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CLS 3311 Advanced Clinical Immunohematology

CLS 3311 Advanced Clinical Immunohematology. Rh Blood Group System Lecture #2. 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

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CLS 3311 Advanced Clinical Immunohematology

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  1. CLS 3311Advanced Clinical Immunohematology Rh Blood Group System Lecture #2

  2. 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.

  3. 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 asRh Negativeif CC cells agglutinate.If POSITIVE report asWeak 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”.

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

  5. GeneticallyTransmissible • 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.

  6. Position Effect (Gene interaction effect) • Callele in transpositionto 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 cisposition • Example: DCe/dce • Can safely transfuse D positive blood components.

  7. 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.

  8. 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 cell’s using routine anti-D typing reagents..

  9. 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.

  10. 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.

  11. 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.

  12. 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?

  13. 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

  14. React optimally RBC Immune Clinically Significant 37oC and AHG Phases Transfusion or pregnancy, IgG, HDN, HTR, etc. Will result in shortened red cell survival - need to transfuse antigen negative blood Rh System Antibodies

  15. 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.

  16. 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 RhControl!! 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.

  17. 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.

  18. 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.

  19. 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?

  20. 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 • Demonstratemild 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.

  21. 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

  22. 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.

  23. 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.

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