Karrie Hovis, MHS, MT(ASCP) CM , CLS(NCA)

1. 2009 Immunology and Immunohematolgy Student Review Karrie Hovis, MHS, MT(ASCP)CM, CLS(NCA)

2. ASCP • Immunohematology—17% • ABO, Rh • Antibody screen and ID • Duffy/Ii/Kell/Kidd/Lewis/Lutheran • MNS/P/Rh/other • Compatibility Testing and Special Tests • DAT • Phenotyping/Genotyping • Elution/absorption • Titer • Pre-warm technique • Fetal rosette and Kleihauer-Betke

3. ASCP, cont. • Blood Donation • Testing and Requirements • Transfusion Therapy • RBC, PLT, FFP, Cryo, RhIG • Transfusion Reactions • HDFN

4. ASCP, cont. • Immunology—8% of total exam • Immunity • Autoimmunity • ANA, anti-DNA • CRP/RF • Thyroid antibodies • Other autoimmunes (e.g., extractable nuclear antigen) • Pre-Analytical, Test Principles

5. ASCP, cont. • Immunology • Infectious Diseases • Viral • EBV/infectious mononucleosis • Hepatitis • HIV/HTLV/CMV • Rubella/measles • Microbial • Cold agglutinins • Syphilis • Other microorganisms

6. NCA • Immunohematology • 26 questions • Donors and Donor Blood • Selection criteria • Preparation, etc. • Basic Techniques in Blood Group Serology • Issue Blood and Blood Products

7. NCA • Immunology • 19 questions • Serologic Assays • Precipitation assays • RID • Immunoelectrophoresis • Flocculation tests • Complement fixation • Etc. • Cellular Assays • Flow Cytometry

8. Immunohematology

9. Antibodies • IgG • Clinically significant • Transfusion Reactions • HDN • IgM • Generally clinically insignificant, except ABO • Cold reacting • No HDN • Activate complement

10. Genetics • Glossary: • Allele—one parent is KK and the other is kk, all the children will be Kk • Homozygous—M+N= • Heterozygous—M+N+ • Recessive • Dominant • Phenotype • Genotype

11. Genetics, cont. • Most blood group antigens are co-dominant; example ABO expression • A and B genes if present will be expressed • O gene – silent – amorph • Xga • Only sex-linked blood group • Gene on X chromosome so incidence higher in females • Inheritance--Mendalian laws

12. ABO System • 4 major phenotypes of ABO System • Immunodominant Sugars added to the precursor substance: • A—N-acetylgalactosamine • B—D-galactose • O—No addition of terminal sugar to H structure; L-fucose is terminal sugar • AB – N-acetylgalactoseamine, D-galactose

13. ABO System Genetic Pathway

14. H Substance • Decreasing order of H substance: • O > A2 > B > A2B > A1 > A1B > Oh • Anti-H lectin • Ulex europaeus • Negative reactions with Bombay phenotype

15. Relationship of H, Se, and Le • 78% of random adults possess Se gene • 22% of random adults lack Se gene • ABH expression is dependent on Se gene • Allows expression of A, B, H and Leb in saliva • Lea expression is not dependent on Se gene

16. Relationship of H, Se, and Le • Lewis antigens-plasma antigens • Lewis gene (lack Se gene)—Lea (RBCs and saliva) • H gene + Le gene (lack Se gene)—H and Lea (RBC); Lea (saliva) • H gene + Le gene + Se gene—H, Leb (RBC); H, Leb, Lea (saliva)

17. ABO Grouping • Patient Cell grouping • Forward type • Anti-A • Anti-B • Patient Serum grouping • Reverse type • A1 cell • B cell

18. ABO Discrepancies • General causes: • Clerical Errors • Technical Errors

19. ABO Discrepancies • Problems with patient’s cells • Cell mixture • A or B transfused with many units of O packed cells • Acquired B • Intestinal obstruction or carcinoma • Weak antigens • Weak subgroups • Effects of disease such as leukemia

20. ABO Discrepancies, cont. • Problems with patient’s cells • Polyagglutination • Results from inherited or acquired abnormalities of the red cell membrane • Heavy coating of red cells by potent autoagglutinins

21. ABO Discrepancies, cont. • Problems with serum • Rouleaux • Atypical cold antibodies—anti- M, N, P1, A1 • No antibody • Elderly • Newborn • hypogammaglobulinemia

22. Subgroups of A • A1 • A2 • A1 Lectin (Dolichos biflorus)

23. Anti-A –B –D A1 cell B cell Antibody screen Group AB Group O • Anti- A1 (lectin) • Dolichos biflorus Group AB Group O Group AB Group O ABO Oddities What’s different about a cis-AB person? 80% of type A persons have A1 antigen; others are subgroups such as A2

24. Rh System • D positive • Weak D positive—NOT Du! • D reactive at AHG only • D negative • Rh control • Must be negative for a valid test • Required if using albumin based reagents • Monoclonal/polyclonal blend—AB types only

25. Rh antibodies • IgG; clinically significant • May react at 37C, as well as AHG • Show dosage, except for anti-D • Enhanced with enzymes

26. Weiner/Fisher-Race • Ro – Dce r – dce • R1 – DCe r’ – dCe • R2 – DcE r” – dcE • RZ – DCE ry – dCE • Weiner (Rr) deleted from ASCP • R – D • r – d • o or nothing – ce • 1or ’ – C • 2 or ” – E • z or y - CE

27. Other Blood Groups • Lewis • Plasma antigens • Cord cells negative for Lewis antigens •  expression of antigen in pregnant females • Can be hemolytic (if complement is activated) • Ii • Anti-I is IgM • I absent or weak on cord cells • i absent on adult cells

28. Other Blood Groups • P • IgM cold antibody • Antigens—P, P1 and Pk • P1 deteriorates with storage • Anti-P1 neutralizable by P1 substance in hydatid cyst fluid • Anti-P is autoantibody of PCH

29. Other Blood Groups • MN • IgM, anti-M can be IgG • Anti-M—acidification • Dosage • Destroyed by enzymes • Ss • IgG • Destroyed by enzymes • U negative also Ss negative (African Americans)

30. Other Blood Groups • Kell • IgG • Kell (K) and cellano (k) • K second most immunogenic • 90% of population are K negative; most are k positive • McLeod phenotype- x-linked recessive • Weak K antigens associated with CGD • Kidd—Jka and Jkb • IgG • Enhanced by enzymes • Dosage • Rapid rise and fall of titers • Jk(a-b-) phenotype very rare, some form anti-JK3

31. Other Blood Groups • Duffy– Fya and Fyb • IgG • Destroyed by enzymes • Dosage • Malaria • Sda • Clinically insignificant • Refractile • Urine neutralization

32. Miscellaneous Blood Group Antigens • HTLA- antigens present on > 90% of cells • Cha, Rga, JMH, Kna, McCa, Yka, Csa, Bg, Sda • Antibodies react weakly at AHG and persist through extensive dilutions (as high as 2048) • Limited clinical significance but may mask other clinically significant antibodies present • Cha, Rga, JMH antigens denatured by enzymes

33. Ab Temp of Reactivity

34. Ab Temp of Reactivity

35. Enhanced

36. Destroyed

37. Dosage

38. Enzymes Papain Bromelin Ficin Trypsin Destroy some antigens

39. Antibody Detection and ID • 3 methods: • Tube • Gel • Solid phase red cell adherence testing

40. Antibody Detection and ID • Indirect antiglobulin test (IAT) • Performed in tube method • antibody screen, panel, etc. • 37C • AHG • Coombs check cells

41. Antibody Detection and ID • Principle of Gel • Controlled centrifugation of RBC’s through dextran-acrylamide gel. • Appropriate reagents predispensed in specifically designed microtubes

42. Gel Reactions • Large agglutinates do not travel through gel • Smaller agglutinates become trapped in gel • Unagglutinated cells form pellet at bottom of the microtube

43. Antibody Detection and ID • Solid Phase Procedure • Intact reagent RBC’s bound to chemically modified polystyrene microplate test wells • Serum or plasma and LISS added • Incubation • Wash • Anti-IgG coated RBC added • Centrifugation

44. Solid Phase Reactions Positive-adherence of indicator RBCs to all of well bottom Weak Positive -indicator RBCs will adhere to part of the well bottom Negative-indicator cells will form a clearly delineated button at center of well.

45. Antibody Detection and ID, cont. • Panels: • Untreated • Enzyme • Rule out antigens based on negative reactions • Use “rule of three” • 3 positive cells, 3 negative cells • Confirms antibody with 95% certainty • Consider dosage • Homozygous vs heterozygous cells

46. Resolution Techniques • Techniques for suppressing antibody • ZZAP, AET • Neutralization • Prewarm technique • Used for the detection of alloantibodies in the presence of cold-reactive autoantibodies • Serum and cells are prewarmed to 37C before they are combined • Titer • Quantifies antibody • Useful in pregnancy

47. Resolution Techniques • Adsorption • Used interchangeably with “absorption” • Removing antibody from serum sample by adsorption onto RBCs that express corresponding antigen • Autoadsorption cannot be used if patient has been transfused within the last 3 months.

48. Method 1 Adsorb out Anti-C, A and B and leave anti-U in serum Method 2 Adsorb out anti-U only then elute off of adsorbing cell Anti-A, B and C to cell Anti-U to cell Eluate anti-U from cell AB cell U- BUT C+ O cell U+ BUT C- Anti- U left in serum Anti-A, B, and C left in serum Adsorption/ Combined Adsorption-Elution Example: Anti-U and C mix in serum with anti-A and anti-B Goal- Pure anti-U Special Techniques

49. Resolution Techniques • Elutions • Use when DAT is positive • Antibody coating the cell is removed to allow identification • Last wash must be negative

50. Direct Antiglobulin Test (DAT) • Antiglobulin added to washed cells • Polyspecific (anti-IgG & anti-C3d) • anti-IgG • anti-complement (anti-C3b, C3d) • Detects in vivo coating of RBCs • Specimen