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Antibody Identification.  Most important blood group system in blood transfusion medicine.  (after ABO)

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Presentation on theme: "Antibody Identification.  Most important blood group system in blood transfusion medicine.  (after ABO)"— Presentation transcript:

1 Antibody Identification

2  Most important blood group system in blood transfusion medicine.  (after ABO)

3  1939 – Levine and Stetson 1 st discovered antibody  1940 – Landsteiner and Weiner discovered antibody developed by using Rhesus monkey cells.  1960’s- Discovered the antibody produced by the pregnant woman and the Rhesus monkeys were actually two different, distinct antibodies.

4  Fisher Race – D, C, E, d, c, e ◦ 3 sets of genes produce the antigens  Examples: ◦ DCe/dce ◦ DcE/DCE  Weiner – R 1, R 2, r ◦ Inheritance of all Rh antigens lies under control of one gene  Examples: ◦ R 1 R 2 ◦ R 1 r’ ◦ R 0 r

5  International Society of Blood Transfusion ◦ Uniform nomenclature both eye and machine readable  D = RH1  C = RH2  E = RH3  c = RH4  e = RH5

6  RHD and RHCE genes located on Chromosome 1  Over 100 RHD and 50 RHCE alleles have been identified  RHAG (RHAG) Rh associated glycoprotein located on Chromosome 6  LW gene located on Chromosome 4

7  PS1 RHAG PS2 CDE LW RH genes LW genes

8  Extends 12 spans of the RBC membrane  Integral part of the red cell membrane  Linked to membrane skeleton

9  D antigen is comprised of multiple epitopes  Persons with one or more epitopes missing from the red cells can produce an immune response when exposed to the common form of the D antigen  Cells generally type normally as D+ since typing reagents are designed to detect multiple epitopes

10  Classification of Partial D Epitopes Category II IIIa IIIb IIIc Iva Ivb Va Vb Vc VI

11  Early reagents relied on antibodies produced by women sensitized by pregnancy or in hyperimmunized volunteers.  Monoclonal antibody technology was introduced in the 1980’s. ◦ But monoclonal antibodies are specific for a single D epitope  Does not detect all D-positive red cells.

12  Current reagents are blends containing monoclonal IgM antibody plus monoclonal or polyclonal IgG antibody ◦ IgM allows for RT reactivity ◦ IgG allows for AHG testing and detection of Weak D  Must read package insert to see which variants it detects ◦ Gammaclone – reacts at AHG with DVI, DBT, D Har, Crawford ◦ Immucor Series 4 and 5 reagents do not react with Crawford ◦ OrthoBioclone does not react with D har or Crawford

13  Enhanced by enzymes  Not affected by DTT, Chloroquine, EGA treatment

14  Anti-D vs Anti-LW  Anti-LW reacts with all adult cells ◦ Reacts stronger with Rh+ cells ◦ Reacts weaker with Rh- cells  Anti-LW reacts strongly with Rh+ or Rh- Cord cells  Anti-LW destroyed by DTT

15 Cell DCEceKkFy a Fy b Jk a Jk b MNSsAHG Gel DTT 1 ++00+0++0+0+++02+0 2 ++00+++0+0+0+0+ 0 3 +0++00+0+++++0+ 0 4 +00++++00+00+0+ 0 5 0+0++0++0+0++0+w/00 6 00+++0++0+++0+0 0 7 000++++0++++0++ 0 8 000++0+0+00++0+ 0 9 ++00+0++++00++02+0 PC 00 Rh= cord 2+0

16  Anti-f  f antigen is expressed on RBCs having c and e on the same haplotype (cis). ◦ R1r DCe/dce  f antigen is not expressed when c and e occur on separate haplotypes (trans). ◦ R1R2 DCe/DcE  65% Caucasian population, 92% African Americans, 12% Asians

17 Cell DCEceKkFy a Fy b JkaJk b MNSs AHG Gel 1++00+0++0+0+++00 2++00+++0+0+0+0+0 3+0++00+0+++++0+0 4+00++++00+00+0+2+ 50+0++0++0+0++0+ 600+++0++0+++0+0 7000++++0++++0++ 8000++0+0+00++0+ 9000++0++++00++0 PC0

18  Anti-G  Inseparable anti-CD  G antigen is present on ANY cell with the C or D antigen, or both  But there have been cases of D-C-G+ and D+G-  Must perform adsorb/elution studies to confirm presence  Patient can have both anti-G plus anti-D or anti-C.

19 Cell DCEceKkFy a Fy b JkaJk b MNSs AHG Gel 1++00+0++0+0+++03+ 2++00+++0+0+0+0+ 3+0++00+0+++++0+ 4+00++++00+00+0+ 50+0++0++0+0++0+ 600+++0++0+++0+00 7000++++0++++0++0 8000++0+0+00++0+0 9++0++0++++00++0 PC0

20  R o (Dce)  Will adsorb out true anti-D while leaving separate anti-C  Antibody will be coating cells after adsorption  Perform elution to harvest coating antibody  Perform antibody identification on eluate  If shows anti-D plus anti-C pattern = ANTI-G  If shows only anti-D, then have separate anti-D and anti-C antibodies

21 e mosaic hr s hr b V/VS e

22  The e antigen is considered to be a mosaic ◦ Correct terminology is Partial  The antigens in the mosaic are  e, hr s,hr b, V, and VS

23  hr s antibody is similar to anti-ce  hr b antibody is similar to anti-Ce  Anti-VS can be naturally occurring

24 CellDCEceKkFy a Fy b Jk a Jk b MNSsAHG Gel 1++00+0++0+0+++02+ 2++00+++0+0+0+0+ 3+0++00+0+++++0+w+ 4+00++++00+00+0+2+ 50+0++0++0+0++0+ 600+++0++0+++0+0 7000++++0++++0++ 8000+00+0+00++0+w+ 9++0++0++++00++02+ PC0

25  Saline  Albumin  Enzyme  37°C Incubation  AHG

26 AHG SampleSalineAlbuminLISSPeGGel Anti-Dw+ 1+2+ Anti-E2+ 3+4+ Anti-C1+w+1+2+

27  Enzymes: Ficin, Papain, Bromelin  Rh antibodies show enhanced reactivity with enzyme-treated cells  Enzyme treatment removes structures from the red cell membrane that otherwise interfere with the antigen-antibody complex

28  Other sources of Rh antibodies ◦ RhIg ◦ WinRho ◦ IVIg ◦ Rutuximab/Rutixan  Other sources of red cell stimulation ◦ Renal transplantation  Red cells still in organ  B cells in graft producing anti-D ◦ Bone grafts ◦ Needle sharing

29  RhIg/WinRho  Used for the treatment of ITP  Used as a prophylaxis in Rh negative mothers  May contain anti-A, anti-B, anti-C, anti-E, Duffy and Kidd antibodies

30  IVIg  Manufactured from a pool of 1000 to 100,000 donors  May contain anti-A, anti-B, anti-D, and other antibodies  Used for treatment of WAIHA  Cost - $10,000 a dose (220 lb person @ 2g/Kg)

31  Rutuximab (Rituxan)  Antibodies directed against CD20 (B cell marker)  Used for treatment of TTP, lymphoma, leukemia, transplant rejection, autoimmune diseases

32  Partial RhD typing kit  Bioarray Rh Variant BeadChip  CICBC should have the new Bioarray kit available by end of next year.


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