Rh Blood Group System

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

Rosenfield
Rh RBC - Aims to bridge the confusion between first 2
- A number in chronological discovery
- No genetic basis
Rabbit - Presence or absence only of Ag
- D–1
- C–2
Produce Anti-Rh - E–3
antibodies - e–4
against Rh RBC - c–5
ISBT (International Society for Blood Transfusions)
- 6 numerical numbers
Ab reaction - 004 = Rh
- D – 004001
with human - C – 004002
RBC - E – 004003
- c – 004004
- e – 004005

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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 2. Partial or Mosaic
- Cf - Because Rh is a low MW compound (174,000
- Cw daltons), it is said that the D-antigen is a mosaic
- Eu structure composed of 4 fragments and if one part
- Du – Weakened expression of D-Antigen or others is/are missing, it shows weak expression of
(Blacks>Caucasians) detectable only by Indirect D-antigen.
Method. It is few in number A B A B
- D+w is the phenotype
C D D
Mechanisms of Weak D Antigen
No Weak D Weak D with Anti-C
1. c-Trans Disadvantage – if you are Rh(-), you can receive only
- C is in cis position with D Rh(-)
- if C is in trans position it causes weak D
3. Genetic Weak D
- All fragments are present but only in few number
D d D d and thus weakly expressed.
Cis Trans
C c c C 2 types of Du
e E e 1. Low Grade
E
- Direct product of inherited gene detectable only by
Normal Weak-D IAG, and can be passed unto future generations.
2. High Grade
- Cannot be passed unto future generations, rarely
need Anti-globulin test

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3 Genes that control Rh expression 3 Varieties of Rh Antibodies
1. Rh D – Presence of D antigen (Chromosome 1) 1. 1st Order – Saline agglutinins, Bivalent, Complete
2. Rh CE – Presence of C, E, c, and e antigen – React in Saline medium
(Chromosome 1) 2. 2nd Order – Albumin reacting, Monovalent,
3. RhAG – Rh Associated glycoprotein, coexpressor Incomplete – React in protein medium
(Chromosome 6) 3. 3rd Order – Typical antibodies, Anti-globulin
- Expresses Rh AG but by itself does not antibodies – React in antiglobulin medium
display any antigen
- If there is a mutation in this gene = Different Anti Sera to detect Rh Antigens
Missing or alteration of Rh D and Rh CE 1. Anti-C
1. Anti-E
Unusual Phenotypes of Rh 2. Anti-c
1. Rh Deleted – no C and E (D _ _ / D _ _ ) 3. Anti-e
2. Rh Null – none at all ( _ _ _ / _ _ _ ) 4. Anti-D
3. Rh Moderate – weak expression
Types of Anti Sera to detect Rh Antigens
Effects of no presence of D, C, E, c, and e
1. Low Protein Based
Rh Deleted Rh Null 2. High Protein Based
RBC Normal Spherocytes, 3. Saline Based
morphology Stomatocytes 4. Chemically Modified
Rh Antigens Increase D per No D, C, E, c, 5. Monoclonal
RBC and e 6. Polyclonal
In vivo survival Normal (120 Reduced
days)  Saline Reactive Anti-sera
Effect Impaired N and - Low Protein Based
K ion transport - Has IgM
Hemolytic - Can test red cells coated with IgG
Anemia - Cannot detect weak D
Decrease OFT - Requires long incubation time (30-60mins)

 Chemically Modified IgG Sera

Genetic Mechanisms of Rh Null Syndrome - Low protein based
1. Regulator Type – Mutation in Rh AG thus there is - No high MW potentiators
Rh AG in RBC but has normal complement of Rh D - Can be used for slide and tube
and Rh CE - Antibody molecules are treated with
2. Amorphic Type - Mutation in Rh CE, Deletion in reducing agents
Rh D, Normal Rh AG
 Modified Tube Anti-D
General Characteristics of Rh Antibodies - Contains
1. Majority are IgG (1 and 3 most significant) 1. IgG
2. Not natural antibodies but immune antibodies 2. High Concentration of protein (20-30%
3. React best at 37C Serum Albumin)
4. Crosses placenta 3. Macromolecular additives – Dextran,
5. Do not bind complement Polyvinyl Pyrollidone
6. Do not react in saline solution
7. Commonly found antibodies in
immunocompromised patients = Anti-D and Anti-G
(Abs found in immunized patients)

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Anti Globulin Test Indirect Anti-globulin Test
- Used to detect incomplete or non- - Detects presence of unexpected antibodies
agglutinating antibodies in the patient’s serum that are able to coat
- Tool to detect IgG, 7s, or complement. antigens in type “O” red cells in vitro (in
- Not reactive in saline or high protein vitro sensitization)
medium - It primarily detects antibodies other than
- Principle – Anti globulin agglutinate with the naturally occurring anti-A and anti-B.
cells with gamma globulin - It is useful in detecting weak variants of D
antigens, red cell phenotyping, in antibody
screening, and identification and in minor
crossmatching.
RBC IgG - React at 37 C
IgG coated cells or sensitized Importance:
RBCs 1. Du Testing
2. Compatibility Testing
Anti-globulin types 3. Antibody Screening Test
- Polyspecific – Polyclonals – can agglutinate 4. RBC Phenotyping
with IgG and anti-complement (Anti-C3D) 5. Investigation of Transfusion
- Monospecific – Monoclonals – specific for Reaction
only one 6. Antibody Titration
- These bridge together to agglutinize
sensitized RBC Factors affecting Anti-globulin Test
1. Ratio of serum to cells
- Higher ratio, higher sensitivity
- Minimum ratio is 40:1 (2 drops serum and
Sensitized RBC Anti-globulin
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
Direct Anti-globulin Test LISS, 1 drop 3-5% RCS)
- In-vivo reaction - Polyethylene Glycol – water soluble
- It is not normal in the body to have polymer used as an additive which increase
sensitization antibody uptake by removing water thus
- Detects antibodies for Hemolytic Disease of increasing antibody concentration
the Newborn as Maternal Anti-D coats fetal 3. Incubation Time – LISS 10-15 minutes only
RBC 4. Temperature
- Used to demonstrate in vivo coating of red 5. Washing – 3 times with NSS, inadequate
cells with antibodies or complement, in washing and excess NSS – False (-)
particular IgG and C3d. 6. Centrifuge
- Cephalexin – Causes sensitization of RBC X 7. Manner of reading result
and Y 8. Reagents used
- Expected in patients with: Hemolytic
Disease of the newborn, Drug Induced
Hemolytic Anemia, Autoimmune Hemolytic
Anemia, and Hemolytic Transfusion
Reaction.

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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
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.
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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
final check, only when there is no clinically
significant antibodies.
Broad Spectrum Compatibility
- Method of Choice
- Phases
1. Protein or Immediate Spin
- For ABO, Cold antibodies, and Prozone Rh
M MA N NA
2 drops patient serum 2 drops patient serum 2 drops patient red cell 2 drops patient red cell
2drops donor red cell 2drops donor red cell 2drops donor serum 2drops donor serum
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
2. Panagglutination – spontaneous clumping
of cells against given serum caused by:
- bacteriologic (20°C Thomsen Friedenrich)
- non-bacteriologic (37°C, Acute Hemolytic
Anemia, Rare specific antibodies)
4. Prozone
5. Polyagglutination
6. Wharton’s Jelly
7. Ag-Ab degradation

Procedure to Shorten Compatibility Time

1. LISS
2. Enhancing Agents
- Albumin
-Papin
-Trypsin
-Polybrene Polycation
-2% RCS of donor

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