IMMUNOHEMATOLOGY
Week 3
Rh Blood group System & Other Blood Group
Systems
HISTORY OF THE Rh System
 O = ABO, + Rh
 Levine & Stetson described a hemolytic
transfusion reaction in an obstetrical patient
○ Can now transfuse correctly,
○ hemolytic transfusion rxn = baby dies;
 Landsteiner & Weiner reported an antibody
that agglutinates 85% of human cells
○ May cause agglutination rxn
 Primary cause of hemolytic disease of the
Fetus & Newborn (HDFN)
○ also known as erythroblastosis fetalis
 Of next umportance is the Rh type
○ Bld grp sys w/ many Ag, one of which is
D 
○ Presence or absence of D antigen on
RBC
○ Unlike ABO sys, ind who lack D Ag do
not naturally produce anti-D
○ Prod of Ab to D req exposure to Ag
 D antigenic = very immunogenic (Ag very
strong, cause agglutination)
○ Ind will likely make Ab to it
 All ind are typed for D, if neg must receive
Rh(D) neg bld
○ ABO + Rh system
 Rh Ag = integral part of red cell membrane
 Protein in nature, w/ active phospholipid
component
RH Typing
 N typing = includes typing for Rh D only
 (+), (-) Rh status
 Some Rh typing sera = diluted in high
protein sol’n, may req (-) control
○ D1 and D2 to confirm Rh neg
○ 2 monoclonal anti-d sera from 2 diff
manufacturers; Fil = likely (+) 99.9%
Fisher-Race: The DCE Terminology
 Ag produced by 3 closely linked sets of
alleles
○ D, C, and E = 1 set
 Each person inherits a set of Rh genes
from each parent
 DCE or dce = reported as
○ C locus lies b/w D and E
○ “d” represents absence of D Ag
Weiner : The Rh-Hr Terminology
 Weiner believed that the gene responsible
for defining Rh, actually produced an
agglutinogen that contained a series of
blood factors.
○ When describing agglutinogen, the
upper case denotes presence of Ag
○ When referring to Rh Ag, single prime (‘)
refers to either C or c and (“) to either E
or e.
Rh = presence; Hr = negative / no Ag
Rosenfield & Coworkers – Alpha/Numeric
Terminology
 This system has no genetic basis
 Demonstrates the presence or absence of
the antigen on the red cell
 A minus sign preceding the # designates
the absence of the antigen
 If an antigen has not been typed for, its #
will not appear in the sequence
 e.g.Rh: 1,2 -3,4,5
WEINE FISHER - ROSENFIELD
R RACE
1 DCe / dce Rh: 1,2,-3,4,5
R r
1 1 DCe / DCe Rh: 1,2,-3,-4, 5
R R
 ○ C = must be in middle
 Rh = normal, arrangement = interferes
 Haplotype Dce / dce = interference cannot
rr dce/dce Rh: -1, -2, -3, 4,
be observed; fully expressed D Ag = not
5
weakened
1 2  Dca / dCe = see if Ab rxn bc of D
R R DCe / DcE Rh: 1,2,3,4,5

3. D Mosaic or Partial D – occurs when one

2 or more parts of the D antigen is missing
R r DcE/dce Rh: 1, -2, 3, 4, 5
o Ab cannot bind = weaken
 Weiner & Unger postulated that the D
dCe/dcE Rh: -1, 2,3,4,5 antigen is made of antigenic subparts,
r’r” genetically determined, that could be
absent in rare cases.
 If an individual lacked one or more pieces
of the total D antigen, alloantibody can be
made to the missing fractions if exposed
 Capital = presence, Small = absence to red cells that possess the complete D
 ‘=C antigen
 R1 = C; R2 = E
 1 or 2 = prime symbol kay Harmening Determination of D status

VARIATIONS OF Rh0 (D) Antigen  Although understanding the difference

 Due to weakened D antigen
o Rate?
o Agglutination = + / - even small amt
3 different mechanisms that can explain the
weakened expression of the D antigen
1. Genetic weak D – inheritance
(mostly encountered in black people)
2. C trans – weakened expression of the D
antigen is described as a position effect or
gene interaction effect.
 Ind. Carrying Dce/dCE –the Rh antigen on
the red cell is normal, but the steric
arrangement of the C antigen in
relationship to the D antigen appears to
interfere
 This interference with D expression does
not occur when the C gene is in the cis
position to D (Dce/dce)
between the genetic weak D, weak D
caused by C trans and the Mosaic of partial
D helps explained why some people with
the weak D phenotype develop anti D and
others do not, still no differentiation was
made in the routine blood bank.
○ Some develop D, some not
○ Weakened = expose = still prod Ab to D
 The donors and patients are classified
simply Rh (+) or Rh (-)
 Any donor blood sample that is typed Rh (-)
by a slide or rapid tube method must be
further tested by the indirect antihuman
globulin technique.
○ 2 monoclonal sera
○ If both (-) = antihuman globulin
○ Sometimes, incubate at 37C to see if
there is agglutination or none
 If both tests are negative, the donor sample
is considered Rh (-)
 If the donor sample tests are positive in any
 phase, the sample is considered Rh (+).
 Determining of Rh D status is critical in
obstretical px
○ All Rh - / weak D neg obstretic px are
candidates for Rh immune globulin
(RhIG)
 Drug injected to prevent Rh (-) ind
who are exposed to Rh (+) red cells
from developing anti-D
 Blocking phenomena
○ NB cells coated w/ maternal IgG anti-D
in utero, very few D Ag sites are avail to
react w/ reagent anti D
○ Mother A (-), baby A (+); Father A (+)
○ 2nd baby: dead  bc of hemolytic dse of
fetus and NB (HDFN)
 Cord cut = exposure of bld to baby
from mother
 Mother = Anti-D; there is
agglutination
○ If mother is (+); mother no Anti-D = no
sensitization = SAFE!
 Solution for Blocking phenomena
○ Elution of sensitizing Ab (removing
Ab)
 Supposedly no Anti-D, but once exposed =
produce Anti-D (from transfusion or
pregnancy when cord is cut)
 Rh = IgG only! = can cross placenta
CHARACTERISTICS OF Rh Antibodies
 Most Rh antibodies are IgG
 Reacts at 37C
o Ab reacts best at 37C
 Usually produced following exposure of the
individual’s immune system to foreign
RBCs, either thru transfusion of pregnancy.
 IgG1, IgG2, IgG3, IgG4 = subclasses of Rh
antibodies
 IgG 1 and IgG 3 are the most clinically
significant
o For HDFN
 Rh antibodies are formed by Rh negative
pregnant women
FALSE POSITIVE (+) w/ Rh Typing Reagents
 Cell suspension too high
 Cold agglutinins
 Test incubated too long or drying
 Rouleaux
 Fibrin interference
 Bacterial contamination
 Incorrect reagent selected
FALSE NEGATIVE (-)
 Immunoglobulin coated cells
o Cannot read Ag
o Ig = in plasma / serum
 Saline suspended cells
o Indirect Anti-globulin chuchu look sa
taas
 Failure to follow manufacturer’s direction
o New brands = tested first to report if
there is false + or -
 Resuspension too vigorous
 Incorrect reagent selected
 Variant antigen
 Reagent deterioration
 For Rogan medicine??? = given to
pregnant women
Lewis Blood Group System
 Unique
 The only system that is not manufactured
by red cells
 Lewis antigens are not synthesized by the
red cells
 These antigens are manufactured by tissue
cells and secreted into body fluids
 Found primarily in the secretions and
plasma
 These Ag are then adsorbed onto the red
cell membrane from plasma, but are not
integral part of the membrane structure.
o ABO + Rh = part
 Since the Ag are manufactured by tissue
cells, Ag production depends not only on
the inheritance of Lewis genes but also on
the inheritance of the secretor genes
 Lewis Ag sys is intimately associated w/
secretor sys + ABO bld grp sys
 ***Sugar = L-fucose; Enzyme = fucosyl
transferase
 Le = dominant
 Not part of red cell (20%), seen in saliva +
body fluids (90%)
 Le and H interacts
The Lewis phenotypes
 2 phenotypes : secretor and non secretor
 Antigens : Lea Leb
Lewis (a-b+) phenotype : Secretors
 Characteristics of Lewis Antibodies
 The Lewis (a+b-) Phenotype: Nonsecretors
Le a
 Individuals with this antigen are mostly non
secretors of ABH
 Both Lea and Leb can be found in the
secretions, however only the Le b adsorbs
onto the red cells from the plasma
Biochemistry of Lewis Antigen
 Lewis antigens found in the secretions are
glycoprotein
 Glycoproteins are composed of 80%
carbohydrates & 15% amino acid
 Lewis Ag in plasma are glycolipids
 These Ag are carried by lipoproteins
present in plasma that adhere to red cell
membranes, forming glycosylceramides
 The exact site for the synthesis of Lewis
glycolipids in the plasma is not known, but,
it has been postulated that they may
originate from the intestinal tract epithelial
cells.
Characteristics of Lewis Antigen
 Poorly developed at birth
o Bc sensitized Ab
o ABO = fully developed Ag
o Rh = fully developed
 Reversibly adsorbed onto red cells from
plasma
 Not found on cord blood or newborn red
cells
 Lewis glycolipids detectable in plasma
after approx. 10 days of life
 Do not show dosage in serologic
reactions
 Usually naturally occuring
 Predominantly IgM
o Same w/ ABO
 May cause in vivo hemolysis of red cells
 Sometimes reacts at 370C and coombs
phase more weakly than at room temp
 Enhanced by enzymes
o BB enzymes to enhance presence w/
agglutination
o If other enzyme = destroyed
LEWIS ANTIBODIES
 Generally produced by Le(a-b-) persons.
o vAnti Lea- most commonly encountered
Ab
o vAb is often IgM, however, some may
have IgG components
o IgG anti Lea - has been detected on red
 cells using enzyme-linked
immunosorbent assay (ELISA)
 Not clinically significant
Anti Lea
 frequently detected w/ saline-suspended
cells at room temp.
 it sometimes react at 37 0C and coombs
phase and can cause hemolytic transfusion
 easily neutralized with plasma or saliva that
contains Lea substance.
Anti Leb
 not as common & not as strong as anti Lea.
 usually an IgM
 does not fix complement
MNSs Blood Group System
 CODOMINANT
 M & N Antigens – found on a well-
characterized glycoprotein called MN-
sialoglycoprotein(MN-SGP), α –
sialoglycoprotein, or glycophorin A.
 Glycophorin A –found in human
erythrocyte membrane
 ---- Antigens are defined by the first and
fifth amino acid
 ----- Antigens can be detected as early as 9
weeks gestational age and they are well
developed at birth
 ----- useful in paternity exclusion cases
involving newborns or very young individual
 Because MN antigens are at the outer end
of GPA (glycophorin A), they are easily
destroyed or removed by the blood bank
enzymes such as ficin, papain, bromelain
and by less common enzymes like trypsin
and pronase.
 M & N are primarily red cell antigens.
 Ss Antigens are located on a smaller
glycoprotein that is very similar to MN
called Ss-sialoglyprotein ,δ- SGP /
glycophorin B
 Ss antigens – appear as early as 12
weeks gestational age and well developed
at birth
 Ficin, papain, bromelain, pronase and
chymotrypsin can destroy Ss activity but
the amount of degradation may depend
on:
o strength of the enzyme solution
o Length of treatment
o Enzyme to cell ratio
 Like MN, Ss are considered red cell
antigens. They are not found in
lymphocytes, monocytes, granulocytes
and platelets.
 Anti M – naturally occurring, cold reactive
saline agglutinins, 50-80% of them are
IgG or have IgG component.
o don’t bind complement
o don’t react with enzyme treated red cells
o pH dependent, reacting best at 6.5 0 C
 Rh = warm reactive (37C)
 Room temp = ABO, Lewis
 As long as anti-M does not react at 37 0 C it
is not clinically significant in transfusion.
 Anti N – cold reactive IgM or IgG saline
agglutinin that does not bind complement or
react with enzyme treated red cells.
o not clinically significant (does not cause
transfusion rxn) unless it reacts at 370 C
 Anti S & Anti s – IgG, reactive at 370 C
o reactive at antiglobulin test phase
o most likely to be clinically significant
 A number of plant lectins have proved
useful in studying biochemistry, some with
practical application.
 Those having N reactivity include:
1. Bauhinia variegata
2. B. candicans
3. B. bonatiana
4. B. purpura
5. Vicea graminea – not commonly used
 Anti M reactivity has been found on the
seeds from the ff plants:
1. Iberis amara
2. I. umbellate
3. I. semperivens
4. Japanese turnip
Maclura aurantiaca – made from osage
orange, reacts to both M-SGP & N-SGP

FORWARD = TEST TUBE = CELL

SUSPENSION; USE RED CELLS

REVERSE = PREPARED A+B (IN PH, WE

PREPARE KZE U KNO WE IZ LAPAERS);
USE SERUM

MIX + CENTRI