Anti-Dob are described as IgG, red cell-stimulated antibodies that react
primarilty in IAT with polyethylene glycol (PEG) or enzyme enhancement - not associated
with HDN, but reported to cause delayed transfusion reactions
Antibodies to holley, joseph, and Gregory antigen have been characterized as IgG
ISBT 015 COLTON Antigen: Coa , Cob , Co3(formerly Coab)
CO antigens have been located on the transport protein known as channel forming integral
protein (CHIP) , which forms the primary RBC water channel and is responsible for water
permeability
CHIP and the CO antigens are expressed in the tissues of the proximal and descending
tubules and the collecting ducts of the kidney and are believed to account 80% of the water
reabsorption
Antibodies:
Both anti-Coa and Anti-Cob are IgG, red cell stimulated and reactive in IAT, and associated
with DHTR
Anti-Co3 is characterized as IgG, red cell stimulated, complement binding, and reacting in
the IAT, and associated with severe HDN
ISBT 017 Antigens: 6 Ch antigens, 2 Rodgers antigen, WH antigen
CHIDO/RODGERS Postulated that CH/RG antigens were associated with HLA system
Alleles for RG and CH have been located on two closely linked genes known as
C4A and C4B on ch#6
Composed of nine antigens,they are on the fourth component of complement (C4), and
are adsorbed onto RBCs from plasma
Serologically, they were both characterized as nebulous because antigen strength on
different samples of RBCs was variable
Antibodies:
Anti-Ch/Rg antibodies were collectively grouped as HTLA (high titer, Low avidity), along with
other antibodies
ISBT 020 GERBICH Antigens: High incidence Ge2 , Ge3 and Ge4 / Low incidence Wb, Ls a ,Ana , Dha
Ge antigens are inherited on chromosome 2 and are expressed on glycophorins C and/or
Glycophorin D
Individuals of the leach phenotype (GE: -2, -3, -4) present with a change in RBC morphology
in the form of Elliptocytes
Antibodies:
Anti-Ge 2 is the most common Gerbich antibody
Anti-GE2 and anti-GE3 have also been noted as naturally occurring IgM and as
autoantibodies, implicating in cause of DHTR but not in HDN
Antibodies to the low incidence Ge antigens: Anti-Wb, Anti-Lsa , Anti-Ana , Anti-Dha are
described as predominantly IgG with and IgM component and unable to bind complement
ISBT 021 CROMER Antigens: High incidence Cra , Tca , Dra, Esa , IFC, UMC, WESb / Low incidence Tcb , Tcc ,
WESa
Antigens are carried by Decay accelerating factor (DAF) , which is involved with the
regulation of complement activation by accelerating the decay of C3 and C5 convertases
Antibodies:
Anti- CROM antibodies are described as predominantly IgG1, red cell stimulated, and
reactive in IAT
CROM antibodies are rarely observed, with majority examples are on black individuals
ISBT 022 KNOPS Antigens: Kna ,Knb , McC (Mccoy) , SIa , and Yka(York)
Alleles for the KN blood group have been located on chromosome 1, with the antigens
residing on complement receptor 1 (CR1)
 Antibodies:
KN antibodies are characterized as IgG, red cell stimulated, and reacting weakly and variably
at the IAT phase, viewed to be of little clinical importance NO HDN AND HTR
ISBT 023 INDIAN Antigens: Ina , Inb
IN antigens are carried on the hematopoietic isoform of the CD44 marker, which is known
for its immune adhesion properties
Antibodies:
IN antibodies are characterized as IgG, red cell stimulated ,and reactive in IAT
ISBT 024 Ok The OK antigens are carried on CD147, or basigin, a member of the immunoglobulin
superfamily that mainly functions as receptors and adhesion molecules
Basigin is a receptor essential for invasion by Plasmodium falciparum
Oka is well developed on RBCs from newborns and is resistant to treatment with ficin and
papain, DTT, and glycine-acid EDTA
ISBT 025 RAPH The only antigen in the Raph system is MER2
It was shown that MER2 is located on CD151, a tetraspanin, which appears to be essential
for the assembly of basement membranes in the kidney and skin
Three examples of alloanti-MER2 were found in Jews originating from India and living in
Israel; two were related. All three had end-stage renal disease
ISBT 026 JOHN JMH is a high-prevalence antigen
MILTON HAGEN Anti-JMH is usually IgG (predominantly IgG4 in acquired JMH-negative people).
ISBT 029 GILL The ISBT Gill system symbol is GIL and number 029. There is only one antigen, GIL.
This antigen is found on the glycerol transporter aquaporin 3 (AQP3), a member of the
major intrinsic protein family of water and glycerol channels
ISBT 030 RHAG The Rh-associated glycoprotein (RhAG) does not have Rh blood group antigens; however, its
presence in a complex with the Rh proteins is essential for Rh antigen expression
Absence of RhAG due to inactivating mutations in RHAG results in the Rhnull phenotype;
some missense mutations in RHAG result in the Rhmod phenotype.
Four antigens have been assigned to the RHAG system: Duclos (high prevalence), Ola (very
rare, low prevalence), the high prevalence DSLK (for Duclos-like), and RHAG4
ISBT 031 FORS In 1987, the Forssman glycosphingolipid was first thought to be a subgroup of A called Apae
The ISBT symbol is FORS and the number 31; there is only one antigen, FORS1
The GBGTI gene produces glycosyltransferase, which causes the formation of the Forssman
glycolipid by the addition of N-acetylgalactosamine (GalNAc) to the P antigen.
Healthy and malignant human tissue such as gastric and colonic mucosa, lung, and kidney
have been reported to express the Forssman glycolipid
The Forssman glycolipid can serve as a receptor for pathogens such as Escherichia coli,
making one believe that human cells that express the FORS1 may have an increased
susceptibility to E. coli infection.
ISBT 032 JR Jra is a high-prevalence antigen in most populations; the Jr(a ) phenotype is found more
commonly in Japanese
The gene ABCG2 is located on chromosome 4 at position 4q22.1. The encoded protein,
ABCG2, is a member of the adenosine triphosphate (ATP) binding cassette transporters
broadly distributed throughout the body. It is involved in multidrug resistance in tumor cells,
presenting a problem in chemotherapy
Anti-Jra is usually IgG
ISBT O33 LAN There is only one antigen, Lan, with an occurrence of greater than 99% in all populations.
The Lan phenotype occurs in about 1 in 20,000 people.
The Lan gene, ABCB6, located on chromosome 2 at position 2q36, encodes another of the
ATP-binding cassette transporters.4 ABCB6 functions in heme synthesis with the ATP-
dependent uptake of heme and porphyrins into mitochondria.
 Anti-Lan is considered clinically significant; the first anti-Lan caused an immediate HTR. Lan
RBCs should be selected for transfusion
ISBT 035 CD59 CD59 is a GPI-linked complement-regulatory glycoprotein also known as the membrane
inhibitor of lysis (MIRL).
CD59 plays a key role in protecting against complement regulated hemolysis by binding to
C8 and C9 thus interfering with the formation of the membrane-attach complex (MAC).
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused
by a mutation in the GPI-linker gene. PNH patients are deficient in all GPI-linked proteins,
including CD59.
Note! ADDENDUM High Incidence antigens- occurring 99% of the population
Examples: Augustine (Ata), Crome (Cra), Ena, Gerbich, Gregory (Gya), Holley (Hy), Jacobs
(Jra), Joseph (Joa), Langereis (Lan) , and Vel (Ve)

*Augustine, Cromer, and Joseph are phenotypes predominant in blacks

LUKE(LKE) Antigen Associated with , and metastasis in renal carcinoma

Decreased expression of the LKE is seen in individuals with the Se gene with secretors having a 3-
to 4-fold decreased risk of E.coli infections
it was thought to be phenotypically related to P Blood group system, because the antibody
reacted with all RBCs except 2% of P1 and P2 phenotypes and those having the rare p and Pk
phenotypes. All individuals with the p and Pk phenotype are Luke .
Antibodies are IgM and is not associated with HTR and HDN
SDa Sda = The Sda antigen is a high-prevalence antigen named for Sid, who was the head of the
maintenance department at the Lister Institute in London. The soluble form of Sda is Tamm-
Horsfall glycoprotein found in urine. The antigen is not expressed on RBCs of newborns but
is in their saliva, urine, and meconium
Anti-Sda is usually an IgM agglutinin that is reactive at room temperature, but it can be detected
in the indirect antiglobulin test and does not react with cord RBCs. Reactivity is described as
small, refractile (shiny) agglutinates in a sea of free RBCs.
Bennett good - a, Bgb, and Bgc) was given to HLA class I antigens that are detectable on
speed antigens mature RBCs.
1. Bga = Represents HLA B-7
2. Bgb = Represents HLA B-17
3. Bgc = Represents HLA A-28

NOTE
Low incidence Antigens- occurs less than 1% of the population
Examples are Wright , Swann (Swa), Mta, and Tra

Private antigen: an antigenic characteristic of the red blood cell membrane that is unique to an individual
or a related family of individuals and therefore is not commonly found on all cells( usually <1% of
population)
High Titer, Low Avidity Antibodies
Antibodies exhibit reactivity at high dilutions of serum, but the strength of agglutination is weak at any dilution

E.g., anti-Ch/Rg , Anti-Kn, Anti-Yk (york) , Anti-Cs (Cost), Anti-JMH , and Anti-Mccoy
When an uncommon antibody is detected, understanding antigen and antibody characteristics and how they relate to frequency,
ethnicity, enzymes, and chemical treatments is critical in the identification process
ANTIBODY CHARACTERISTICS
Naturally Occuring /Cold antibodies LIPMAN! - Lewis, I, P, MNs, AB, Lutheran
IgM
Clinically significant RKDAKS ABO, Rh, Kell, Duffy, Kidd, SsU
Warm antibodies RKD Rh, Kell, Duffy, Kidd
IgG
React only in AHG KKD Kell, Duffy, Kidd
Can react in any phase of testing Lewis
Enhanced by enzyme PRICK Kidd, Lewis, I, Rh, P
Destroyed by enzyme CDMY MN, Duffy, Ch, Yt
Enhanced by acidification M
Dosage Effect RKMLD Kidd, Rh, Duffy, MN, Lutheran*(rare) DR KIMNS.

Bind Complement PILAR P, I, Lewis, ABO, Kidd

Most common cause of DHTR Kidd

SOURCES OF SUBSTANCES FOR NEUTRALIZATION FOR CERTAIN ANTIBODIES

Anti I
Anti-H Saliva
Anti-Sda Guinea pig urine
Anti- Chido, Anti-Rodgers Plasma/serum
Anti-Lewis Secretor saliva, plasma/serum
Anti-P1 Hydatid cyst fluid, pigeon droppings, turtle dove egg white

LECTIN REAGENTS
Dolichos biflorus. Source of anti-A1 lectin
Ulex europeus Source of anti-H lectin
Bandeiraea simplicifolia. Source of anti B lectin
Iberis amara. Source of anti-M lectin
Vicia graminea. Source of anti-N lectin
 DETECTION AND IDENTIFICATION OF ANTIBODIES

of the ABO system.

Clinically significant alloantibodies are those that cause decreased survival of RBCs possessing the target antigen. These
antibodies are typically IgG antibodies that react at 37°C and/or at the antihuman globulin (AHG) phase of the indirect
antiglobulin test (IAT).

TYPES OF UNEXPECTED ANTIBODIES

immune alloantibodies which are produced in response to RBC stimulation through transfusion, transplantation, or pregnancy
Naturally occurring -produced without RBC stimulation
alloantibodies -Result of exposure to environmental sources, such as pollen, fungus, and bacteria, which have
structures similar to some RBC antigens
Passively acquired Antibodies produced in one individual and then transmitted to another individual via plasma containing
antibodies blood components or derivatives such as intravenous immunoglobulin (IVIG)
Auto-antibodies
generally react with all RBCs tested
NOTE: When an unexpected antibody is detected in the antibody screen, an antibody identification panel is performed to determine the specificity of the
antibody present

ANTIBODY SCREEN

1. TUBE METHOD

Traditional testing method used to detect clinically significant antibodies is the IAT performed in a test tube

Procedure:
I 3- 4
2 .

1drop of Reagent Known RBC suspension (2 to 5%) + 2 drops Patient plasma --- Incubate wash (3to4x) --
centrifuge and observe for hemolysis or agglutination

This test may include an immediate spin (IS) phase to detect antibodies reacting at room temperature

This test also includes

sensitize any reagent RBCs that possess the target antigen. Enhancement reagent may be added to the test tube prior to
incubation at 37°C to increase the degree of sensitization

Use of the tube test remains popular due to the flexibility of the test system, the availability of required testing
equipment, and the relative low cost

Disadvantages: instability of the reactions and subjective nature of grading by the technologist, the amount of hands-
on time for the technologist, and problems related to the failure of the washing phase to remove all unbound antibody.

Reagents Used
Screen cells/ RBC The RBC reagents /screen cells used in antibody screen testing come from group O individuals who have
reagents been typed for the most common and the most significant RBC antigens.
Enhancement Various enhancement reagents, or potentiators, may be added to the test system prior to the 37°C
reagents incubation phase to increase sensitivity.
Examples are LISS, 22% Albumin, and PEG
AHG reagents AHG reagent (monospecific or polyspecific) is added to/ included in the test system as it allows for
agglutination of incomplete antibodies.

cells, which are Group O Rh-positive RBCs that have been coated with anti-D
2.GEL TECHNOLOGYAND SOLID PHASE TECHNOLOGY

GEL TECHNOLOGY Yves Lapiarre -

Inventor

Controlled centrifugation red cells through dextran-acrylamide gel and appropriate reagents, predispensed in
specially designed microtube
Can do-
ABO forward and reverse grouping,
- Rh typing,
r DAT, Antibody
- screening,r
Antibody identification, andr
Compatibility
testing
A gel card measures approximately 5× 7 centimeters and consists of six microtubes. Each microtube contains predispensed
gel, diluent, and reagents, if applicable.
Major advantage: Standardization
Disadvantage: Need special instrument/ equipment
Centrifugation time: 10 minutes at 910 rpm
Incubation time: 15 minutes
Principle: Hemagglutination

AGGLUTINATION REACTION
4+ Solid band of agglutinated red cells at the top of the gel column. Usually no red cells are visible in the
bottom of the microtube
3+ Predominant amount of agglutinated red cells towards the top of the gel column with a few
agglutinates staggered below the thicker band. The majority of agglutinates are observed in the
tophalf of the gel
2+ Red cell agglutinates dispersed throughout the gel column with few agglutinates at the bottom of the
microtubes.
Agglutinates should be distributed through the upper and lower halves of the gel.
1+ Red cell agglutinates predominantly observed in the lower half of the gel column with red cells also in
the bottom. These reactions may be weak, with a few agglutinates remaining in the gel area above the
red cell pellet in the bottom of the microtube
Mixed Field Layer of red cell agglutinates at the top of the gel column accompanied by a pellet of unagglutinated
cells in the bottom of the microtube
Negative Red cells forming a well-delineated pellet in the bottom of the microtube. The gel above the
red cell pellet is clear and free of agglutinates

SOLID PHASE TECHNOLOGY

Based on solid phase red cell adherence
Plapp and coworkers reported using solid-phase red cell adherence (SPRCA) for detecting RBC antigens and
antibodies.
Can do antibody screening, antibody identification, and compatibility testing

AFFINITY COLUMN TECHNOLOGY (Gamma React)

Principle of affinity adherence of IgG sensitized erythrocytes to an immunologically active matrix
Can do antibody screening, antibody identification, and compatibility testing

INTERPRETATION OF RESULTS
Positive test result at any phase of testing indicates the need for antibody identification studies. Evaluation of antibody screen
results, including the autologous control if tested at this time, and patient history (i.e., age, sex, race, diagnosis,
pregnancy and transfusion history, current medications, and intravenous solutions) can provide clues and give direction for
identification and resolution of the antibody or antibodies present
 IgM Antibodies of the IgM class react best at room temperature or lower and are capable of causing
agglutination of saline-suspended RBCs (immediate spin reaction)
IgG Antibodies of the IgG class react best at the AHG phase
Autologous control The autologous control is
antibody screen
NOTE: A positive antibody screen and a negative autologous control indicates that an alloantibody has been
detected
Multiple antibodies More than one screen cell may be positive when the patient has multiple antibodies, when a single
n more than one screen cell, or
autoantibody
Immediate spin AHG Phase Ab :

Dosage effect i DK - KIMNS Abi RKDKSLUB

bt) Lela)PMN Rh Duffy Ss
(Fya b -11
KIDD / Jka
Duffy
-
-

↑ ↑ Kell Kidd hub

MNSLMTN ) (eluapmivs
-

Rh

ANTIBODY PANEL IDENTIFICATION

Once an antibody has been detected in the antibody screen, additional testing/review is necessary to identify the
antibody and determine its clinical significance

and intravenous solutions may provide valuable clues in antibody identification studies, particularly in complex
cases
REAGENT= An antibody identification panel is a collection of 11 to 20 group O reagent RBCs, with various antigen expression.

CASE NUMBER 1
Hntigram confirmation
↓
/ / 1/1/11 / / _
/ / / / / / // / / / /
O O
- - - - - - - - o - - - - - - -
O
O O
O O
- - r - r - O r - r - r / O
O O
O O
r r r r -
O- r - r - r - r - O
- - - -
- - -
O - - - - - - - -
O
O O
O O

WHAT IS THE ANTIBODY DETECTED? Alloanti -fya

CASE NUMBER 2

WHAT IS THE ANTIBODY DETECTED? Alloanti-jkb

AET, 2-Aminoethylisothiouronium bromide; AHG, antihuman globulin; DAT, direct antiglobulin test; DHTR, delayed hemolytic transfusion reaction; DTT, dithiothreitol;
HDFN, hemolytic disease of the fetus and newborn; HTR, hemolytic transfusion reaction; Ig, immunoglobulin; ZZAP, combination of DTT and cysteine-activated papain.

ANTIBODY Adsorption is a process used to physically remove antibodies from sera. The adsorption procedure may be
performed using washed and/or enzyme-treated autologous RBCs, phenotypically matched allogeneic cells,
ADSORPTION
cells selected to be positive for a specific combination of blood group antigens, or RBC stroma.
ANTIBODY ELUTION Elution is the process used to remove antibodies bound to RBCs, as indicated by a positive DAT. Elution is
commonly used to concentrate and solubilize antibodies from RBCs for subsequent identification studies.
The elution of an antibody with known blood group and/or drug specificity can be diagnostic in the
evaluation of acute and delayed hemolytic transfusion reactions, HDFN, autoimmune hemolytic anemia, and
immune hemolytic anemia induced by medication. -

A process whereby cells (RBCs) that are coated with antibody are treated in such a manner as to disrupt
the bonds between the antigen and antibody. The freed antibody is collected in an inert diluent such as
saline or 6% albumin. This antibody serum then can be tested to identify its specificity using routine
methods. The mechanism to free the antibody may be physical (heating, shaking) or chemical (ether, acid),
and the harvested antibody-containing fluid is called an eluate - Harmening
 BLOOD PRESERVATIVES/ ADDITIVES/REJUVENATION/ FREEZING
The goal of blood preservation is to provide viable and functional blood components for patients requiring blood
transfusion. RBC viability is a measure of in vivo RBC survival following transfusion
The FDA requires an average 24-hour post-transfusion RBC survival of more than 75%.

I.PRESERVATIVES
APPROVED PRESERVATIVES SHELF LIFE The loss of RBC viability has been correlated with
Acid citrate dextrose (ACD) 21days
the storage lesion, which is associated with
Citrate-phosphate-dextrose (CPD) 21 days
various biochemical changes
Citrate-phosphate double dextrose (CP2D) 21days
Citrate phosphate adenine (CPDA-1) 35 days
Mr

CPDA-2 42 day

COMPONENTS OF BLOOD PRESERVATIVES

Citrate Acts as anticoagulant, chelates calcium
Dextrose Provides energy or glucose to RBC;
Substrate of ATP
Citric acid Lowers pH thus prevent caramelization
Monobasic sodium Maintains pH during storage; necessary for
phosphate maintenance of adequate levels of 2,3-DPG
Increase ATP levels
Adenine Production of ATP (Improve RBC survival from
21 to 35 days)

The most commonly used anticoagulant-preservative solution for RBC is CPDA-1.

II.ADDITIVES
These are preserving solutions that are added to the RBCs after removal of the plasma with or without platelets.
Additive solutions (100 mL to the packed RBCs prepared from a 450-mL blood collection) reduce hematocrits
from around 65% to 80% to around 55% to 65% with a volume of approximately 300 to 400 mL.
BENEFITS OF RBC ADDITIVE SOLUTIONS include:
a. Extends the shelf-life of RBCs to/
42 days by adding nutrients
b. Allows for the✓
harvesting of more plasma and platelets from the unit
c. Produces a packed RBC of lower viscosity that is easier to infuse
RBCs stored for 42 days in AS-1, AS-3, or AS-5 demonstrated a mean 24-hour post-infusion survival of greater than 75 percent, the
minimum requirement for satisfactory RBC survival

NAME STORAGE TIME SNAG COMPONENTS

Adsol (AS-1) 42 days Saline
Nutricel (AS-3) 42 days Adenine
Optisol (AS-5) 42 days Glucose
SOLX (AS-7) 42 days Mannitol =Acts as a RBC membrane stabilizer

III.REJUVENATION SOLUTIONS
Used in some blood centers to regenerate ATP and 2,3DPG
Red cells stored in the liquid state for fewer than 3 days after their outdate are rejuvenated for 1 to 4 hours
<3
solution
Rejuvesol- only FDA approved rejuvenation solution
 Components: PIGPA /PIPA (Phosphate, Inosine, Glucose, Pyruvate, Adenine)
Currently, rejuvenated RBCs must be washed before infusion to remove the inosine (which may be toxic) and transfused
within 24 hours or frozen for long-term storage

Stored RBCs regain the ability to synthesize 2,3-DPG after transfusion, but levels necessary for optimal hemoglobinoxygen delivery
are not reached immediately. Approximately 24 hours are required to restore normal levels of 2,3-DPG after transfusion.
The 2,3-DPG concentrations after transfusion have been reported to reach normal levels as early as 6 hours post-transfusion. The rate
acid-base status of the recipient, the /
of restoration of 2,3-DPG is influenced by the - phosphorus metabolism, the/
degree of anemia,
/overall severity of the disorder
and the

Approximately 220 to 250 mg of iron are contained in one RBC unit

IV.RBC FREEZING
RBC freezing is primarily used for autologous units and the storage of rare blood types.
Frozen RBC Shelf life: 10 years (longest shelf life)
Commonly uses Glycerol, a freezing agent

Advantage High Glycerol (40% Weight per vol) Low Glycerol (20% Weight per vol)
1. Initial freezing temperature 80°C 196°C
2. Need to control freezing rate No Yes
3. Type of freezer Mechanical Liquid nitrogen
4. Maximum storage temperature 65°C 120°C
5. Shipping requirements Dry ice Liquid nitrogen
6. Effect of changes in storage Can be thawed and refrozen Critical
temperature

Advantages Disadvantages
1.Long-term storage (10 years) 1. A time-consuming process
2.Maintenance of RBC viability and function 2. Higher cost of equipment and materials
3. Low residual leukocytes and platelets 3. Storage requirements ( 65°C)
4. Removal of significant amounts of plasma proteins 4. Higher cost of product

DEGYLCEROLIZATION
Removal of glycerol from a unit of red blood cells after thawing has been performed; required to return the cells to a normal
osmolality
Blood unit is exposed to decreasing osmolarity, from hypertonic solution then followed by isotonic solution
The thawing process takes approximately 45 minutes and involves immersing units into a 30° to 37°C water bath and washing
the RBCs with solutions of decreasing osmolarity (e.g., 12% NaCl, 1.6% NaCl, 0.9% NaCl, with 0.2% dextrose)
Shelf-Life /Expiration date: 24HOURS
Deglycerolized blood product should ensure 80% RBC MASS RECOVERY
Glycerol must be removed to a level of less than 1% residual.
Measure the osmolarity of the unit using an osmometer. The osmolarity should be about 420 mOsm (maximum 500 mOsm).

Deglycerolization
High glycerol 12%NaCl 1.6%NaCl 0.9%NaCl with 0.2% dextrose
* An exception to this rule is a donor with sickle trait in which RBCs would hemolyze upon
suspension in hypertonic solution, the 1.6% NaCl should be omitted
Low glycerol 45%NaCl in 15%mannitol 0.9%NaCl
VI.BLOOD SUBSTITUTES
Blood Substitutes
The current research on blood substitutes is focused on two areas: hemoglobin-based oxygen carriers (HBOCs) and
perfluorocarbons (PFCs).
Perfluorocarbons are synthetic hydrocarbon structures in which all hydrogen atoms have been replaced with fluorine. They are
chemically
- inert, are excellent
✓ gas solvents, and r
carry O2 and CO2 by dissolving them. Because of their small size (about 0.2 um
in diameter), they are able to pass through areas of vasoconstriction and deliver oxygen to tissues that are inaccessible to RBCs.

VII.PLATELET PRESERVATION
Platelets are stored at 20°C to 24°C with maintaining continuous gentle agitation throughout the storage period of 5 days.
Agitation has been shown to facilitate oxygen transfer into the platelet bag and oxygen consumption by the platelets
The positive role for oxygen has been associated with the maintenance of platelet component Ph
Maintaining pH was determined to be a key parameter for retaining platelet viability in vivo when platelets were stored at 20°C
to 24°C
When the bicarbonate buffers are depleted during platelet concentrate storage, the pH rapidly falls to less than 6.2, which is
associated with a loss of platelet viability. In addition, when pH falls below 6.2, the platelets swell and there is a disk-
to-sphere transformation in morphology that is associated with a loss of membrane integrity. The platelets then
become irreversibly swollen, aggregate together, or lyse, and when infused, will not circulate or function. This change is
irreversible when the pH falls to less than 6.2.

QUALITY CONTROL MEASUREMENTS REQUIRED FOR PLATELET CONCENTRATES

1. Platelet concentrate volume
2. Platelet count
3. pH
4. Residual leukocyte count (from leukoreduction)
5. Platelet swirl (no visible aggregation)
Immediately before distribution to hospitals, a visual inspection is made that often includes an assessment of platelet swirl
(no visible aggregation). The absence of platelet swirling is associated with the loss of membrane integrity during storage,
resulting in the loss of discoid shape with irreversible sphering

PLATELET STORAGE LESIONS

Increased Decreased
-Lactate -pH
-Degranulation (Beta-thromboglobulin, PF4) -ATP
-Platelet activation markers(P-selectin) -Morphology (from discoid to spherical)
loss of swirling effect
-Platelet aggregation
 DONOR SCREENING
DONATION PROCESS
1. Donor registration
2. Interview and physical exam
3. Donor selection and blood collection

DONOR BLEEDING

Standard needle used: 16 gauge

Needle insertion:
For blood collection, most blood centers use an iodine compound such as PVP-iodine or polymer iodine complex. Using a
tourniquet or blood pressure cuff, the venipuncture site is identified, and the area is scrubbed at least 4 cm in all
directions from the site for a minimum of seconds
For donor blood collection, Scrub the site (2 × 2 inches) for 30 seconds using an aqueous iodophor scrub solution
(0.7%). Iodophor is a polyvinyl pyrrolidone- iodine or poloxamer iodine complex
Donations of whole blood where the bleed time exceeded 15 minutes are not suitable for the production of plasma components,
platelet products, and cryoprecipitate for direct clinical use.

QUALIFICATION OF THE POTENTIAL BLOOD DONOR (ALLOGENIEC)

General appearance
Age
Body weight
Volume of blood to be drawn
and volume of Anticoagulant
needed in cases of a donor
with a less than 50kg weight
Appears to be in good health, no sign of intake of drugs, or alcohol, no skin lesions
18-65 years old
50 kg or 110 lbs
All donors should weigh at least 110 lbs.Standards mandates a maximum of 10.5 mL of blood/kg
of donor weight for whole blood collection, inclusive of pilot tubes for testing. If the donor weighs
less than 100 pounds, the amount of blood collected must be proportionately reduced as well as
that of the anticoagulant. The following formulas can be used to calculate the adjusted volume of
blood to be collected and anticoagulant (CPD and CPDA-1) to be used.

B.Reduced volume of Anticoagulant = Volume to collect (Answer in A) x 63mL

450

C. Amount of Anticoagulant to be removed = 63mL Answer in B

 Temperature Do not exceed 37.5 C or 99.5 F
Pulse 50-100 beats, lower pulse rate for athletes
Blood pressure Systolic: not greater than 180 mm Hg, Diastolic: not greater than 100 mmHg 180/100
Hemoglobin 12.5 gldl ( women ) , 13.091dL ( men )

Hematocrit 38 % ( women) ,
39% (men )

ALLOGENEIC BLOOD DONOR SCREENING

Every unit of blood is tested for the ABO
- type, r Infectious marker for T.cruzi, HIV 1/ 2, HTLV 1 / 2,r
RH type, and- Hepatitis B and
C, Syphilis,
- - West nile virus,-
Zika virus,-
Babesiosis, etc.

NOTE: Screening of donation for other infections, such as those causingr r disease, orr
malaria, chaga HTLV, should be based on
local epidemiological evidence - WHO

AUTOLOGOUS BLOOD DONATION

Someone who donates blood for his/her own for future purposes.
It is the safest blood to be transfused to individual
The disadvantages of autologous donation or transfusion include a higher cost due to added administrative processes and
special labelling requirements to ensure that units get transfused to the proper patient.
The collecting facility must determine the ABO and Rh of the blood, but antibody screening is optional. If the
collecting and the transfusing facilities are the same, then viral marker testing is not required; however, if they are different,
10 the collecting facility must test forr
HBV DNA,rHBsAg, anti-HBc,
r anti-HCV,/
r /
HCV RNA, anti-HIV-1/2,✓HIV-1 RNA,✓
anti-HTLV-I/II,
,
WNV RNA, and/
STS on at least the first unit collected from the donor in every 30-day period. If any of the markers yield a

Autologous units also generally have a distinct green label and tag

Methods/Techniques for obtaining Autologous Blood

Pre-operative collection
Acute Normovolemic
hemodilution
Intra operative collection
Postoperative Blood salvage
-Blood that is withdrawn before (72 hours) an anticipated transfusion and stored until use
-Results in the collection of whole blood with the concurrent infusion of crystalloid or colloid
solutions
-The ratio of replacement is 3:1 for crystalloids and 1:1 for colloids.
-involves collecting shed blood from the surgical site; processing the blood through an
instrument that washes it with saline to remove tissue debris, free hemoglobin, and plasma that
may contain activated coagulation factors; concentrating the residual red cells (to a hematocrit of
50% to 60%); and then reinfusing those cells immediately.
-in which a drainage tube is placed in the surgical site and postoperative bleeding is
salvaged, cleaned and reinfused

General Requirements for Autologous Donation

Age No age limit
Weight No strict weight requirements
Hemoglobin 11.0 gldl
Hematocrit 33%
Frequency NOT MORE THAN EVERY 3 DAYS

DIRECTED BLOOD DONATION

A directed donation is a unit collected under the same requirements as those for allogeneic donors, except that the unit
collected is directed toward a specific patient
The tag for the directed unit is a distinct color (e.g., yellow, salmon) to differentiate it from autologous tags

Temporary deferral
Indefinite Deferral
Permanent Deferral
ALLOGENEIC DONOR DEFERRAL
Prospective donor is unable to donate blood for a limited period of time.
Prospective donor is unable to donate blood for someone else for an unspecified period of time due
to current regulatory requirements. This donor would not be able to donate blood until the current
requirement changes. These donors may be eligible to donate autologous blood.
Prospective donor will never be eligible to donate blood for someone else. These donors may be
eligible to donate autologous blood. Some permanent deferrals may result from the testing performed
on a previous donation.

PERMANENT OR INDEFINITE 1 YEAR 1 MONTH

Haemophiliacs Rape victim German measles(rubella)
IV drugs User After Hepa B immunoglobulin administration vaccination
Positive in AIDS Healthcare worker exposed to blood or body Chicken pox vaccination
Positive HBsAg fluids Drug:
Positive HTLV Tattoo a. Isotretinoin
Malignant solid tumors Needle stick injury (Accutane)- acne
Drugs Ear or body piercing treatment
Etretinate (Tegison)- treatment for Sexual contact with a prostitute, with AIDS, b. Finasteride
psoriasis Hepatitis, with hemophiliacs, or with IV drug (Proscar)- for
History of Babesiosis users benign prostate
Female who had sexual contact of men who hyperplasia
Chronic cardiopulmonary ever had sex with another men
Renal disease Travel to endemic areas with malaria
Liver disease Syphilis / gonorrhoea
recipient of cornea and duramater Received transfusion of blood components
transplant its components or other human tissues (organ,
Recipient of HUMAN pituitary tissue, bone marrow transplant, or bone or skin
derived growth hormone graft) known to be possible sources of
bovine insulin bloodborne pathogens
Leukemia, lymphoma and Rabies vaccination
myeloproliferative disorder Major operation including dental surgery
Have been in juvenile detention, lockup, or
prison for more than 72 hours

Note: Tooth extraction 3 days deferral

OTHERS 1. MMR vaccination: 8 WEEKS (NBVSP), 4weeks (book)

2. Childbirth (AABB: 6 WEEKS after delivery), Philippines: 9 Months after childbirth
NOTE A first-trimester or second trimester abortion or miscarriage is not cause for
deferral. A 12-month deferral would apply if the woman received a transfusion during her pregnancy.
3. 2 weeks deferral due to vaccination
Measles (rubeola), Mumps, Oral polio, Typhoid, and Yellow fever
4. Aspirin or Piroxicam= 48 hours
5. Plavix (clopidogrel) or Ticlid (ticlopidine) = 14 DAYS
6. Individuals with a history of dengue or chikungunya virus: defer for 6 months following full recovery
from infection
7. Individuals with tuberculosis: defer for 2 years following confirmation of cure
8. Small pox vaccination
-14 to 21 days or until the scab has fallen off
 9. There is no deferral for toxoids or killed or synthetic viral, bacterial, or rickettsial vaccines such as
diphtheria, hepatitis A, hepatitis B, -
- pneumococcal polysaccharide, -
influenza, Lyme disease, - polio
=
injection (Salk), anthrax, cholera, _ =
pertussis, plague, -
paratyphoid, -
rabies, -
Rocky Mountain spotted
fever, tetanus, or typhoid injection if the donor is symptom-free and afebrile
10. Men who have sex with men = 12 months or indefinite
11. Sexual contact or living with a person who has acute or chronic hepatitis B (test
positive for HBsAg or HBV) or who has symptomatic hepatitis C or other hepatitis virus requires a 12-
month
residing in the same dwelling (house, apartment, or dormitory).

MALARIA AABB:
DEFERRAL a. 1year Travelers to endemic areas
b. 3 years- Immigrants, refuges, citizens who resided on endemic areas, and diagnosed/infected with
malaria

PHILIPPINES:
a. Stayed for less than6 months: 6 months
b. Stayed for more than6months/ Resident: 1 year
c. Infected: 3 years
Blood Donation Whole Blood Donation
Frequencies a. AABB: 8weeks/ 2 month
b. PHL: 12 weeks/ 3 months
Hemapheresis- 2days

APHERESIS/ HEMAPHERESIS
Apheresis (plural aphereses; from the ancient Greek aphairesis
from the body and passed through an apparatus that separates out one (or more) particular blood constituent. It then returns

Includes Plasmapheresis, Erythrocytapheresis, Leukapheresis, Plateletpheresis

The idea of apheresis was developed by Dr.Edwin J. Cohn

Anticoagulant: Citrate (ACD) is used as the primary anticoagulant in apheresis procedures

POSSIBLE ADVERSE REACTION/SIDE EFFECTS

Citrate toxicity -Most common reaction. Donor may feel numbness or tingling around the mouth
(paresthesias). If unattended, the symptoms can lead to tetany (due to hypocalcemia) and cardiac arrhythmia.
Vascular access complications (hematoma, sepsis, phlebitis, neuropathy)
Vasovagal reactions - vasovagal syncope, which is the most
common type of fainting.
Hypovolemia
Allergic reactions
Hemolysis
Air embolus
Depletion of clotting factors
Circulatory and respiratory distress
Transfusion-transmitted diseases
Lymphocyte loss
Depletion of proteins and immunoglobulins
The variables that are considered during an apheresis procedure include:
1. Centrifuge speed and diameter
2. Duration of dwell time of the blood in the centrifuge
3. Type of solutions added, such as anticoagulants or sedimenting agents (E.g., Hydroxy ethyl starch)
4. Cellular content or plasma volume of the patient or donor

CELLULAR LOSS
20% to 29% A platelet donor typically experiences an acute fall in platelet count of __ following apheresis donation
3% Typically, a drop in hematocrit of __ and a fall in platelet count of 22% occur after each granulocyte donation.

METHODS OF SEPARATION
The donor or patient remains attached to the apheresis instrument for the duration of the procedure; the amount of time for a particular
procedure can range from 45 to 120 minutes. Collection of hematopoietic progenitor cells takes considerably longer.
Intermittent flow centrifugation Requires only one venipuncture, in which blood is withdrawn and reinfused
through the same needle. Once the desired component is separated, the
remaining components are reinfused to the donor, and one cycle is complete
Continuous flow centrifugation Procedures withdraw, process, and return the blood to the individual
simultaneously.
Two venipuncture sites are necessary
Membrane filtration Membrane separators are typically composed of bundles of hollow fibers or flat
plate membranes with specific pore sizes
As whole blood flows over the fibers or membrane, plasma passes through the
pores and is collected, while the remainder of the cellular components is
returned to the donor.

DOUBLE RBC PHERESIS

Criteria Male Female
Weight 130 lbs 150 lbs

Height 515
"

51
"

Hematocrit at least 40% at least 40%

DONATION FREQUENCY
APHERESIS COMPONENT FREQUENCY OF DONATION
COLLECTED
Double RBC 16 Weeks 4 Months
Plasma(frequent) Every 48 hours (no more than two times in 7 days)
Plasma (Infrequent) Every 4 weeks (no more than 13 times a year)
Platelet single apheresis Every 2 days (no more than 2 times in 7 days; no more than 24 times in 12 months)
Granulocytes Every 2 days

ADDITIONAL INFORMATION
Donor Criteria for
plasmapheresis
a. Serum total protein should be at least 6g/dl
b. If the donor weighs 50 to 80 kg = not more than 500ml WB should be removed at one time
c. If the donor weights more than 80 kg = not more than 600ml WB should be removed at one time
d. FDA guidelines recommend 12 L (14.4 L for donors weighing more than 175 pounds) as the maximum
allowable plasma volume donated per year
Donor criteria for Donor selection criteria for the plateletpheresis donor are the same as for whole blood donation, with two
Plateletpheresis additional requirements.
1. The platelet count must be at least 150,000/uL or 150x109/L
2. Donor should not take medications that interfere with platelet function
a. Aspirin 48 hours deferral
b. Feldene -48 hours deferral
c. Clopidogrel (Plavix), ticlopidine (Ticlid), ticagrelor (Brilinta), vorapaxar (Zontivity), and
prasugrel (Effient) = 14 days deferral
6 to 8 units derived
platelets (random-donor platelets)
Hydroxyl ethyl starch A common sedimentating agent. It enhances the separation of the white cells from the red cells during
centrifugation, which increases the amount of leukocytes collected and decreases the amount of red cell
contamination in the final product for leukapheresis
Physician A qualified, licensed ____ must be responsible for all aspects of the apheresis program.
Operators of Operators of apheresis instruments may be medical technologists (clinical laboratory scientists), nurses, or
apheresis technicians trained on the job and should be friendly and outgoing
Therapeutic pheresis The rationale of therapeutic apheresis (TA) is based on the following:

mechanisms.
Therefore, therapeutic apheresis, like donor apheresis, involves the removal of a specific blood component,
with return of the remaining blood constituents to the patient
The effectiveness of TPE is related to the volume of plasma removed and the concentration of the
pathological substance in the blood. Apheresis is most efficient at removing the substance at the
beginning of the procedure and least efficient at the end
Therapeutic plasma The removal and retention of the plasma, with return of all cellular components to the patient. This is the
exchange (TPE) most common TA procedure performed. The purpose is to remove the agent in the plasma, such as an
antibody, toxin, or abnormal protein, that is causing the clinical symptoms.

plasma
Therapeutic Therapeutic plateletpheresis can be used to treat patients who have abnormally elevated platelet counts with
plateletpheresis related symptoms.
Therapeutic Therapeutic leukapheresis has been used to treat patients with hyperleukocytosis, defined as a WBC or
leukapheresis circulating blast count of over 100,000/uL. A single procedure should reduce the WBC count by 30% to 60%
Therapeutic
erythrocytapheresis compatible allogeneic donor RBCs. Commonly performed in patients with sickle cell disease in order
(RBC exchange) to decrease the number of hemoglobin S containing RBCs, thereby treating or preventing the
complications (acute chest syndrome, impending stroke, unrelenting painful crisis) associated with
the disease. The therapeutic goal is to decrease the level of hemoglobin S to less than 30%.

ADDITIONAL INFORMATION ABOUT APHERESIS

10.5 mL/kg What is the regulatory restriction limit for extracorporeal volume during apheresis to avoid hypovolemic
reaction?
Hypotension during Hypotension may also occur during an apheresis procedure due to a vasovagal reaction, particularly in
apheresis apheresis donors
Factors that have been associated with vasovagal reactions in apheresis donors include younger age and the
sex of the donor.
Teenaged donors have higher vasovagal reaction rates compared with adults, and female donors have a
higher incidence of these reactions than male apheresis donors.

Insertion of large IV In plasmapheresis, Insertion of a rather large intravenous catheter can lead to bleeding, lung puncture
catheter (depending on the site of catheter insertion), and if the catheter is left in too long, infection.
Therapeutic pheresis The TA procedure is classified according to the blood component removed: a cytapheresis procedure may
(TA) be used to selectively remove RBCs, WBCs, or platelets; a plasmapheresis procedure is used to remove
plasma when the pathological substance is found in the circulation. Therapeutic apheresis has become an
accepted and standard therapy for many hematologic, neurological, renal, metabolic, autoimmune, and
rheumatic diseases, among others

Categories of TA Category I Apheresis is a first-line treatment, alone or in conjunction with other therapies
Category II Apheresis is a second-line treatment, alone or in conjunction with other therapies
Category III The optimal role for apheresis has not been established. Treatment should be
individualized based on clinical evaluation and assessment of the anticipated risks and
benefits
Category IV Apheresis is reported as either of no benefit or harmful in these conditions. Clinical
applications should be undertaken only under an approved research protocol.

Physiological d volume
consideration before (TBV) in order to Minimize the risk of hypovolemia
performing TA
exchange in order to appropriately remove sufficient amounts of plasma during the TA procedure.
This is eas s hematocrit,
the higher the PV.
A blood warmer is often used as part of the apheresis circuit to avoid hypothermia due to the rapid
infusion of room temperature fluids

Plasmapheresis
(Plasma exchange)
Therapeutic plasma exchange (TPE) is the removal and retention of the plasma, with
return of all cellular components to the patient
This is the most common TA procedure
The purpose is to remove the agent in the plasma, such as an antibody, toxin, or abnormal protein,
that is causing the clinical symptoms
It is also used to replace a normal factor or substance that may be missing or deficient in the

The effectiveness of TPE is related to the volume of plasma removed and the concentration of the
pathological substance in the blood. Apheresis is most efficient at removing the substance
at the beginning (first portion) of the procedure and least efficient at the end.
TPE show that a one-volume exchange should reduce the unwanted plasma component to
approximately 30% of its initial value
For abnormal IgM TPE can be an effective therapeutic tool, since IgM antibodies tend to be located
primarily in the intravascular space
TPE performed to remove IgG antibodies is most effective when combined with immunosuppressive
drugs.

Therapeutic Therapeutic plateletpheresis can be used to treat patients who have abnormally elevated platelet
plateletpheresis counts with related symptoms
The preferred method for lowering the platelet count is medication; however, therapeutic
apheresis may be indicated during an acute event to rapidly reduce the platelet count until
pharmacological therapy takes effect.
In this procedure, platelet count will be decreased by 30% to 60%.

Therapeutic Used to treat patients with hyperleukocytosis, defined as a WBC or circulating blast count of over
leukapheresis 100,000/uL.
These elevated WBC levels place the patient at risk for complications associated with leukostasis
including organ dysfunction due to the formation of microthrombi in the pulmonary and cerebral
microvasculature
A single procedure should reduce the WBC count by 30% to 60%; however, more than one
procedure may be necessary due to rapid mobilization of cells from the extravascular
compartment.
To achieve an adequate reduction in the WBC count, up to 1 L of fluid may be removed,
necessitating the use of a replacement fluid

Erythrocytapheresis Removes a large number of RBCs

(Red Blood Cell along with compatible allogeneic donor RBCs
Exchange) The procedure is most commonly performed in patients with sickle cell disease in order to
decrease the number of hemoglobin S containing RBCs, thereby treating or preventing the
complications (acute chest syndrome, impending stroke, unrelenting painful crisis) associated with
the disease.
The therapeutic goal is to decrease the level of hemoglobin S to less than 30%. This
usually accomplished with a single red blood cell exchange procedure, requiring from 6 to 10 RBC
blood cell volume
The donor RBCs selected for transfusion should be ABO- and Rh-compatible, relatively fresh (less
than 10 days is preferable to allow maximum in vivo survival), leukocyte-reduced, and negative
for hemoglobin S (donor does not have sickle cell trait).
Other indications:
- Treatment of overwhelming malaria or Babesia infections. A 1.5- to 2-volume red blood
cell exchange should significantly decrease the parasite load
- Can be used to remove incompatible
 In TA, fluid must be replaced to maintain appropriate intravascular volume and oncotic pressure
Fluid Replacement
for TA LISTS OF FLUID REPLACEMENT
5 % Human serum albumin (HSA) The most common replacement fluid
Crystalloid (NSS) Used for up to one third of the replacement volume
FFP Contains all the constituents of the removed plasma and thus would
appear to be the optimal replacement fluid for TPE procedures
Usually reserved for treatment of thrombotic thrombocytopenic
purpura (TTP) and related disorders
Used for patients with a preexisting coagulopathy (severe liver disease)
or on those who are scheduled to undergo an invasive procedure (such
as a pretransplant antibody reduction)
May cause citrate toxicity is because of the combined effects of the
anticoagulant in the FFP and the citrate used in the apheresis procedure
itself.
Associated with allergic reaction
HPCs collection HPCs (Hematopoietic progenitor cells), also known as peripheral blood stem cells (PBSCs), are more
through Leukapheresis frequently harvested from peripheral blood by leukapheresis
Because few HPCs are in circulating blood (0.05%), numerous days would be required to collect

marrow to the peripheral blood with cytokines, the number of leukapheresis procedures can usually
be reduced to one or two
The donors receive the cytokine analog, granulocyte colony-stimulating factor (G-CSF)
filgrastim to stimulate the release of more HPCs into the peripheral blood. Less commonly,
granulocyte-macrophage colony-stimulating factor (GM-CSF) is used. The usual dose of

HPCs express the cell surface glycoprotein CD34, and measurement of CD34+ cells in the peripheral
blood prior to collection is typically performed to ensure adequate mobilization has occurred
HES (Hydroxylethyl A common RBC sedimenting agent used to facilitate leukocyte withdrawal during leukapheresis.
starch) During centrifugation of whole blood, granulocytes are found in the buffy coat between the RBC
and plasma layers.
Adding the red cell sedimenting agent, hydroxyethyl starch (HES), allows better separation of layers,
resulting in an improved yield with reduced RBC contamination. It enhances the separation of the
white cells from the red cells during centrifugation, which increases the amount of leukocytes
collected and decreases the amount of red cell contamination in the final product
it allows better separation of layers, resulting in an improved WBC yield with reduced RBC
contamination.
Fatalities associated Rare fatalities occurring during therapeutic apheresis procedures have been reported. The majority of
with apheresis these have been caused by circulatory (cardiac arrest or arrhythmia) or respiratory complications (acute
pulmonary edema or adult respiratory distress syndrome

PREVENTION AND MANAGEMENT OF COMMON DONOR REACTIONS

COMMON DONOR REACTIONS COMMONLY ATTRIBUTED PREVENTION/MANAGEMENT
TO
Lightheadedness Anxiety Reassuring conversation
Weakness Hypoglycemia Try any of the following:
Tingling sensation a.
Palpitations
b. Apply cold, wet towels to neck and forehead
c. Have donor breath into paper bag
d. Provide juice even before donation
Last resort: Discontinue donation
Fainting Anxiety Discontinue donation
Hypoglycemia If necessary, administer glucose solution
Provide juice even donation
Position donor in place protected from a possible fall
Convulsion Anxiety or underlying disease Discontinue donation
Elevate feet
Restrain gently to prevent injury
Maintain airway
Reassure after recovering consciousness
Inform about possible involuntary loss of control of urine or
stool during convulsion
 Cardiopulmonary emergency Underlying heart disease Ventilation
Cardiopulmonary resuscitation
Transfer donor to emergency medical facility
Hematoma Very fragile veins Discontinue if large
Unskilled phlebotomist Apply pressure to site for at least 15 minutes
Uncooperative donor Apply cold packs
Reassure donor

Jet like pulsating bleeding with Inadvertent puncture of artery Discontinue immediately
bright red blood when deep vein punctures are Apply firm pressure over puncture site (10mins)
attempted Apply dressing on site
Follow up donor for additional care
Shooting pain followed by Inadvertent puncture of median Reassure
numbness and tingling in the nerve or cutaneous branches Apply support to the arm
forearm

BLOOD COMPONENTS
*
Heavy/Hard spin
a. 5000g for 5mins (packed RBC, platelet Concentrate)
b. 5000g for 7 mins (cryoprecipitate, cell free plasma)
c.
Light Spin/ soft spin
a. 2000g for 3 minutes (platelet rich plasma)

NOTE
1. Blood Components must be processed within 6 to 8 hours of
collection
2. Blood components are prepared using refrigerated centrifuge
except platelet concentrate

3. Modified Whole blood = A Fresh Whole Blood (FWB) minus

cryoprecipitate and/ or platelets

PRBC
Heavy /
(FNB) Hard spin
*
(PPP) stored @
, FFP
Plasma
Fresh
-18°C
Whole
Blood PRBC
Light / ✓ Platelet 40 -1070mL plasma
w/
Soft Spin •
(PRP ) Hard Concentrate

Plasma spin *
ppp

↓stored @
- 18°C

FFP = Fresh Fmen Plasma FFP

*
Ppp = Platelet Poor Plasma
pRP= Platelet Rich Plasma
•
 Procedure for Preparing Random-Donor Platelets
and Plasma from Whole Blood
1. Maintain the whole blood at 20°C to 24°C before and during
platelet preparation.
2. Set the centrifuge temperature at 22°C. The rpm and time must be
specifically calculated for each centrifuge. It will generally be a short (2 to
3 minute), light (3,200 rpm) spin. This spin should separate most of the
RBCs but leave most of the platelets suspended in the plasma.
3. Platelet preparation should be done in a closed, multibag system.
4. Express off the platelet-rich plasma into one of the satellite bags.
Enough plasma must remain on the RBCs to maintain a 70% to 80%
hematocrit level.
5. Seal the tubing between the RBC and the plasma. Disconnect the
RBC and store it at 4°C.
6. Recentrifuge the platelet-rich plasma at 22°C using a heavy spin
(approximately3,600 rpm for 5 minutes). This will separate the platelets
from the plasma.
7. Express the majority of the plasma into the second satellite bag, leaving
approximately 50 to 70 mL on the platelets. The volume is important to
Procedure for Preparing Random-Donor Platelets
and Plasma from Whole Blood
11. Place the plasma in a protective container and freeze. The plasma
must be frozen in such a way that evidence of thawing can be determined.
Freezing some sort of indentation into the bag, which is visible as long as
the plasma remains frozen, is an easy way to accomplish this. The freezing
container is important because the plastic bag becomes quite brittle when
frozen at low temperatures and can be cracked or broken easily.
12. The plasma must be frozen solid within 8 hours or 24 hours,
depending on which frozen plasma product is to be made. The rate of
freezing for the plasma will depend on the temperature of the
freezer and the amount of air circulation around the plasma.
13. Before freezing, be sure that any tubing segments and the transfusion
ports (ears) of the bag are tucked in or placed in such a manner as to
prevent or minimize possible breakage.
14. The label on the frozen plasma must include all of the standard
information. (See the following section on labeling).
15. The plasma can be stored as FFP, '
_
single-donor plasma frozen within
24 hours (PF24), or liquid recovered plasma. Be sure to record the plasma
-

maintain the pH above 6.2 during storage.
8. Seal the tubing between the bags and separate. Make segments for
both the platelets and the plasma for testing purposes.
9. Allow the platelets to rest undisturbed for 1 to 2 hours at 20°C to 24°C
or until all platelet clumps have been resuspended in the residual plasma.
Be sure the platelet button is covered with the plasma. Gentle
manipulation can be used if needed.
10. Weigh the plasma bag and determine the volume. Record the
volume on the bag.
volume on the bag. Shelf-life is 12 months when stored at 18°C or colder.
16. Once the platelet concentrate has rested, the unit should be placed
on an agitator to maintain gentle agitation during storage.
17. Platelet concentrate shelf-life is 5 days from the date of collection. If
the system is opened, transfusion must occur within 6 hours. The volume,
expiration date, and time (if indicated) must be on the label.
18. All of the units for a single platelet dose (typically 6 to 8 units for an
adult) can be pooled into a single bag before transfusion. Once pooled,
the product must be transfused within 4 hours of pooling. The pooled
unit must be given a unique pool number, which must be placed on the

* label

WHOLE BLOOD Indication:

provides blood volume expansion and RBC mass in acute blood loss
Storage: 1- 6°C

Transport: I -
10°C

Shelf-life: Depends on the anticoagulant (closed system), 24 hours open system

PACKED RBCs Indication:

Increase RBC mass of symptomatic, normovolemic patients
Storage: 1- 6°C
Transport: 1- 10°C
Shelf-life: Depends on the anticoagulant (closed system) , 24 hours open system

LEUKOCYTE-REDUCED Indication:
RBCs -Increase RBC mass in patients with severe and or recurrent febrile transfusion reactions due to
leukocyte antibodies
↳ For px w/ history of
TR All -Increase RBC mass in patients at risk for HLA alloimmunization to HLA antigens or susceptible to
f- NHTR &

CMV
↳ achieved thru centrifugation ,
-absolute WBC count in the unit is reduced to less than 5 × 106 and contains at least 85% of the
washing t filtration → most common
original RBC mass
↳ used to
prevent CMV infx .
Storage: 1-
Shelf-life: Depends on the anticoagulant (closed system), 24 hours open system

WASHED RBCs Indication:

↳ used to
prevent : Increase RBC mass of symptomatic anemic patients with history of allergic, febrile, urticarial and
i. Allergic Transfusion Reaction
2. Anaphylactic Transfusion Reaction anaphylactic reaction
3. Px w/ Aplastic Anemia
4. PX w/ PNH
Storage: 1-
Shelf-life: 24 hours (open system)
FROZEN RBCs Indication:
Storage of rare blood and autologous units
Storage life: - -
Shelf-life: 10 years

PLATELET CONCENTRATE Indication:

For bleeding due to thrombocytopenia or thrombocytopathy
For cancer patient receiving chemotherapy because of induced thrombocytopenia
Storage: 20-
Shelf-life: 5
days
PLATELET PHERESIS Indication:
For thrombocytopenic patients alloimmunized to HLA or platelet antigen
Limit donor exposure in thrombocytopenic patients who acquire long term platelet transfusions
Storage: 20-
Shelf-life: 5 days

FRESH FROZEN PLASMA Indication:

(FFP) Correct multiple coagulation factor deficiency
Replace isolated factor deficiencies when specific component is not available
Reverse effects of warfarin anticoagulant drug
Storage - or -
Shelf-life: 1 Year @ - -

CRYOPRECIPITATE Indication: primary or major use

secondary use

↳ product formed from

thawing FFP PF24 under

or
ref.lt -6°C)
temp followed by hard spin .
deficiency
cryo poor
Plasma
→ gfBBsgMRy Storage -
unite
ryoppt
Components of Cryoppt: Shelf-life:
.

I year
80 U AHF Anti hemophilic factor Condition Shelf-life
F- 11111

150-250mg fibrinogen If stored at - 1 year

VwF If thawed 6 hours
Factor XIII If pooled 4 hours
Fibronectin If thawed and pooled 4 hours

GRANULOCYTE PHERESIS Indication:

Patients with granulocyte dysfunction or myeloid hypoplasia who are unresponsive to antibiotics
Storage: 20 -24°C w/out agitation
Shelf-life: 24 hours

FACTOR IX CONCENTRATE Indication:

primary indication
Prothrombinase concentrate Prevent or control bleeding in patients with hemophilia B or with specific factor deficiencies

present IX. × , V11 , 11 Storage: 1-

Shelf-life varies

FACTOR VIII Indication:

CONCENTRATE Prevent or control bleeding in hemophilia A patients. Used for multiple clotting factor deficiencies
Storage: 1-
Shelf-life: Varies
PLASMA PROTEIN Indication:
FRACTION Replace loss of colloids in hypovolemic shock, severe burns, or for pressure support during
ALBUMIN hypotensive episodes
Storage 2-
Shelf-Life: 5 years

IRADDIATED BLOOD Indication: Essential for patients at risk from TA- GVHD
↳ inactivate T-cells
Recipients of platelets selected for HLA or platelet compatibility
Recipients of donor units from blood relatives
Uses: cesium 137 and cobalt 60 irradiated at 25-35 Gy dose
Expiration: RBC expires 28 days after irradiation or at the end of the storage period, whichever
comes first
Storage temp: 1-

ADDITIONAL INFORMATION
FFP Must be frozen within 8 hours of collection -375mL
7th Ed 200
-

Store up to 1 year at -18C, or 7 years at -65C (this is less common)

A single unit of FFP or PF24, from whole blood collection, should contain 150 to 250 mL
of plasma, approximately/
400 mg of fibrinogen, and -
1 unit of activity per mL of each of the stable
clotting factors. FFP also contains the same level (1 unit/mL) of factors V and VIII. FFP or PF24
prepared from apheresis collections may contain from 400 to 600 mL.
Can store for up to 24 hours at 1-6C after thawing at 37 C
-6C for up to 5 days after thawing. Thawed Plasma
products have reduced FVIII and FV levels. Provides a readily available source of plasma without
needing to thaw (Thawing takes ~20-30 min)
Platelet product
Single donor Platelet Random donor platelet
Prepared from apheresis Prepared from Whole blood Is → Its )

Contains at least 3 × 1011 platelets Contains at least 5.5 × 1010 platelets

(equivalent to 6 to 8 RDP)
Plasma content: 300ml Plasma content: 40-70ml
Can raise platelet count by 30,000 Can raise platelet count by 5,000 -10,000 platelet
-60,000,1¥ ,e+
Note! Platelet percent component retention should be at least 85%
Note! Pla
Component Shelf-life Frozen platelets

At room temp with 5 days Platelets are collected by apheresis, the

agitation cryopreservative dimethyl sulfoxide (DMSO)

2 days is added, and the platelets are frozen at

Frozen platelet 2 years -80°C. The frozen platelets can be stored for
up to 2 years. Prior to transfusion, the
Pooled 4 hours
platelets are thawed and centrifuged to
In an open system 4hours
remove the DMSO
Washed platelets 4 hours

Cryopoor plasma or The supernatant remaining from the production of cryoprecipitate.

cryoprecipitate It is relatively deficient in high molecular weight forms of vWF but retains normal levels of the
plasma vWF-cleaving metalloprotease ADAMTS 13
Cryo-poor plasma must be refrozen within 24 hours and stored at 18°C or colder for 1 year from
the time of collection. This product still contains albumin; factors II, V, VII, IX, X, XI; and
10,917,2 5,11
ADAMTS13 ,

Used to treat patient with TTP

Rhogam
 PRE-TRANSFUSION TESTING
identification and Pretransfusion testing begins and ends with the proper _______ of the patient sample
collection
clerical error A major cause of transfusion-associated fatalities is _____ resulting in incorrect ABO groupings and
transfusion of ABO incompatible blood. Clerical error is the greatest threat to safe transfusion therapy. The
most common cause of error is misidentification of the recipient
Disadvantage of using Serum or plasma may be used for pretransfusion testing. Disadvantages to using plasma include the
plasma /
possible formation of small fibrin clots, /
which may be difficult to distinguish from true agglutination
10ml About ___ of blood is usually sufficient for all testing procedures if there are no known serologic problems.
2 to 5% A ___ saline suspension of RBCs is used for most serologic testing procedures
7 days Both donor and recipient samples must be stored for a minimum of ___ following transfusion
O Rh-negative packed In extreme emergencies, when there is no time to obtain and test a pretransfusion sample, group __ can
cells be used
72 HOURS According to AABB Standards, a pretransfusion specimen for testing and red cell transfusion is valid for
____Hours. After ____ hours, a new sample must be drawn.

COMPATIBILITY TESTING
Serologic aspect of pretransfusion testing; it includes every serologic facet, beginning with donor blood and ending with the
recipient blood sample

COMPONENTS OF COMTABILITY TESTING

1. Identification of the patient and donor and collection of appropriate samples for testing
2. Testing of the donor blood sample
3. Testing of the patient sample and review of past blood bank records
4. Selection of appropriate donor units
5. Crossmatching
6. Re-identification of the patient before infusion of blood

Testing A Donor Sample

ABO Typing, Rh typing, and serological test for different blood transmitted diseases
AABB Standards requires a screening test for unexpected antibodies to RBC antigens on samples from donors who have a history
of transfusion or pregnancy
AABB Standards also requires that the transfusing facility confirm the ABO cell grouping on all units and Rh typing on units
labeled Rh-negative. Repeat weak-D testing is not required

Testing the Patient Sample

A record must be maintained of all results obtained in testing patient samples.
Any discrepancies between previous and current results of the patient must be resolved before transfusion is initiated. A new
sample should be collected from the patient, if necessary, to resolve the problem
ABO, Rh, and unexpected antibody screening test results should be included in the record
If the patient has had a transfusion or has been pregnant within the last 3 months or if the history is unavailable or uncertain,
the sample must be obtained from the patient within 3 days of the scheduled transfusion

An Rh control is indicated when spontaneous agglutination of red blood cells is suspected, such as in patients
typing as AB-positive
In Rh typing, If the diluent Control is used in parallel and gives a positive result, then result of the Rh typing test is invalid. In
er uptake of
autoantibodies or alloantibodies (if the recipient has been recently transfused) is responsible for the positive control. If the DAT
 is positive, accurate Rh typing can sometimes be performed using saline-active or chemically modified Rh blood typing serum
with an appropriate diluent or 8% albumin control
The test for weak D is unnecessary when testing transfusion recipients
dy
screening test is to detect as many clinically significant unexpected antibodies as possible

Selection of Appropriate Donor Blood

When a recipient must be given blood of a different ABO group, only packed RBCs can be given.
Rh-positive blood should not be given to Rh-negative female patients of childbearing age
Transfusion of Rh-negative male patients and female patients beyond menopause with Rh-positive blood is acceptable as long
as no preformed anti-D is demonstrable in their sera. In these

There is no need to provide antigen-negative RBCs for patients whose sera contain antibodies that are reactive only below 37°C,
because these antibodies are incapable of causing significant RBC destruction in vivo.
No compatibility testing is required for the transfusion of platelets, thawed plasma, and cryoprecipitate. Either a current or a
historical ABO grouping is sufficient

CROSSMATCHING
The Purpose of crossmatch is a final check of ABO compatibility and, to a lesser extent, detection of unidentified
Part of compatibility testing, and it is the final step of pretransfusion compatibility testing
It is a final check of ABO compatibility between donor and patient
It may detect the presence of an antibody
Computer crossmatch = This is applicable when at least two group have been
made, at least one on a current sample, and there are no unexpected antibodies
Blood donor and recipient samples used in cross-matching must be stored for a minimum of _____
7 day

Major crossmatching Donor: RBC DROPS

Patient
Patient: SERUM Donor
RBC Serum

Minor Crossmatching Donor: SERUM

PRODS
Patient: RBC Patient Donor
RBC Serum

Example Question
Predict compatibility or incompatibility for the following situation. Assume that the patient has a negative antibody screen and auto-
control
Patient: Group B positive Donor Group O positive
a. Compatible major side crossmatch

b. Compatible minor side crossmatch

c. Incompatible major side crossmatch

d. Not enough information to predict

PHASES OF CROSSMATCHING

A. Immediate Spin Crossmatch / Saline phase/Initial phase

When no clinically significant unexpected antibodies are detected and there are no previous records of such
antibodies, a serologic test (immediate spin) to detect ABO incompatibility is sufficient. This is
accomplis (i.e., immediate spin)
Done at room temp

Uses NSS or 22% albumin enhancement media 30 minutes (30 to 60 minutes)

Can be used for Detection of naturally occurring antibodies/Cold reacting antibodies

The type-and-
unexpected antibodies. The patient sample is then stored in the blood bank refrigerator for future crossmatch if blood
is needed for transfusion

The type and screen, coupled with an immediate spin crossmatch, is referred to as an abbreviated crossmatch.

False reactions may be seen in the presence of other immediate spin-reactive antibodies (e.g., autoanti-I) or in patients
with hyperimmune ABO antibodies, or may be seen when the procedure is not performed correctly (e.g., delay in

ethylenediaminetetraacetic acid to the test system reportedly eliminates some of the false-positive reactions
EDTA
Incompatible immediate spin crossmatches may occur for many reasons and must be resolved prior to issuing units for
transfusion. Possible reasons for incompatibilities and subsequent resolutions include the following:

a) Incorrect ABO grouping of recipient or of donor unit selected

b) Cold-reactive allo- or autoantibody in the plasma not detected in antibody detection tests

c) detected in antibody detection tests Abnormalities in the recipie

-Perform saline replacement and repeat the immediate spin crossmatch. Saline replacement technique involves
removing the plasma from the immediate spin crossmatch and replacing with saline. Rouleaux will disappear while
true agglutination will not

B. Incubation Phase

Done

Usage of enhancement media such as: LISS -> 15 minutes (5 to 15minutes)

Detection of clinically significant antibodies such as IgG

C.Antiglobulin Crossmatching Phase

The antiglobulin crossmatch procedure begins in the same manner as the immediate spin crossmatch, continues to a
37°C incubation, and finishes with an antiglobulin test

Several enhancement media may be applied to boost antigen-antibody reactions. These may include/ /
albumin, low ionic
strength solution (LISS), -
polyethylene glycol, and-
polybrene

crossmatch test. Although current AABB Standards no longer requires an autocontrol, some technologists still find it
useful
 Possible reasons for incompatibilities include the following:

a)

b) Alloantibody to a low-incidence present is on the donor unit red blood cells

c) Warm-

d) Donor unit has a positive direct antiglobulin test (DAT).

Enhancement media /Protein Media/ Potentiators

1. 22% Albumin= works by reducing the zeta potential and dispersing the charges, thus allowing the RBCs to approach each
other.
2. Low ionic strength solution 0.2% sodium chloride = LISS increases the uptake of antibody onto the RBC during the
sensitization phase.
3. Polyethylene glycol = removes water, thereby concentrating any antibodies present.
4. Polybrene, polyvinyl pyrolidone
5. Enzymes

Tipiaz
Zeta potential
The difference in charge density between the inner and outer layers of the ionic cloud that surrounds red blood cells in an
electrolyte solution.
Natural RBC repulsive effect, can be reduced using potentiators for better Antigen-Antibody binding

PAK PAG BINASA MO TO!

Blood components are infused slowly for the first 10 to 15 minutes while the patient is observed closely for signs of a
transfusion reaction
The blood components should then be infused as quickly as tolerated, or at most within 4 hours
Massive transfusion is arbitrarily defined either as the administration of 8 to 10 RBC units to an adult patient
in less than 24 hours, or as the acute administration of 4 to 5 RBC units in 1 hour.
The ure, and temperature) should be monitored periodically during the
transfusion to detect signs of transfusion reaction
Delayed hemolytic transfusion diagnosed 5 to 10 days after transfusion
Intravenous fluid- only (0.9%) isotonic saline or 5% albumin should be used to dilute blood components
Blood filters
First generation fliters 170-260 microns pore size
Screen filter
Standard blood filter- removes gross debris, used for all blood components
Second generation filters 20-40 microns pore size
Micropore screen filter
Removes 75-90% of leukocytes; used only for red blood cells
Third generation filters Adhesion
An adsorption filter; removes 99 to 99.9% of leukocytes; used for both red blood cells
and platelets
Made of polyester or cellulose acetate and will produce a 2- to 4- log reduction of
WBC (<5x106) or platelets, or both

Blood warmers
-
Speed of infusion
-about 200ml blood /hr
-infusion must be completed within 4 hours
MONITORING OF INSTRUMENTS OR EQUIPMENT
Daily When in Use Heating blocks, Water Baths, Donor unit agitators, Scales, Balances, Hemoglobinometer,
8 Microhematocrit centrifuges, Refrigerator and Freezers (continuous monitoring)
Monthly Alarm activation (freezers and refrigerators), centrifuge temperature(refrigerated)
Quarterly Blood warmers, Cell washers (speed, timer), Centrifuge speed timer
Annually Mercury thermometers
Every 4 hours Platelet incubators (enclosed, monitored chambers)

SYNTHETIC VOLUME EXPANDERS

CHARACTERISTIC CRYSTALLOID COLLOID
Intravascular retention Poor Good
Peripheral edema Common Possible
Pulmonary edema Possible Possible
Easily excreted Yes No
Allergic reactions Absent Rare
Cost Inexpensive Expensive
Examples Albumin, Dextran, Hydroxylethyl starch
 TRANSFUSION REACTIONS /TRANSFUSION ADVERSE REACTIONS

A transfusion reaction is defined as any transfusion-related adverse event that occurs during or after the transfusion of whole
blood, blood components, or human-derived plasma products.
A transfusion reaction with signs or symptoms presenting during or within 24 hours of transfusion is defined as an acute
transfusion reaction

A transfusion reaction with signs or symptoms presenting after 24 hours of transfusion is defined as a delayed transfusion
reaction
I
The definition of a DHTR includes a positive DAT 24 hours to 28 days after transfusion with either a positive eluate or a newly
identified alloantibody in the plasma or serum and evidence of hemolysis (Harmening, 7th edition)
2

CATEGORIES/CLASSIFICATION OF TRANSFUSION REACTION

Immediate Delayed
Immunologic Non-Immunologic Immunologic Non-Immunologic
IHTR Immune hemolytic TR
TACO Post transfusion purpura TA-hemosiderosis
Febrile non hemolytic TR Bacterial contamination TA-GVHD Disease transmission
Allergic/ Anaphylactic DHTR
TRALI

IMMEDIATE TRANSFUSION REACTIONS

AHTR Cause: Incompatible blood transfused (commonly ABO)

:
Sign and Symptoms: Fever (most common), chills, flushing, nausea. Dyspnea, chest pain, flank pain, pain
at infusion site, hypotension, shock, hemoglobinemia, hemoglobinuria, DIC, renal failure

Prevention: -Avoid human or clerical error, use a well written procedure manuals, use highly trained clinical
and laboratory staff
FNHTR Cause: Anti-leukocyte antibody I
2
-Another mechanism is closely related to platelet storage changes, which involve the production and release
of biologically active cytokines by the white cells present in the component during storage

Sign and Symptoms: A febrile transfusion reaction is defined as a rise in temperature of 1° C or

greater, possibly accompanied by chills rigor, nausea or vomiting, tachycardia, increase BP, and tachypnea
6
Prevention and Treatment:
1. Transfuse of leukocyte reduced products to patients who have experienced two or more FNHTR.
2. Administer antipyretics to resolve fever
3. Meperidine to resolve rigor but it must be caused with extreme caution
Allergic/Urticarial Cause: ALTR occurs as a response of recipient antibodies to an allergen present in the blood component.
transfusion Due to donor plasma proteins (allergens). ALTR can range from minor urticarial effects to fulminant
reaction anaphylactic shock and death.

Sign and Symptoms: Hives and wheals, etching, erythema, or pruritus

Prevention and Treatment:

1. Use washed RBC
2. Administer anti-histamine
Anaphylactic/ Cause: Attributed to IgA deficiency or absolute IgA deficiency (IgA levels less than 0.05 mg/dL) in patients
anaphylactoid who have developed anti-IgA antibodies by sensitization from transfusion or pregnancy
transfusion ¢
reaction Sign and Symptoms:-Bronchoconstriction (wheezes), r
angioedema (periorbital edema, tongue swelling),
-gastrointestinal symptoms (diarrhea), and cardiovascular
- instability (hypotension, cardiac arrhythmia, loss of
consciousness, shock,cardiac arrest). Anaphylaxis can range from mild urticaria (hives) and pruritus
to severe shock and death.

Two significant features distinguish anaphylactic and anaphylactoid reactions from other types
of transfusion reactions:
1. Fever is absent
2. Clinical signs and symptoms occur after transfusion of just a few milliliters of plasma or plasma-containing
blood components

Prevention and Treatment:

1. Transfuse washed RBCs or platelets or frozen RBCs and plasma from IgA deficient donors
2. Give epinephrine (usually about 0.5 mL of 1:1000 solution) immediately
3. For severe reactions, corticosteroids or aminophylline, or both, may be indicated
TRALI / Cause: Anti-leukocyte antibodies, Anti-HLA class I, and Anti-neutrophil antigen that are present in plasma of
Non-cardiogenic the transfused unit.
pulmonary
edema Transfusion-related acute lung injury (TRALI) consists of an acute transfusion reaction presenting with
✓respiratory distress and severe hypoxemia during or within 6 hours of transfusion in the absence of other
apt 1ns Smb acct
causes of acute lung injury (e.g., aspiration, pneumonia, toxic inhalation, lung contusion, near drowning, severe
sepsis, shock, multiple trauma, burn injury, acute pancreatitis, cardiopulmonary bypass, drug overdose).
This syndrome is now considered the leading cause of transfusion-associated fatalities,
surpassing ABO incompatibility and bacterial contamination (Harmening, 6th edition)
9
Signs and Symptoms: Chills, fever, non-productive cough, acute respiratory distress including dyspnea,
cyanosis, bilateral pulmonary edema on chest x-ray, severe hypoxemia, tachycardia, hypotension

Prevention and Treatment: Use of proper donor selection and Use of leukopoor RBCs unit
Transfusion Cause: Iatrogenic (physician-induced)
associated with
Circulatory Signs and Symptoms: It presents as congestive heart failure during or shortly after transfusion. Signs and
overload (TACO) symptoms can include dyspnea, orthopnea, cyanosis, tachycardia, elevated blood pressure, pulmonary
edema, jugular venous distention, pedal edema, and headache 9

The laboratory assay of brain natriuretic peptide (BNP), consisting of the measurement of a peptide
secreted from the ventricles in response to increased filling pressures and a marker of congestive heart failure,
may be used to aid in the diagnosis of TACO. A post-transfusion to pretransfusion BNP ratio of 1.5, with a
post-transfusion level equal or greater than 100 picograms per milliliter as a cutoff point, provides
a sensitivity of 81% and a specificity of 89% for diagnosis of TACO.
TACO is the second most common cause of transfusion related deaths reported to the FDA, and the incidence
appears to be increasing (Harmening, 7th edition)

Prevention and Treatment:

1. Identify people at risk: Children, Elderly, Patient with cardiac disorder
2. Transfuse blood slowly
3. Treatment consists of putting the patient in a sitting position, administering supplemental oxygenation,
and starting diuresis.
Bacterial Cause: Caused by psychrophilic bacteria
contamination / Transfusion-associated sepsis occurs when bacteria are introduced to the patient via a contaminated blood
Transfusion product, manifested by an
•increase in body temperature or more than 2°C,- /hypotension
rigors, and
associated Sepsis
Bacterial contamination can occur during, ,
phlebotomy, preparation and processing of blood components, and
during
/ thawing.

Common sources of bacterial contamination are from donor skin or from asymptomatic donor blood.
Platelets have been the most frequent source of septic transfusion reactions because room temperature
storage promotes bacterial growth.

Yersinia enterocolitica
Pseudomonas spp.
Propionobacterium acnes
Bacillus cerues,
Staphylococcus epidermidis
According to CDC, it is the Most common cause of death by bacterial
contaminated blood components
Most common isolate found in RBC units
Second most common isolate found of RBC units
Common isolate of human skin, was the most common bacterial
contaminants in RBC
Most frequently recovered from donated blood and contamination of
platelet
*Staphylococcus epidermidis or Staphylococcus aureus are the most
common bacterial contaminants of blood (Harmening, 7th edition)

Staphylococcus aureus accounts for the greatest number of deaths due to blood product contamination
reported to the FDA from FY2011 to 2015, all of which were platelet products (Harmening, 7th edition)

Signs and Symptoms: Fever, rigor,chills, hypotension, tachycardia, shock, DIC, hemoglobinuria,
hemoglobinuria, renal failure

Prevention and Treatment:

1. Administer IV antibiotics
2. Carefully inspect blood products before distribution for transfusion
3. Proper sterilization technique
4. Visual inspection of units (check for clots, and changes of color of plasma, or hemolysis)
5. Phlebotomy diversion consists of collecting the first 20 to 30 mL of blood in a separate container to
be used for testing. This reduces the quantity of skin contaminants entering the unit during
phlebotomy and appears to be very effective in reducing Staphylococcus species contamination.

NOTE:
1. Brown plasma, purple plasma, Clots, and Hemolysis, blood unit should not be used for transfusion because
they are signs of bacterial contamination
2. Green color plasma in blood unit is usually associated with biliverdin
3.Platelet concentrate is the most common blood component associated with transfusion sepsis.

Transfusion-related Cause:
adverse events -Mechanical damage through infusion of blood in small bore needle
during massive -Thermal trauma
transfusion and -osmotic or chemical change by addition of hypotonic or hypertonic solutions or drugs
Physical, or -Citrate toxicity
-Potassium toxicity
Chemical Hemolysis NOTE:
during transfusion 1. Massive transfusion is defined as the replacement of one total blood volume in 24 hours or the
replacement of 50% of the blood volume in 3 hours
2. The chief metabolic effects of transfusion involve citrate toxicity and hyperkalemia (increased
potassium).
3. hypocalcemia and/
Citrate toxicity can cause✓ hypomagnesemia. In addition, metabolic alkalosis can
develop from bicarbonate produced from citrate metabolism.
4. Hyperkalemia is very uncommon in massive transfusion in adults, but it is of special concern in
neonatal transfusions, especially in premature infants
5.
units (less than 7 10 days old)
Signs and Symptoms: Asymptomatic, hemoglobinuria, DIC and renal failure (rare)
1. Citrate toxicity: Hypocalcemia, tingling of lips or fingertips, Twitching or tremors, Shivering, Muscle
contractions (carpopedal spasms involving hands or feet), EKG abnormalities: prolonged QT interval
2. Potassium toxicity: Muscular weakness, , EKG abnormalities: peaking of
T waves, prolonged P-R interval, Ventricular fibrillation, Cardiac arrest

DELAYED TRANSFUSION REACTION

DHTR Cause: Incompatible blood transfusion especially KIDD BGS (most common)

Signs and symptoms: Fever, decreased haptoglobin level, mild jaundice, flu-like symptoms, pallor

Prevention and Treatment

-
- tibody status in permanent laboratory protocol
-Administer antigen negative blood for all subsequent transfusion
TA-GVHD Cause: delayed immune transfusion reaction due to an immunologic attack by viable donor lymphocytes
Transfusion (T cells) contained in the transfused blood component against the transfusion recipient.
associated-graft -Recipient who are immunocompromised or with immunodeficiencies, with cancer, or infants are at risk
versus host disease
Signs and Symptoms: Fever, maculopapular skin rash, scleroderma-like, sicca syndrome,
interstitial pneumonitis, pancytopenia, abnormal liver function (elevated liver function test such as
enzymes), diarrhea with bloody stool

Prevention and Treatment:

1. Usage of irradiated blood products. AABB Standards requires a minimum dose of 25 Gy (2,500 cGy)
delivered to the central portion of the container and a minimum of 15 Gy (1,500 cGy) dose elsewhere
in the container to the products given to at-risk patient populations
2. Usage of immunosuppressive medications
3. Hematopoietic stem cell transplantation

Three conditions must exist for TA-GVHD to develop in a recipient:

1. HLA antigen difference between donor and recipient
2. presence of donor immunocompetent cells in the blood component
3. recipient incapable of rejecting the donor immunocompetent cells
Post transfusion Cause: Anti-platelet antibodies / anti-HPA1(human platelet antigen1) antibodies
purpura Post-transfusion purpura (PTP) is a rare transfusion reaction in which there is a severe and sudden drop in
the platelet count, usually occurring 5 to 10 days after transfusion due to alloimmunization to platelet-
specific antibodies from prior transfusion or pregnancy.
 Signs and Symptoms: Profound self-liming thrombocytopenia, generalized purpura, bleeding. PTP is a
self-limiting syndrome with the platelet count usually returning to normal within 2 weeks

Prevention and Treatment:

1. Difficult to prevent
2. Clinicians should have thorough patient history and history of adverse reactions on previous
transfusion
Iron overload / Cause: Iron overload is a delayed, nonimmune complication of transfusion, presenting with multiorgan
Transfusioninduced (i.e., liver, heart, endocrine organs) damage secondary to excessive iron accumulation
Hemosiderosis
-Population at risk:r
sickle cell anemia,rhemoglobinopathies,
/thalassemia,✓patient who are transfusion
dependent

Note: Each unit of red blood cells contains approximately 250 mg of iron. After 10 to 15 red cell
transfusions, excess iron is present in the liver, heart, and endocrine organs

Signs and Symptoms: Muscle weakness, weight loss, mild jaundice, fatigue, cardiac arrhythmias, mild
diabetes, and multi-organ failure

Prevention and Treatment:

1. Administration of Iron chelating agent such as deferroxamine or desferioxamine
2. Transfusion of neocytes (young RBCs)

Transfusion Cause: Microorganisms such as HBV (most common), HIV, HTLV, Syphilis, Malaria, west Nile virus, EBV
Transmitted
diseases Must know
1. HBV DNA is the first marker to appear and can be detected by polymerase chain reaction (PCR)
testing before HBsAg reaches detectable levels
2. Hepatitis B immune globulin injections (HBIG) and the vaccine given soon after exposure or within
hepatitis 12 hours of birth, if the mother is infected, may prevent infection.
3. HCV is the most common hepatitis associated with chronic carriers and chronic liver disease
4. Hepatitis D (HDV) is a defective, single-stranded RNA virus that is found only in patients with HBV
infection. It requires HBsAg in order to synthesize an envelope protein and replicate
5. HTLV-I was the first retrovirus to be associated with a human disease. That association
was with adult T-cell lymphoma/ leukemia (ATL), a highly aggressive, mature T-cell non-
lymphoma with a leukemic phase
HTW
6. HTLV-I is also associated with the progressive neurological disorder known as HTLV-I-associated
myelopathy or tropical spastic paraparesis (HAM/TSP).
7. The risk of transmitting HTLV through infected blood is estimated to be between 10% to 30%.
Leukocyte reduction and serologic testing greatly reduces the risk of HTLV transmission
8. WNV is capable of crossing the blood-brain barrier and can cause what is known as West Nile
WNV
encephalitis, West Nile meningitis, or West Nile meningoencephalitis
9. Those at the highest risk of a CMV infection are fetuses and individuals receiving allogeneic marrow
transplants, Intrauterine transfusions with CMV-positive components is also a high risk to the fetus
10. Approximately 1% of all newborns are infected with CMV, but most are asymptomatic at birth
CMU 11. CMV infection from transfusion is between 1% to 3%.
12. CMV is the most frequently transmitted virus from mother to fetus.
13. Blood and blood components are not universally screened for CMV because of the
generally benign course of this disease and the high percentage of virus carriers. To
prevent CMV transmission, leukocyte-reduced blood or blood from seronegative donors
may be used.
 14. In adolescence and young adulthood, EBV causes infectious mononucleosis in 30% to 50% of
patients
EBV 15. EBV has been called the
the oropharynx, possibly in infected B cells. The virus is shed in the saliva and is most frequently
associated with infectious mononucleosis
16. Human B19 parvovirus (B19) is a small, single-stranded DNA nonenveloped virus.3 It causes a

the face and a lacy red rash when occurring on the trunk and limbs.
17. Common sources of bacterial contamination include donor skin and blood. Less common
sources are the environment and disposables
18. Most cases of babesiosis are asymptomatic. Symptomatic patients usually develop a malaria-
type illness characterized by fever, chills, lethargy, and hemolytic anemia
19. Currently there is no screening test for blood donors with Babesiosis.
20. smears
stained with Giemsa or Wright stain may be examined for the characteristic C- or U-shaped
trypomastigote
21.
immunofluorescence, and ELISA.
22. The test, called the Abbott Prism Chagas, is highly sensitive and specific for the detection of
antibodies to T. cruzi
23. Plasmodium can survive in blood components stored at room temperature or 4°C for at least a week,
and deglycerolized RBCs can transmit disease
24. Some individuals have a natural immunity to certain species of malaria, caused by a
genetic alteration in their RBCs. These include persons who have "
sickle cell anemia or
G6PD deficiency, or /
trait,- RBCs that lack the Duffy blood group antigen
25. Creutzfeldt-Jakob disease (CJD) is one of the transmissible spongiform encephalopathies (TSE).
These are rare diseases characterized by fatal neurodegeneration that results in spongelike lesions
in the brain.
Trypanosoma cruzi, -
26. Parasite that can be transmitted thru blood transfusion=- Babesia
microti, - , Leishmania spp., and-
Toxoplasmosis, Microfilaria- Plasmodium spp. (malaria)
a-
TRANSFUSION REACTION INVESTIGATION
During transfusion reactions

1. Stop the transfusion

2. Keep IV open with saline

3. Perform clerical check for ID errors

4. Contact treating physician

5. Monitor /record vital signs

WORK UP TRANSFUSION INVESTIGATION

1. Examination of pretransfusion clotted blood specimen, an EDTA anti-coagulated post transfusion blood specimen (perform
DAT), and the blood bag
2. Perform gram stain on the blood in the bag and culture
3. Repeat the ABO/Rh typing, Antibody screen, and crossmatching
4. Examination of post transfusion urine
5. Measurement of Hct/Hb at frequent interval
 HEMOLYTIC DISEASE OF THE FETUS AND NEWBORN (HDFN)
Clinical condition caused by the immune destruction of fetal or neonatal RBCs due to attachment of maternal antibody(IgG) to
an antigen present on the membrane of RBCs

The mother can be stimulated to form the antibodies by previous pregnancy or transfusion and sometimes during the second
and third trimester of pregnancy

Rh BGS is considered as the most severe cause of HDNF.

Rh BGS was considered as the most common cause of HDNF but the incidence of the disease caused by anti-D has steadily
decreased since 1968 with the introduction of Rh-immuneglobulin (RhIG).

In addition to the use of RhIG(Rh immuneglobulins) / rhogam , many other advances have been made in the diagnosis and
treatment of HDFN. Ultrasonography, Doppler assessment of middle cerebral artery peak systolic velocity, cordocentesis, allele-
specific gene amplification studies on fetal cells in amniotic fluid, fetal DNA analysis in maternal plasma, and intravascular
intrauterine transfusion have greatly increased the success of accurately diagnosing and adequately treating this disease.

There are several factors that affect immunization and severity of HDFN, including antigenic exposure, host factors,
immunoglobulin class, antibody specificity, and influence of the ABO group

BLOOD SYSTEMS CAUSING HDN

1. Anti-D (most immunogenic), either alone or in combination
with Anti-C or and Anti-E
2. Other Rh antibodies, such as anti c
3. Other blood groups: Anti- Kell, Anti- Duffy, Anti- Kidd
4. ABO antibodies (especially from blood type O mother)

Of the non Rh system antibodies, anti-Kell is considered the most clinically significant in its ability to cause HDFN. Kell antigens are
present on immature erythroid cells in the bone marrow, so severe anemia occurs not only by destruction of circulating RBCs but also
by precursors
RH BLOOD GROUP SYSTEM HDFN
In Rh(D) HDFN, the Rh-positive firstborn infant of an Rh-negative mother usually is unaffected because the mother has not yet
been immunized
When D antigen is inherited from the father, these fetal cells immunize the mother and stimulate the production of anti-D. Once
the mother is immunized to D antigen, all subsequent offspring who inherit the D antigen will be affected
As little as 1 mL of fetal RBCs can immunize the mother
The maternal anti-D crosses the placenta and binds to the fetal Rh-positive cells. The sensitized RBCs are destroyed (hemolyzed)
by the fetal monocyte-macrophage system, resulting in anemia.
When the mother is ABO incompatible with the fetus (major incompatibility), the incidence of detectable fetomaternal
hemorrhage is decreased. Investigators noted many years ago that the incidence of D immunization is less in mothers with
major ABO incompatibility with the fetus. The ABO incompatibility protects somewhat against Rh immunization.

RHOGAM or RhIg
Rh-immune globulin (RhIg) is a solution of concentrated anti-Rho(D).
Prepared from pooled human plasma of patients who have been hyperimmunized and contains predominantly IgG anti-D
RhIg has two primary uses: treatment of ITP and prevention of Rh hemolytic disease of the newborn.
RhIg should be administered ___
72 hours after childbirth

The administered RhIG attaches to the fetal Rh-positive RBCs in the maternal circulation. The antibody-coated RBCs are
removed by the macrophages in the maternal spleen.
There is no risk of transmission of the viral disease hepatitis A and B and HIV with the administration of RhIG
Full-dose Micro-dose
Contains 300ug of Anti-D Contains 50 ug anti-D
Protects up to 30ml of D+ Whole blood Protects up to 5ml of D+ Whole blood
dose (300 ug) is During the first 12 weeks of pregnancy, a 50-ug dose
indicated for abortion or miscarriage in D-negative of RhIg is indicated for D-negative females for abortion
women or miscarriage, or ectopic pregnancies
Computation!!
NOTE: A Kleihauer-Betke test or flow cytometry is used to quantitate the number of fetal Rh-

circulation as a result of a fetomaternal hemorrhage.

Problem: Kleihauer betke reveals 3% fetal cells. Determine the RhIg vials # fetal cells
✗ Maternal blood voi .

Formula: Volume of fetomaternal hemorrhage (ml) % fetal # of Maternal cells

go or
cells ✗

30

1.Vol of FMH = %fetal cells x 50

2. Alternative formula for the Volume of FMH = # of fetal cells x Maternal blood volume
Number of Maternal cells

2. Number of RhIg vials = (3x50) / 30 = 5 vials +1 vial for safety = 6 VIALS

Kliehauer Betke test (Acid Elution Test)

In the Kleihauer-Betke test, a maternal blood smear is treated with acid and then stained with counterstain. Fetal cells contain fetal
hemoglobin (Hgb F), which is resistant to acid and will remain pink. The maternal cells will appear as ghosts. After 2,000 cells are
counted, the percentage of fetal cells is determined, and the volume of fetal hemorrhage is calculated
ABO BLOOD GROUP SYSTEM HDFN
ABO incompatibility between the mother and newborn infant can
cause HDFN. Maternal ABO antibodies that are IgG can cross the
placenta and attach to the ABO-incompatible antigens of the fetal
RBCs.
Example is when a Mother is Blood type O, and the Fetus/Newborn
is either Blood type A, B, or AB.
Destruction of fetal RBCs leading to severe anemia is extremely rare
The disease is manifested by the onset of hyperbilirubinemia and
jaundice within 12 to 48 hours of birth. The increasing levels of
bilirubin can be treated with phototherapy.
As the incidence of HDFN caused by Rh(D) has declined, ABO
incompatibility has become the most common cause of
HDFN
Microspherocytes and increased RBC fragility in the infant are
characteristic of ABO HDFN, but not of Rh HDFN
When a newborn develops jaundice within 12 to 48 hours after birth, various causes of jaundice need to be investigated, and
ABO HDFN is only one. The DAT on the cord or neonatal RBCs is the most important diagnostic test

DIAGNOSIS AND MANAGEMENT

ABO, Rh, and Antibody screen The prenatal specimen must be typed for ABO and Rh. The antibody screening method must be
of the Mother able to detect clinically significant IgG alloantibodies that are reactive at 37°C and in the
antiglobulin phase
Antibody identification If the antibody screen is reactive, the antibody identity must be determined. Follow-up testing will
depend on the antibody specificity. Cold reactive IgM antibodies such as anti-I, anti-IH, anti-Lea,
anti-Leb, and anti-P1 can be ignored. Lewis system antibodies are rather common in pregnant women
but have not been reported to cause HDFN.
Paternal genotype and
Genotype corresponding antigen. If the mother has anti-D and the father is D-positive, a complete Rh
phenotype can help determine his chance of being homozygous or heterozygous for the D antigen
Fetal DNA testing If the mother has anti-D and the father is likely to be heterozygous for the D antigen, amniocentesis
determine
whether the fetus has the gene for the D antigen.
Antibody titers The relative concentration of all antibodies capable of crossing the placenta and causing HDFN is
determined by antibody titration.
For the recommended method, 16 is considered the critical titer.
If the initial titer is 16 or higher, a second titer should be done at about 18 to 20 weeks gestation.
A titer reproducibly and repeatedly at 32 or above is an indication for color Doppler imaging to assess
middle cerebral artery peak systolic velocity (MCA-PSV) after 16 weeks gestation

DIRECT ANTIGLOBULIN TEST

The most important serologic test for diagnosing HDFN in the Newborn or infant is the DAT with anti-IgG reagent. A positive
e well with
the severity of the HDFN
Cordocentesis /
Percutaneous
Umbilical cord Blood
sampling (PUBS)
MCA-PSV
Cordocentesis, intrauterine transfusion, and amniocentesis have several risks, including infection, premature labor,
and trauma to the placenta, which may cause increased antibody titers because of antigenic challenge to the mother through
fetomaternal hemorrhage
INTERVENTIONS / TREATMENT OF HDN
Intrauterine transfusion
Intrauterine transfusion
performed by accessing the fetal
umbilical vein (cordocentesis)
and injecting donor RBCs directly
into the vein
Exchange Transfusion
The use of whole blood or
equivalent to replace
Exchange transfusion is rarely
required because of advances in
phototherapy and the use of
IVIG.
PHOTOTHERAPY
Using high-resolution ultrasound with color Doppler enhancement of blood flow, the umbilical vein is
visualized at the level of the cord insertion into the placenta. A spinal needle is inserted into the
umbilical vein, and a sample of the fetal blood is obtained
-
hemoglobin,-
The fetal blood sample can then be tested for- hematocrit, -
bilirubin, blood type,
direct - phenotype and genotype.
-antiglobulin test (DAT), and antigen
The measurement of the fetal middle cerebral artery peak systolic velocity (MCA-PSV) with color Doppler
ultrasonography can reliably predict anemia in the fetus.
MCA-PSV is noninvasive and poses no adverse effects for the fetus.
Transfusion of packed RBCs to the fetus
Performed in Utero
is
The goal of intrauterine transfusion is to maintain fetal hemoglobin above 10 g/dL
SELECTION OF RBCs
Fresh as possible (Less than 5 or 7 days old), Group O negative, CMV negative, Hgb S negative,
irradiated
Criteria in Performing Intrauterine Transfusion
1. MCA-PSV indicates anemia.
2. Fetal hydrops is noted on ultrasound examination
3. Cordocentesis blood sample has hemoglobin level less than 10 g/dL.
4.
Removal of infant RBCs coated with maternal antibody and replacement with antigen
negative RBCs
Performed in neonatal period
the
Exchange transfusions are used primarily to remove high levels of unconjugated
bilirubin and thus prevent kernicterus. Premature newborns are more likely than full-term
infants to require exchange transfusions for elevated bilirubin because their livers are less
able to conjugate bilirubin.
Other advantages of exchange transfusion include the -
removal of part of the circulating
removal of sensitized RBCs, and-
maternal antibody,- replacement of incompatible RBCs with
compatible RBC
SELECTION OF RBCs
Fresh as possible (less than 5 or 7days old), Group O negative or group specific, CMV negative, Hgb
S negative, irradiated
After delivery, the neonate can develop hyperbilirubinemia of unconjugated bilirubin. Phototherapy at
460 to 490 nm is used to change the unconjugated bilirubin to isomers, which are less lipophilic and
less toxic to the brain. In infants with mild-to-moderate hemolysis or history of intrauterine transfusion,
phototherapy is generally sufficient
 Aliquotted red cell is the product most often transfused during the neonatal period or in infants younger than 4 months of
age.

The anticoagulant most often used for neonate transfusions is CPDA-1.

Group O Rh-negative
Neonatal transfusion
Blood for intrauterine transfusion must be compatible with maternal antibodies capable of crossing
the placenta. If the ABO and Rh groups of the fetus have been determined, group specific blood could be
given provided that there is no fetomaternal ABO or Rh incompatibility. If the ABO and Rh groups of the
fetus are not known, then _____ RBCs should be selected for the intrauterine transfusion
Blood for an exchange or regular transfusion of a neonate (younger than 4 months of age) should be

or AHG

RETENTION OF DONOR RECORDS

Donor Records Retention Time (Years)
Donor ABO/Rh 10 years
Donor antibody screen 10 years
Informed consent for donation 10 years
Physical examination 10 years
Medical history information 10 years
Identification number of donor unit 10 years
Viral marker testing results 10 years
Quarantine of donor unit 10 years
Repeat testing of donor blood 10 years
ID of donor-processing tech 10 years
Plt count for frequent platelet apheresis 10 years
Sedimenting agent of leukapheresis 10 years
Notification of abnormal results 10 years / indefinite
Medical director approval for donation interval 5 years

QUALITY MANAGEMENT AND COMPLIANCE IN BLOODBANK (Harmening 7th Edition)

Blood banks must provide quality to their customers in many forms, including;
1. Safe, satisfying donation experiences for blood donors.
2. Accurately labeled and tested blood components provided to transfusion services.
3. Timely, accurate transfusion services provided to physicians and other health-care personnel.
4. Safe and efficacious blood transfusions to patients

Compliance programs evaluate how effectively the facility meets regulatory and accreditation requirements by detecting errors,
deficiencies, and deviations
Compliance also called surveys or assessments
inspections applicable requirements and are usually conducted every 2 years
Quality management Activities of the management function that determine the quality policy
The fundamental components of quality management are quality control (QC), quality assurance
(QA), and the quality management system (QMS).
Quality System Organizational structure, procedures, processes, and resources needed to implement quality
management
Quality Assurance Planned, systematic activities implemented within the quality system to provide confidence that
requirements for quality will be fulfilled
Quality Control Operational techniques and activities used to fulfill the requirements for quality Operational techniques
and activities used to fulfill the requirements for quality
Medical director The ____ is responsible for review, approval, and signing the Quality control plan before patient testing
and reporting of results commences
Quality management The QMS essentials are in a logical order that can be explained as follows for any new or changed product
essentials or service. The blood center or transfusion service organization develops its mission, vision, values, goals,
and objectives around the products and services it plans to offer

QUALITY MANAGEMENT SYSTEM ESSENTIALS

The QMS essentials are in a logical order that can be explained as follows for any new or changed product or service. The blood
center or transfusion service organization develops its mission, vision, values, goals, and objectives around the products and
services it plans to offer
1. Organization There should be an organizational chart showing the following:
personnel by job title

2. Customer focus Although both blood centers and transfusion services work on behalf of patients, the real customer of
these facilities is the entity or person who receives and must be satisfied with a product or service
a. For blood centers the customers are the donors
b. For transfusion services, the customers are the physicians and nurses
-Physicians want the blood transfusions they order to occur in a timely manner
-Nurses want the correctly issued blood components in a timely manner for administration to
patients
3. Facilities and safety -In hospitals, the Joint Commission mandates an environmental control program that addresses all
significant environmental issues for facility management and maintenance, such as temperature
control, electrical safety, fire protection, and so forth
-any blood bank that performs irradiation of blood components must also have a radiation safety
program and document appropriate training.
4. People Quality begins and ends with people.
Rather, a quality problem is almost always due to a faulty process
5. Purchasing and In hospital-
inventory responsible for vendor contracts and purchasing decisions.
6. Equipment Before putting equipment into operation, the blood bank needs a process to ensure that installation,
operational, and performance qualifications for the piece of equipment have been met. Installation
qualification demonstrates that the equipment is correctly installed; operational qualification ensures
the equipment operates as intended; and performance qualification evaluates the personnel.

7. -A process can be defined as a set of interrelated resources and activities that transforms inputs into
Process Management/ outputs
Control -Process control is a set of activities that ensures a given work process will keep operating in a state
that is continuously able to meet process goals without compromising the process itself
-Total process control is the evaluation of process performance, comparison of actual performance to
a goal, and action taken on any significant difference
8. Flow charts is a sequential flow of the elements of total process control. An effective tool for understanding any
process is the flowchart.
9. Standard operating -Provide instructions for each activity in the larger process.
procedures (SOPs) -Orientation to a blood bank laboratory begins with reading the standard operating procedure
In Blood bank (SOP) manuals. These manuals, usually located at the workbench and accessible to all personnel,
contain information outlining the operations of the laboratory; details on how, when, and why
particular activities are done; and procedures for all tests performed. SOP manuals are integral
components of any blood bank -assurance program
 -SOPs are reviewed at least annually (YEARLY) and updated on a regular basis to reflect changes
in operations and implementation of new regulations
10. Documents Are approved information contained in a written or electronic format
11. Records capture the results or outcomes of performing procedures and testing on written forms or electronic
media, such as manual worksheets, instrument printouts, tags, or labels
12. Assessments
requirements at a single point in time. Internal and external assessments are used for measuring and
monitoring performance and identifying opportunities for improvement
13. Internal audit A very effective assessment tool
14. LEAN a process improvement methodology that helps to identify non-value-added activities that are
considered waste.

A computer system includes three major components: hardware, software, and people. Hardware components are the
physical pieces of equipment. Software is a set of instructions written in computer language that tells the computer how to operate
and manipulate the data. The people interface with the hardware to enter the data that are manipulated by the software.

Common Classifications of Various Types of Non-Conformances

Type Definition
Accident

Adverse reaction Complications that occurred to the donor during or after the donation process or to the recipient of
transfused blood components
Complaint Expression of dissatisfaction from internal customers (physicians, employees) or external customers
(donors, patients)
Discrepancy Difference or inconsistency in the outcomes of a process, procedure, or test results
Error Nonconformance attributable to a human or system problem, such as a problem from failure to follow
established procedure or a part of a process that did not work as expected
Postdonation The receipt of information (call or information letter) from a donor with additional details regarding his or
her donation, such as subsequent illness or neglecting to mention an illness or medication

S MORE

--GOBLESS FUTURE RMTs

-Irekevin T. Aytona