Immunohematology

Our Lady of Fatima University – Pampanga

College of Medical Laboratory Science
MIDTERM

COMMON BLOOD GROUP (Part 1) LEWIS PHENOTYPES

1. Le(a+b-): Non-secretors
LEWIS SYSTEM ● All are non-secretors of the ABH substance did not
● ISBB NO.:007 inherit Se gene: sese genes are only present
● SYSTEM SYMBOL: LE ● Lea substance is secreted regardless of secretor status
LE GENES ● Common in Asians
● Genes: Chromosome 19 2. Le(a-b+): secretors
● Gene does not actually code for the production of Lewis ● Result in the genetic interaction of the Lele and
antigens but the production of L-fucosyltransferase to SeSe/Sese genes
Type I precursor substance ● Both Lea and Leb soluble antigens can be found in
● Synthesis of Lewis antigens are based from two secretions and plasma but only Leb adsorbs onto the
independent genes: RBC membrane
Le gene (FUT3 gene): 3. Le(a-b-): Secretors or Non-secretors
■ Lea antigen, gene is located on chromosome 19 at
● Le Null; 80% ABH secretors and 20% ABH non
position 19p13.3 (p=short arm)
secretors
Se gene (FUT2 gene):
● Frequently found in Africans
■ Leb antigen, gene is located on chromosome 19 at
4. Le (a+b+):
position 19q13.3 (q=long arm)
LEWIS ANTIGENS ● Frequently found in Asians
● Antigens are manufactured by tissue cells and secreted PREVALENCE OF THE LEWIS PHENOTYPES
into the body fluids and then ADSORBED onto the RBC PHENOTYPE WHITES (%) BLACKS (%)
membrane Le (a+b-) 22 23
● Antigen differs from all other blood groups because it is Le (a-b+) 72 55
soluble (present in secretions) and plasma Le (a-b-) 6 22
○ Lewis substances (in secretions): GLYCOPROTEINS Le (a+b+) Rare Rare
■ In newborns, glycoproteins are present in their
saliva, but glycolipids are not detectable in the LEWIS ANTIBODIES
plasma until about 10 days after the birth 1. Anti-Lea and Anti Leb
○ Lewis antigens (RBC membrane): GLYCOLIPIDS ● Are usually naturally occurring IgM (some react at 37), react
Interaction of ABO, Lewis, Se genes best at room temperature or lower; clinically insignificant
(not causing HDN because cant cross placenta)
Substance RBC Le
Genes (secretions) phenotype Le antigens antibodies ● Enhanced by enzyme treatment
ABH, lele, ABH, Le Anti-Lea, ● Readily neutralized by Lewis blood group substances
None None ● Activate complement at can cause in vivo and in vitro
sese (a-b-) anti-Leb
ABH, lele, ABH, Le Anti-Lea, hemolysis
ABH None
SeSe/Sese (a-b-) anti-Leb
ABH,
ABH, Le
LeLe/Lele, ABH, Lea Lea Anti-Leb
(a+b-)
sese
ABH, Le
ABH, a (a-b+) (Lea is
ABH, Le , None (no
LeLe/Lele, not adsorbed Leb
Leb anti-Lea
SeSe/Sese in the RBC
membrane)
● Cord blood and red cells from newborn infants ; poorly
developed at birth (a-b-)
○ Development of Le antigens:
■ Le and Se genes: Le (a-b-) → Le (a+b-) → Le
(a+b+) → Le (a-b+) (true Lewis phenotype)
■ Le and sese genes: Le (a-b-) → Le (a+b-)
■ lele genes: Le (a-b-) at birth and for the rest of
their lives
● For pregnant women, Lewis antigens disappear during
pregnancy Le (a-b-)
 THE P BLOOD GROUP SYSTEM ● Weakly cold reactive saline agglutinin
● ISBB NO.:003 ● Strong Anti-P1 observed in individuals with: Hydatid disease
● (P1PK ) (003) (Echynococcus granulosus)
● Globoside (028) System ● Associated with other parasitic infections: Fasioliasis,
● Related (209) antigens Clonorcrhis sinensis, Opisthorchris viverini infections
● Introduced by Landsteiner and Levine In 1927 using rabbit 2. Anti-P
blood, they injected rabbits with human RBCs then initially ● Naturally occurring alloantibody in the serum of Pk
produce anti-P individuals
● Genes: Chromosome 22 (P1Pk gene) ● Alloanti-P
Chromosome 3 (P gene) ○ Rarely seen; it is hemolytic with a wide thermal range of
● Currently, in ISBT nomenclature, P1, Pk and NOR are reactivity (clinically significant)
assigned to the P1PK blood group system (003, symbol ○ IgG alloanti-P: is associated with early abortion
P1PK) ● Autoanti-P (Donath-Landsteiner antibody)
● P and PX2 ARE ASSIGNED TO THE Globoside blood ○ Cold reactive; IgG autoanti-P which is a biphasic
group system (028, symbol GLOB) hemolysin (in vitro: binds to RBC in the cold; coated
● LKE is assigned to the Globuside collection (209, symbol RBCs are lysed in warm temp)
GLOB) ○ Seen in patients with: Paroxysmal Cold
P ANTIGENS Hemoglobinemia
● Synthesized by sequential action of glycosyltransferases, ○ Detected only using: Donath-Landsteiner Test
which adds sugar to precursor substances 3. Anti-PP1Pk
● Consist of 4 antigens: P, P1, Pk, LKE (Luke Erythrocyte ● Formerly known as: anti-Tja
Antigen) ● Serum of Mrs. Jay (adenocarcinoma in the stomach;
T=tumor)
P BLOOD GROUP PHENOTYPES, ANTIGENS, AND ● Predominantly IgM, some are IgG (wide thermal range;
ANTIBODIES spontaneous abortion (some are igG) and pregnancy)
PHENOTYP ANTIBODIE BLACKS ● Associated with: spontaneous abortions in early pregnancy
E ANTIGENS S WHITES (%) (%)
● Has potential to cause HDN and HTR
P1 P1, P, Pk None 79 94 4. Anti-Pk
● Serum of P1 individuals with biliary cirrhosis and AIHA
P2 P, Pk Anti-P1 21 6

p(P null) None Anti-PP1Pk Rare Rare

P1k P1, Pk Anti-P Very rare Very rare

Anti-P,
P2k Pk Very rare Very rare
Anti-P1
1. P1 antigen
● Poorly expressed at birth and may take up 7 years to be
fully expressed
● Deteriorates rapidly on storage
● P1 like antigen: found in pigeon and turtledove droppings,
and egg white of turtle doves
● P1 substance: seen in hydatid cyst fluid, extracts of
Lubricoides terristris (earthworm) and Ascaris suum (pigs)
2. P antigen
● Receptor for parvovirus B19
3. Pk antigen
● Protection against HIV infection of peripheral blood
mononuclear cells
● Receptor for Shiga toxins and E. coli associated Hemolytic
Urine Syndrome (HUS)
4. Luke antigen
● 1965 by Tipett
● Described an antibody in the serum of patient with Hodgkin’s
Lymphoma
P ANTIBODIES
1. Anti-P1
● Naturally occurring IgM antibodies present in the serum of P2
individuals

Immunohematology 2
 THE I BLOOD GROUP SYSTEM THE MNS BLOOD GROUP SYSTEM
● ISBB NO.: I - 027 ● ISBB NO.:002
i - 208 ● SYSTEM SYMBOL: MNS
● Two antigens: I and I antigen; not antithetical antigens (I ● Two loci system: MN, Ss
stands for individuality) ● Antigens are considered antithetical
● They can coexist ● Genes: Chromosome 4
● Genes: IGnT (also known as GCNT2) gene on chromosome MNS ANTIGENS
6 at position 6p24.2 1. M and N antigens
● Consequently, I has been raised to blood group system status
● Well developed at birth; important for paternity testing
(system number 027, symbol I) and the I antigen remains in
● Found on: Glycophorin A (GPA)
the li collection (collection number 207, symbol I)
● Easily removed by enzymes (found at the outer end of GPA)
I AND I ANTIGENS
● Differs in their amino acid residue at positions 1 and 5
● At birth, infant RBC’s are rich in i antigen (small i) ○ M antigens: 1-Serine and 5-Glycine
○ Capital I is almost undetectable ○ N antigens: 1-Leucine And 5-Glutamic acid
● During 18 months of life, the quantity of i antigen slowly 2. S and s antigens
decreases
● Adult RBC’s are rich in I antigen and trace amount of i ● Found on: Glycophorin B (GPB)
antigen ● Differ on the amino acid at the 29th position on GPB:
● Increased i antigen is seen in: HEMPAS or Type II CDA ○ S (methionine)
(chronic dyserythropoietic anemia) ○ s (threonine)
● Well developed at birth, less degraded by enzymes
○ The enzyme sensitive sides are less accessible
I and i PHENOTYPES
● Located further down the glycoprotein
Adult cells (adult I) Cord cells (cord)
2. U antigen
Anti-I strong -/weak ● U stands for “universal”
● RBCs with the S or s antigen also has the U antigen
Anti-i -/weak strong
● co-expressed with S/s
PREVALENCE OF MNS PHENOTYPES
I AND I ANTIBODIES
PHENOTYPE WHITES (%) BLACKS (%)
1. Anti-I
● IgM, common cold reactive autoantibody M+N- 28 26
● Not associated with HDN because antigen is poorly
expressed on infants RBC’s M+N+ 50 44
● 2 types: Benign anti-I and Pathogenic anti-I
● Benign anti-I M-N+ 22 30
○ Found in serum of normal, healthy individuals
S+s- 11 3
○ Commonly interferes with ABO reverse typing
○ Not associated with in vivo RBC hemolysis S+s+ 44 28
○ Weakly, naturally occurring saline reactive IgM agglutinin
(reacts at 4C) S-s+ 45 69
● Pathogenic anti-I
○ Associated with: CAD (COLD AGGLUTININ DISEASE) S-s-U- 0 <1
○ Strong IgM agglutinins that demonstrate high titer ● In whites, the common haplotypes were calculated to appear
reactivity with a broad thermal range (0-32C) in the following order of relative frequency:
○ Attaches in vivo; causes autoagglutination, vascular Ns > Ms > MS > NS
occlusion (acrocyanosis), hemolytic anemia MNS ANTIBODIES
○ Seen in patients with: Primary atypical pneumonia 1. Anti-M
(Mycoplasma pneumoniae)
● Naturally occurring cold reactive saline agglutinins that react
2. Anti-i
below 37C
● Most example of autoanti-i are IgM reacts best with saline ● Can be IgM or IgG (50-80%)
suspended RBC’s at 4C ● Do not bind complement; reaction enhanced by acidification
● Seen in patients with: infectious mononucleosis (Epstein (pH 6.5; acidified serum or plasma)
barr virus) ● Anti-M appears to be more common in children than in adults
● IgG anti-I is associated with HDFN and is particularly common in patients with bacterial infections
● Do not react with enzyme treated RBC’s (destroyed by
enzymes)
● Rarely causes HTR and HDN (if reacts at 37C)
2. Anti-N
● Naturally occurring cold reactive saline agglutinins that react
below 37C
Immunohematology 3
 ● Less common than anti-M ● Made in response to antigen exposure through pregnancy
● Rarely causes HDN (if reacts at 37C) and transfusion and can persist for many years
● Seen in renal patients who are dialyzed on equipment ● in vivo red cell destruction is usually extravascular via the
sterilized with formaldehyde (anti-Nf) (F induced) macrophages in the spleen
○ anti -Nf - dialysis associated anti-N, because it react with ● associated with severe HTR and HDFN
N positive or negative RBCs treated with formaldehyde 2. Anti-k
3. Anti-S and Anti-s with anti-U ● Rare but can cause HTR and HDN .
● Mostly IgG, reactive at 37C and the antiglobulin test ● It is also a IgG
● Bind complement and associated with severe HTR, HTN, and 3. Antibodies to Kpa, Jsa, and other low prevalence Kell
hemoglobinuria antigens
● Rare because so few people are exposed to the antigen
THE KELL BLOOD GROUP SYSTEM ● Clinical significance parallel to anti-K
● ISBB NO.:006 4. Antibodies to k, Kpb, Jsb, and other high prevalence Kell
● SYSTEM SYMBOL: KEL antigens
● Anti-K was discovered from the serum of Mrs. Kelleher in ● Rare because few people lack these antigens
1946 ● Clinical significance parallel to anti-K
○ Because her RBC reacts to her newborn infant, older
daughter and her husband The McLeod Phenotype
● Genes: Chromosome 7 (KEL gene) ● Medical student - initially appeared to be Kell null but
● Expression is very weak on McLeod phenotype cells demonstrated weak expression of k, Kpb, Jsb detected
● First blood group discovered after the introduction of through adsorption and elution (Allen and coworkers, 1965)
Antiglobulin testing (AGT test) ● RBC’s lack the: Kx ‘antigen and Kell system high prevalence
KELL ANTIGENS: antigen, Km, and have marked depression of all other Kell
● Found only in red cells, well developed at birth antigen
● Considered antithetical (K,k) ● X-linked inheritance associated with CGD; common only in
● K antigen: detected on fetal RBCs as early 10 weeks males
● k antigen: detected at 7 weeks ○ originates from a carrier mother
● K antigen is rated second only to D in terms of ● RBC morphology: Acanthocytic (decreased formability and
immunogenicity (3rd in immunogenicity if ABO is included) increased hemolysis)
● Destroyed or inactivated by sulfhydryl reagents ● In 1971, Giblett and colleagues made and association
○ Acid Ethyl Thiol (AET), between the rare Kell phenotypes, including the McLeod
○ Dithiothreitol (DTT), phenotype, and the rare X-linked chronic granulomatous
○ DTT + Papain-which is proteolytic enzyme (ZZAP), disease (CGD)
○ 2-Mercaptoethanol (2-ME) ○ CGD is characterized by inability of phagocyte to make
PHENOTYPES OF THE KELL BLOOD GROUP SYSTEM NADH that is responsible for generating H202 that is
necessary for bacterial infection
PHENOTYPE WHITES (%) BLACKS (%)
● McLeod males with CGD make anti-Kx + Km (sometimes
K-k+ 91 96.5 called anti-KL)
● Anti-Km is made by McLeod males without CGD
K+k+ 8.8 3.5 ● McLeod syndrome:
○ Chronic hemolytic anemia with reticulocytosis,
K+k- 0.2 <0.1 bilirubinemia, splenomegaly, and reduced haptoglobin
levels
Kp(a+b-) <0.1 0
○ Slow progressive form of muscular dystrophy between
Kp(a+b+) 2.3 Rare ages 40-50 and Cardiomegaly (leading to
cardiomyopathy) which is associated with areflexia (lack
Kp(a-b+) 97.7 100 of deep tendon flexes), choreiform movements
(well-coordinated but involuntary movements)
Js(a+b-) 0 1 ○ Increased: serum CK-MM and carbonic anhydrase III
levels
Js(a+b+) Rare 19

Js(a-b+) 100 80 K null (K0 phenotype) McLeod Phenotype

Kx antigens Present in abundance Lacking

KELL ANTIBODIES
1. Anti-K Autosomal Kell Decreased
Lacking/-
antigens expression
● Aside from ABO and Rh antibodies; it is the most common
antibody seen IgG and reactive in the antiglobulin phase Red cell abnormality None Acanthocytes
○ 3rd most common antibody found in human

Immunohematology 4
 THE DUFFY BLOOD GROUP SYSTEM
● ISBB NO.:008 PREVALENCE OF KIDD PHENOTYPES
● SYSTEM SYMBOL: FY
● Name after Mr. Duffy, a hemophiliac with multiple PHENOTYPE WHITES (%) BLACKS (%) ASIANS (%)
transfusions who in 1950 was found to have the first Jk(a+b-) 28 57 23
described example of anti-Fya
● Fyb antigen was found in the serum of a woman who had 3 Jk(a+b+) 49 34 50
pregnancies (1951)
Jk(a-b+) 23 9 27
● Antibody is present in his serum
● Genes: Chromosome 1 Jk(a-b-) Exceedingly Exceedingly 0.9 polynesians
DUFFY ANTIGENS rare rare
1. Fya and Fyb antigens
● Well developed at birth; detected on fetal RBCs as early as 6 KIDD ANTIBODIES
weeks 1. Anti-Jka and Anti-Jkb
● Destroyed by enzymes ● Difficult to detect in blood banking (require presence of
● Receptor for: Plasmodium vivax and Plasmodium complement, cannot be detected in old serum)
knowlesi ● Usually IgG; immune antibodies (response to pregnancy or
● Fy(a-b-) phenotype blood transfusion)
○ Common marker for American Black race ● Have a notorious reputation in blood bank
○ Confer resistance to malaria ● found in combination with other antibodies; Demonstrate
PREVALENCE OF THE COMMON DUFFY PHENOTYPES dosage (most significant dosage effect)
● mild HDN; severe delayed HTR’s (bind the complement)
AFRICAN-AME ● can be drug induced (Methyldopa / Aldomet) (Kidd
PHENOTYPE WHITES (%) CHINESE (%)
RICANS (%) autoantibody)
Fy(a+b-) 17 9 90.8
LUTHERAN BLOOD GROUP SYSTEM (LU) (005)
Fy(a+b+) 49 1 8.9 ● ISBB NO.:005
● SYSTEM SYMBOL: LU
Fy(a-b+) 34 22 0.3
● 1945, Anti-Lua was found in the serum of a patient with
Fy(a-b-) (null lupus erythematosus
Very rare 68 0
phenotype) ● The new antibody was named Lutheran for the donor; the
DUFFY ANTIBODIES donor last name was Lutteran but the donor blood sample
1. Anti-Fya and Anti-Fyb was incorrectly labeled
● Usually IgG antibodies and react best at the antiglobulin ● Cutbush and Chanarin described anti-Lub, which defined
phase the antithetical partner to Lua
● Activity is enhanced in a LISS medium ● Genes: Chromosome 19 (secretor locus; closely linked with
● Complement binding is rare (only to C3) Se gene) (LU gene)
● Do not react with enzyme treated cells LUTHERAN ANTIGENS
a b
● Associated with delayed HTR and HDN 1. Lu and Lu antigens
● Poorly developed at birth; do not reach adult levels until
KIDD BLOOD GROUP SYSTEM (JK) (009) age 15
● ISBB NO.:009 ● Lu (a-b-) phenotype: may result from three different genetic
● SYSTEM SYMBOL: JK backgrounds
● Genes: Chromosome 18 (JK gene) ○ Dominant Type Lu(a-b-)
● Simple and straightforward system because it only has 3 ○ Recessive Type Lu(a-b-)
antigen ○ Recessive Sex-linked inhibitor Type
● In 1951, Allen and colleagues reported finding an antibody in PREVALENCE OF LUTHERAN PHENOTYPES
the serum of Mrs. Kidd with an infant who had HDN
○ The antibody, named anti-JKa (for the infant John Kidd) PHENOTYPES WHITES (%) BLACKS (%)
KIDD ANTIGENS
a b
1. Jk and Jk Lu(a+b-) 0.15 0.1
● Well developed at birth and RBC’s of neonates; found on
RBCs of most individuals
Lu(a+b+) 7.5 5.2
● Jka antigen: detected on fetal RBCs as early as 11 weeks
● Jkb antigen: detected at 7 weeks
● Reactivity enhanced by enzymes Lu(a-b+) 92.35 94.7
● Jk(a-b-) phenotype: resist lysis in 2M urea
2. Jk3 Lu(a-b-) Very rare Very rare
● common antigen found in rbc of Jk null (a-b-) of Jk null

Immunohematology 5
 LUTHERAN ANTIBODIES
1. Anti-Lua
● Most are naturally occurring IgM saline agglutinins that react
best at room temperature
● Few react at 37 C by indirect antiglobulin test
● Associated with rare and mild related HTR
2. Anti-Lub
● Most are IgG (often IgG4) although IgM and IgA antibodies
have been note
● Reactive at 37 C and the antiglobulin phase
● Made in response to pregnancy or transfusion
● Shortened survival of transfused cells and post transfusion
jaundice

Immunohematology 6
 Optimum reaction
BGS Symbol ISBT Chromosome # Gene Antibody class Reaction phase Enzyme treatment
temp

GPYA IgM (MN) 4C or 37C IS, 37C and AHG Destroyed

MNS MNS 002 4
GPYB IgG (Ss) 37C
AHG Variable effect

P1PK 003 22 P1PK IgM (anti-P1) 4C

P IS, 37C, AHG Enhanced
P (GLOB) 028 3 P IgG (anti-P) 4C or 37C

Anti-Lua: IgM 4C Lua: room temp

Lutheran LU 005 19 LU Variable effect
Anti-Lub: IgG 37C Lub: AHG

Kell KEL 006 7 KEL IgG 37C AHG No effect

Often 4C, Room temp,

Lewis LE 007 19 LE IgM Enhanced
sometimes 37C 37C, AHG

Duffy FY 008 1 FY IgG 37C AHG destroyed

Kidd JK 009 18 JK IgG 37C AHG Enhanced

I 027
Immediate spin
I 6 IGNT IgM 4C Enhanced
(IS) and occ 37C
i 207

Immunohematology 7
 MINOR BLOOD GROUP SYSTEM (UNCOMMON BLOOD SCIANNA SYSTEM ISBT (013)
GROUPS) ● Sc system is composed of the three antigen:
DIEGO SYSTEM (DI) ISBT (010) 1. Sc1 antigen
● Composed of 22 antigens; three sets of high and low pairs 2. Sc2 antigen
of: 3. Sc3 antigen
○ Antithetical antigens: Dia/Dib , Wra /Wrb and Wu/DISK ● Most anti-Sc1 and anti-Sc2 antibodies are IgG, red cell
and 16 low-prevalence antigens. stimulated and react in indirect antiglobulin testing; not
● Reported in 1955, anti-Dia had caused HDFN in a associated with HTR
Venezuelan baby. Anti-Dib was described 2 years later. ● Anti-Sc3 is an IgG that reacts in the IAT; associated with
● Genes: Chromosome 17 mild HTR
● Dia - rare in most population, but is polymorphic in people of ● The SC gene ERMAP (Product of sc gene; For RBC
Mongoloid ancestry adhesion protein) is located on chromosome 1 at position
● Antibody are usually IgG sometimes IgM reactive at 1p34.2
antiglobulin test can cause HDFN ● ERMAP function is RBC adhesion
● Dia and Dib antigens are located on the Band 3 (red blood DOMBROCK SYSTEM (DO) ISBT (014)
cell exchanger or AE1 or SLC41) which is an integral ● Antigens: Doa and Dob ,Gregory (Gya), Holley (Hy) and
transport protein involved in the anion exchange of Joseph (Joa )
bicarbonate for chloride in the red cell membrane. ● Anti-Doa and Anti-Dob
● The Dia/Dib polymorphism is located on the last (seventh) ○ Described as IgG, red cell-stimulated antibodies that
extracellular loop of the protein. react primarily in indirect antiglobulin test w/ PEG or
● anti-Dia and anti-Dib are IgG that do not bind complement and Enzyme enhancement
are reactive in IAT ○ Not associated with HDFN
● anti-Dia and anti-Dib have caused hemolytic transfusion ○ Reported to cause acute to delayed HTR
reactions (HTRs) and HDFN. ● Antibodies to Gya, Hy and Joa
● Anti-Wra has caused severe HTRs ○ all are characterized as IgG, red cell stimulated
Yt SYSTEM ISBT (011) ○ IAT: reactive
● Cartwright Blood System ○ Do not cause HDFN, but has been described to cause
● Composed of two antigens: moderate transfusion reaction
○ Yta and Ytb ● Dombrock antigens are carried on a
● 1956 – named the first antibody marker and used the last mono-ADP-ribosyltransferase 4 (ART4)
letter ”t” in the patient’s last name: “Cartwright” ○ Attached to the RBC membrane by a GPI anchor
● Yta is the high-prevalence antigen in all populations ● Gene: Chromosome 12
● Ytb is the low-prevalence antigen COLTON SYSTEM (CO) ISBT (015)
● Three phenotype are observed: ● Consists of four antigens:
○ Yt(a+b-), Yt(a+b+) (common) ○ Coa, Cob, Co3 (formerly Coab), Co4
○ Yt(a-b+) (rare) ○ Coa - high prevalence antithetical antigens
● Yt antigens are represented by an amino acid substitution ○ Cob - low prevalence antithetical antigens
on the glycosylphosphatidylinositol (GPI)-linked RBC ○ Co3 is present on all RBCs except those of the very rare
glycoprotein acetylcholinesterase (AChE) Co(a–b–) phenotype.
● AChe - located on long arm of chromosome 7 (7q22) ○ Co4 a high-prevalence antigen, has been identified on
○ Plays a role in neuro-transmission but when bound on RBCs from two individuals with the Co(a–b–) phenotype
a red cell it is not known. ● Colton antigens are carried on an integral membrane
● anti-Yta and anti-Ytb are IgG reactive in the IAT and are protein, aquaporin 1 (AQP1)
considered to be clinically important because they are ○ The geneAQP1 is located on chromosome 7 at
predominantly red cell stimulated. position 7p14.
● Yt antibodies HAVE NOT CAUSED HDFN. ● Antibodies are usually IgG and are enhanced with enzyme
Xg SYSTEM ISBT (012) treated RBCs
● 1962 – discovered in the serum of multiply transfused man ● anti-Coa and anti-Cob are IgG, reactive in IAT, associated
● High prevalence in female than male with acute to delayed HTR
● Named after the X Chromosome and g “Grand rapids” ● Anti-Co3, which reacts with all Co(a+) and Co(b+) RBCs,
where the patient was treated has been reported to cause mild HTR and severe HDFN.
● There are two antigens in the Xg system: CHIDO/ROGERS BLOOD GROUP SYSTEM (CH/RG) ISBT (017)
○ Xga and CD99 ● Includes 9 antigens, which are subdivided into:
● Only the Xga antigen has been identified with no antithetical ○ Six Ch antigens
partner ○ Two Rh antigens
● Gene: X chromosome ○ WH antigen
● Frequency: males (66%) females (89%) ● Postulated that the CH/RG antigens were associated with
● Anti-Xga is an IgG reactive in the IAT and sensitive to human leukocyte antigen (HLA) system
enzymes but not DTT treatment; not been associated with
HTR or HDN

Immunohematology 8
● Alleles for RG and CH have been located on two closely ○ are usually IgG
linked genes known as C4A (Express Rogers antigen) and ○ reactive in the antiglobulin phase of testing
C4B (Carries Chido antigen) on Chromosome 6 ○ do not bind complement.
● Antigens product have been demonstrated on the C4d ○ Positive DATs
fragments of the C4A (Rodgers) and C4B (Chido) ○ no clinical HDFN have been reported for anti-Ina and
glycoproteins of the C4 complement components anti-Inb
● Anti-Ch and anti-Rg are usually IgG and react weakly, ○ an immediate HTR
often to moderate or high titration endpoints. OK BLOOD Grp SYSTEM ISBT (024)
● Anti-Ch and anti-Rg are clinically insignificant for ● Anti-Oka
transfusion. ○ Identified in 1979 and named after the marker found in
GERBICH BLOOD GROUP SYSTEM (GE) ISBT (020) Mrs. Kobutso
● There are currently six high-prevalence Gerbich antigens ● Additional antigen
(Ge2, Ge3, Ge4, GEPL, GEAT, and GETI) and five ○ OKGV and OK VM
low-prevalence antigens (Wb, Lsa, Ana, Dha, and GEIS). ● OK antigen
● GE antigens are inherited on chromosome 2 and are ○ Carried on CD147
expressed on glycophorine C (GPC) and/or glycophorine D ○ Function as receptor and adhesion molecules
(GPD) they are sialoglycoprotein structures ● Gene: Chromosome 19
● There are three Gerbich-negative phenotypes in which the ● Well developed on RBCs from newborn
RBCs lack one or more of the high-prevalence antigens: ● Has not been reported to cause HDFN
■ Ge:–2,3,4 (Yus type) Raph BLOOD Grp SYSTEM ISBT (025)
■ Ge:–2,–3,4 (Gerbich type) ● MER2 (only antigen)
■ Ge:–2,–3,–4 (Leach type) ○ Antigen is derived from monoclonal, and Eleanor
● Individuals of the Leach phenotype (GE:-2,-3,-4) present Roosevelt, the laboratory where the antibody was
with a change in erythrocyte morphology in the form of produced
Elliptocyte (Mild anemia) ● Raph - first person with this kind of blood type
● Anti-GE2 and Anti-GE3 ● Gene: Chromosome 11
○ Have also been noted as naturally occurring IgM and ● MER2 located at CD151
as autoantibodies, ○ A tetraspanin, which appears to be essential for the
○ Implicated causing acute to delayed transfusion assembly of basement membranes in the Kidney and
reactions skin
○ No clinical HDFN ● Three examples of Alloanti-MER2
CROMER BLOOD GROUP SYSTEM (CROM) ISBT (021) ● Found in JEWS originating from India and living in ISRAEL
● Antigen are carried on decay accelerating factor (DAF/CD55) ○ With end stage renal disease
○ Involves with the regulation of complement activation ● Fourth example:
by accelerating the decay of the C3 and C5 convertase ○ Healthy, Turkish blood donor.
● Antigens are resistant to treatment with ficin and papain JOHN MILTON HAGEN BLOOD Grp SYSTEM ISBT (026)
but are destroyed by α-chymotrypsin ● Anti-JMH – found in patients 50 years old and older.
● CROM Antibodies ● JMH protein
○ Are described as predominantly IgG1 (reactive in IAT) ○ The GPI-linked glycoprotein CD108
○ do not cause HDFN ● Gene: Chromosome 15
○ Present majority in blacks individuals ● Usually IgG (Predominantly IgG4)
KNOPS BLOOD GROUP SYSTEM (KN) ISBT (022) ● Clinically insignificant
● Composed of NINE antigens: GILL BLOOD Grp SYSTEM ISBT (029)
● Alleles for the KN blood group have been located on ● 1980 – Anti-GIL was identified
chromosome1, with the antigens residing on complement ● Gil antigen – found on the glycerol transporter aquaporin
receptor one (CR1) 3 (AQP3)
● KN antibodies ● A major intrinsic protein family of water channels
○ Are characterized as IgG ● Gene: Chromosome 9
○ Weakly reacting in IAT RH-Associated Glycorpotein BLOOD Grp SYSTEM ISBT (030)
○ Not associated with HDFN or HTR ● Newest blood group system
● Helgeson Phenotype ● Does not have Rh blood group antigen
○ Represents the serologic null phenotype for the Knops ● Essential for Rh antigen expression
blood group ● Absence of RhAG due to mutations result in the Rhnull
INDIAN BLOOD GROUP SYSTEM (IN) ISBT (023) phenotype
● There are now five antigens in the system; Composed of ● Two antigens:
two antithetical antigens: ○ Duclos (RHAG1) – (previously 901013) high
○ Ina (relatively low incidence) and Inb (high incidence) prevalence
● IN antigens are carried on the hematopoietic isoform of the ○ Ola (RHAG2) – (previously 700043) low prevalence
CD44 marker located on chromosome 11 at position 11p13
● IN antibodies

Immunohematology 9
 DONOR SELECTION/ DONOR SCREENING ● Those with malignant solid tumors (except basal cell
carcinoma of skin and inside of cervix)
3 CATEGORIES OF DONORS ● Hematologic malignancies; chemotherapeutic agents
1. Voluntary/ non numerated administered for malignancy
2. Family/replacement ● Chronic cardiopulmonary, liver, or renal diseases
3. Paid/professional/commercial ● Serious abnormal bleeding tendencies
● Acquired viral hepatitis after age of 11
VOLUNTARY
● Taking tegison
● Receives nothing in return (any form of payment)
● Diagnosed with Babesiosis and Chaga’s disease
● Lower incidence & prevalence of transfusion transmissible
● MSM since 1977 (does increased risk on unprotected sex)
infections
● Blood-associated diseases
● Absence of risk of anemia on the part of the donors through
B. 3 YEARS
depletion of their iron source
● Donors are more willing to donate blood more regularly ● Diagnosed and treated with malaria; refugees or immigrants
● Donors have expressed a commitment to donate blood from a Malaria endemic place
during emergency C. 1 YEAR
FAMILY ● Close contact with a Hepatitis infected individual
● Require to give blood when a member of patient’s family or ● Received blood transfusion or injections of blood products,
community requires it organ or tissue transplant
● Usually first to donate ● Tattoo, piercing, acupuncture
● Received Hb Ig
PAID
● Traveled to an endemic place for Malaria
● They give blood but receives money in return
● Received rabies vaccine
DISADVANTAGES OF PAID DONORS ● Risk exposure to hepatitis
1. High incidence & prevalence of transfusion transmissible ● Positive for syphilis
infection ● Sexual contact with a person with high risk to HIV infection
2. Often donors are undernourished and of poor health ● Female contact with a bisexual male
STANDARD PROCEDURES AND GUIDELINES IN DONOR ● Treated with syphilis or gonorrhea
SCREENING ● Underwent major operation
1. Registration ● Incarceration in a jail
2. Medical History Questionnaire D. 3 MONTHS
3. Physical Examination ● Pregnant women
4. Blood Unit Collection E. 2 MONTHS
5. Post-donation instructions ● Previous blood donation
1. REGISTRATION ○ DOH - 3mos or 12 weeks
● Name ○ ABB - 2mos or 8 weeks
● Date and Time of Donation ● Treatment for retinoids
● Address F. 1 MONTH
● Telephone ● German measles vaccine (Rubella)
● Gender (self-identified) ● Taking Acutane (acne treatment) and Proscar (prostatic
● Age or Date of Birth hyperplasia)
○ Allogenic donation: greater than or equal to 16 years G. 2 WEEKS
○ Autologous donation: no age limit ● Acute febrile illness
● Consent to donate ● Measles, oral polio, mumps and yellow fever vaccines, BCG,
○ Donors will be informed with procedure and potential smallpox vaccination (uses synthetic vaccine)
risks.
H. 5 DAYS
○ Educational materials for signs and symptoms of HIV and
● Antibiotic for infection, oral antifungal drugs
how it spreads. Usually given at the end of
I. 48 HOURS
questionnaires.
● Hemapheresis donation
2. MEDICAL HISTORY QUESTIONNAIRE
J. 1 DAY
● There are three types of deferrals:
○ Temporary - cannot donate for specific amount of period ● Allergic drugs like penicillin
such as after a vaccine K. 12 HOURS
○ Indefinite - unspecified period ● Recent alcohol intake
○ Permanent L. MAY DONATE ANYTIME IF WITHOUT VACCINE
Donor deferral ASSOCIATED SYMPTOMS (FEVER)
● Killed vaccines and toxoids, DPT, polio vaccine, cholera,
A. PERMANENT
Hepa B, Typhoid, paratyphoid, typhus, influenza
● Diagnosed with AIDS
M. NO DEFERRAL
● Hemophiliacs, intravenous drug abusers (always look on
arms) ● Aspirin
● 3 days deferral if needed for platelet concentrate

Immunohematology 10
 3. PHYSICAL EXAMINATION ● Purpose:
A. General Appearance ○ Treat surgical blood loss
B. Weight ○ Decreased risk of disease transmission
● 10.5ml/kg of donor weight inclusive of the pilot tube ○ Dec. transfusion reaction
○ if less than 110 lbs: blood to be collected must ○ And alloimmunization
proportionately reduced as well as the anticoagulant ○ Greatest advantage for those very rare blood types
● Amount of blood to be drawn: ○ Those with multiple antibodies
𝐷𝑜𝑛𝑜𝑟’𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑙𝑏𝑠) 𝑥 450 𝑚𝐿 ● Phlebotomy process stimulates the body mass to increase
= allowable amount (mL)
110 𝑙𝑏𝑠 cell production
● Amount of AC needed: ● Disadvantage:
𝐴𝑙𝑙𝑜𝑤𝑎𝑏𝑙𝑒 𝑎𝑚𝑜𝑢𝑛𝑡
100
𝑥 14 = 𝑎𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 𝑛𝑒𝑒𝑑𝑒𝑑 ○ Higher cost because of added administrative processes
● Amount of anticoagulant to be removed ○ Special labelling
63 𝑚𝐿 − 𝑎𝑚𝑜𝑢𝑛𝑡 𝑛𝑒𝑒𝑑𝑒𝑑 9𝑚𝐿) = 𝑎𝑛𝑡𝑖𝑐𝑜𝑎𝑔𝑢𝑙𝑎𝑛𝑡 𝑡𝑜 𝑏𝑒 𝑟𝑒𝑚𝑜𝑣𝑒𝑑 ○ High percentage of wasted units
○ Ratio: 1:7 ● Criteria:
C. Temperature : ○ No age limit
○ No strict weight limit requirements
● 37.5 ̊C or 99.5 ̊F
○ Hemoglobin/hematocrit – should not be less than
D. Pulse :
11g/dL and 33%
● 50-100 bpm ; < 50 bpm: cases of athletic donors
● Frequency – donations should not be more frequent than
E.Blood Pressure:
every 3 days and the final donation must be completed at
● Less than or equal to 180 mmHg: systolic least 3 days prior to the scheduled surgical procedure
● Less than or equal to 100 mmHg: diastolic
Types of Autologous Donation
F. Hemoglobin
1. Predeposit donations
● greater than or equal to 12.5 g/dL
● Refers to the blood that is drawn some time before the
● Hematocrit: 38% for allogeneic donation
anticipated transfusion and stored.
○ F.1 Copper sulfate method (sp. gravity: 1.053)
● Usually liquid but occasionally frozen
○ F.2 POCT devices
2. Intraoperative Autologous transfusion
G. Skin lesions
● Occurs when blood is collected during the surgical
4. BLOOD UNIT COLLECTION
procedure and usually reinfused immediately.
● Aseptic technique (Povidone-iodine or polymeriodine
3. Immediate preoperative hemodilution
complex)
● Takes place in the operating room when 1-3 units of WB are
5. POST-DONATION INSTRUCTIONS
collected and the patient’s volume is replaced with colloid
● Mild reactions: syncope, nausea or vomiting,
or crystalloid.
hyperventilation, twitching, muscle spasms
● The blood is reinfused during the surgical procedure
● Moderate reactions: mild loss of consciousness, decreased
4. Post-operative salvage
pulse rate, decrease systolic pressure to 60 mmHg
● An autologous donation in which a drainage tube is placed
● Severe reactions: convulsions
in the surgical site and postoperative bleeding is salvaged,
● Hematoma: localized collection of extravascular blood under
cleaned and reinfused.
the skin, resulting in bluish discoloration
○ Apply pressure after collecting since it is a 16g needle
● Donors must not be left unattended
● Mix blood and anticoagulant every: 45 seconds
● Duration of blood collection: 7-10 minutes
○ 15mins it is only used for Whole Blood
DONOR BLOOD UNIT PROCESSING
● All blood units must be tested and processed before releasing
1. ABO/Rh grouping
2. Ab screen especially those with previous pregnancies
and transfusion
3. Serologic test for syphilis
4. HBsAg
5. Anti-HCV
6. Anti-HIV1/2
7. Anti-HTLV I/II
8. HIV 1&2, p24 Ag
9. Malaria* (optional)
Autologous Donation
● Donor who donates blood for his or her own use
● Donor referred as: Donor-patient
○ Rare blood type (such as Rh - in Philippines)

Immunohematology 11
 THE ANTIHUMAN GLOBULIN TEST
● Also called as the: Coombs test
● For transfusion medicine
● Used for detecting clinically significant antibodies that have
coated cells
PRINCIPLE:
● Anti-human globulins obtained from immunized non-human
species bind to human globulins such as IgG or complement
components which are free in serum or attached to antigens
on red blood cells.
○ Since IgM is a large pentametric structure, it directly
agglutinates RBC in saline.
○ IgG are termed incomplete antibody since they are too
small to direct agglutinate, addition of AHG reagent
containing anti-IgG allowing hemagglutination.
HISTORY OF THE ANTIHUMAN GLOBULIN TEST
● 1945: Coombs and associates used antiglobulin test for
detection of weak and non-agglutinating Rh antibodies in the
serum
● 1946: Coombs and co-workers used AHG to detect in vivo
sensitization of RBC of babies with HDN
● 1947: Coombs and Mourant demonstrated that antibody
activity that detected Rh antibodies was associated with the
anti-gamma globulin fraction of the reagent
● 1957: Dacie and co-workers stated that reactivity of AHG to
cells sensitized by warm antibodies resulted from
anti-gamma globulin fraction and anti-non gamma globulin
fraction was responsible for the reactivity of cells sensitized
by cold antibodies and had specificity for complement
THE ANTIHUMAN GLOBULIN REAGENTS
● Antihuman globulin reagents can either be:
○ Polyspecific (polyclonal)
○ Monospecific (monoclonal)
1. POLYSPECIFIC AHG
● Contains: anti-IgG and anti-C3d
● Other complement antibodies are: anti-C3b, anti-C4b, and
anti-C4d
● Contains little activity against IgA and IgM heavy chains
● May contain antibody activity to kappa and lambda light
chains common to all immunoglobulin class
2. MONOSPECIFIC AHG
● Contains only one antibody specificity: either anti-IgG or
antibody to specific complement components such as C3b
or C3d
● Anti-IgG: no anti-complement activity; contain antibodies
specific for the Fc fragment of the gamma heavy chain of
the IgG molecule
● Anti-complement: reactive against only the designated
complement components and contain no activity against
human immunoglobulins;
PREPARATION OF ANTIHUMAN GLOBULINS REAGENTS
1. CLASSIC METHOD (Hyperimmunization of rabbits)
● Produces: polyclonal antibodies
● Can recognize different epitopes
PROCEDURE:
1. Injecting human serum or purified globulin which contains
IgG and C3d into rabbits
Immunohematology
2. The rabbits will then produce antibodies to human
globulins (anti-IgG or anti-C3d).
a. For large quantities can use sheep of goat
2. HYBRIDOMA TECHNOLOGY (Kohler and Milstein technique)
● Produces: monoclonal antibodies (derived from one clone
of plasma cells and recognized a single epitope)
PROCEDURE:
1. Mice are immunized with purified human globulin
2. After suitable immune response, mouse spleen cells
containing antibody-secreting lymphocytes are fused with
myeloma cells (Hybridoma cells)
3. The resulting hybridomas are screened for antibodies with
the required specificity and affinity
4. Antibody secreting clones are then propagated in tissue
culture or by inoculation into mice in which the antibody is
collected as ascites
DIRECT ANTIGLOBULIN TEST (DAT)
● Detects: in vivo sensitization of RBC’s with IgG or
complement components
● Clinical conditions associated with in vivo sensitization of
RBC’s:
1. HDN - maternal antibody coating fetal RBC
2. HTR - recipient antibody coating donor RBC
3. AIHA - autoantibody coating own RBC
PROCEDURE:
Specimen: EDTA (clotted specimen can cause false positive)
● Initial DAT: one drop of 3-5% RCS with polyspecific AHG
reagent (w/anti-IgG and anti-C3d)
● If the test is positive: perform a DAT panel (to detect if IgG
or complement components sensitized the RBC) and saline
control (detect spontaneous agglutination without the addition
of the AHG reagent)
● DAT panel: monospecific anti-IgG and anti-C3d reagents
INDIRECT ANTIHUMAN GLOBULIN TEST (IAT)
● Detects: in vitro sensitization of RBC’s with IgG or
complement components (require incubation at 37C)
Applied in the following situations:
● Detection of incomplete antibodies to potential donor RBCs or
to screening cells in serum
● Determination of RBC phenotype using known antisera
● Titration of incomplete antibodies
PROCEDURE:
1. Incubate RBC’s with antisera
➔ Allows antibody molecules to attach to antigen
2. Perform a minimum of three washings
➔ To remove free globulin molecule
3. Add AHG reagent
➔ Serves as a bridge producing hemagglutination
4. Centrifuge
➔ Accelerates by bringing cells together
5. Examine for agglutination
➔ Pos or neg
6. Grade agglutination reactions
IF RESULT IS NEGATIVE:
● Add Check cells (Group O RBC’s coated with IgG; negative
reactions should demonstrate hemeagglutination of these
RBC’s with the anti-IgG in the AHG reagent)
● If still negative after addition of check cells: invalid
test/repeat procedure
12
 FACTORS AFFECTING THE ANTIHUMAN GLOBULIN TEST NOTE:
● DAT: can detect a level of 100 to 500 IgG molecules per ● Saline stored for long period in plastic containers decreases
RBC and 400 to 1,100 molecules of C3d per RBC pH (causes elution of antibodies; false negative result)
● IAT: there must be between 100 and 200 IgG or C3 ● Bacterial contamination in saline causes false positive results
molecules on the cell to obtain a positive reaction 7. ADDITION OF AHG
1. RATIO OF SERUM TO CELLS ● Addition of AHG: immediately after washing to minimize
● Increasing ratio of serum to cells increases the the chance of antibody elution that can neutralizing the
reactivity of the test AHG reagent (does not affect anti-complement antibodies:
● Minimum ratio: 2 drops of serum and 1 drop of 5% RCS anti- C3d, etc)
● When using cells suspended in saline: 133:1 (4 drops of 8. CENTRIFUGATION FOR READING
serum and 1 drop 3% RCS) ● Centrifugation for AHG: 1,000 RCF for 20 seconds
2. REACTION MEDIUM
a. Albumin SOURCES OF ERRORS IN THE ANTIHUMAN GLOBULIN
● Macromolecules of albumin allow antibody coated cells to TESTING
come into closer contact with each other
● Optimum reaction: 2 drops of serum, 2 drops of 22% False positive results False negative results
bovine albumin, 1 drop 3-5% RCS Improper specimen (refrigerated, Inadequate or improper washing
b. LISS (Low ionic strength solution) clotted samples) of cells
● Introduced by Low and Messeter Over centrifugation and Failure to wash additional times
● Enhances antibody uptake and allow incubation time to be overreading when increased volume of serum
decreased by: reducing zeta potential of RBC’s (0.2% is used
NaCL) Centrifugation after incubation Contamination of AHG by
● LISS can shorten the time to 10-15mins (when PEG is used) extraneous protein
● Optimum reaction: 2 drops of serum, 2 drops of 3% Bacterial contamination of saline High concentration of IgG
RCS’ in LISS (or addition of LISS reagent) used for washing paraproteins in test serum
c. Polyethylene glycol (PEG) Dirty glasswares Elution of antibodies from RBC’s
● Water soluble linear polymer; increases antibody uptake due to interruption in testing
by removal of water effectively concentration antibody Presence of fibrin Elution of antibodies from RBC’s
3. TEMPERATURE (pseudoagglutination) due to improper testing
● Optimal temperature: 37C for IgG antibodies and temperatures
complement activation (usual incubation temperature for Cells with positive DAT will yield Improper reagent storage
IAT) positive IAT (deterioration or neutralization of
4. INCUBATION TIME reagent)
● Normal incubation time for cells suspended saline: 30-120 Polyagglutinable cells Excessive heat, repeated
minutes; majority of clinically significant antibodies are freezing and thawing of test
detected after 30 minutes of incubation serum
● LISS and PEG: 10-15 minutes Saline contaminated by heavy Non-reactive serum because of
○ For LISS technique, it is incubated for up to 40 metals deterioration of complement
mins. It shows antibodies to elute from RBC
Using serum as a sample for AHG reagent, test serum,
causing decreased sensitivity.
DAT enhancement media is not added
5. WASHING OF RBC
● Requires a minimum of: 3 washings
False Negative
● Inadequate washing: false negative reaction because of
neutralization of the AHG reagent by residual unbound ● Rare antibodies are present that are only detectable with
serum globulins polyspecific AHG when active complement is present
NOTE: ● Under Centrifugation
● Serum:cell ratio not ideal
● Washing should be performed immediately after removal ● Low pH of saline
of the tube from the incubator (to minimize the elution of ● Poor reading technique
low-affinity antibodies) ● Inadequate incubation in IAT
● The cell button should be completely resuspended before
adding the next saline wash
○ Dislodging is very important
● All saline should be discarded after the final wash because
residual saline dilutes the AHG reagent and therefore
decreases the sensitivity of the test
6. SALINE FOR WASHING
● Freshly buffered with a pH of 7.2-7.4

Immunohematology 13