CLS 3311 Advanced Clinical Immunohematology

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CLS 3311Advanced Clinical Immunohematology

• Rh Blood Group System
• Lecture 2

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Weak D Phenotype

• Most D positive rbcs react macroscopically with
  Reagent anti-D at immediate spin
• These patients are referred to as Rh positive
• Reacting from 1 to 3 or greater
• HOWEVER, some D-positive rbcs DO NOT react (do
  NOT agglutinate) at Immediate Spin using Reagent
  Anti-D. These require further testing (37oC
  and/or AHG) to determine the D status of the
  patient.

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• Further testing of Patients Cells for
• Weak D Status
• If negative at Immediate Spin, patient cells and
  anti-D reagent are incubated at 37o C for 20
  mins. (Do not add enhancement media.) After
  incubation, Centrifuge, observe for
  agglutination. If positive, report as Rh
  Positive.
• If negative wash three times and add AntiHuman
  Globulin. Centrifuge. If NEGATIVE add CC cells
  and report as Rh Negative if CC cells
  agglutinate. If POSITIVE report as Weak D
  Positive.
• Patients/Recipients who require AHG testing to
  determine the presence of the D antigen, and have
  the D antigen are designated Weak D Positive.

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Weak D Mechanisms

• There are three mechanisms that account for the
  Weak D antigen.
• Genetically Transmissible
• Position Effect
• Partial D (D Mosaic)

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Genetically Transmissible

• The RHD gene codes for weakened expression of D
  antigen in this mechanism.
• D antigen is complete, there are just fewer D Ag
  sites on the rbc. Quantitative!
• Common in Black population (usually Dce
  haplotype). Very rare in White population.
• Agglutinate weakly or not at all at immediate
  spin phase.
• Agglutinate strongly at AHG phase.
• Can safely transfuse D positive blood components.

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Position Effect (Gene interaction effect)

• C allele in trans position to D allele
• Example Dce/dCe, DcE/dCE In both of these cases
  the C allele is in the trans position in relation
  to the D allele.
• D antigen is normal, C antigen appears to be
  crowding the D antigen. (Steric hindrance)
• Does NOT happen when C is in cis position
• Example DCe/dce
• Can safely transfuse D positive blood components.

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Partial D (D Mosaic)

• Missing one or more PARTS of the D antigen
• D antigen comprises many epitopes Table 6-8 Page
  136
• PROBLEM
• Person types D positive but forms alloanti-D that
  reacts with all D positive RBCs except their OWN.

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Partial D Multiple epitopes make up D antigen.
Each color represents a different epitope of the
D antigen.
A.
Patient B lacks one D epitope.
B.
The difference between Patient A and Patient B is
a single epitope of the D antigen. The problem is
that Patient B can make an antibody to Patient A
even though both appear to have the entire D
antigen present on their red blood cells using
routine anti-D typing reagents..
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• No Differentiation In Weak-D Status Is Made
  Serologically In The Routine Blood Bank
• In the routine blood bank we cannot differentiate
  which mechanism accounts for the patients Weak
  D status.

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Weak-D Determination Donor Blood

• When testing Donor Blood for the D antigen,
  testing is required through all phases.
• Weak-D testing is REQUIRED
• We need to know the D Status of all Donor Blood.
  Why?
• Main problem is Rh Negative women of child
  bearing age and pediatric patients.
• Donor RBCs are labelled Rh positive if any part
  of the D antigen is present on the red blood cell
  membrane.

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Recipient Blood

• Controversy
• AABB Standards state that you do NOT have to
  perform complete D typing of recipient blood.
• Most weak-D patients can receive D positive blood
  without forming anti-D.
• Partial D is very rare, BUT these patients are
  capable of making alloanti-D even though they are
  Weak D positive.
• So, some blood banks ONLY perform immediate spin
  D and if it is negative they do NO further D
  testing and label the patient (recipient) Rh (D)
  negative and transfuse Rh Negative blood
  components.

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• Some consider it wasteful to transfuse Rh
  Negative blood into Weak-D recipient. The testing
  policy is up to each individual facility.
• Recipients who need complete testing
• Obstetric patients Weak D status MUST be
  determined on all obstetric patients. Why? What
  will you transfuse?
• Newborn Need to determine D status on all
  newborns. Why?

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Rh Antibodies

• RBC Immune IgG (anti-D, anti-C, anti-c, etc.)
• Rh antibodies do NOT bind complete
• Only in extremely rare cases
• Cause extravascular hemolysis
• Cross the placenta
• Cause Hemolytic Disease of the Newborn (HDN)
• Rh antigens are well developed at birth
• Rh antibody reactivity is ENHANCED using enzyme
  treated red blood cells

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Rh System Antibodies

• React optimally
• RBC Immune
• Clinically Significant

1. 37oC and AHG Phases
2. Transfusion or pregnancy, IgG, HDN, HTR, etc.
3. Will result in shortened red cell survival - need
   to transfuse antigen negative blood

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Rh Antigen Typing Reagents

• Routine Rh typing for donors and patients
  involves typing for only the D antigen.We dont
  routinely type for E, e, C or c.
• Historically speaking Original D typing tests
  require long saline incubation times because it
  is IgG antibody. The goal was to produce an
  antisera that reacts at I.S.
• Saline Anti-D (IgM) Reagent
• Reacts strong at immediate spin (I.S.)
• Low protein reagent.
• Can be used to test antibody coated cells
• Very expensive!! Cost prohibitive.
• One of the first Immediate Spin anti-D reagents.

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D Antigen Typing Reagents

• High protein anti-D
• High protein reagent with macromolecular
  additives
• Protein enhanced reactivity of IgG anti-D reagent
  so it would react at immediate spin.
• Must run an Rh Control!! Why?
• The control reagent is the suspending media in
  which the anti-D antibodies swim.
• Enabled reduced incubation times. Both slide and
  tube testing can be performed.

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D Antigen Typing Reagents

• Chemically Modified Anti-D
• Reagent antibodies with broken disulfide bonds so
  IgG anti-D can span distance between RBCs
• Low protein suspending media
• Slide and tube method testing
• No need for Rh Control when patient is A, B or O
  positive
• Need control for AB Pos, Why?
• This applies to all the remaining anti-D reagents.

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D Antigen Typing Reagents

• Monoclonal Polyclonal Blend Anti-D
• Monoclonal anti-D reagents are too specific and
  may miss some partial D categories so
• Mix monoclonal IgM and polyclonal IgG into one
  anti-D reagent
• Increase reaction strength at room temperature
• Able to test Weak-D at AHG phase
• Low protein suspending media No control
  necessary.

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D Antigen Typing Reagents

• Monoclonal Blend
• Blend monoclonal IgM with monoclonal IgG anti-D
• Added multiple clones to increase reactivity with
  Partial D patients
• Low protein reagent No need for a control unless
  patient is what ABO group?

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Rh Null Phenotype

• Persons lack ALL Rh antigens
• Lack both the RHD and RHCE genes
• No D, C, c, E, e antigens present on the RBC
  membrane
• Demonstrate mild hemolytic anemia (Rh antigens
  are integral part of RBC membrane and absence
  results in loss of membrane integrity)
• Reticulocytosis, stomatocytosis, slight decrease
  in hemoglobin and hematocrit, etc.
• When transfusion is necessary ONLY Rh Null blood
  can be used to transfuse.

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Other Rh Antigens

• Cw Antigen
• Usually found in combination with C or c antigens
• 2 whites, rare in blacks
• Anti-Cw seen in BOTH RBC Immune (Transfusion and
  pregnancy) and NON RBC Immune situations.
• f (ce) Antigen
• c and e in cis position, same haplotype
• Compound antigen (ce), however f is a single Ag
• anti-f test with R1R2 (f negative) and R1r (f
  positive) red cells

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Other Rh Antigens

• rhi (Ce) antigen
• Also a compound antigen
• C and e in the cis position
• R1R2 is positive for the rhi antigen
• R0Rz is negative for the rhi antigen
• G antigen
• G antigen is generally weakly expressed and is
  associated with the presence of the VS antigen.
• Almost invariably present on RBCs possessing the
  either the C or D antigens
• Antibodies to G appear to be anti-CD, but the
  anti-G activity CANNOT be separated into anti-C
  and Anti-D.

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Other Rh Antigens

• V, VS antigens
• Page 140 Harmening
• These little guys I will let you read about.
• Deletion Phenotype D-- or -D-
• Both designations indicate the same phenotype
• C, c, E, e antigens are absent from the RBC
  membrane in this phenotype.
• Very strong D antigen expression STRONGEST
• CAN make antibodies to all missing antigens.
  Usually make anti-Rh17 antibody.