MEDICAL TECHNOLOGY LICENSURE EXAM - ISBB

BLOOD BANKING AND TRANSFUSION MEDICINE

Lecturer: Sir Edmund Francisco, RMT, MLS (ASCPi)
By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King

BLOOD BANKING AND TRANSFUSION MEDICINE ABO AND H BLOOD GROUP SYSTEM (ISBT No. 001 and 018)
- Blood Banking - Discovered by Karl Landsteiner
o includes activities, procedures, and tests done to ensure blood for - The most important of all blood groups
transfusion is properly collected, preserved, stored, and dispensed for - The ONLY blood group system in which individuals have antibodies in their
later use in blood transfusion. serum to antigens that are absent from their RBCs (naturally occurring
- Transfusion Medicine antibodies)
o is a branch of medicine that is concerned with transfusion of blood
components, including proper selection and utilization of the BLOOD GROUP ANTIGENS ON CELL ANTIBODIES IN PLASMA
aforementioned in the treatment or prevention of disease. A A Anti-B
- Food & Drug Administration (FDA) B B Anti-A
o the governing body that inspects blood banks every year (annually) AB A and B None
because blood is considered both as a biologic product and as a drug O None Anti-A and B

HISTORY - Transfusion of the wrong ABO group remains the leading cause of death in
hemolytic transfusion reaction fatalities (FDA)
PERSONAGE - ABO incompatibility can cause the most severe HTR
YEAR/S SIGNIFICANT EVENTS
INVOLVED - In 2009, according to the FDA, TRALI was the most common cause of death
In relation to Transfusion
First recorded blood transfusion ABO GENES
Pope Innocent VII
performed - Genes code for specific glycosyl transferases that add sugars to a basic
(died of severe massive
1492 (PS) precursor substance (paragloboside/glycan).
intravascular
Physician: Giacomo di San - The sugars added to the PS determine what blood group an individual
coagulopathy)
Genesio acquires:
1616 William Harvey Discovery of the circulatory system o O gene: Amorphic; It does NOT code for any enzymes and is merely
Animal to Animal transfusion (Dog a representation of the absence of A and B genes/antigens
1665 Richard Lower
to Dog) o A gene: Higher concentrations of transferases than B gene. Has
Jean Baptiste Animal to Human transfusion 810,000 to 1,170,000 Ag sites on an A1 adult RBC.
1667
Denis/Denys (Sheep to Human) o B gene: has 610,000 to 830,000 Ag sites
First successful human blood o A&B genes: B enzyme compete more efficiently for the H substance
1818/ transfusion performed on a woman than the A enzyme; A-600,000 sites & B - 720,000 sites
James Blundell
1829 suffering from postpartum
hemorrhage INHERITANCE OF ABO BLOOD GROUPS
First to work on blood transfusion
and blood preservation techniques
1941 Charles Drew
Director of first American Red
Cross blood bank at Presbyterian
Hospital
In relation to Anticoagulants / Devices
Sodium Phosphate (Na3PO4) as
1869 Braxton Hicks
anticoagulant
Sodium Citrate (Na2C6H3O7) as
1914 Albert Hustin
anticoagulant
Determined minimum non-toxic
1915 Richard Lewisohn amount of Citrate needed to
prevent coagulation
Citrate-Dextrose (CD) as - Bernstein (1924) – described the theory for the inheritance of the ABO
1916 Rous and Turner
anticoagulant groups
Acid Citrate Dextrose (ACD) as - Codominance expression - an individual inherits one ABO type B gene
1943 Loutit and Mollison
anticoagulant from each parent and that these two genes determine which ABO antigens
Utilized glycerol to extend RBC are present on the RBC membrane (follows simple Mendelian genetics)
1950 Audrey Smith
lifespan by 10 years
Citrate Phosphate Dextrose (CPD) Father’s blood type
1957 Gibson A B AB O
as standard preservative at present
First Vein-Vein Transfusion using A, O
Edward Lindemann A A, B, AB, O A, B, AB A, O
special cannulas and syringes
Mother’s blood type

Child’s blood type

A, B, AB, O
Unger Syringe valve apparatus B B, O A, B, AB B, O
In relation to major Blood Group Systems A, B, AB
ABO blood groups discovered AB A, B, AB A, B, AB A, B

1901 Karl Landsteiner

Wrote “The specificity of serologic O A, O B, O A, B O
reactions”
Alfred von Decastello Defined the fourth ABO blood
1902
and Adriano Sturli group: AB - The ABO gene is: autosomal
Karl Landsteiner and MN and P systems discovered - A and B blood groups are: dominant
1927
Philip Levine - A and B group genes are: codominant
Karl Landsteiner Rh blood group discovered - The O gene is considered as an amorph, as no detectable antigen is
1940
Alexander Wiener produced in response to the inheritance of this gene
Others - The group O phenotype is an autosomal recessive trait with the
1937 Cook County Hospital 1st blood bank (Chicago) inheritance of two genes that are non-functional
Irvin Memorial Blood 1st community based blood center
1941
Bank (San Francisco)
PHENOTYPE VS GENOTYPE NOMENCLATURE
- Genotype: is the sequence of DNA that is inherited (individual’s pair of allele BLOOD GROUP JANSKY (CZECH) MOSS (US)
genes together). O I IV
o Homozygous genotype: the presence of two identical alleles A II II
o Heterozygous phenotype: the inheritance of different alleles B III III
- Phenotype: is anything that is produced by the genotype, including an AB IV I
enzyme to control a blood group antigen (individual’s outward
characteristics). ABO ANTIGENS
- Form as early as the 37th day of fetal life, though RBCs of neonates carry
GENE CHROMOSOME only 25-50% of the number of antigenic sites found on adult RBCs
ABO 9 - Full expression of antigens is found at 2-4 years of age and remains
H 19 constant throughout life
Se 19 - The antigens can be found on RBCs, endothelial cells, platelets,
lymphocytes, epithelial cells, and secretions (plasma soluble substances)
PHENOTYPE POSSIBLE GENOTYPES
O OO ABO Antigens ABO Antigens
A1 A1A1, A1A2, A1O on RBCS on Soluble Substances
A2 A2A2, A2O Glycoproteins, Glycolipids or Glycoproteins
B BB, BO Glycosphingolipids
A1B A1B Synthesized only on Primarily synthesized on
A2B A2B type 2 precursor chains - type 1 precursor chains - refer to a
- Example: refer to a β1→4 linkage β1→3 linkage
o Phenotype: Type B Blood Controlled by A,B,H genes Controlled by A, B, H, Se, & Lewis genes
o Genotype: Could be BO or BB depending on parents

- The number one carbon of the galactose is attached to the number three
(type 1) / four (type 2) carbon of the N-acetylglucosamine sugar of the
precursor substance

- Most common to least common blood types: Type O > A > B > AB SECRETOR GENE
- Responsible for the secretion of ABH antigens in the body fluids
FORMATION OF A, B, AND H ANTIGENS - Individuals with the secretor gene (Sese or SeSe) aptly called ‘secretors’,
comprise 80% of the population
- Those who do not have the gene (sese) are called “non-secretors” and
Immunodominant
Gene

Glycosyltransferase Ag comprise of the remaining 20%

Sugar
- ABH substances can be found in DUBSTAMP
H α-2-L-fucosyltransferase L-fucose H o Digestive juices
A α-3-N-acetylgalactosaminyltransferase N-acetyl-D- A o Urine
galactosamine o Bile
(GalNac) o Saliva
B α-3-D-galactosyltransferase D-galactose B o Tears
o Amniotic fluid
- These genes (H, A, & B genes) DO NOT actually code for the production of o Milk
antigens but rather PRODUCE specific glycosyltransferases that ADD o Pathological fluids (pleural, peritoneal, pericardial, ovarian cyst fluid)
SUGARS to a basic precursor substance - Excessive ABH substances in secretions can be observed in:
o Pseudomucinous ovarian cyst
o Intestinal obstruction
o Carcinoma of stomach and pancreas
- Determination of Secretor Status: Hemagglutination Inhibition
o Specimen: saliva
o The principle of the test is that if ABH antigens are present in a soluble
form in a fluid they will neutralize their corresponding antibodies and
the antibodies will no longer be able to agglutinate red cells possessing
the same antigens
 If antigen is present = no agglutination
 If antigen is absent = agglutination

No No
(+) A Ag Anti-A + (+) B Ag Anti-B +
Saliva

Saliva

agglutination agglutination
A cells B cells
(-) A Ag Agglutination (-) B Ag Agglutination

H ANTIGEN
- The H gene must be inherited to form the ABO antigens on the RBCs
- H antigen
o Produced by the HH or Hh gene
o H gene is found in > 99.99% of the population
o Forms of H antigens:
- A, B, and H antigens are formed from the same basic precursor material-  H1 and H2: unbranched straight chains
paragloboside or glycan, to which sugars are attached in response to  H3 and H4: complex branched chains
specific enzyme transferases elicited by an inherited gene
- The H gene is needed for the formation of the H antigen. The H antigen, in
turn, is needed for the formation of A & B antigens. Without the H Antigen,
A & B antigens will NOT be expressed
 Reactivity of anti-H Antisera or anti-H Lectin with ABO Blood Groups - No H antigens formed (absence of H enzyme) = no A or B antigens
formed
o NO AGGLUTINATION with anti-H, anti-A, or anti-B lectin
- Presence of A or B enzymes in serum
- Anti-A, Anti-B, Anti-AB, and Anti-H present in the serum
- Bombay phenotype can donate to any ABO blood group but can only be
transfused with blood from another Bombay (Oh)
Mnemonic: Oh (O), Eto (A2) ba (B), Eto ba (A2B), Ewan (A1), Ewan ba (A1B) - Phenotypes as blood group O
Greatest amount of H antigen: has the strongest reaction with anti-H lectin Group O (OH) vs Bombay Phenotype (Oh)
Lowest amount of H antigen: has the weakest reaction with anti-H lectin Blood group Blood Forward Grouping Reverse Grouping
Anti- Anti-B Anti- Anti-H A1 B O
ABO SUBGROUPS A A,B cells cells cells
O (OH) - - - + + + -
A SUBGROUPS Bombay (Oh) - - - - + + +

Subgroup Notable characteristics Para-Bombay Phenotype (hh secretor)

A1 React with anti-A, and anti A - Absent or only trace A, B and H antigens on RBCs with normal expression
A2 React with anti-A only in secretions and body fluids
A3 MF reaction with anti-A and anti-AB 1. hh genotype
Ax React with Anti-AB, No reaction with anti-A 2. Caused by:
MF reaction with anti-A and anti-AB; but with only few a. Silenced/Mutated FUT 1 gene (H gene) with or without an active
Aend FUT2 gene (Se gene); or
agglutinates (<10% of cells)
Am Weak/No reaction with anti-A and anti-AB b. Silenced FUT1 gene (H gene) with an active FUT2 gene (Se
gene)
Ay & Ael No reaction with anti A and anti-AB
3. RBCs are completely devoid of H antigens or have small amounts of
No reaction with anti A and anti-AB
Ay H antigen present = RBCs express weak forms of A and B antigens
Observed in siblings; A germline mutation of an A gene
4. Absence of H enzyme in serum even though there is limited production
*Mixed field is defined as small agglutinates within predominantly unagglutinated
of H antigen on the RBC; Presence of A or B enzymes in serum
red cells
5. Anti-H is present in the serum
- Adsorption and Elution of anti-A is the method used to confirm the
6. Ah serum: anti-B, anti-A1, and no anti-A
presence of Am, Ay, and Ael antigens 7. Bh serum: anti-A and anti-B
A1 VS A2 PHENOTYPES
ABO ANTIBODIES
- A1 cells contain "A1" antigen and "A" antigen.
- A2 is not really a unique antigen. It is thought to be simply "A" antigen with - Naturally occurring antibodies: individuals normally produce antibodies
no "A1" antigen. directed against the A and/or B antigens absent from their RBCs
- Produced at birth but detected only at 3-6 months of age
- Not normally present or detected at birth
Anti-A Anti-
Antigenic Antigen - Predominantly IgM (can be IgM, IgG, IgA)
Abs

Blood Group (Anti-A with A1

sites Present Anti-A1) - React at room temperature (20-24oC) or below after an immediate spin
Lectin
- Activate complement at 37°C and can cause in-vitro/vivo hemolysis
More
A1
antigenic
80% of all A (or
Anti-B

Anti-A & Anti-B Anti-A,B

sites for A A & A1 + +
AB) are A1 (or Primarily IgM IgG
and less for
A1B) IgG may be found in O individuals Found in O individuals only
H
*Anti-A,B - is not a combination of anti-A and anti-B but is a "cross-reacting"
Less antibody
Anti-B, Anti-A1

A2
antigenic
20% are A2 (or LECTINS
sites for A A + -
A2B) or weaker
and more for - Lectins are plants or seed extracts that agglutinate human cells with some
subgroups
H degree of specificity.
o Prolectins: derived from snails
- These proteins bind specifically to carbohydrate determinants and
Structural Characteristics of A1 and A2 RBCs agglutinate RBCs through their cell surface of oligosaccharide determinants
- A2 RBCs: Predominantly Aa and Ab and unconverted H3 and H4 antigen
sites LECTINS USED IN BLOOD BANKING
- A1 RBCs: Aa, Ab, Ac, and Ad determinants and no unconverted H3 and H4 Anti-A1 Dolichos biflorus
antigen sites Anti-B Bandeiraea (Griffonia) simplicifolia
Anti-H Ulex Europaeus
B SUBGROUPS Anti-N Vicia graminea
- Subgroups of B are very rare and much less frequent than A subgroups Anti-M Iberis amara
Anti-T,Th Arachis hypogea
Subgroup Notable characteristics Anti-Tn Salvia sclarea
MF reaction with anti-B and anti-AB
B3
Most frequent B subtype
ABO ANTIGEN AND ANTIBODY DETECTION
Bx Weak reaction with anti-B and anti-AB
No/Weak reaction with anti-B and anti-AB
Bm Appear as early as Peak at Decline
Converted to B if incubated with Uracil diphosphate
2-4 Remains
No reaction with anti-B and anti-AB ABO
Bel 37th day of fetal life years constant
Extremely rare phenotype Antigens
old throughout life
- Adsorption and Elution of anti-B is the method used to confirm the
Appear as early as BIRTH 5-10
presence of Bm and Bel antigen ABO After 10 years
BUT detected only 3-6 years
Antibodies old
months after birth old
NULL PHENOTYPES

Bombay Phenotype (Oh/Hnull/hh non-secretor)

- First reported by Dr. Y.M. Bhende in 1952 in Bombay (Mumbai), India
- Homozygous inheritance (hh genes) of the h gene results in the inability to
form the H antigen and subsequently the A or B antigens.
- hh genotype
- Autosomal recessive; caused by:
o Mutation in FUT1 gene - silenced H gene
o Mutation in FUT2 gene - silenced Se gene
ABO GROUPING ANTISERA QC

REAGENTS & SPECIMENS FOR FT & RT POSITIVE CONTROL

- To ensure the reactivity of the antisera
1. Forward Typing - Should be weakly reactive for the antigen being tested
o Reagent: Typing Sera - Heterozygous cells in order to provide accurate indication of antisera
 Anti-A (Blue; Trypan blue dye) potency
 Anti-B (Yellow; Acriflavin yellow)
 Antisera must be: IgM, Highly specific, and NEGATIVE CONTROL:
Monoclonal - To ensure the specificity of the antisera
o Specimen: Whole Blood or 2-5% RCS - Should lack the target antigen to confirm the reactivity with only the
o Expected Reaction: 3+ to 4+ target antigen
2. Reverse Typing
GRADING AGGLUTINATION REACTIONS
o Reagent: Reagent Red Cells – from a human source (4-5%)
o Specimen: Patient Serum
ACCORDING TO HARMENING:
o Expected Reaction: 2+ to 4+

FORWARD GROUPING/ DIRECT TYPING/ CELL TYPING/ FRONT TYPING Grading Macroscopic evaluation
- Using antisera (Anti-A, Anti-B) to detect antigens on patient's RBCs 4+ One solid agglutinate
- Procedure: 3+ Several large agglutinates – clear background
1. Label test tubes 2+ Medium-sized agglutinates – clear background
2. Make a 2-5% patient red cell suspension 1+ Small agglutinates turbid background
3. Add reagent antisera W+ Tiny agglutinates turbid background
a. Add reagent anti-A antisera to one tube (1-2 drops) 0 No agglutination or hemolysis
b. Add reagent anti-B antisera to another tube (1-2 drops) *Note: Partial or complete hemolysis is a positive reaction
4. Add one drop of 2-5% suspension of patient red cells to each tube
5. Mix and centrifuge (approximately 20 seconds)

REVERSE GROUPING/ INDIRECT TYPING/ SERUM TYPING/ BACK TYPING

- Using reagent RBCs (A1 and B cells) to detect ABO antibodies in the
patient's serum
- Procedure:
1. Label test tubes
2. Add two drops of patient serum to each tube
3. Add one drop of reagent cells
a. Add 1 drop of reagent A1 cells
b. Add 1 drop of reagent B cells
4. Mix and centrifuge (approximately 20 seconds)
ACCORDING TO HENRY:

RED BLOOD CELLS SERUM

Can donate blood to
Can receive blood

Reactions Reactions
with with
ABO Type

Antibodies
Antigen/s

reagents reagents
from
present

present
Anti-A,B

A cells

B cells
Anti-A

Anti-B

*H = Hemolysis

A A + - + Anti-B - + A,O A, AB
B B - + + Anti-A + - B, O B, AB
A, B,
AB AB + + + None - - AB
AB, O
Anti- A, B,
O None - - - + + O
A,B AB, O

- Universal RBC Donor: Type O

- Universal Plasma Donor: Type AB
- Universal RBC Recipient: Type AB
- Universal Plasma Recipient: Type O
- Most dangerous ABO transfusion: Type A blood to Type O recipient
ABO DISCREPANCIES Problem: FORWARD GROUPING
Causes: Weakly reacting or Missing antigens
A. MAY BE CAUSED BY DISEASES
Disease may alter red cell antigens resulting in weaker reactions or
o 1. ABO subgroups
additional acquired antigens (Pseudoantigens) 2. Weakened/Depression of A or B antigens
1. Conditions that cause weaker reactions: a. Leukemias
 Leukemia (mixed field reaction in forward grouping) b. Hodgkin's disease
 Chromosome Translocations (chromosome 19) 3. Pseudoantigens (Acquired A/B phenomenon)
 Hemolytic Diseases
 Hodgkin's Disease
 Hypogammaglobulinemia (CLL) / Immunodeficiency
2. Conditions that cause pseudoantigens:
Acquired A phenomenon Acquired B phenomenon
- Proteus mirabilis infection - E. coli O86
- T-cell inactivation - Proteus vulgaris infection
- Intestinal obstruction
- Carcinoma of the colon and rectum RESOLUTION:
- Clostridium tetani infection 1. Incubate test mixture at ROOM TEMP for 30 minutes.
o Weakly reacting antigens + Reagent antisera
2. If still (-), incubate at REFRIGERATOR TEMP (4oC) for 15-30
minutes
3. Include O cell control and auto control
4. Enzyme treatment (RBCs pre-treated with enzymes and retested with
reagent antisera)
How to differentiate acquired B from B?
- Acquired B cells will NOT react with pH <6 or >8.5. RARE GROUP II DISCREPANCIES
RESOLUTION: 1. BGSS (Blood group-specific soluble) substances
1. RBCs treated with acetic anhydride; this –reacetylates- surface molecules; - Will neutralize the reagent anti-A or anti-B, leaving no unbound
decrease reactivity with anti B antibody to react with the patient cells
2. Use of Monoclonal Anti-B (detects true B antigens)
3. Use of Acidified Anti-B (detects true B antigens only) FORWARD REVERSE
Anti-A Anti-B A1 cells B cells
Group II

4. Detection of secretor status (only the A substance is secreted)

1+ 1+ 0 0
5. Use an autocontrol Type AB (weak reactions)
o patient’s serum [anti-B antibodies] + patient’s RBCs [pseudo B
antigens]
RESOLUTION:
o no reaction if the patient has an acquired B phenomenon
- Washing the patient cells free of the BGSS substances with saline
B. MAY BE CAUSED BY TECHNICAL ERRORS
1. Incorrect specimen/labelling/recording of results 2. Antibodies to low-incidence antigens in reagent Anti-A or anti-B
o Clerical errors: most common source of ABO discrepancies
2. Failure to add reagents/sample (false negative)
3. Over/Undercentrifugation
o Overcentrifugation: false positive
o Undercentrifugation: false negative
4. Cell suspension too heavy or light (false positive)
5. Contaminated reagents / materials (Pseudoagglutination; false [+])
6. Mix-up of samples
7. Failure to follow manufacturer's instructions RESOLUTION:
8. Uncalibrated centrifuge; Warming during centrifugation (if incubation - Repeat forward typing using different lot number antisera
temperature is higher than room temperature = false negative) - Use monoclonal antibodies
THINGS TO REMEMBER: 3. Chimerism
- Always add serum before reagent red cells (because serum is colorless) 1. True Chimerism
- In emergency situations, Group O, Rh-compatible (or Rh negative) o Twins - two cell populations are both recognized as self, and the
PRBCs may be issued individuals do not make anti-A or anti-B
- ABO reactions are considered strong (3+, 4+), if weak (1+, 2+), it denotes 2. Artificial Chimerism
the presence of a discrepancy o Blood transfusions (group O to A or B patient)
o Bone marrow or stem cells transplantation
Problem: REVERSE GROUPING o Exchange transfusions
Causes: Weakly reacting or missing antibodies o Feto-maternal bleeding

1. Newborns (antibodies detectable 3-6 or 4-6 months of age)

2. Elderly patients (depressed antibody production)
3. Hypogammaglobulinemia (↓ antibody production)
a. Leukemia (CLL)
b. Malignant lymphoma
c. Immunosuppressive drugs usage
d. Bone marrow or stem cell transplantations
4. Agammaglobulinemia
a. Congenital or acquired
b. Immunodeficiency disease
5. Patients undergone plasma transfusion or exchange transfusion
Group I

(antibody present might be diluted)

6. ABO subgroups

RESOLUTION:
1. Incubate patient serum with reagent at ROOM TEMP for 15-30
minutes.
o Patient serum + A1 cells/B cells or add 1-2 drops plasma
or serum
2. If still (-), incubate at REFRIGERATOR TEMP (4°C) for 15
minutes (to enhance antibody reaction)
3. Include O cell control and auto control
4. Determine patient age and history
 Problem: FORWARD AND REVERSE GROUPINGS Rh BLOOD GROUP SYSTEM (ISBT No. 004)
Causes: Protein or Plasma abnormalities
Rh GENES
1. Elevated globulin levels
a. Multiple myeloma (↑ IgG antibody production) - Autosomal codominant
b. Waldenstrom's macroglobulinemia (↑ IgM antibody production)
c. Plasma cell dyscrasia 1. RHD and RHCE genes
d. Advanced cases of Hodgkin's lymphomas o Proposed by: Tippett
2. Elevated fibrinogen levels o Located on chromosome 1
3. Plasma expanders o RHD: controls expression of RhD
a. Dextran o RHCE: controls expression of RhCe, RhcE, RhCE, Rhce
b. Polyvinylpyrrolidone o Codominance is observed
4. Wharton's jelly in cord blood samples

ROULEAUX
Group III

- is a stacking of erythrocytes that adhere in a coin-like fashion, giving

the appearance of agglutination

2. RHAG gene
o located on chromosome 6
o Co-expressor, but by itself cannot express any Rh antigen
RESOLUTION: o Mutations can cause significantly altered RhD and RhCE proteins
1. Microscopic examination
2. Saline replacement technique
 Remove serum, replace with equal volume of saline
3. Wash cells with saline (3x)
 Wash 6-8x to remove Wharton's jelly
4. Run antibody screen

Problem: FORWARD AND REVERSE GROUPINGS

Causes: Miscellaneous problems

1. Cold reactive autoantibodies - RH genes are inherited as codominant alleles.

2. Unexpected ABO isoagglutinins - Rh-positive individuals inherit one or two RHD genes, which result in
3. Unexpected non-ABO alloantibodies expression of RhD antigen and are typed Rh-positive.
4. More than one ABO group RBCs (transfusion, BM or stem cell - In addition to the RHD gene(s), two RHCE genes are inherited, one from
transplant) each parent.
5. Polyagglutination
RH SYSTEM NOMENCLATURE

RESOLUTION:
For RBCs (forward typing):
- Incubate patient’s RBCs at body temperature, wash with saline at
body temperature, 3x and retype
- If still unresolved, patient’s RBCs can be treated with DTT
(Dithiothreitol)

For serum (reverse typing): 1. FISHER-RACE (DCE NOMENCLATURE)

- The reagent RBCs and serum can be warmed to 37oC, then mixed, - Involved the presence of 3 separate genes D, C, and E and their alleles c
tested, and read at 37oC and e - linked on the same chromosome and are inherited as group of 3
Group IV

- If still (-), cold autoadsorption could be performed - Each inherited gene expresses it corresponding antigen to the RBC surface

Genes Antigens (RBC surface)

D gene D antigen The absence of D antigen is
C/c gene C/c antigen designated as "d"
E/e gene E/e antigen

RESOLUTION: 2. WIENER (Rh-Hr TERMINOLOGY)

- Determine the specificity of the antibody by examining the pattern of
reactivity (test cells with anti-A1 lectin and serum with A1, A2, and O
cells)
- Based on the notion that a single locus on a gene carries entire Rh system
- Each gene produces an agglutinogen that carried a series of 3 blood factors
o Agglutinogen: is a substance that stimulates production of agglutinin
o Agglutinogen = antigen
o Agglutinin = antibody
- Each factor is an antigen recognized by an antibody

Gene Agglutinogen Rh Factor (RBC surface)

RESOLUTION: Rho
- Run antibody screen and panel Rho gene →→ Rho hr'
hr'’
RARE GROUP IV DISCREPANCIES - Each agglutinogen represents the presence of a single haplotype composed
1. Antibody to acriflavine of 3 different antigens
o Resolution: Wash RBCs with saline 3x, then retype
2. cis-AB phenotype FISHER-RACE MAY BE CONVERTED TO
o Offspring inherits AB genes and O gene leading to weak WIENER NOMENCLATURE AND VICE VERSA
antigen expression D R
d r
C Single prime (') /1 R1 or r’
c No single prime (') /1
E Double prime (") /2 R2 or r’’
e No double prime (") /2
CE z/y Rz or ry
ce o Ro or r
 CONVERT FISHER-RACE TO CONVERT WIENER TO 4. International Society of Blood Transfusion (ISBT) Nomenclature
WIENER FISHER-RACE - A system that adopted a 6-digit number for each authenticated antigen
1. DCe = R1 1. Ror’ = Dce/dCe - First 3 numbers represent the: system
2. DcE = R2 2. R1R2 = DCe/DcE - Last 3 numbers represent the: antigenic specificity
3. DCE = Rz 3. R1r" = DCe/dcE
4. dcE = r” 4. Rzr = DCE/dce FISHER-RACE WIENER ROSENFIELD ISBT
5. dCe = r’ 5. R2r' = DcE/dCe D Rho Rh1 004001
6. dCE = ry 6. r'r" = dCe/dcE C rh' Rh2 004002
7. Dce = Ro E rh'’ Rh3 004003
8. dce = r c hr' Rh4 004004
e hr'’ Rh5 004005

RH ANTIGENS
- Antigens are polypeptide in nature and reside on transmembrane proteins
- They are not soluble and are not expressed on the tissues
- They are well developed at birth and therefore can easily cause HDN
- They are very good immunogens. D antigen is the most immunogenic after
A and B antigens.
- There are 5 major antigens that may be found in most individuals:
o D found in 85% of the population
o C found in 70% of the population
BLOOD FACTORS DESIGNATION o E found in 30% of the population
D antigen Rho o c found in 80% of the population
C/E (capital letter) r precedes h (rh) o e found in 98% of the population
c/e (small letter) h precedes r (hr) o d refers to the 15% of the population that has NO D antigen
C/c Single prime (') (rh'/hr')
E/e Double prime (“) (rh"/hr") IMMUNOGENICITY
*No designation for d (absence of D antigen)

WEAK D (Du)
- Common in blacks
- No/Weak reactions with anti-D
- Detected by IAT/Indirect Antihuman Globulin Test

CAUSES OF WEAK D
- Allele carrying RHD is in trans position to the allele carrying C
1. C in trans

Positional

- Steric hindrance causes the anti-D reagent to weakly attach

to RHD/

- Rh antigen is normal
D

- Can receive D+ RBCs with no adverse effects

- Most common in Blacks: R0r (Dce/dce) - Ex. Dce/dCe
o 2nd most common: R0R0 (Dce/Dce)
- Most common in Asians/Whites: R1r (DCe/dce) - RHD gene inheritance that codes for a weak expression of D
2. Genetic
Weak D

- The Big 4: R1, R2, r, R0 (four most common genotypes in the Rh system) antigen
- African-Americans: R0 > r > R1 > R2 - D antigen is expressed completely but fewer in number
- Caucasians: R1 > r > R2 > R0 - Rarely makes anti-D since changes are INSIDE of the RBC
3. Rosenfield (Alpha-numeric Terminology) - One or more epitopes within the D protein is missing / altered
- A system that assigns a number to each antigen of the Rh system in order
D Mosaic
3. Partial D/

- D antigen made of subparts that are antigenic

of its discovery or recognized relationship to the Rh system - Alloantibodies can be formed against the missing or altered
- It demonstrates the presence of the antigen on the RBC (no genetic basis) epitopes; can cause HDN
- The minus sign (-) preceding a number designates the absence of the o As Donor: Partial D Individual is considered Rh (+)
antigen (antigen was tested for but was not present) o As Recipient: Partial D individual is considered Rh (-)
ANTIGEN DESIGNATION EXAMPLES OTHER RH PHENOTYPES / RARE ALLELES
D Rh1 1. D+, C+, E+, C+, e+ = 1, 2, 3, 4, 5 1. Rh Deletion - C & E are not expressed on RBCs
C Rh2 2. D+, C-, E+, C+, e- = 1, -2, 3, 4, -5 (D--/D--)- Increased number of D antigens on RBC
E Rh3 3. D+, C+, E+, C-, e- = 1, 2, 3, -4, -5 2. Rh Null - Antigen not expressed on RBC
c Rh4 4. D-, C-, E-, C-, e+ = -1, -2, -3, -4, 5 (---/---) - Caused by abnormalities in transmembrane proteins
e Rh5 - Characterized by hemolytic anemia, reticulocytosis,
- If an antigen has not been typed, its number will not appear in the sequence stomatocytosis
- Example: - Decreased: Hb, Hct, Haptoglobin
o D+, C+, E+, c- (e was not tested) - Increased: Bilirubin, HbF
o Rh: 1, 2, 3, 4 (5 is not reported) Two types of Rh Null:
REGULATOR TYPE AMORPHIC TYPE
- Mutation in RHAG gene - Mutation in RHCE
- Normal RHD and RHCE genes gene
- No RHAG expression - Deleted RHD gene
- No RHCE and RHD expression on RBCs - Normal RHAG gene
- Can pass normal RHD and RHCE genes
3. Rh Mod - Partial suppression of RH gene; weakened Rh antigen
expression
- Caused by RHAG mutation
4. f (ce) - A compound antigen; expressed when 'c' and 'e' are in cis
position = f antigen
- All 'c' and 'e' negative individuals are ‘f’ negative
5. Cw - Caused by single amino acid change on RhCe protein
6. rhi (Ce) - A compound antigen; expressed when 'C' and 'e' are
present
7. G (DC) - Present on most D+ and C+ RBCs
- Present on D+C- / D-C+ / D+C+ RBCs
8. Exalted D - Strong D expression because of no Cc/Ee
RH ANTIBODIES 2. Tube Method
- NOT naturally occurring; will only form upon exposure to corresponding o Prepare 2-5% RCS
antigens (transfusion or pregnancy) o Add anti-D, centrifuge, dislodge
- IgG: Warm Reacting antibodies and react best at 37oC  Check for agglutination (If + stop & report, If [-] continue)
o Most clinically significant: IgG1 & 3 o Incubate at 37oC, spin and dislodge
o Complement fixation and opsonization: IgG3 > IgG1 > IgG2  Check for agglutination (If + stop & report, If [-] continue)
o Cannot bind complement: IgG4 o Add Coomb’s reagent (AHG reagent)
o Best at crossing placenta: IgG1 > IgG4 > IgG3  Check for agglutination (If + stop & report, If [-] continue)
o Cannot cross placenta: IgG2 o Add Check cells (for quality control; RBCs coated with IgG
o Greatest significance because RBCs coated with these are cleared in antibodies)
the RES: IgG1/IgG2  Must be positive; otherwise test must be repeated
- Can cross the placenta and cause HDN  Most commonly used: Rh+ RBCs coated with anti-D Abs)
- HTRs due to Rh incompatibilities usually result in extravascular hemolysis o Agglutination indicates Rh+; no agglutination indicates Rh- BUT
- Exposure to less than 0.1 mL of Rh+ RBCs can stimulate antibody production should be confirmed with weak D testing
in an Rh- person o No reverse typing is done since antibodies are not naturally-
- Warm autoantibodies often appear to have anti-e like specificity occurring

LW (LANDSTEINER-WIENER) BLOOD GROUP SYSTEM (ISBT No. 016) Specimen: Whole Blood / RBC suspension
- Was demonstrated as an antigen present on Rhesus monkey and on Reagents:
majority of human RBCs 1. BSA (Bovine Serum Albumin; serves as control)
o Antibodies react with 85% of human RBCs 2. Anti-D
- Gene is on Chromosome 19 a. Saline Anti-D
- Antigen is carried by ICAM-4 (Intracellular Adhesion Molecule 4) o IgM
- Was originally thought to be the same as the Rh family; now recognized as o Low protein based
distinct from D antigen o Used for testing cells that are already coated with IgG antibody
- To differentiate D Ag from LW: Treat cells with DTT (Dithiothreitol) o Cannot be used for weak D typing
o Lengthy incubation
DIFFERENTIATION BETWEEN D AND LW ANTIGENS b. High Protein Anti-D
D Antigen LW Antigen o IgG
On D(+) adult cells Obviously present Strongly present o Base material used was human plasma containing high titer D-
On D(-) adult cells Obviously absent Weakly present specific antibody
On D(-) cord cells Absent Still present o Potentiators (dextran or PVP) were added
o Cause RBCs to be in closer proximity to each other
Enzyme treatment Resistant Resistant allowing IgG anti-D to crosslink and cause direct
(ficin/papain) agglutination
DTT treatment Resistant Destroyed o Increased False (+) due to high protein
(Sulfhydryl reagent) concentrations
In pregnancy Unaffected Decreased expression c. Chemically modified Anti-D
o IgG molecule modified via breakage of disulphide
- Antibodies were produced in rabbits and guinea pigs exposed to Rhesus bonds
monkey RBCS o Low protein based
- Anti-LW: o Used for both slide and tube testing
o React strongly with most D+ cells
o React weakly with Rh- cells B. WEAK D TYPING
o Never react with Rh null cells o A form of: IAT/Indirect Antihuman Globulin Test
Reacts EQUALLY with CORD Cells regardless of D Type o Specimen: Taken from a negative result from the Rh typing
o Reagent: AHG
BLOOD BANKING PROCEDURE: RH TYPING o Procedure:
 Wash tube with (-) result with isotonic saline 3x
A. ROUTINE RH TYPING  To remove plasma proteins
o Detects for the presence of D antigen on patient's RBC  Inadequate washing of cells will result in neutralization of the
antiglobulin serum by trace amount of residual globulin (false
2 Methods: negative)
1. Slide Method  Add AHG and check for agglutinations
o Place anti-D and patient specimen on one slide o As a Patient: Individual should be considered D- or Rh-
o Warm Slide o As a Donor: Individual should be considered D+ or Rh+
 Temperature in Rh Viewbox: 45-50oC
o Read agglutination within 2 minutes
MNEMONIC!

001 Ang - ABO

002 Mens - MNS
003 Po ni - P
004 Rhea - Rh
005 Lumabas - Lutheran
006 Kaya - Kell
007 Lang -Lewis
008 Di - Duffy
009 Kita ni - Kidd
010 Diego – Diego

011 whY -Yt

012 eX? - Xg
013 She - Scianna
014 Doesn't - Dombrock
015 Call anymore! - Colton
016 Let - Landsteiner-Weiner
017 Rodger and - Rodger/Chido
020 Gerbich - Gerbich
021 Cry - Cromer
022 Know - Knops
023 I'm - Indian
024 Ok – Ok

025 Raspberry - RAPH

026 Juice - JMH
027 Is - I
028 Good - Globoside
029 Grabe - Gill
030 Rawr - RHAG
 MAJOR (NON-ABO, NON-RH) BLOOD GROUPS - Antibodies:
- Public antigens: High-frequency or High-incidence antigens (>98% of the Anti-M Anti-N
population) - Cold reacting; naturally occurring - Cold reacting; naturally occurring
- Private antigens: Low-frequency or Low-incidence antigens (<1% of the - IgG/IgM - IgG/IgM
population) - Reaction enhanced at pH: 6.5 - Reaction enhanced at: alkaline
(Acidic) pH
- Seen in multiparous women - Seen in renal dialysis patients
I. LEWIS (ISBT No. 007) (women with multiple where dialysis machine is sterilized
- ONLY major system not produced by RBCs pregnancies) with formaldehyde (Anti-Nf)
o Not intrinsic to RBCs Anti-S & anti-s Anti-U
o Produced by tissue cells and secreted into body fluids - IgG in nature - Found in S-s- individuals
o Passively adsorbed onto the RBC membrane from the plasma - May cause HDN &HTRs - May cause HDN &HTRs
o #1 antigen source: Gastrointestinal tract
- Le gene: chromosome 19 & codes for fucosyltransferases - Disease associations
- Chain: Type 1 (β1→3) o GPA - receptor for: Escherichia coli
o GPA & GPB - receptor for: Plasmodium falciparum
- Antigens: Lea, Leb → found in lymphocytes, platelets, pancreas, stomach,
intestine, skeletal muscle, renal cortex, and adrenal glands III. P BLOOD GROUP: P1Pk (ISBT No. 003)
o Le antigens in secretions are glycoproteins and Le antigens absorbed - The P blood group was introduced in 1927 by Landsteiner and Levine.
onto RBCs are glycolipids - In their search for new antigens, they injected rabbits with human RBCs and
o Possible Genotypes: Le (a+b-), Le (a-b+), Le (a-b-), Le (a+b+) produced an antibody, initially called anti-P, that divided human RBCs into
 Le (a+b+) possible in: Asians two groups: P+ and P–.
 lele common in: Blacks - Antigens:
 Phenotype changes can be caused by: o P1 antigen: deteriorates rapidly upon storage
 Pregnancy (decreased expression)  May cause false negative result in old specimens
 Cancer o Poorly expressed at birth (Full expression: 7 years old)
 Viral and parasitic infections
 Alcoholic Cirrhosis
 Development:

Secretor Non-secretor
Le (a-b-) → Le (a+b-) → Le (a+b+) → Le (a-b+) Le (a-b-) → Le (a+b-)
at birth after 10 days 6 years at birth after 10 days

o Expression:
 Affected by presence or absence of H and Secretor Gene *p (null) is slightly more common in Japan, North Sweden, and in an Amish group in Ohio
 Leb is present only if both Le and Se are present
- Antibodies:
o Anti-P1
 Naturally occurring, IgM
 Neutralized by hydatid cyst fluid from E. granulosus
 Found in patients with fascioliasis
o Anti-PP1Pk (Anti-Tja)
 IgM and IgG
 First described in a patient's serum suffering from
adenocarcinoma of the stomach; patient name is Mrs. Jay
o Autoanti-P
 IgG autoantibody
 Biphasic hemolysin: in vitro, the antibody binds to RBCs in the
cold, and, via complement activation, the coated RBCs lyse as
they are warmed to 37°C
 A.k.a. Donath-Landsteiner antibody
 Produced in patients with: Paroxysmal cold hemoglobinuria
- Disease Associations:
o P antigen - receptor for Parvovirus B19 - antigen
o Pk antigen- receptor for Shiga toxin
o All P antigens - receptor for P. fimbriated uropathogenic E. coli
BOMBAY PARABOMBAY o Anti-P1 - Hydatid cysts & other parasitic infections
- No A, B, and H antigens on red - Absent or trace A, B, and H o Anti-PP1Pk (Anti-Tja) - spontaneous abortions in early pregnancy
cells and secretions/body fluids antigens on red cells, normal in o Autoanti-P – PCH
secretions/body fluids
IV. I, I (ISBT No. 027)
- Antibodies: Anti-Lea, Anti-Leb - Antigens:
o Related to ABH and Lewis
o IgM; Naturally occurring; Cold Reactive o Chain: Type 2 (β1→4)
 Not clinically significant because antibodies rarely cause o i = ↑ in infants (i antigens are converted to I antigens after 18 months)
hemolysis or agglutination (neutralized by the recipient’s plasma) o I = low in infants, high in adults
o Can be neutralized by Lewis substances in secretions o Also found on the membranes of leukocytes and platelets
o Le (a-b+) individuals do NOT produce Anti-Lea o Antigens are neutralized by human milk
o Anti-Leb – produced by Le (a+b-) individuals (non-secretor) o Found in secretions & ovarian cyst fluid
- Disease associations: - Antibodies:
o Anti-l and anti-i
o Leb is associated with Helicobacter pylori infection
 IgM
II. MNSs (ISBT No. 002)  Can agglutinate all adult cells at RT (Anti-I)
 Can agglutinate cord cells or adult i RBCs (Anti-i)
- Gene: Chromosome 4 - Disease Association:
- Antigens o HEMPAS (Hereditary Erythroblastic Multinuclearity with (+) acidified
o Well developed at birth (may cause HDN) serum test OR Chronic Dyserythropoeitic Anemia II): for individuals
o Demonstrates dosage effect that do not convert i to I (after 18 months)
 Dosage effect: antibodies react more strongly with homozygous o Anti-I / Pathogenic Autoanti-I
RBCs (MM) than heterozygous RBCs (MN)  Associated with Cold Agglutinin Disease & Primary Atypical
o Used in paternity testing Pneumonia (Walking pneumonia by Mycoplasma pneumoniae)
 Cause acrocyanosis (vascular occlusion)
MN Ss o Anti-i - associated with Infectious Mononucleosis, EBV infection,
- On Glycophorin A - On Glycophorin B Alcoholic cirrhosis
- MN: differ at amino acids at - Ss: differ at amino acid at position o Adult i – Congenital cataracts
position 1 & 5 29
o M: 1-serine, 5-glycine o S: Methionine
o M: 1-leucine, 5-glutamic acid o s: Threonine
*U phenotype: formed when individual is S+s+ (U = Universal)
 V. KELL (ISBT No. 006) - Most common phenotypes:
- Mrs. Kellaher = 1st isolated anti-K o Whites: Fy(a+b+), Fy(a-b+)
- First blood group to be discovered after antiglobulin testing o Blacks: Fy(a-b-)
- Second only to D in terms of immunogenicity o Asians: Fy(a+b-)
o Fya + Fyb = Fy3 (common in Orientals and many Filipinos)

- Antibodies:
o Anti-Fya and Anti-Fyb
 IgG
 Implicated in HTR and HDN; not severe cases however
 Acute or delayed HTR and Mild to severe HDN
o Anti-Fy3
 Made by Duffy null individuals

- Disease associations:
o Duffy null: associated with resistance to Plasmodium vivax or
Plasmodium knowlesi infections; seen in Blacks
 Because merozoites attach to Fya and Fyb

VII. KIDD (ISBT No. 009)

- History
o Mrs. Kidd = HDN in her 6th child
o Jk = initials of the baby (John Kidd)
- Antigen: (Jka, Jkb)
- Gene: KEL Gene; chromosome 7 o WEAKLY immunogenic
- Antigens: o Well developed at birth (may cause HDN)
o Come from the precursor substance on WBCs / RBCs o Phenotypes:
 K - Kell  Jk (a+b-): common in blacks
 k - Cellano  Jk (a+b+), Jk (a-b+): common in Asians and whites
 Kpa – Penney  Jk (a-b-): found in 0.9% of Polynesians
 Kpb – Rautenberg
 Jsa – Sutter - Antibodies:
 Jsb – Matthews o Anti-Jka, Jkb
 Notorious in the blood bank; cause delayed severe and fatal
- Antibodies: HTR
o Anti-K  Can cause HDFN (rarely)
 IgG; reactive in AHG phase; warm-reacting  Drug induced HTR in patients receiving Aldomet/Methyldopa
 Implicated in severe HDN and HTR (Anti-hypertensive drugs)
 Production stimulated by transfusion or pregnancy o Anti-Jk3
o Anti-Ku  Found in Jk (a-b-)
 Produced by immunized individuals with the Ko phenotype  Associated with severe immediate and delayed HTRs and mild
 Causes HDN and HTRs HDFN
MINOR BLOOD GROUPS
- Disease Associations:
o Acquired K: infection from Streptococcus/Enterococcus faecium
o Anti-K: associated with E. coli O125 B15 infection I. LUTHERAN (ISBT No. 005)
o CGD (Chronic Granulomatous Disease): Absence of Kx on WBCs - History
o McLeod Phenotype: o Lutteran was the donor (mistyped name of the donor)
 Absence of Kx and Km on RBCs - Chromosome 19
 Depressed expression of all Kell antigens - Antigens (Lua, Lub, Lu3):
 X-linked pattern of inheritance; males are affected (females are o Poorly developed at birth (cannot cause HDN)
carriers) o Found in tissues such as brain, lungs, pancreas, placenta, skeletal
 RBCs are primarily – acanthocytes (thorn cells) muscle, and hepatocytes
 Well compensated hemolytic anemia, reticulocytosis, o Bind to laminin in sickle cell disease
bilirubinemia, splenomegaly, and reduced haptoglobin levels - Antibodies:
o McLeod Syndrome: o Anti-Lua
 Neuroacanthocytosis  Found in patient with Lupus Erythematosus
 Muscular and nerve disorders + serologic and hematologic  RT reacting; IgM, naturally occurring
picture o Anti-Lub
 Cardiomegaly and muscular dystrophy at age 40-50 leading to  React at 37oC / AHG; IgM, IgA, IgG
cardiomyopathy  Associated with shortened survival of transfused cells and post-
 Lack of deep tendon reflexes: areflexia transfusion jaundice
*Progresses to well-coordinated, involuntary movement o Anti-Lu3
(choreiform movement)  Made only by individuals with the recessive type of Lu(a-b-)
 Increased Creatine Phosphokinase (CK3, CKMM)  Usually IgG; antiglobulin reactive
 Increased Carbonic Anhydrase III
II. DIEGO (ISBT No. 010)
- Antigens: (Dia/ Dib, Wra / Wrb, Wu/WISK)
VI. DUFFY (ISBT No. 008)
o Dia used for anthropologic marker for Mongolian ancestry
- History  Located on Band 3/AE1 (Anion exchange for bicarbonate and
o Mr. Duffy = a multiply transfused patient chloride)
o Anti-Fya = hemophiliac patient  Defects in AE-1 cause RBCs to be: ASO
o Anti-Fyb = woman who had 3 pregnancies  Acanthocytes = Congenital Acanthocytosis
 Spherocytes = Hereditary spherocytosis
- Gene: Found on chromosome 1
 Ovalocytes = Southeast Asian ovalocytosis
- Antigens: (Fya and Fyb)
o Antibodies
o Well developed at birth (may cause HDN)
 Usually IgG
o Do NOT store well in saline suspension
 Anti-Dia and anti-Dib have caused HTRs and HDFN
o Fya is more immunogenic
 Anti-Wra has caused severe HTRs
o Found on DARC (Duffy Antigen Receptor for Chemokine)
 Autoanti-Wrb is relatively common in the serum of patients with
o Phenotypes: Fy(a+b+), Fy(a+b-), Fy (a-b+), Fy(a-b-)
WAIHA
 III. CARTWRIGHT (ISBT No. 011) XI. KNOPS (ISBT No. 022)
- Cartwright = the person in whom antibodies to the Yt antigens were first - Mrs. Knops = 1st antibody maker
discovered - Antigens are located on CR1 (Complement receptor 1); associated with
- However, all the letters in the individual’s name, with the exception of T, membrane band 4.1
were already used in the names of other blood group antigens. The - Decreased in Systemic Lupus Erythematosus (SLE) and Coronary Artery
researchers who discovered the Yt blood group then reasoned “Why not T?” Disease (CAD)
and hence Yt became the official name. - Knops null: Helgeson phenotype [Kn(a-b-), McC(a-), SI(a-), and Yk(a-)]
- Antigens: (Yta & Ytb)
o Located on glycophosphatidylinositol (GPI)-linked RBC glycoprotein: XII. INDIAN (ISBT No. 023)
acetylcholinesterase (AChe) - Antigens (Ina, Inb) are located on: CD44
o Absent from PNH III RBCs - Ina is more prevalent in Arab and Iranian populations
- Antibodies: Anti-Yta, Anti-Ytb
o IgG and are stimulated by pregnancy or transfusion
XIII. OK (ISBT No. 024)
- Named after the antibody maker, Mrs. Kobutso. Because Ko was already in
IV. XG (ISBT No. 012) use, the first two letter were switched to OK.
- Anti-Xga was discovered in 1962 in the serum of a multiply transfused man - Antigens are carried on CD 147
- Higher frequency in females (89%); Males (66%) - Well developed at birth but has not been reported to cause HDFN
- X-linked; on short arm of X chromosome
o X = X-linked
XIV. RAPH (ISBT No. 025)
o G = Grand Rapids (where the patient was treated)
- Associated with CD99 - The only antigen is MER2 (M = Monoclonal; ER = Eleanor Roosevelt)
- Located on CD151 (Tetraspanin; essential for membrane assembly in the
kidneys and skin)
V. SCIANNA (ISBT No. 013) - Abundant on platelets and is expressed on erythroid precursors and
- Scianna = 1st antibody maker decreases over time with increasing maturation
- Sc Gene: Chromosome 1 - Antibody is associated with renal disease
- Expressed on RBC adhesion protein called: ERMAP (Erythrocyte
Membrane Associated Proteins)
XV. JOHN MILTON HAGEN - JMH (ISBT No. 026)
- Antigens: Sc1 (Sm), Sc2 (Bua), Sc3, Sc4 (Rd), Sc5 (STAR), Sc6 (SCER,
Sc7 (SCAN) - Antigens (JMH1 through 6) are located on SEMA7A or Semaphorin 7A/
o SC2 in Northern Europeans is 1% but is higher in the Mennonite CDw108
population - No JMH antigen is observed in PNH III

VI. DOMBROCK (ISBT No. 014) XVI. GILL (ISBT No. 029)
- DO Gene: Chromosome 12 - The antigen is found on the glycerol transporter aquaporin 3, a member of
- Antigens: Doa, Dob, Gya, Hy, Joa the major intrinsic protein family of water channels.
o DO Antigens are carried on: ART4 (Mono-ADP ribosyltransferase 4)
- Absent from PNH III RBCs XVII. RHAG (ISBT No. 030)
- Antibodies (anti-Doa, anti-Dob) have caused delayed HTRs but no clinical - NEWEST Blood Group System
HDFN - Antigens: Duclos (RHAG1), Ola (RHAG2), DSLK (Duclos-like), RHAG4

VII. COLTON (ISBT No. 015) MISCELLANEOUS ANTIGENS

- Calton = 1st antibody maker
- Antigens (Coa, Cob, Co3, Co4) located on aquaporin-1 (Integral Membrane Sda (ISBT No. 901 012)
Protein); Accounts for 80% reabsorption in kidneys - Sid = head of maintenance department of the University of London
- Antibodies (anti-Coa, anti-Cob) cause HTRs and HDFN - The antigen (Sda) is found in saliva, urine, & meconium
- Anti-Co3 has been reported to cause severe HDFN - Soluble form is Tamm-Horsfall protein/Uromodulin
- Decreased in pregnancy
VIII. CHIDO/ROGERS (ISBT No. 017) - Antibodies (anti-Sda) are neutralized by urine
- Antigens (Ch, Rg) are not intrinsic to RBC membrane - Reactivity is described as shiny, refractile agglutinates in a sea of free RBCs
- Found on C4 complement component (mixed-field agglutination).
o C4a = Rodgers
o C4b = Chido HUMAN LEUKOCYTE ANTIGENS – HLA
- Antibodies (Anti-Ch, Anti-Rg) are usually IgG and react weakly - Bennett-Goodspeed blood group system; described the 1st antibody
- Nebulous: antigen strengths vary between samples - Antigens
o Bga = HLA B7
IX. GERBICH (ISBT No. 020) o Bgb = HLA B17
- Antigens are carried on sialoglycoprotein structures: Glycophorin C and D o Bgc = HLA A28
(Associated with membrane band 4.1)
- Gerbich-negative phenotypes: OTHER MINOR BLOOD GROUPS
o Leach = (Ge: -2,-3,-4); Gerbich Null - Jra = High prevalence antigen in most population; The Jr(a-) phenotype is
o Yus = (Ge: -2,3,4) found more commonly in Japanese
o Gerbich = (Ge: -2,-3,4) - Ata = High prevalence antigen, and all At(a-) individuals have been black.
- High prevalence: Ge2, Ge3, Ge4, GEPL, GEAT, and GETI Anti-Ata was first described in the serum of a black woman named Mrs.
- Low prevalence: Wb, Lsa, Ana, Dha, and GEIS Augustine
- Antibodies may be IgM, but most are IgG - Vel = High prevalence antigen
o Anti-Ge2: most common antibody produced by Gerbich-null
phenotypes
o Anti-IFC is made by the Cromer null Inab phenotype

X. CROMER (ISBT No. 021)

- Mrs. Cromer = Black prenatal patient
- Antigens (Cra, Dra, Tca, Tcb, Tcc) are carried on the protein DAF (Decay
Accelerating Factor) or CD55
- No Cromer antigen is observed in patients with PNH III
- Antibodies are usually IgG, but do not cause HDFN
- Anti-Cra and Anti-Tca have been implicated in HTRs
 NOTABLE CHARACTERISTICS OTHER BLOOD BANKING TECHNIQUES

IgM ANTIBODIES IgG ANTIBODIES A. RED CELL SUSPENSION (RCS)

“LIPMAN” 1. Preparation:
- Lewis - Rh (DCEce) o Wash red cells 3x to remove traces of plasma and excess
- I - SsU antibodies
- P1 - Duffy (Fy) o 50% RCS = Slide method
- M - Kell (K) o 5% RCS = Test Tube method
- A, B, H - Kidd (Jk) o 1% RCS = Gel technology
- N - Lutheran (Lub) o 1% RCS = Microplate
- Lutheran 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑃𝑅𝐵𝐶𝑠
Mixed-Field Reaction % 𝑅𝐶𝑆 = 𝑥 100
- Sda 𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 (𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑃𝑅𝐵𝐶𝑠 + 𝑁𝑆𝑆)

- JMH 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑃𝑅𝐵𝐶𝑠 𝑥 100

𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 =
- Cromer % 𝑅𝑒𝑑 𝑐𝑒𝑙𝑙 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛
Absent in PNH III
- Cartwright
- Dombrock 2. Determination of amount of NSS to be added
o Formula: mL (of NSS) = Total Volume - PRBC volume
- Lewis
Carbohydrate o Sample Problem:
- I
antigen  How much NSS must be added to 0.3 mL of PRBCs to
- P
make a 5% RCS?
0.3 𝑥 100 30
POORLY DEVELOPED AT BIRTH WELL DEVELOPED AT BIRTH  𝑇𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 =
5
=
5
=𝟔
- Lewis - Duffy  𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑁𝑆𝑆 = 6 − 0.3 = 𝟓. 𝟕 𝒎𝑳 𝒐𝒇 𝑵𝑺𝑺
- Lutheran - MNSs
- P1 - Kell B. ANTIGLOBULIN TEST / COOMB’S TEST
- Kidd
- REAGENT: ANTIHUMAN GLOBULIN (AHG)
- Anti-Lea/Leb o A.k.a. Coomb’s serum/reagent
In-Vitro Hemolysis - Anti-Tja  The reagent is dyed green by the addition of patent
- Vel blue and ariavit tartrazine.
o Antibodies to human globulin
- Rh (CcEe) o Serves as a bridge to connect two non-agglutinating
- Duffy antibodies (IgG)
- MNSs o Either Monoclonal or Polyclonal
Dosage
- Kidd  Monoclonal - produced through hybridoma
- Kell (sometimes) technology using mice
- Lutheran  Polyclonal - produced through conventional methods of AHG
production using rabbits (most common), goats, sheep
- Chido/Rogers o Either Monospecific or Polyspecific
- Kn  Polyspecific - contain a blend of anti-IgG and anti-C3d (C3b)
HTLA  Monospecific - contain only one antibody: either anti-IgG and anti-
- JMH
(High-Titer, Low- C3d (C3b)
- York
Avidity) Antibodies
- Cost TYPES
- McCoy DIRECT ANTIGLOBULIN TEST INDIRECT ANTIGLOBULIN TEST
(DAT) (IAT)
Decreased antibody - Lewis
- Detects in-vivo sensitization - Detects in-vitro sensitization
in pregnancy - Decreased antigen in pregnancy: Sda - Specimen: RBCs - Specimen: Serum
(Post-partum) - Incubation Phase: None - Incubation Phase: Yes
Destroyed by Enhanced by Unaffected by enzymes
Enzymes Enzymes

“KALIPER”
- Duffy - Kidd - U
- MNS - ABO - Kell (destroyed by
- Xga - Lewis sulfhydryl agents)
- Minor: JMH, - I - Lutheran (variable)
Ch/Rg, Pr - P1 - Scianna
- Rh

- Anti-P1: Hydatid Cyst Fluid, Turtle Dove Egg White, Pigeon

Dropping
Neutralization

- Anti-Le, Anti-H: Saliva

- Anti-Ch/Rg: Plasma/Serum
- Anti-Sda: Guinea Pig Urine (soluble form (Tamm-Horsfall
protein)
- I antigen: Mother's milk

1. DIRECT ANTIGLOBULIN TEST (DAT)

- Steps:
o Prepare a 5% RCS of the patients red cells
o Add one drop of the prepared cell suspension to a small tube
o Wash 3x with NSS to remove all the traces of serum
o Decant completely after the last washing
o Add two drops of Anti-human serum
o Mix well and centrifuge for one minute at 1500 RPM
o Re-suspend the cells by gentle agitation and examine macroscopically
and microscopically for agglutination
o If (-), add Coombs Control Cells / Check Cells for confirmation
 Coomb’s Check cells: RBCs coated with IgG antibodies (Rh+
RBCs coated with anti-D)
 Positive for agglutination = Valid result
- Associated conditions: SOURCES OF ERROR
o HDN: Maternal Ab coating Fetal RBCs
o HTR: Recipient Ab coating Donor RBCs CAUSES OF FALSE (+) CAUSES OF FALSE (-)
o AIHA (Autoimmune Hemolytic Anemia): Autoantibody coating - Improper specimen (clotted, fridged) - Inadequate washing
individual RBCs - Autoagglutinable cells - Reagent (AHG) deterioration
 AIHA = WAIHA, CAS (Cold Agglutinin Syndrome), PCH - Cells with (+) AHG test used for IAT - Reagent not added
o DIHA (Drug-induced hemolytic anemia): commonly caused by - Cells/Saline contaminated by bacteria, - Serum not added
Cephalosporins heavy metals or colloidal silica - Serum non-reactive b/c of
2. INDIRECT ANTIGLOBULIN TEST (IAT) - Dirty glassware complement deterioration
- Overcentrifugation and over reading - Inadequate incubation
- Steps:
- Polyagglutinable cells - Undercentrifugation
o Place two drops of patient's serum in a tube
- Contaminating antibodies in AHG - Low pH of saline
o Add one drop of reagent red cells
- Centrifugation of test with PEG prior to - Prozone or Postzone with
o Incubate tube for one hour at 37°C
washing RCS
o Wash the cells three times in NSS
o Add two drops of Coombs serum/AHG to each tube
o Keep for 5 minutes and then centrifuge at 1,500 RPM for one minute
o Re-suspend the cells and examine macroscopically as well as
microscopically
o If (-), add Coombs Control Cells / Check Cells for confirmation
- Applications:
1. Antibody Detection
 Compatibility Testing - Recipient Antibodies with Donor RBCs
 Antibody Screening - Antibodies reacting with Screening cells
2. Antibody Identification
 Antibody Panel – Antibody reacting with Panel cells
3. Antibody titer
 Rh antibody titer – Antibody and selected Rh cells
4. RBC phenotyping
 RBC antigen detection - specific antisera + RBCs to detect antigen
 Weak D, Kell, paternity testing

FACTORS AFFECTING AHG TESTS C. ANTIBODY SCREEN & IDENTIFICATION

A. Serum-Cell Ratio
o Increased serum to cell ratio = Increased sensitivity of the test
o Minimum ratio for routine tests: 40:1
 Achieved by adding 2 drops of serum to 1 drop of 5% RCS
o Minimum ratio for detecting weak antibodies: 133:1
 Achieved by adding 4 drops of serum to 1 drop of 3% RCS
B. Reaction Medium/Potentiators
o Albumin (22%): allows antibody coated cells to come into closer
contact with each other by reducing the zeta potential. Incubation time
is 30-60 minutes.
 Zeta potential: is the degree of negative charge on the surface of
a red blood cell-electrostatic repulsion between RBCs
 ↑ Dielectric constant = ↓ Zeta potential
1. ANTIBODY SCREEN
- Performed on the patient's or the donor's serum to check for the presence
of unexpected clinically significant antibodies
- It often serves as the replacement to minor crossmatch
- Done via tube method or the gel method
- Specimen: Serum/Plasma
- Reagent: O red cells with known antigen typing (Screening cells)

- ANTIBODY SCREEN PHASES

o Immediate Spin (IS)
 Detects clinically insignificant cold antibodies (IgM)
 IS phase may not be performed
o Low Ionic Strength Saline (0.2% Sodium chloride): enhance o 37oC incubation with enhancement medium
antibody uptake and allow incubation times to be decreased from 30-  Detects for warm reactive antibodies (IgG)
60 minutes to 10-15 minutes by reducing the zeta potential  Enhancement media (potentiators) are added before incubating to
surrounding an RBC. increase sensitivity of test (Ex. albumin, LISS, PEG)
 ↓ Ionic strength = ↓ Zeta potential o Antiglobulin Phase
o Polyethylene glycol (PEG): increase antibody uptake by removing  Detects non-agglutinating warm antibodies (IgG) that are possibly
water molecules surrounding RBCs. More sensitive than LISS or not detected in the incubation phase
albumin. NOT for patients with high protein levels  AHG serves as a bridge between sensitized RBCs producing
agglutination
C. Temperature: 37oC  Coomb's check cells are RBCs coated with IgG antibodies (Rh+
D. pH RBCs coated with anti-D)
o For Ag-Ab reaction: 6.5-7.5
 For anti-M and M = <6.5 (acidic) - STEPS IN PERFORMING ANTIBODY SCREEN (TUBE METHOD)
 For anti-N and N = alkaline o Mix 2 drops of serum & 1 drop of reagent cells
o For saline washing: 7.2-7.4 o Spin; Observe for hemolysis / agglutination
o Add potentiators and incubate at 37oC
E. Centrifugation: 1000 RCF (20 seconds) /1500 RPM (1 minute) o Wash with saline (3x); to remove serum and free antibodies which may
F. Specimen: EDTA-anticoagulated blood (to avoid in vitro complement react with AHG and lead to false negative result
attachment to RBCs when refrigerated) o Add 2 drops of AHG
o Spin; Observe for hemolysis / agglutination
o If (-), add Coomb's control cells for confirmation. Agglutination
indicates a valid result.
- FACTORS AFFECTING AGGLUTINATION: 2. Circle antigens not crossed out
o Antigen-Antibody ratio (2 drops serum, 1 drop cells)
o Dosage effect
 Antibody reacts more strongly with homozygous RBCs than
heterozygous RBCs
o pH (6.8 – 7.2)
 <6.5 = Anti-D, Anti-M, Anti-PR
o Incubation time (depends on the potentiator used; 1 hour is optimum)
o Centrifugation
o Type & temperature range of antibody
o Number and location of antigen

- INTERPRETATION OF ANTIBODY SCREEN

o Phase of Reaction
 Reaction in the IS phase indicates a cold reactive antibody (IgM)
 Reaction in the 37°C and AHG phase indicates a warm reactive
antibody (IgG) 3. Consider antibody’s usual reactivity
o Autocontrol
 A (+) autocontrol indicates the presence of an autoantibody
 If reactive in IS and (+) in autocontrol = cold-reactive
autoantibodies
 If reactive in 37oC and AHG phase and (+) in autocontrol = warm-
reactive autoantibodies

- STRENGTH OF THE REACTION

o A stronger the reaction = more clinically significant antibody
o Hemolysis and MF reactions should be noted
o When all cells react at the same phase with the same strength expect
a single antibody specificity
o When cells react at different phases with varying strengths expect
multiple antibodies present

- ROULEAUX FORMATION VS. TRUE AGGLUTINATION

o If patient Antibody Screen is NEGATIVE, proceed to crossmatching
o If patient Antibody Screen is POSITIVE, proceed to antibody panel for
antibody identification 4. Look for a matching pattern

2. ANTIBODY IDENTIFICATION/ANTIBODY PANEL

- Done to identify the antibody that was detected in the antibody screen
- Procedure is mostly similar to antibody screen except that 11-20 reagent
red cells are used instead of the 2-3 reagent cells in the antibody screen
- Steps in performing Antibody Identification
o Immediate Spin Phase
 2 drops patient’s serum + 1 drop each panel cell
 Check for agglutination
o 37oC with potentiators Phase
 2 drops LISS, incubate for 10-15 minutes
 Check for agglutination
o AHG Phase
 Wash with saline, add 2 drops of AHG serum
 Check for agglutination
o Checking: Our previous example fulfills the “rule of three”
 Coomb’s check cells is added to all negative units
- Interpretation of Antibody Identification
1. Ruling Out – means crossing out antigens that did not react

ADDITIONAL PROCEDURES FOR ANTIBODY IDENTIFICATION

PROCEDURES INVOLVING PROCEDURES INVOLVING RED

SERUM CELLS
- Hemagglutination Inhibition - Enzyme Treatment
- Serum Adsorption - Use of AET/DTT
- Use of Sulfhydryl Reagents - Elution
- Titration - Chloroquine diphosphate
- Pre-warming - Use of ZZAP
- Saline Replacement - Lectin Typing
- Cell Separation Techniques
 FOR SERUM: D. COMPATIBILITY TESTING
- Substances (BGSS) in the body and in nature that have similar
Neutralization
antigenic structures to RBC antigens can neutralize antibodies in the
(Inhibition)
HOMOLOGOUS - ABO & Rh Typing on donor and recipient
serum TRANSFUSION - Screening of the Donor's and Recipient serum for
- This allows for separation; serum is incubated with neutralizing Allogeneic unexpected antibodies
agent, antibody identification is then performed on the treated serum - Crossmatch
transfusion
- Purpose: to neutralize and separate clinically insignificant antibodies
- ABO & Rh typing on autologous units and recipient
- Removal of antibodies from serum AUTOLOGOUS
- Antibody Screening
- Steps: TRANSFUSION - Major Crossmatch
o Addition of the target antigen and allowing the antibody present in
the serum to bind the antigen, forming antigen-antibody complex - ABO & Rh Typing on the infant (Forward typing only
Adsorption

o Complexes are removed via centrifugation

for ABO)
o Adsorbed serum is then tested against RBC panel
- Antibody screen using maternal serum, infant's
NEONATAL serum, or eluate prepared from infant's serum
- Ex. A patient is suspected to have anti-E or anti-c TRANSFUSION
o Treat serum with cells having E antigen
- If donor cells are NOT group O, infant must be
o (-) reaction = anti-E is the antibody in question
tested for Anti-A & Anti-B antibodies. If antibodies
o (+) reaction = rule in anti-c
are present, ABO compatible blood must be used
- Includes:
Use of Sulfhydryl

o 2ME (2-mercaptoethanol) E. CROSSMATCHING

o DTT (Dithiothreitol) - Donor and patient blood is mixed to check for compatibility in 3 phases
Agents

- Destroys pentameric structure of agglutinating IgM; bypassing cold
reactive autoantibodies and alloantibodies
- This allows for easier detection of underlying IgG alloantibodies
- Maintain all test components (tubes, reagents, specimens) at 37°C
prior to testing.
warming
Pre-
- May detect an unexpected antibody in the patient's blood against the donor
that wasn't detected in the antibody screen
- Serves as a final check of ABO compatibility between donor and patient
o In-vitro crossmatch is most commonly performed
o In-vivo crossmatch can also be done
- Type & Screen procedure: an acceptable alternative to crossmatching

- Bypasses cold reactive autoantibodies and alloantibodies of limited

titer and thermal amplitude
- Prevents complement activation in antibody screens
- Serum is removed and equivalent amounts of saline is added
Replacement
TUBES USED IN CROSSMATCHING
- Major Crossmatch: Donor’s RBCs + Patient’s serum (DRPS)
- Minor Crossmatch: Donor’s Serum + Patient’s RBCs (DSPR); has been
replaced by antibody screening tests

- Done to remove formed rouleaux

- Autocontrol: Patient’s RBCs + Patient’s serum
Saline

PHASES OF CROSSMATCHING
1. Immediate Spin / Room temperature phase
- Method to semi-quantitatively determine the amount of antibody o Mix 2-3 drops serum with 1 drop wash 2-5% RCS
present in a given serum/plasma sample o Spin; check for hemolysis. Re-suspend cell button; check for agglutination
Antibody Titer
Determination

- Done after knowing the identity of the antibody 2. 37oC incubation with enhancement media
- Aids in identification of HTLA antibodies o Add 2 drops enhancement media to the mixture of serum & RBCs
o HTLA (High-Titer, Low-Avidity) antibodies are those Abs that o Mix & incubate. Spin; check for hemolysis or agglutination
react weakly at high dilutions (>1:64)
- Used to measure the production of IgG antibodies during pregnancy 3. AHG phase
o Wash 3x with NSS; decant saline completely
o Add 1-2 drops AHG
FOR RED CELLS:
o Spin; check for any agglutination
- Enzymes are used to enhance or destroy certain antigens in red cells
o Add 1 drop Coomb’s check cells to (-) test
- Proteolytic enzymes commonly used:
o Ficin (fig tree), Papain (papaya), Trypsin (lining of a pig's
o Spin; check for any reaction
Treatment
Enzyme

stomach), Bromelain (pineapple)
- Muraminidase is used to remove sialic acid
- Steps:
o Incubate test RBCs with enzyme solution at 37°C
o Wash cells thoroughly; retest cells with serum
- AET (2-aminoethylisothiouronium) & DTT are used to create RBCs
AET/
*Test should be (+) for results to be considered valid
ALTERNATIVES TO CROSSMATCHING
- TYPE AND SCREEN
o Test patient blood for ABO, Rh, & clinically significant unexpected Abs
o Patient sample is then stored in blood bank refrigerator for future

negative for all antigens of the Kell blood group system EXCEPT Kx
DTT

crossmatch of blood is needed for transfusion

- Destroy the disulfide bonds on IgM, does not affect IgG
- ABBREVIATED CROSSMATCH
- Comprised of cysteine-activated papain & DTT o Immediate Spin crossmatch + Type Screen
- Removes antibodies attached to RBCs - COMPUTER CROSSMATCH
ZZAP

o In cases wherein autoantibodies are attached to RBCs – Add

o Compares recent ABO serologic results and interpretation of file for
ZZAP; more free sites for antibodies to attach
- Destroys Kell, Cartwright, Gerbich, & Dombrock both patient and recipient
- Dissociates IgG from patient cells with (+) DAT so that they may be o Can only be used for detecting ABO incompatibility
typed with blood grouping reagents that require an indirect o Patient should have had no unexpected Abs or any records for such
diphosphate
Chloroquine

antiglobulin technique DETERMINING NUMBER OF UNITS TO CROSSMATCH FOR COMPATIBLE UNITS

o Removes IgG antibodies from IgG-coated RBCs
- Only anti-IgG should be used as the AHG reagent; as Complement
proteins are not dissociated with chloroquine
o Monospecific AHG: Anti-IgG
o C3d proteins are not dissociated
- Removal of antibodies from the surface of RBCs
- Methods:
o Heat: used for recovering ABO antibodies in case of ABO HDN
o Freeze-Thaw: also done in cases of HDN
Elution
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑢𝑛𝑖𝑡𝑠 𝑡𝑜 𝑐𝑟𝑜𝑠𝑠𝑚𝑎𝑡𝑐ℎ =
# 𝑜𝑓 𝑢𝑛𝑖𝑡𝑠 𝑛𝑒𝑒𝑑𝑒𝑑 𝑏𝑦 𝑡ℎ𝑒 𝑝𝑎𝑡𝑖𝑒𝑛𝑡
=
𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝐴𝑔 #1 𝑥 𝑓𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑖𝑡𝑦 𝑜𝑓 𝐴𝑔 #2
Sample problem #1:
- Four c-negative donors are required for a patient with anti-c.
- The frequency of the c antigen is 80%.
- The patient needs 4 units of compatible blood.

 Lui Freeze Method: Washed packed red blood cells are 4 4 4

treated with subsequent rapid freezing and thawing to =
(100 − 80)
=
20%
=
0.20
= 𝟐𝟎
cause lysis of the red blood cells. This will result in the
freeing of cell bound antibodies. Answer: a minimum of 20 donors should be antigen typed
o Acid: Use of glycine acid-EDTA, citric acid, digitonin acid
o Organic Solvents: chloroform, xylene, ether Sample problem #2:
- Reagents derived from plant seeds that are to test for the presence Patient needs few units of whole blood for tomorrow's surgery. The patient
of a specific blood group antigen antibody screen reveals two antibodies: Anti-Jka and Anti K. The frequency of
Lectins and Prolectins

- Lectins are derived from: plants antigen negative Jka in the population is 75% and antigen positive K in the
- Prolectins are derived from: snails population is 25%. How many units of blood are needed to screen to find 5 units
of compatible a blood
LECTINS USED IN BLOOD BANKING
Anti-A1 Dolichos biflorus Anti-N Vicia graminea a. 8 b. 9 c. 26 d. 27
Anti-B Bandeiraea (Griffonia) Anti-M Iberis amara Given:
simplicifolia - Negative frequency Jka = 75%
Anti-H Ulex Europaeus Anti-T,Th Arachis hypogea - Negative frequency K = 75% (100-25)
Anti-Tn Salvia sclarea 5 5
= = = 8.88 𝑜𝑟 𝟗
0.75 𝑥 0.75 0.5620
DONOR SELECTION & SCREENING GENERAL REQUIREMENTS FOR AUTOLOGOUS DONATION
1. Age: No age requirement
TYPES OF DONATION: 2. Hemoglobin: >11 g/dL
A. Allogeneic/Homologous donation 3. Hematocrit: >33%
B. Autologous donation 4. General Condition: Patient should have no condition predisposing to
C. Apheresis bacteremia or any form of severe CV/Pulmonary condition
5. Last donation: at least 72 hours (3 days) before the surgery
A. ALLOGENEIC DONATION 6. Prescription or order from the patient's physician
- Careful selection and screening of donors is done to ensure the recipient of
the blood component will benefit from the transfusion CONTRAINDICATIONS OF AUTOLOGOUS DONATION
- It is also done to determine:
1. Conditions presenting risk of bacteremia
o If a donation of 450 mL blood at this time would be harmful to the donor
2. Unstable angina
o If the blood collected from the donor at this time may contain
transmissible diseases 3. Recent Myocardial infarction or Cerebrovascular attack
4. Significant cardiac or pulmonary disease with on-going symptoms that have
GENERAL DONOR REQUIREMENTS not been evaluated by the treating physician
5. Untreated aortic stenosis (narrowing of the aortic valve opening)
1. Identification Card (ID) for photographic identification
6. Pregnancy is NOT a contraindication unless certain risky circumstances
2. Donor Registration Information - Full name, date and time of donation,
address, telephone, age, sex and date of birth exists
3. Consent form (<16 or 17 years old)
4. Donor's physiological values should be acceptable or within range: *Autologous units must be used for the donor-patient only
o General Appearance: Appears Healthy
o Age: 18 years old & above - can donate C. APHERESIS
 16-17 years old - require written consent from guardian - Derived from the Greek word "aphairos" meaning to "to separate / to remove
 60 years old and above - may donate at the discretion of the BB / to take from"
physician - Process wherein blood is withdrawn from the donor and separated into its
o Weight: >50kg or 110 lbs components; one or more of the components is retained and the remaining
o Hematocrit: >38% components are recombined and returned to the individual
o Hemoglobin: >12.5 g/dL - Uses anticoagulants to prevent blood from clotting as it enters the
o Temperature: <37.5oC/99oF separation machine
o Blood Pressure:
o Most commonly used anticoagulant: ACD
 Systolic: <180 mmHg
 Diastolic: <100 mmHg
o Pulse Rate: 50-100 bpm TYPES OF APHERESIS
 Athlete donors = <50 bpm is acceptable - Donor should have a platelet count of at least 150 x 103/uL or
150 x109/L
- In cases wherein donor weighs less than 50 kgs / 100 lbs, adjustments can - Interval of donation is at least 2 days (48 hours)
be made to the amount of blood to be collected, likewise, the amount of - Donation should NOT exceed more than 2 times a week or 24
Plateletpheresis

anticoagulant must also be adjusted. times a year

- Formulas to be used: - Platelet unit from apheresis is called Single Donor Platelets
𝐷𝑜𝑛𝑜𝑟 ′ 𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 𝑥 450 o SDP = derived from apheresis
𝐴𝑙𝑙𝑜𝑤𝑎𝑏𝑙𝑒 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑 𝑡𝑜 𝑏𝑒 𝑐𝑜𝑙𝑙𝑒𝑐𝑡𝑒𝑑 =
50 𝑘𝑔 o RDP = derived from whole blood/platelet concentrates
𝐴𝐴𝑂𝐵𝑇𝐵𝐶 𝑥 14 o 1 SDP = 6-10 RDPs
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑎𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 𝑛𝑒𝑒𝑑𝑒𝑑 =
100 - 75% of plateletpheresis products should contain a minimum of
3.0 x 1011 platelets (SDP)
𝐴𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑎𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 = 63 𝑚𝐿 − 𝑎𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 𝑛𝑒𝑒𝑑𝑒𝑑
o RDP = 5.5 x 1010 platelets
- pH at the end of the storage period should be 6.2 or greater
- Direct Donation: a unit collected under the same requirements as those for
allogeneic donation, except that the unit is directed toward a specific patient - 2-day interval between donations
Plasmapheresis

o Infrequent plasmapheresis: 4-weeks interval

- Maximum of 3 donations in a 7-day period
B. AUTOLOGOUS DONATION
- Used to collect immune plasma from donors with increased
- Safer because it poses no risk of disease transmission, transfusion concentrations of certain plasma immunoglobulins for
reactions or alloimmunization to WBCs, RBCs, platelets, plasma proteins immunocompromised patients
TYPES OF AUTOLOGOUS DONATION
- For patients who are severely neutropenic, with severe bacterial
- Refers to removal and storage of blood from a
Leukapheresis

infection, and are unresponsive to antibiotics

donor patient before an elective procedure for
- HES (Hydroxyethyl starch; a sedimenting agent which ↑ separation
reinfusion during or after the procedure
- Potential problems of WBCs & RBCs), Corticosteroids (↑ WBCs if administered 12-24
o Presurgical anemia & hypovolemia hours before collection) & G-CSF (↑ demargination of WBCs) are
o Clerical Identification errors used to increase the granulocyte yield
PREOPERATIVE
o Outdating of liquid stored blood
COLLECTION
Erythrocytapheresis

o Homologous blood transfused instead of - Requirements:

autologous o Female: 5'5" height; 150 lbs weight; 40% Hematocrit
o Patient-donor unable to donate (ex. Poor o Male: 5'1" height, 130 lbs weight; 40% Hematocrit
veins, or pre-existing medical conditions) - Two standard units of RBCs can be collected OR One unit of
o Inadequate amount of time to collect RBCs collected concurrently with plasma and/or platelets
suitable number of units
- Blood is collected after administration of
ACUTE
anesthesia and before a surgical procedure. This - Done to remove substances that exist in blood that contributes
NORMOVOLEMIC
is done with simultaneous replacement of a
HEMODILUTION to a disease process or symptoms
comparable volume of crystalloid or colloid
(Intraoperative
Therapeutic cytapheresis and

- Therapeutic cytapheresis: refers to the removal of cellular

Therapeutic plasmapheresis

- Blood collected during hemodilution can be save

Hemodilution) components as a form of treatment or to alleviate patient's
and re-transfused as whole blood
- Collection and reinfusion of blood lost by a symptoms.
INTRAOPERATIVE - Therapeutic plasmapheresis: refers to the removal of plasma
patient during the surgery
COLLECTION components; can be used to remove immune complexes,
- A device that utilizes vacuum is used to collect
(Intraoperative antibodies, inflammatory mediators, toxins, lipoproteins and
shed blood. Blood is then washed with saline
Salvage) platelet aggregating factors
and then concentrated to a Hct of 50-60%
- Blood is collected from a drainage tube at the - Therapeutic thrombocytopheresis: is used to treat patients
POST-OPERATIVE surgical site with abnormally high platelet counts such as myeloproliferative
COLLECTION - It is reinfused with or without processing via a disorders
microaggregate filter to screen out any debris - Therapeutic leukopheresis: can be used to treat patients with
leukemia
 ADVERSE EFFECT OF APHERESIS OTHER DEFERRALS
- Citrate toxicity Previous donation (whole blood)
- Vasovagal reactions - AABB 8 weeks
- Hypovolemia - Philippines
- Hemolysis o 450 mL 12 weeks (3 months)
- Air embolus o 200 mL 6-8 weeks
- Vascular access complications Apheresis donation (WBC/Platelet Apheresis) 2 days (48 hours)
- Depletion of clotting factors *Max of 24 platelet apheresis per year
- Circulatory and respiratory distress Infrequent Plasma apheresis 4 weeks
- Transfusion-transmitted diseases Double Red Cell Unit Apheresis 16 weeks
- Lymphocyte loss Childbirth
- Depletion of proteins and immunoglobulins/antibodies - AABB 6 weeks (after giving birth)
- Philippines 9 months (after giving birth) or 3
TYPES OF APHERESIS MACHINES months (after weaning)
Tooth extraction 3 days
1. BY CENTRIFUGATION: separates components based on density Acute Febrile episode 2-3 weeks
o Intermittent Flow Centrifugation Alcohol intake 12-24 hours
 Performed in cycles or passes Visit to countries with SARS 14 days (no infection)
 One cycle involves withdrawal of blood through a pump, addition 28 days (if infected)
of anticoagulant and separation processes, which is then followed - Malaria confirmed diagnosis 3 years
by reinfusion back into the donor - Resident in an area endemic for (after being asymptomatic)
 Equipment is smaller and mobile compared to continuous flow malaria for at least 5 consecutive years
centrifugation - Travel to endemic area (prior resident
 Can be: of endemic country <3 years after
 One-arm procedure: blood is drawn & reinfused through leaving)
same needle - Travel to endemic area for more than 1 year (if asymptomatic)
 Two-arm procedure: blood is drawn on one arm and 24 hours and less than 5 years
reinfused on the other arm - Travel to endemic area (prior resident
o Continuous Flow Centrifugation of endemic country >3 years after
 Involves withdrawal, processing and reinfusion of blood to the leaving)
individual simultaneously Travel in endemic country but not endemic No deferral
 Does NOT involve cycles or passes area for more than 24 hours
 Blood is withdrawn and returned continuously; would require two
arms for the procedure DRUGS
Tegison/ Etretinate (treatment for psoriasis) Permanent
2. BY FILTRATION: separates components based on size Soriatane (treatment for psoriasis) 3 years
3. BY CENTRIFUGATION & FILTRATION
Avodart (treatment for Benign Prostatic Hyperplasia) 6 months
Accutane/ Isotretinoin (treatment for acne) 1 month
BLOOD DONOR DEFERRALS Propecia (treatment for baldness)
Proscar/ Finasteride (treatment for BPH)
PERMANENT DEFERRAL Aspirin 3 days
- History of parenteral drug use (if for platelet concentrate
- Men-to-men sexual contact since 1977 production/
- Received clotting factor concentrate (donor may be hemophiliac) plateletpheresis)
- Men & women engaged in sex for money/ drug since 1977
- High risk occupation (i.e, prostitution) 36 hours (AABB/DOH)
- Positive Viral Hepatitis since 11 years of age
No deferral
- Who spent 3 months in UK from 1980 through 1996 (if for whole blood)
- Spent 5 years in Europe from 1980 to the present
- Positive test for HBsAg
VACCINES
- Reactive test for anti-HBc on more than one occasion
- Rabies vaccine (following bite from rabid animal) 1 year (12 months)
- Babesiosis/Chagas Disease
- Hepatitis B Immune globulin (HBlg)
- Have Leukemia/Lymphoma or other types of Cancer
- Gamma globulin
- Presence of HCV, HTLV, HIV infection or previous donation associated with
- Rh Immune globulin (RhoGam) for HDN
HCV, HIV, HTLV transmission.
- Risk factors for Creutzfeldt-Jakob disease (prions) or who has an immediate prevention
family with CJD Live attenuated Vaccines of: 4 weeks
- Received human derived growth hormone/ corneal/ dura mater - German measles (Rubella)
- Received bovine insulin (Mad cow disease) - Chicken pox (Varicella Zoster Virus)
- Tegison (treatment for psoriasis; considered teratogenic or may cause Live attenuated Vaccines of: 2 weeks
developmental or congenital malformation) - Measles (Rubeola)
- Oral Polio (Sabin vaccine)
- Mumps
ONE (1) YEAR DEFERRAL
- Oral typhoid
- Mucous membranes exposed to blood - Yellow fever Henry: 2 weeks
- Nonsterile skin or needle penetration - Small pox (Henry's) Harmening: 8 weeks
- Sexual contact with an individual (+) for HbsAg / with viral hepatitis / with Toxoids/killed/synthetic viral, bacterial, rickettsial No deferral
HIV infection or at risk for HIV infection vaccines, especially if donor is asymptomatic
- Incarceration / Imprisonment for >72 hours (3 days) - Cholera
- History of Syphilis or Gonorrhea - Anthrax
- Received blood transfusion or tissue transplant - DPT (Diphtheria, Pertussis, Tetanus)
- Dental operations - Influenza
- Tattoo & Skin Piercing - Paratyphoid & Typhoid
- Rape Victims - Polio (Injection/ Salk Vaccine)
- RMSF
NO DEFERRAL - Typhus
- Recipients of Recombinant GH - Non-replicating, inactivated, or mRNA-based Asymptomatic: No
- History of TB that has been fully treated and is no longer active COVID-19 vaccine (AztraZeneca, Janssen/J&J, deferral
Moderna, Novovax, Pfizer)
- Live-attenuated viral COVID-19 vaccine 28 days (DOH/WHO)
- Uncertain which COVID-19 vaccine was
administered 14 days (US
- With symptoms FDA/AABB)
DONOR MANAGEMENT, BLOOD COLLECTION & PROCESSING - ACD/CPD: 9:1
- Currently, the maximum amount of blood that can be collected from a donor
is: 525 mL (500 mL for transfusion, 25 mL for testing, 70 mL anticoagulant)
DONOR MANAGEMENT
- If collection exceeds 8 minutes; unit may not be suitable for platelets
o Blood is then stored at 1-6oC (refrigerator temperature); for units as a
ADVERSE DONOR REACTIONS MANAGEMENT TO BE DONE source of platelet store at 20-24oC (room temperature)
Fainting & vasovagal reactions 1. Apply cold compress on donor's o Blood must be processed within 6-8 hours of collection
(leads to blood vessel dilation) forehead or at the back of the neck
- Characterized by general (nape) BLOOD PROCESSING PROCEDURES
weakness, sweating, 2. Raise donor's legs above the level of 1. ABO/Rh
dizziness, pallor, & loss of the head 2. Antibody Screen
consciousness 3. Loosen tight clothing and secure
3. Serology (prevents transmission of disease)
- Blood pressure is low airway
o HBsAg o Hepatitis C (NAT) o Syphilis
4. Administer aromatic spirit of
- Pulse rate falls below normal ammonia by inhalation. o Anti-HBc o HIV-1 (NAT) o West Nile Virus (NAT)
range *Donor should respond by coughing o Anti-HCV o Anti-HIV ½ o Anti-T. cruzi
5. Monitor vital signs o Anti-HTLV I/II
6. Last resort: discontinue Anti-HCV
7. Administer glucose solution if
- 90% of post transfusion hepatitis is caused by non-A, non-B hepatitis and
necessary
only 10% is caused by hepatitis B
Hyperventilation
- Supporting tests that could help screen blood for Hepatitis C include:
- Twitching or muscle spasms 1. Talk to the patient to disrupt his/her
o ALT (Alanine aminotransferase)
are the initial signs of breathing patterns
2. Have the donor breathe into a paper o Anti-HBc (used for ruling out Hepatitis B)
hyperventilation
- May induce tonic-clonic bag Anti-HIV 1/2
movements similar to - If initial screening test is (-), the unit is suitable for transfusion
convulsion or grand mal - If initial screening test is (+), repeat test and if any test is (+), unit should be
seizure discarded
Nausea and vomiting 1. Instruct the patient to breathe slowly - ELISA is used for screening, Western Blot Technique is used as the
- Stomach distress and urge and deeply. Instruct them to turn their confirmatory test
to vomit head to one side - Donor units or blood bag confirmatory testing for HIV: send to RITM
2. Provide an emesis basin and clean (Research Institute for Tropical Medicine)
towels
- Patient confirmatory testing for HIV: send to SACCL/SLH (STD AIDS
Cardiogenic or Hypovolemic 1. Pharmacologic therapy to maintain
Cooperative Central Laboratory/San Lazaro Hospital)
Shock blood pressure and cardiac output
- Characterized by increase in 2. Admission to an intensive care Anti-HTLV I/II
Pulse Rate facility/unit - HTLV: Human T-cell Lymphotropic Virus
3. Restoration of coronary blood flow - HTLV-1 is the causative agent of adult T-cell Leukemia
4. Electrolyte and acid-base correction - HTLV-2 is similar to type 1 but is more prevalent in IV drug users.
Hematoma - EIA/ELISA serves as screening, Western Blot, RIPA
- It is a localized collection of 1. Remove tourniquet & withdraw needle
(Radioimmunoprecipitation assay), NAT (Nucleic Acid Test) serve as
extravasated blood under 2. Apply pressure with sterile gauze for 7-
confirmatory
the skin resulting in a bluish 10 minutes
discoloration 3. Apply ice/cold compress to the area Syphilis
- Caused by needle going for 5 minutes - Causative agent: Treponema pallidum
through a vein, with - Spirochetes cannot live in blood stored for 3-4 days at 1-6oC; making
subsequent leakage of blood
platelets the only unit capable of transmitting it
Convulsion/Seizure 1. Discontinue
- RPR (Rapid Plasma Reagin) and VDRL (Venereal Disease Research
- Possible involuntary loss of 2. Maintain open airway
Laboratory) serve as screening tests. They detect reagin or antibody
control of urine/stool during 3. Restrain gently
episode directed toward cardiolipin particles
Cardiopulmonary Emergency / 1. Ventilation (CPR if necessary) - FTA-ABS (Fluorescent Treponemal Antibody Absorption) serve as the
Arrest 2. Transfer to emergency medical facility confirmatory test
Jet-like bleeding with bright red 1. Discontinue immediately BLOOD PRESERVATION
blood 2. Apply pressure on site for 10 minutes
- Inadvertent puncture of 3. Apply dressing on site A. ANTICOAGULANTS
artery
Shooting pain followed by 1. Apply support to arm 1. Composition
numbness and tingling on o All of the main anticoagulant preservatives utilize a combination of the
forearm following constituents:
- Inadvertent puncture of Components Function
median nerve/cutaneous
Citrate - Chelates Calcium (prevents clotting; anticoagulant)
branches
Adenine - Increases ADP levels, driving glycolysis toward ATP
synthesis
POST-DONATION CARE - Prolongs shelf-life for up to 21-35 days
1. Raise arm and apply pressure on the puncture site after removal of the Glucose - Serves as energy source for cells; a.k.a. Dextrose
needle - Substrate for ATP production
2. Rest after blood collection, recline for a few minutes, then sit upright and
Phosphate - Maintains pH during storage
allow to walk
3. Instruct the donor to drink lots of water, refrain from smoking and avoid - Monobasic phosphate; serves as 2,3 DPG source,
strenuous works promoting O2 release to tissues
Citric acid - Prevents caramelization of glucose
BLOOD COLLECTION PROCEDURES 2. Types
- Use of aseptic technique:
o Iodine Anticoagulant Storage Time Notes
o Chlorhexidine gluconate with Isopropanol (If patient is allergic to Heparin 2 days Only for priming heart-lung machines
iodine) ACD-A Used for apheresis components
o Scrub site at least 4 cm in all directions for 30 seconds CPD 21 days
- Apply tourniquet or BP cuffs (inflated to 40-60 mmHg) CP2D
- Gauge of needle for blood donation is 16g CPDA-1 35 days Current standard anticoagulant
- Insertion angle is 20 degrees
- Patient should open & close his/her hand every 10-12 seconds during the
collection
- Mix bag periodically: 1-2x per minute or every 30 – 45 seconds
- 450 mL +10% blood is drawn over 7-10 minutes; anticoagulant is 63 mL
- Blood to anticoagulant ratio (450 mL): 7:1
- Blood to anticoagulant ratio (250 mL): 8:1
B. ADDITIVE SOLUTIONS (AS) F. RBC SUBSTITUTES
- These are solutions that are added to RBCs -after- removal of plasma. - Described as oxygen therapeutics and as Artificial Oxygen Carriers (AOC)
- AS are composed of: SAGM (Saline, Adenine, Glucose, Mannitol) 1. HBOC (Hemoglobin-based Oxygen Carriers)
- Mannitol acts as a stabilizing agent; retards in-vitro storage lysis of cells o Stroma-free hemoglobin
- Units with AS are stored up to 42 days o Polymerized hemoglobin
- Added to RBC concentrates with high hematocrit (viscous), Decreasing o Liposome-encapsulated hemoglobin
hematocrit from 70-85% to 50-60% 2. PFC (Perfluorocarbons)
Storage
Additive Solution Abbrev. Solution G. CONSIDERATIONS
Time
Adsol 1. 70% is the minimum amount of the RBCs must remain viable at the end of
(Baxter Healthcare)
AS-1 SAGM the permitted storage period
Saline, Adenine, 2. 75% of cells that have been transfused should remain viable for 24 hours
Nutricel AS-3 Glucose, Citrate- (post transfusion viability).
(Pal Corp.) 3. 1-10°C is the transport storage temperature. Ice or other cooling devices
phosphate 42 days
Optisol should not be in physical contact with the unit to prevent hemolysis
(Terumo Corporation)
AS-5 SAGM
SOLX AS-7 SAGM BLOOD COMPONENTS & COMPONENT PREPARATION
(Haemonetics)
- AS-1, AS-5, and AS-7 contain saline, adenine, and glucose. They also
A. BLOOD BAG
contain mannitol, which protects against storage-related hemolysis,
whereas AS-3 contains citrate and phosphate for the same purpose. Color Coding:
FDA 1985 RA 1517
Blood Type Color Color Label
A Yellow Blue
B Pink Yellow
AB White Pink
O Blue White
C. REJUVENATING SOLUTIONS (RS)
- These are solutions used to restore ATP and 2,3 DPG levels. RS are Additional Colors:
composed of: PIGPA/PIPA (Phosphate, Inosine, Glucose, Pyruvate, - Tan - Hold for further testing - Orange - For emergency use only
Adenine or Phosphate, Inosine, Pyruvate, Adenine) - Green - For autologous use only - Purple - Irradiated
- RBCS can be rejuvenated at up to 3 days after outdate. - Gray - NOT for transfusion - Red - Biohazard
- Manner: Done by placing RBC unit w/ 50 mL rejuvenating solution, for 1 - Chartreuse (yellow-green) - From
hour @ 37oC therapeutic phlebotomy
- Lifespan: Within 24 hours
- Purpose: For saving O type and rare blood Materials:
1. PVC (Polyvinyl chloride; most commonly used)
D. RBC FREEZING 2. Polyolefin containers
- Use of cryoprotective agents 3. Latex-free plastics
- Penetrating: Glycerol, DMSO (Dimethylsulfoxide)
o These are small molecules that enter cells and prevent dehydration Blood bag materials must have certain qualities:
- Non penetrating: HES (Hydroxyethyl starch), PVP (Polyvinylpyrrolidone), - Adequate flexibility and strength to withstand centrifugation and handling
glucose - Temperature resistance for both steam sterilization and freezing
o These are large molecules that form shells around the cells and - Limited toxicity to the transfusion recipient
prevent escape of water - Compatibility with cells and plasma to reduce component adulteration
- Selective permeability for cellular gas exchange
1. GLYCEROLIZATION - Water and pathogen impermeability
 RBCs are added with a cryoprotective agent, commonly glycerol, - Transparency
and frozen to prolong their lifespan. RBCs to be frozen should be
less than 6 days old.
Types:
 Manner: Glycerol is added by vigorously shaking unit so that
1. Single
glycerol may permeate the cell membranes
2. Double
 Lifespan: 10 years (Autologous donor units and rare blood units)
3. Triple
 Lifespan after thawing: 24 hours (lf regular method; OPEN
4. Quadruple
System)
5. Penta bags
HIGH LOW
CONCENTRATION CONCENTRATION B. COMPONENT PREPARATION
GLYCEROL GLYCEROL
(40% w/v) (20% w/v)
Initial Freezing Temperature -80oC -196oC
Need to control freezing rate No Yes
Freezer Type Mechanical Liquid Nitrogen
Shipping requirement Dry ice Liquid Nitrogen
Maximum Storage -65oC -120oC
Temperature (can be thawed and (critical to temp
refrozen) changes)
2. DEGLYCEROLIZATION
 This is done before infusion to prevent cell lysis. This is achieved
by replacing glycerol with decreasing amounts of saline
Saline Concentration (HIGH) Saline Concentration (LOW)
1. 12% Saline 1. 45% Saline in 15% Mannitol
2. 1.6% Saline 2. 0.2% dextrose w/ NSS (0.9%)
3. 0.2% dextrose w/ NSS (0.9%)

E. STORAGE LESION

STORAGE LESIONS
2,3-DPG (decreased at 2nd week of storage; reform or attain
normal levels upon 24 hours in vivo circulation) - Centrifuge:
pH 1. LIGHT SPIN - @ 3,200 RPM or 2,000 g for 3 minutes
DECREASE o separates RBCs and Plasma
Glucose
ATP 2. HARD SPIN - @ 3,500 RPM or 5,000 for 5-8 minutes
o separates RBCs, plasma, and buffy coat (WBCs and Platelets)
Sodium
Hemoglobin Lactic acid
INCREASE
Ammonium Potassium, Phosphate
C. BLOOD PRODUCTS 10. SINGLE DONOR - Bleeding due to quali/quantitative platelet defect
PLATELET - Prevent HLA alloimmunization & platelet
BLOOD PRODUCTS FUNCTIONS/INDICATIONS refractoriness
Derived from - One unit contains minimum number of platelets:
- To restore blood mass & volume
WHOLE BLOOD plateletpheresis 3.0 x 1011 platelets
1. a. Rapid blood loss
- Transfusion of this unit can increase platelet
b. Symptomatic anemia with large volume
count by: 30,000-60,000 platelets per unit
450mL = 63 mL AC deficit
500mL = 70 mL AC c. Neonatal exchange transfusion - For von Willebrand's disease,
525mL = 70 mL AC - Each unit should increase the Hypofibrinogenemia, Hemophilia A (Factor VIII
o Hematocrit: 3-5% 11. CRYOPRECIPITATE deficiency), Factor XIII deficiency, and DIC
o Hemoglobin: 1-1.5 g/dL - Used as fibrin glue (cryoprecipitate + thrombin)
- To increase the RBC mass in patients who have Prepared by thawing - Contains
2. PACKED RED symptomatic anemia. FFP @ 4oC, HARD o 150-250 mg fibrinogen (Factor I)
- For patients unable to tolerate a sudden increase Spin, leave only 10-15 o 80-120 U Factor VIII
BLOOD CELLS
in blood volume. mL plasma o 40-70 U vWF
(PRBCs) 20-30 U Factor XIII
- RBC aliquot (10-25 mL) - for neonates suffering o
from anemia due to FMH, obstetric accidents and o Fibronectin
Remove 200-250 mL of - Given to severely neutropenic patients
internal hemorrhage
plasma; PRBC Hct - Each unit should increase the - Patients who have overwhelming sepsis and
should be o Hematocrit: 3% 12. GRANULOCYTE those at risk of life threatening infections
60-80%. o Hemoglobin: 1 g/dL CONCENTRATES unresponsive to antibiotics
NEVER over 80% - 1-unit aliquot should increase the - Contain:
o Hemoglobin: 2-3 g/dL Obtained via apheresis o 200-600 mL plasma
- To treat symptomatic anemia on patients with o 1 x 1010 WBCs if corticosteroids and HES
severe allergic/ anaphylactic reaction are used
3. WASHED RED 13. PROTHROMBIN - For Factor II, VII, IX, X deficiencies
- To remove plasma proteins
BLOOD CELLS - Used for rare patients who have anti-IgA Ab COMPLEX - For Hemophilia B (Factor IX deficiency)
because of IgA deficiency (Factor IX Concentrate)
- Prevent non-hemolytic febrile transfusion 14. FACTOR VIII - For Hemophilia A (Factor VIII deficiency)
FROZEN &
4.
reactions, plasma allergies, & exposure to CMV CONCENTRATE
DEGLYCEROLIZED
- For long-term storage of rare donor units or - For patients with congenital
RBCs 15. IMMUNE SERUM
autologous units hypogammaglobulinemia / immunodeficiencies
GLOBULIN (ISG)
- Used to extend time between transfusions in (monthly administration)
NEOCYTE-
5. periodically/chronically transfused individuals 16. SINGLE DONOR - For hypovolemic shocks & severe burns
ENRICHED RBCs (patients with thalassemia) PLASMA
- Neocytes: young RBCs 17. VOLUME - For hemorrhagic shock and burn patients
- To minimize exposure to donor WBCs EXPANDERS
6. LEUKOREDUCED - Prevent febrile transfusion reaction caused by - For maintaining blood volume and colloidal
OR LEUKO-POOR antibodies against WBCs/anti-leukocyte oncotic pressure
18. 5% ALBUMIN
COMPONENTS antibodies - For patients under hypovolemic shock,
Prevent HLA alloimmunization, TRALI, and hypotensive episodes, severe burns
Remove 70% of WBCs o
Leave at least 85% GVHD SHELF-LIFE & STORAGE TEMPERATURE
pRBC - To reduce CMV transmission
2 x 109 – Normal WBC - Modes of leukocyte depletion BLOOD STORAGE TEMP. STORAGE TEMP/SHELF-
5 x 108 – prevent FNHTR 1. Leukofiltration COMPONENT LIFE
5 x 106 – prevent HLA 2. Centrifugation and buffy coat removal 1. Whole blood 1-6oC Anticoagulant-dependent
alloimmunization 3. Washed red cell concentrate 2. Packed RBCs 1-6oC Anticoagulant-dependent
4. Frozen deglycerolized RBCs Open system: 24 hours
7. FRESH FROZEN - Replenish plasma proteins and all coagulation 3. Washed RBCs 1-6oC Open system: 24 hours
PLASMA (FFP) factors 4. Leuko-reduced / 1-6oC Anticoagulant-dependent
- For patients with multiple coagulation factor Leuko-poor Open system: 24 hours
Contains 400 mg deficiency (Vitamin K Deficiency & Liver Components
fibrinogen Disease) & at risk of bleeding (DIC) 5. Deglycerolized 1-6oC 24 hours
Does not contain - To patients who have received massive RBCs
platelets transfusion 6. Frozen RBCs -65oC (High)
Plasma is frozen within -120oC (Low) 10 years
6-8 hours (otherwise, it 7. Irradiated RBCs 1-6oC 28 days after irradiation or
becomes PF24, plasma original outdate (whichever
frozen within 24 hours) comes first)
- Inactivate the replicative machinery of the donor 8. FFP -18oC 1 year
leukocyte -65oC 7 years
- Prevent GVHD Thawed: 1-6oC 24 hours
- Patients receiving chemotherapy/ radiotherapy 9. Cryoprecipitate -18oC 1 year
- Organ transplantation recipients who have been Pooled 4 hours
immunosuppressed Thawed (20-24oC) 6 hours
8. IRRADIATED 10. Granulocyte 20-24oC without 24 hours
- Low-birth-weight neonates
BLOOD concentrate agitation
- Patients with genetically deficient immune
COMPONENTS systems 11. Platelet 20-24oC with agitation 3-5 days
- Source of radiation (2 most commonly used concentrate 1-6oC 2 days
Open/pooled 4 hours
radioisotopes):
12. Factor VIII 1-6oC Variable shelf-life
o Cesium-137
Concentrate (Lyophilized or
o Cobalt-60 Freeze-dried)
13. Factor IX
- Minimum radiation required = 25-35 Gy Concentrate
- Bleeding due to quali/quantitative platelet 14. Normal Serum 2-10oC 5 years
defects Albumin (NSA) &
- Most prone blood unit to contamination (stored at Plasma Protein Prepared by Cohn
20-24oC; room temperature) Fraction (PPF) Ethanol Fractionation
- Contraindicated in TTP (Moschcowitz and Heat Inactivation
9. RANDOM DONOR Syndrome, spontaneous platelet activation) and NSA: 96% albumin,
PLATELET/ heparin thrombocytopenia 4% globulin
PLATELET - Taken from whole blood PPF: 83% albumin,
CONCENTRATE - Pooled from 6-8 donors 17% globulin
- Prepared 6-8 hours from collection
- One unit contains minimum number of platelets: STORAGE & TRANSPORTATION
5.5 x 1010 platelets
Refrigerator temperature 1-6oC
- Transfusion of this unit can increase platelet
Transport of blood components 1-10oC
count by: 5,000-10,000 platelets per unit
(Whole blood & Packed RBCs)
Platelet/Granulocyte concentrate 20-24oC
TRANSFUSION
TRANSFUSION PROCEDURE
- Checked & monitored by 2 nurses
1. Positive Identification of the patient, patient's blood specimen prior to
transfusion as well as the blood unit for transfusion is done:
o Clerical errors represent main cause of transfusion-related deaths
and acute HTRs
o Final clerical check is done at bedside by the nurse: checking the
blood bank tag attached to the component to be transfused, against
the patient armband
o Specimen must be collected within 3 days of scheduled transfusion
o Stoppered or sealed sample of donor's blood must be stored at 1-6oC
for at least 7 days after transfusion
2. Venous access
o Selection of location and type of access depends on the volume,
timing, and expected duration of transfusion therapy
 Peripheral access with an 18g needle or catheter is typically
sufficient
 Central venous access is desirable for high-volume
administration or long-term therapy
3. IV line
o Only isotonic saline or 5% albumin should be used as IV solutions to
dilute blood components
o Use of 5% dextrose in water (D5W) is PROHIBITED, since it is
hypotonic and will cause RBC lysis
o Use of Ringer’s solution is PROHIBITED, since it contains calcium and
will initiate coagulation
o NO medication must be added to the same line with blood components
4. Filters
a. First generation filters/Clot-screen filters
 Pore size of 170-260 um to remove gross clots and cell debris
b. Second generation filters/Microaggregate filters
 Pore size of 20-40 um to remove microaggregates such as
degenerated WBCs and cell fragments
c. Third generation filters/Leukoreduction filters
 Removes WBC leaving less than 5 x 106
 Pore size of 10 um
5. Vital Signs of patient must be monitored (PR, RR, BP, Temp)
6. Blood warmer
o Cold blood can cause hypothermia in patient = cardiac arrhythmia &
hemorrhage.
o Blood warmers should be set at 37oC. Blood must NOT be warmed
over 38oC.
o Automatic temperature control is set to alarm when blood is warmed
over 42oC.
o Never use water baths or microwaves to warm blood
o For patients receiving many units in a short period of time
7. Speed & Length of Infusion
o Infused slowly for the first 10-15 minutes (approximately 2 mL/minute
for the first 15 minutes) while the patient is observed with signs of
transfusion reaction
o Administration rate may be increased if patient seems normal throughout
o Plasma/platelet transfusion should be done within 30-60 minutes
o Desirable time to complete a transfusion is within 2 hours.
o ANY transfusion should be done within 4 hours
REISSUE OF UNIT
Blood can be reissued after returning from the ward if:
- The closure must not have been entered in any way
- The blood must have been kept between 1-10oC on a continuous basis
- The pilot tube or sealed segment is still attached to the container
- The blood must not have been away from the blood blank for >30 minutes
- Records must be available that verify all inspection criteria
SPECIAL CONSIDERATIONS IN TRANSFUSION
1. Emergency Transfusion
o Group O, Rh negative red cells are given when patient ABO/Rh typing
are unknown
o They contain no antigens
2. Massive Transfusion
o Administration of enough blood or components in less than 24 hours
to constitute a complete volume replacement
 For adults: 8-10 units
 For infant: more than half a unit
o Adverse effects of Massive Transfusion
 Citrate toxicity & Hypocalcemia
 Hypothermia
 2,3 DPG depletion
 Depletion of coagulation factors and platelets
 Accumulation of biochemical and microaggregates
3. Neonatal RBC Transfusion
o Less than 7-days old blood to reduce hyperkalemia and maximize
2,3-DPG
o Anticoagulant: CPDA1
o Irradiated
o CMV Negative/Leukocyte reduced
o HbS (-)
o O (-) or compatible with mother & infant
o Dose: 10mL per kilogram for 2-3 hours
4. Exchange Transfusion
o Done on a newborn infant suffering from anemia and
hyperbilirubinemia due to HDN
o Small amounts of blood are removed from the baby and replaced with
donor blood
o Prevents jaundice and kernicterus
o Purpose
 Replace antibody-coated cells with compatible donor cells
 Remove bilirubin to prevent kernicterus (>20mg/dL of bilirubin)
 Remove circulating antibodies in baby's plasma
 Suppress erythropoiesis (production of incompatible RBCs)
o Indications
 Greater than 0.5 mg/dL/hr rise in bilirubin
 Rise of 10 mg/dL in the first 24 hours
o Selection of blood for exchange transfusion
 Must be negative for RBC antigen to which the mother has the
corresponding antibody
 Must be ABO and Rh type compatible with infant’s blood type
 If mother’s specimen is unavailable, O (-) red cells must be
selected
 CMV (-), HbS (-), and less than 7 days old
 Irradiated to prevent TAGVHD
 Blood can be prepared using RBCs from the whole blood units
and replacing the plasma with group AB plasma to reduce amount
of blood group antibodies
5. Intrauterine Transfusion
o Performed when baby inside the mother's womb is suffering from
severe anemia due to HDN
o Done by:
 Intraperitoneally: injecting the RBCs into the fetal peritoneal
cavity where RBCs can be absorbed into the circulation
 Cordocentesis: donor RBCs are directly injected into fetal
umbilical vein
o Purpose: Maintain fetal Hgb above – 10 g/dL
o Once initiated the procedure must be repeated every 2-4 weeks until
34-36 weeks gestation or until fetal lungs mature
o Indications:
 When amniotic fluid is A450 nm results are in high zone Il/zone III
 When cordocentesis blood sample has Hgb of less than 10 g/dL
 Fetal hydrops is noted on ultrasound examination
o Selection of blood for intrauterine transfusion
 Most centers use O (-) RBCs
 Must be compatible with the mother’s specimen
 Irradiated to prevent TAGVHD
TRANSFUSION REACTIONS
- STOP TRANSFUSION but keep IV lines open with physiologic saline
- Primary or basic testing involves the post-transfusion sample only
- CHAD = Clerical check, Hemolysis check, ABO testing, DAT
TRANSFUSION REACTION INVESTIGATION WORK-UP
1. Clerical Checks
2. Visual inspection of patient's pre & post transfusion specimen
o Pink Plasma = specimen contains 20 mg/dL free hemoglobin
o Red Plasma = specimen contains >100 mg/dL free hemoglobin
o Plasma Hgb over 25 mg/dL can be detected by the naked eye
3. DAT
4. Repeat ABO/Rh Typing
5. Repeat Compatibility Testing (Antibody screen and panel or identification)
6. Repeat Crossmatch with pre & post transfusion specimen
7. Bilirubin test: maximum concentration of bilirubin after hemolysis will be
evident in blood approximately 3-6 hours after transfusion
8. Urine: First voided specimen is checked for free hemoglobin and
urobilinogen
9. Hemosiderin can be found weeks after transfusion reaction
10. Hemoglobin & Hematocrit
TYPES OF TRANSFUSION REACTIONS - Cause: Air allowed into infusion equipment or blood
5. AIR EMBOLISM in open system infused under pressure causing air
1. HEMOLYTIC TRANSFUSION REACTIONS bubble
- Cause:
Possible Causes: o Rapid infusion of large volume of blood
- Antibody binding to RBC products iatrogenic (physician-induced
- Release of Anaphylatoxin (histamine; leads to hypotension) transfusion reaction)
- Activation of Cytokines 6. TRANSFUSION - Prevention:
- Activation of Coagulation Cascade ASSOCIATED o Slower rate of infusion 100 mL/hr (usual is
CIRCULATORY 200mL/hr)
Immediate HTR Delayed HTR OVERLOAD (TACO) o Split into aliquots
- Treatment:
- Occurs very soon during or just - Associated with an anamnestic
o Intravenous Diuretics
after transfusion response in a patient who has
o Therapeutic Phlebotomy (worst case)
- Reaction occurs within the first previously been sensitized by
o O2 therapy (hyperbaric therapy)
24 hours transfusion, pregnancy, or transplant
- Most severe: usually caused by - May be diagnosed in 7-10 days up to - Manifestations:
ABO incompatibility 14 days after transfusion o Shaking chills, Hemoglobinuria, DIC,
- s/s: fever with back pain - s/s: Jaundice and decreasing Oliguria/anuria
- Considered a medical Hematocrit levels o A 2oC temperature rise associated with
emergency; stop transfusion and - Common antibodies implicated in transfusion
induce diuresis by administering DHTR: anti-Jka, anti-E, anti-D, anti-C, - Causative Agents:
mannitol or potassium-sparing anti-K, anti-Fya, and anti-M 7. BACTERIAL 1. Yersinia enterocolitica (most common)
diuretics CONTAMINATION/ 2. Serratia liquefaciens
TRANSFUSION 3. Pseudomonas fluorescens
Effects in Blood: Effects in Blood:
ASSOCIATED 4. Pseudomonas aeruginosa
- ↑ plasma free Hb - ↓ Hb and Hct
SEPSIS (TAS) 5. Escherichia coli
- ↑ Bilirubin - (+) DAT
- Prevention:
- ↓ Haptoglobin - (+) post-transfusion antibody screen
o Visual inspection of unit before transfusion
- (+)/(-) DAT
(Check for Brown or Purple Discolorations,
Hemolysis, Clots, Cloudiness)
2. PHYSICALLY/ CHEMICALLY-INDUCED/ NON-IMMUNE HEMOLYTIC
- Treatment: Introduce broad-spectrum antibiotics
TRANSFUSION REACTION
to intravenous lines
Possible Causes: DELAYED NHTR
- Physical damage due to intravascular lysis by hypertonic or hypotonic Reaction Cause & Prevention Treatment
solutions - Cause: Prior exposure to blood components
- Heat Damage due to blood warmers - Prevention:
- Freeze Damage due to absence of cryopreservative o Matching of donor RBC phenotypes (for red
- Mechanical damage by blood pumps, roller pumps, infusion under higher 1. ALLOIMMUNIZATION
cell antigen alloimmunization)
pressure through small-bore needles o Matching of donor WBC phenotypes (for WBC
antigen alloimmunization)
3. NON-HEMOLYTIC TRANSFUSION REACTIONS - Usually occurs 1 week after transfusion
- Observed in multiparous women
IMMEDIATE NHTR - Associated with platelet refractoriness
REACTION MANIFESTATIONS, CAUSE & PREVENTION/ - Cause:
TREATMENT 2. POST- o Anamnestic production of platelet antibodies
- Manifestations: Fever only TRANSFUSION o Most commonly involved antibody is anti-
- A 1oC temperature rise is associated with PURPURA (PTP) human platelet antigen-1a (HPA-1a or anti-
1. FEBRILE NHTR HPA-1)
transfusion and without any other explanation
- Cause: Anti-leukocyte antibodies - Prevention:
Most commonly o Future transfusion should consist of platelet
- Prevention:
encountered type of TR negative for the antigen
o Administer leukoreduced components
o Remove all plasma before transfusion 3. TRANSFUSION- - Cause: Proliferation of T lymphocytes from donor
- Manifestations: hives (urticaria)/pruritus ASSOCIATED blood responding to HLA in the patient
o Hives: reddish, edematous patches of the skin GRAFT VERSUS - Prevention: Irradiated blood components
and mucous membranes HOST DISEASE
o Pruritus: itching - A.k.a. Transfusion Hemosiderosis
2. ALLERGIC/ - Cause: - In patients with aplastic anemia, congenital
URTICARIAL o Passive transfer of donor plasma which hemolytic anemia, thalassemia, chronically
TRANSFUSION contain foreign protein (allergen) that reacted 4. IRON OVERLOAD transfused patients
REACTION with patient IgE / IgG - Cause: Chronic transfusion
- Prevention: - Prevention: transfuse neocytes instead (neocyte-
o Administer washed components enriched red blood cells)
- Treatment: - Treatment: Iron chelation therapy, Deferoxamine,
o Administer: Anti-histamine Desferrioxamine
- Manifestations: Clinical signs (namely hypotension - Cause: Massive transfusion
& shock) are seen suddenly after infusion of only a 5. CITRATE - Prevention:
few mL of blood component (Often before 10 mL OVERLOAD o Negated by calcium chloride or calcium
3. ANAPHYLACTIC/ of plasma has been infused) gluconate solution
ANAPHYLACTOID - Cause: Transfusion of IgA positive blood to an IgA-
TRANSFUSION deficient recipient with anti-IgA
REACTION - Prevention:
o Remove plasma / wash components
o Transfuse components from IgA deficient
donors
- Manifestations:
4. NONCARDIOGENIC
o Bilateral pulmonary edema, Hypotension
PULMONARY
o Increased respiratory distress shortly after
EDEMA (NCPE)/
transfusion
TRANFUSION-
o Occurs within 6 hours of a plasma-containing
RELATED ACUTE
transfusion
LUNG INJURY
- Cause: Anti-leukocyte antibodies
(TRALI)
- Prevention: Transfuse leukoreduced components
HEMOLYTIC DISEASE OF THE NEWBORN (HDN) PREVENTION
- A.k.a. Erythroblastosis fetalis - Rh antibody production is stimulated during delivery of first child; wherein
- Fetal red cells are coated with maternal antibodies that correspond to Fetomaternal Hemorrhage occurs
specific fetal antigens - At least 30mL of fetal RBCs pass into maternal circulation
- Caused by immune isoantibodies (isoagglutinins) from the mother; crossing - To prevent this, Rh (-) mothers who have delivered Rh (+) babies must be
the placenta and agglutinating fetal red cells. given RhoGam or RhIg
- Conditions for HDN to occur:
o Baby should be (+) for the antigen ADMINISTRATION GUIDELINES
o Mother should be (-) for the antigen - Rh negative woman who is not Rh-alloimmunized should receive anti-D
o Mother must contain the specific antibody globulin
o Antibody must be IgG (crosses the placenta) - At 28 weeks gestation, unless the father of the baby is known to be Rh D
PATHOGENESIS negative; Within 72 hours after deliver of Rh D positive infant
- After a first trimester pregnancy loss
- After invasive procedures (CVS [Chorionic Villus Sampling], Amniocentesis,
1. RBC destruction leads to severe anemia
FBS)
2. Stimulates BM to produce more RBCs; immature cells are released
3. Hepatomegaly and Splenomegaly occur
Anti-D Immunoglobulin Prophylaxis should be considered if patient
4. Severe anemia and hypoproteinemia due to liver damage causes high
experienced:
cardiac output with edema, effusion, ascites
- Threatened abortion
5. Hgb released due to RBC lysis is conjugated by maternal liver
- Second or third trimester antenatal bleeding
6. Once delivery is done, child is at risk because newborn liver cannot
- External cephalic version
conjugate bilirubin leading to hyperbilirubinemia and jaundice
- Abdominal trauma
7. Unconjugated bilirubin levels, if at or beyond 18 mg/dL can cause
- Accidental transfusion
kernicterus
TESTS TO DETERMINE FMH
1. FETAL SCREEN ROSETTE METHOD (Qualitative Determination)

𝑀𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑏𝑙𝑜𝑜𝑑 (𝑤𝑖𝑡ℎ 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠) + 𝐴𝑛𝑡𝑖 − 𝐷 + 𝑅ℎ(+) 𝑖𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 𝑐𝑒𝑙𝑙𝑠

= 𝑅𝑜𝑠𝑒𝑡𝑡𝑒 𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛

2. KLEIHAUER BETKE ACID ELUTION TEST (Quantitative Determination)

o Use of citric acid buffer
o Cause maternal cells (HbA) to burst (ghost cells) while fetal cells
remain intact (stained pink)

CALCULATING FOR FMH & # OF RHOGAM VIALS TO GIVE

Formula:
% 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 𝑥 𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒
𝐹𝑀𝐻 =
100
# 𝑜𝑓 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠
% 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 = 𝑥 100
2000 (𝑡𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑)
- Most COMMON type is ABO HDN # 𝑜𝑓 𝑓𝑒𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 𝑥 𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒
- Most SEVERE type is Rh HDN 𝐹𝑀𝐻 =
2000

ABO HDN Rh HDN Note: If Maternal Blood Volume is NOT given but mother's weight is:
Mother Group O Rh (-)
𝑀𝐵𝑉 = 𝑊𝑒𝑖𝑔ℎ𝑡 (𝑘𝑔) 𝑥 70 𝑚𝐿/𝑘𝑔
Infant Group A, B, AB Rh (+)
Antibody IgG Yes Yes Note: If neither weight or blood volume is given, use average maternal blood
Anti-AB Anti-D volume
Occurrence in first born Yes No/Rare 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑚𝑎𝑡𝑒𝑟𝑛𝑎𝑙 𝑏𝑙𝑜𝑜𝑑 𝑣𝑜𝑙𝑢𝑚𝑒: 5000 𝑚𝐿
45-50% of cases 5% of cases
Stillbirth and/or hydrops Rare Frequent Sample Problem:
Disease predicted by titers No Yes - Kleihauer-Betke acid elution stain for post-partum FMH is reported as 1.3%
Bilirubin at birth Normal Elevated fetal cells. What is the total volume of FMH?
Severe anemia Rare Frequent 1.3% 𝑥 5000
𝐹𝑀𝐻 = = 𝟔𝟓 𝒎𝑳
DAT Weakly +/- (+) 100
Phototherapy Yes Yes - We therefore need to neutralize 65 mL of fetal red cells
(Often)
Exchange transfusion Rare Sometimes
CALCULATING # OF RHOGAM VIALS NEEDED
Intrauterine transfusion None Sometimes
Spherocytosis Yes Rare - Note: A standard dose of Rhogam (300 ug) can neutralize 30 mL D(+) fetal
cells
- Formula:
- HDN can also be caused by: Duffy, MNSs, Kidd, Kell
WB: # of Rhogam vials = FMH / 30
- HDN cannot be caused by: Lewis, I, P1
Or PRBC: # of Rhogam vials = FMH / 15
TREATMENT & ASSESSMENT
- Early delivery (induced labor) - Sample Problem:
- Intrauterine transfusion o Using answer from the previous problem. Considered FMH as whole
- Exchange transfusion blood
- Phototherapy (480-510 nm) 65
- Amniocentesis = = 2.17 𝑜𝑟 𝟑
30
o Assess severity of HDN in utero
o Amniotic fluid absorbance is determined using spectrophotometric - HOWEVER, we always add an additional one vial for protection purposes.
analysis at 365-550 nm; bilirubin peaks at 450 nm (OD 450nm) - Answer is therefore 3 vials
o Plot in Liley graph (absorbance against gestational age)
 Zone 1: mildly affected or unaffected
 Zone 2: moderately affected; requires close monitoring
 Zone 3: severely affected; consider induced/forced labor and
intrauterine transfusion
AUTOMIMMUNE HEMOLYTIC ANEMIA
- Associated with the presence of autoantibodies that agglutinate, sensitize
or lyse one's own red cells

CAN BE CLASSIFIED INTO:

- Most common type (70% of cases) QUALITY ASSURANCE PRACTICES IN BLOOD BANKING
Warm Autoimmune - Antibodies are reactive at warm temperatures;
Hemolytic Anemia IgG 1. Blood banks are inspected annually (every year)
(WAIHA) - Antibodies have Rh-like specificity (simple anti- 2. Antisera should be stored at 2-6oC when not in use
e specificity) 3. Freeze rare antisera for extended storage
Cold Autoimmune - Antibodies are reactive at colder temperatures; o Divide antisera into aliquots to avoid repeated freezing and thawing
Hemolytic Anemia IgM o Thaw at 37oC and mix before use
(CAIHA) 4. Blood banks must check each reagent each day of use for correct reactivity
Drug-Induced - Result of patient's production to antibody from a 5. RBCs selected as + controls for antisera must be heterozygous for the
Hemolytic Anemia particular drug or drug complex (ex. antigen being tested in order to provide accurate indication of antisera
(DIHA) Cephalosporins) potency
6. Positive and Negative controls must be used for each reagent tested
ALTERNATIVE TECHNOLOGIES 7. Anti-A, Anti-B, Anti-AB, Anti-D, Anti-D control, and AHG reagents must be
tested daily
8. Reagent red cells must be checked for presence of hemolysis
GEL TECHNOLOGY 9. Timing of the centrifuge and serofuges must be checked periodically with
- Performed in a specially designed microtube a stopwatch; Speed should be monitored with tachometer every 6 months
- Developed by Yves Lapierre in 1985 10. Water baths & heat blocks should be maintained at 37oC
- Controlled centrifugation of RBCs is done through a Dextran acrylamide gel 11. Refrigerators, Freezers, and platelet incubators should have temperature
that contains predispensed reagents or no reagents at all monitoring every 4 hours
- Each microtube is composed of: 12. Rh view box glass surface temperature should be at 45-50oC (Anti-D and
o Upper reaction chamber wider than the tube itself Red cells will be reacting at 37°C)
o Long, narrow portion called column 13. Donors' hemoglobin can be determined using copper sulfate method.
- Use gel card approximately 5x7 cm with 6 microtubes o Solution has a specific gravity of 1.053 (equivalent to 12.5 g/dL)
- Principle: size exclusion chromatography o Solution should be changed daily
- Advantages: o 25 tests can be performed in a 30 mL container of the solution
o Major advantage: standardization (easy to interpret even for unskilled o Drop of blood is placed at about 1 cm from the surface of the solution
staff)  Drop should sink within 30 seconds; otherwise hemoglobin value
o Offers more objective, consistent, and reproducible interpretation of is unacceptable
the results
o Decreased sample volume needed for testing RETENTION OF DONOR RECORDS
o Enhanced sensitivity and specificity of the results
o Improved productivity
DONOR RECORDS RETENTION TIME
- Disadvantages:
Donor ABO/Rh
o Sample restrictions (hemolyzed, icteric, and lipemic samples)
o Requires special equipment or instruments (special centrifuge to Donor Antibody Screen
accommodate the microtube cards, pipette to dispense 25 uL of Informed consent for donation
serum; expensive) Physical examination
Medical history information
10 years
REPORTING Identification number of donor unit
GRADE DESCRIPTION Viral marker testing results
- Solid band of agglutinated RBCs at the top of the gel column Quarantine of donor unit
4+ ID of donor processing tech
- NO RBCs at the bottom
- Majority of agglutinates are in top/upper half of the column Notification of abnormal results
3+ - Most agglutinated RBCs remain near the top of the gel column Medical director approval for donation interval
- Few agglutinates staggered below the thicker band Repeat testing of donor blood
5 years
- Agglutinates distributed throughout the upper and lower halves Platelet count for frequent plateletpheresis
2+ of column Sedimenting agent of leukopheresis
- Few agglutinates at bottom of tube
- Majority of agglutinates are at the lower half of the column
1+
- Some RBCs at bottom of tube
- RBCs form well-delineated pellet
Negative
- Clear and free of agglutinates
Mixed- - Layer of agglutinates at the top of the gel
field - Pellet of delineated cells at the bottom

SOLID PHASE TECHNOLOGY

- Target antigen (red cells) is bound to solid support (microtiter well)
- If antibody is present in patient serum/plasma, it will bind to the antigen on
the microtiter well
o A monolayer of red cells will be formed when indicator cells are added
- If antibody is NOT present, nothing is attached to the immobilized antigens
o A clear delineated button will be formed at the center of the well