Karrie Hovis, MHS, MLS(ASCP) CM

1. 2011 ImmunohematolgyStudent Review Karrie Hovis, MHS, MLS(ASCP)CM

2. MLS(ASCP) • Immunohematology—17% • ABO, Rh • Antibody screen and ID • Duffy/Ii/Kell/Kidd/Lewis • MNS/P1Pk/Rh/multiple antibodies • Crossmatch and Special Tests • DAT • Phenotyping/Genotyping • Elution/absorption • Titer • Pre-warm technique • Fetal rosette and Kleihauer-Betke

3. MLS(ASCP), cont. • Blood Donation • Donor Testing • Donor Requirements • Transfusion Therapy • RBC, PLT, FFP, Cryo, RhIG • Transfusion Reactions • HDFN

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

5. 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

6. 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 - Mendalianlaws

7. 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

8. ABO System Genetic Pathway

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

10. 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

11. 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)

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

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

14. 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

15. 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

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

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

18. 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

19. 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

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

21. 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

22. 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

23. Other Blood Groups • P1Pk • Formerly known as the P System • IgM cold antibody • Antigens—P1and Pkonly • P1 deteriorates with storage • Anti-P1neutralizable by P1 substance in hydatid cyst fluid

24. Other Blood Groups • The Globoside (GLOB) System includes: P only • Anti-P is autoantibody of PCH • The Globoside (GLOB) Collection includes : LKE and PX2

25. 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)

26. 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

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

28. 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

29. Ab Temp of Reactivity

30. Ab Temp of Reactivity

31. Enhanced

32. Destroyed

33. Dosage

34. Enzymes Papain Bromelin Ficin Trypsin Destroy some antigens

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

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

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

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

39. 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

40. 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.

41. 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

42. 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

43. 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.

44. 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

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

46. 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

47. Applications of the DAT • Hemolytic Transfusion Reactions • First immunohematologic evidence of HTR • Hemolytic Disease of the Fetus and Newborn • Maternal antibody coating fetal cells • Investigation of Autoantibodies • Warm auto- cells coated with IgG or rarely IgG/C3 • Cold auto- Cells coated with C3d only (IgM) • Drug Induced antibodies • Cells coated with IgG, C3d, or both depending on mechanism

48. Drug Induced Hemolytic Anemias • Stimulation of Autoantibody • Methyldopa (Aldomet) • Drug Absorption Mechanism • Penicillin- forms haptenic determinant • Nonimmunologic absorption of proteins • Cephalosporins- adsorb serum/plasma proteins • Immune Complex • Antibody formed to drug-carrier protein complex that attaches to cells

49. Compatibility Testing • Pretransfusion • Crossmatch • Abbreviated • Computerized • Through AHG • Neonatal • Antigen typing • Quality control

50. Probability • What is the probability of finding Kell negative, Jka negative units? • Kell—8% frequency • 92% negative • Jka—75% frequency • 25% negative • 0.92 * 0.25 = 0.23 or 23%