Rh Blood Group System

- 2nd most important Blood Group System after

ABO
- Greatest clinical importance in Hemolytic
Disease of the Newborn (erythroblastosis
fetalis) and Transfusion Reactions
- 50 Rh antigens ( Rh1 -> Rh57)
- Come from Rhesus monkey - 85% Agglutinated = Rh (+)
- Most common based on antigenicity: - 15% Non-agglutinated = Rh (-)
D>c>E>C>e - Ab in Female = Anti-Rh
- D – primary antigen, would be Rh(+) or Rh(-) - Ab in Rabbit = Anti-LW
due to this no matter what
- Location: C. Weiner and Peter
Non-glycosylated protein on RBC membrane - Rh(-) who received Rh(+) blood are compatible
(Chromosome 1) along with
1. genes for elliptocytosis 4 NOMENCLATURES
2. 6-phosphoglucoronic dehydrogenase 1. Fischer-Race – DCE Terminology
3. phosphoglucomutase 2. Weiner – rH, Hr
4. phosphopyruvate hydratase 3. Rosenfield – Alpha/Numeric
- no glucose moity 4. ISBT – Numeric
- Autosomal dominant Fisher-Race
- Difficulty in describing structure - 3 closely linked genes that can be inherited
- 174,000 MW compound from each parent has 3 closely linked loci that
carry the Rh genes
HISTORY - Crossing over is rare cause they are closely
- Discovered by Blood Transfusion linked
A. (1939) Levine and Stetson - MNSS
- Antibody of D Antigen (Anti-D) - CDE -> cde
- In serum of woman whose fetus had HDN Weiner
(second birth) - 1 gene at a single locus controls the entire Rh
- Mother is Rh(-), Father is Rh(+) system
B. (1940) Landsteiner and Weiner - D – Rh0
-Identified the Rh Blood Group System - C – RhI
- E – RhII
- c – hrI
Rh RBC - e – hrII
- Has agglutinogen w/c is made up of 3 blood
factors
Rabbit - Antigen – subscript

Rosenfield
Produce Anti-Rh - Aims to bridge the confusion between first 2
antibodies - A number in chronological discovery
against Rh RBC - No genetic basis
- Presence or absence only of Ag
- D–1
Ab reaction - C–2
- E–3
with human - e–4
RBC

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 - c–5 - E – 004003
ISBT (International Society for Blood Transfusions) - c – 004004
- 6 numerical numbers - e – 004005
- 004 = Rh
- D – 004001
- C – 004002

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Possible Fisher-Race Weiner Gene Agglutinogen 3 blood Rosenfield
combinations Shorthand or Ag factors
Dce Dce Ro Rho Rho Rho, hrI, hrII Rh 1, -2, -3, 4,
5
DCe DCe R1 Rh1 Rh1 Rho, rhI, hrII Rh 1, 2, -3, -4,
5
DcE DcE R2 Rh2 Rh2 Rho, hrI, rhII Rh 1, -2, 3, 4,
-5
DCE DCE Rz Rhz Rhz Rho, rhI, rhII Rh 1, 2, 3, -4,
-5
dce dce r rh rh hri, hrii Rh -1, -2, -3,
4, 5
dCe dCe rI rhI rhI rhi,hrii Rh -1, 2, -3,
-4, 5
dcE dcE rII rhII rhII hri,rhii Rh -1, -2, 3, 4,
-5
dCE dCE rY rhY rhY rhi,rhii Rh -1, 2, 3, -4,
-5

Variants of Rh
- Cu
- Cf
- Cw
- Eu
- Du – Weakened expression of D-Antigen
(Blacks>Caucasians) detectable only by Indirect
Method. It is few in number
- D+w is the phenotype 2. Partial or Mosaic
- Because Rh is a low MW compound (174,000
Mechanisms of Weak D Antigen daltons), it is said that the D-antigen is a mosaic
structure composed of 4 fragments and if one part
or others is/are missing, it shows weak expression of
1. c-Trans
D-antigen.
- C is in cis position with D
A position
- if C is in trans B A weak
it causes B D

C D D
D d D d
No Weak D Weak D with Anti-C
C Cis c Trans
c C Disadvantage – if you are Rh(-), you can receive only
e E Rh(-)
e E
3. Genetic Weak D
Normal Weak-D - All fragments are present but only in few number
and thus weakly expressed.

2 types of Du

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 1. Low Grade 1. Regulator Type – Mutation in Rh AG thus there is
- Direct product of inherited gene detectable only by Rh AG in RBC but has normal complement of Rh D
IAG, and can be passed unto future generations. and Rh CE
2. High Grade 2. Amorphic Type - Mutation in Rh CE, Deletion in Rh
- Cannot be passed unto future generations, rarely D, Normal Rh AG
need Anti-globulin test
General Characteristics of Rh Antibodies
1. Majority are IgG (1 and 3 most significant)
2. Not natural antibodies but immune antibodies
3. React best at 37C
4. Crosses placenta
5. Do not bind complement
6. Do not react in saline solution
7. Commonly found antibodies in
3 Genes that control Rh expression immunocompromised patients = Anti-D and Anti-G
1. Rh D – Presence of D antigen (Chromosome 1) (Abs found in immunized patients)
2. Rh CE – Presence of C, E, c, and e antigen
(Chromosome 1)
3. RhAG – Rh Associated glycoprotein, coexpressor
(Chromosome 6)
- Expresses Rh AG but by itself does not 3 Varieties of Rh Antibodies
display any antigen 1. 1st Order – Saline agglutinins, Bivalent, Complete
- If there is a mutation in this gene = Missing – React in Saline medium
or alteration of Rh D and Rh CE 2. 2nd Order – Albumin reacting, Monovalent,
Incomplete – React in protein medium
3. 3rd Order – Typical antibodies, Anti-globulin
Unusual Phenotypes of Rh
antibodies – React in antiglobulin medium
1. Rh Deleted – no C and E (D _ _ / D _ _ )
2. Rh Null – none at all ( _ _ _ / _ _ _ )
3. Rh Moderate – weak expression Different Anti Sera to detect Rh Antigens
1. Anti-C
1. Anti-E
Effects of no presence of D, C, E, c, and e
2. Anti-c
3. Anti-e
Rh Deleted Rh Null 4. Anti-D
RBC Normal Spherocytes,
morphology Stomatocytes Types of Anti Sera to detect Rh Antigens
Rh Antigens Increase D per No D, C, E, c,
RBC and e
1. Low Protein Based
In vivo survival Normal (120 Reduced 2. High Protein Based
days) 3. Saline Based
Effect Impaired N and 4. Chemically Modified
K ion transport 5. Monoclonal
Hemolytic 6. Polyclonal
Anemia
Decrease OFT
❖ Saline Reactive Anti-sera
- Low Protein Based
- Has IgM
Genetic Mechanisms of Rh Null Syndrome - Can test red cells coated with IgG
- Cannot detect weak D

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 - Requires long incubation time (30-60mins)

❖ Chemically Modified IgG Sera

- Low protein based
- No high MW potentiators
- Can be used for slide and tube
- Antibody molecules are treated with
reducing agents

❖ Modified Tube Anti-D

- Contains
1. IgG Direct Anti-globulin Test
2. High Concentration of protein (20-30% - In-vivo reaction
Serum Albumin) - It is not normal in the body to have
3. Macromolecular additives – Dextran, sensitization
Polyvinyl Pyrollidone - Detects antibodies for Hemolytic Disease of
the Newborn as Maternal Anti-D coats fetal
RBC
- Used to demonstrate in vivo coating of red
cells with antibodies or complement, in
particular IgG and C3d.
- Cephalexin – Causes sensitization of RBC X
and Y
Anti Globulin Test - Expected in patients with: Hemolytic
- Used to detect incomplete or non- Disease of the newborn, Drug Induced
agglutinating antibodies Hemolytic Anemia, Autoimmune Hemolytic
- Tool to detect IgG, 7s, or complement. Anemia, and Hemolytic Transfusion
- Not reactive in saline or high protein Reaction.
medium Indirect Anti-globulin Test
- Principle – Anti globulin agglutinate with - Detects presence of unexpected antibodies
cells with gamma globulin in the patient’s serum that are able to coat
antigens in type “O” red cells in vitro (in
vitro sensitization)
- It primarily detects antibodies other than
RBC IgG the naturally occurring anti-A and anti-B.
IgG coated cells or sensitized - It is useful in detecting weak variants of D
RBCs antigens, red cell phenotyping, in antibody
screening, and identification and in minor
crossmatching.
Anti-globulin types
- React at 37 C
- Polyspecific – Polyclonals – can agglutinate
Importance:
with IgG and anti-complement (Anti-C3D)
1. Du Testing
- Monospecific – Monoclonals – specific for
2. Compatibility Testing
only one
3. Antibody Screening Test
- These bridge together to agglutinize
4. RBC Phenotyping
sensitized RBC
5. Investigation of Transfusion
Reaction
6. Antibody Titration

Sensitized RBC Anti-globulin

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Factors affecting Anti-globulin Test
1. Ratio of serum to cells
- Higher ratio, higher sensitivity
- Minimum ratio is 40:1 (2 drops serum and
1 drop 5% RCS)
- If the reaction of the antibody is weak ->
133:1 (4 drops serum and 1 drop 5% RCS)
2. Reaction Medium – Enhances Ag-Ab rxn
- Albumin – allows sensitized cell to come
closer in contact (2 drops serum, 2 drops
albumin, 1 drop 3-5% RCS)
- LISS (Low Ionic Strength Solution) –
Enhances antibody uptake and reduces
incubation time (2 drops serum, 2 drops
LISS, 1 drop 3-5% RCS)
- Polyethylene Glycol – water soluble polymer
used as an additive which increase antibody
uptake by removing water thus increasing
antibody concentration
3. Incubation Time – LISS 10-15 minutes only
4. Temperature
5. Washing – 3 times with NSS, inadequate
washing and excess NSS – False (-)
6. Centrifuge
7. Manner of reading result
8. Reagents used
Compatibility Testing
- Also called crossmatching
- Composed of procedures intended for
safest blood transfusion
- ABO and Rh Blood groups of donor and
recipient must be compatible
Important because:
1. Ensures max benefits to intended
recipient
2. Prevent transfusion reaction that
might happen to the recipient due
to antibodies
Compatibility Testing for Homologous Transfusions
1. ABO Typing – Direct or Indirect
2. Rh Typing – Direct
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3. Antibody Screening – Detects presence or
absence of unexpected antibodies.
- Done in women with previous
pregnancy and patients with
previous transfusions
4. Compatibility Testing
- Major – Patient Serum, Donor Red Cell
- Minor – Patient Red Cell, Donor Serum
- Antibody in serum that can destroy
transfused red cells
- Tests if transfused red cells can survive in
vivo
- Transfusion Reaction
Autologous Transfusion
- Own blood is transfused to self
- Surgical procedures
Preferred specimen – Fresh, not less than 48 hours,
not inactivated serum or plasma
Blood of donor and recipient must be kept at 1-6°C
at minimum for 7 days first.
IV Tube Patient – Do not get blood cause it is diluted,
can’t detect weak antibodies
Solution: Stop IV for 5-10 minutes, extract 10mL and
discard, use the specimen after.
Different Serological Tests For Cross Matching
1. Immediate Spin Saline Cross Match – Sole
cross match, Simplest
- Mix, centri, dislodge, interpret
- Only for no clinically significant antibodies,
test for ABO incompatibility
2. Abbreviated Cross Match – Type and screen
coupled with Immediate Spin
- For ABO, Rh, and unexpected antibodies
3. Anti Human Globulin Cross Match
 4. Computed Cross Match – use computer for 2. Panagglutination – spontaneous clumping
final check, only when there is no clinically of cells against given serum caused by:
significant antibodies. - bacteriologic (20°C Thomsen Friedenrich)
- non-bacteriologic (37°C, Acute Hemolytic
Broad Spectrum Compatibility Anemia, Rare specific antibodies)
- Method of Choice 4. Prozone
- Phases 5. Polyagglutination
1. Protein or Immediate Spin 6. Wharton’s Jelly
- For ABO, Cold antibodies, and Prozone Rh 7. Ag-Ab degradation
M MA N NA
Procedure to Shorten Compatibility Time
1. LISS
2. Enhancing Agents
- Albumin
-Papin
-Trypsin
2 drops patient serum 2 drops patient serum 2 drops patient red cell 2 drops patient red cell
-Polybrene Polycation
2drops donor red cell 2drops donor red cell 2drops donor serum 2drops donor serum
-2% RCS of donor
2 drops BSA 2drops BSA

- Centri, dislodge, interpret. If no

agglutination, use MA or NA and proceed to
next step.

2. Thermo or 37°C
- 37°C for 10 mins. Centri, dislodge, interpret.
If no agglutination, proceed to next step.
3. AHG
- Wash three times with NSS, add AHG. If no
agglutination:
- Add 1 drop check cells (Cells with IgG) and
should be 4+

Criteria of a Universal Donor

- Titer is less than 1:50
- If titer is high, remove the plasma and give
the RBC
- Use Witebsky substance to neutralize
naturally occurring anti-A and anti-B but not
immune anti-A and anti-B
1. A substance – from Hog’s stomach
2. B substance – from Horse’s stomach

Rh without previous immunization – no

anti-D
Rh with previous immunization – has anti-D

Problems
1. Rouleaux

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