Transcript CLS 3311 Advanced Clinical Immunohematology

Rh/D y grupos débiles de
D, de notas de internet
• Notas amables, sencillas, claras
de apoyo en el estudio del tema
Weak D Phenotype
• Most D positive rbc’s 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 rbc’s 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.
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 min’s. (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”.
Weak D Mechanism’s
There are three mechanisms that account for
the Weak D antigen.
1. Genetically Transmissible
2. Position Effect
3. Partial D (D Mosaic)
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.
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.
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.
Partial D: Multiple epitopes make up D antigen. Each
color represents a different epitope of the D antigen.
A.
B.
Patient B lacks
one D epitope.
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 cell’s using routine antiD typing reagents..
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 patient’s
Weak D status.
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.
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.
• 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?
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
Rh System Antibodies
1. React optimally
1. 37oC and AHG Phases
2. RBC Immune
2. Transfusion or pregnancy,
IgG, HDN, HTR, etc.
3. Clinically
Significant
3. Will result in
shortened red cell
survival - need to
transfuse antigen
negative blood
Rh Antigen: Typing Reagents
• Routine Rh typing for donors and patients involves
typing for only the D antigen.We don’t 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.
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.
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.
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.
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?
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.
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
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 RBC’s possessing the
either the C or D antigens
• Antibodies to G appear to be anti-C+D, but the anti-G
activity CANNOT be separated into anti-C and Anti-D.
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.