Presentation Transcript

1. Immunohematology Review

   Deborah Baudler MS, MT(ASCP)SBB Assistant Professor University of Illinois Springfield Patchwork Conference April 2014
2. University of Illinois-Springfield
3. UIS CLS Students
4. Objectives Quick Review of most blood group systems Highlight Antibody Identification and Special procedures Review donor screening Discuss component collection, testing and clinical indication Discuss HDFN and Prenatal testing Review transfusion reactions End with Review Questions
5. ABO Inheritance and Genetics The ABO gene is autosomal (the gene is not on either sex chromosomes) The ABO gene locusis located on the chromosome 9. A and B blood groups are dominant over the O blood group A and B group genesareco-dominant
6. H Substance RBC The H gene codes for the enzyme fucosyltransferase that adds the sugar fucoseto the terminal sugar of a precursor substance (PS) Glucose H antigen Galactose N-acetylglucosamine Galactose Fucose
7. Group A RBC The A gene codes for the enzyme N-acetyl galactosaminyl-transferase that adds the sugar N-acetyl galactosamineto the terminal sugar of H antigen Glucose Galactose N-acetylglucosamine Galactose N-acetylgalactosamine Fucose
8. Group B RBC The B gene codes for the enzyme D-galactosyltransferase that adds the sugar D-galactoseto the terminal sugar of H antigen Glucose Galactose N-acetylglucosamine Galactose Fucose Galactose
9. ABO Blood Group Group A: you have A antigens on the surface of your RBCs and B antibodies in your plasma. Group B, you have B antigens on the surface of your RBCs and A antibodies in your plasma. Group AB, you have both A and B antigens on the surface of your RBCs and no A or B antibodies in your plasma. Group O, you have neither A nor B antigens on the surface of your RBCs but you have both A and B antibodies in your plasma.
10. ABO Frequencies http://liengdieng.tumblr.com
11. Subgroups of A and B Differ in the amount of antigen present on the red blood cell membrane Subgroups of B are very rare B3 is the most common, shows 2+mf with Anti-B and Anti-A,B
12. Bombay Doesn’t produce L-fucosyltransferase No A, B or H antigens on rbcs Non-secretor Phenotypes as Group O This is the ABO “Null” group
13. Rh Antigens Rh antigens are proteins that are highly immunogenic the D antigen is most antigenic Exposure to less than 1 ml of Rh positive red cells can stimulate Ab production D  c  E  C  e Highly Rarely Immunogenic f [ce]: compound antigen, Dce or dce on same haplotype Rhi [Ce]: compound antigen, DCe or dCe on same haplotype D - - has strongest expression of D Make Anti-Rh17 [anti-Hro] D · · variation of D - - Weak D: quantitative, position or mosaic MUST PERFORM ON Rh neg Units initial testing and Rh Neg newborns
14. Rh Null Rh null = NORh antigens expressed 2 types: Regulator type: RhAG gene is mutated but has normal RhD and RhCE genes so can pass Rh antigens onto off spring Amorphtype: RhAG is normal, but lacks RhD gene and RhCE is mutated , Affects other antigens: S, s, U depressed Negative for Fy5 [Duffy 5] antigen Lacks LW antigen Lack Rh29 antigen [total Rh]
15. Fisher-Race & Weiner Nomenclature Fisher-Race: CDE terminology They theorized that the Rh antigens are controlled by a complex of 3 sets of genes with closely linked loci Weiner: Rh-Hrterminology A single gene at Rh locus leads to the expression of the Rh antigens WeinerFisher-Race R0 =Dce R1 = DCe R2 = DcE r =dce r’ = dCe r” = dcE Rz = DCE [rare] ry = dCE[rare]
16. Rh Antibodies 50- 70% of Rh negative patients will make anti-D Most are IgG React optimally at 37° C or after Antiglobulin testing Clinically significant Don’t bind Complement Anti-D is most potent, followed by Anti-c, Anti-E, anti-C and last Anti-e Anti-LW reacts stronger with D positive cells than D negative cells [mimics Anti-D] Can differentiate from Anti-D: Anti-LW reacts with ALL cord cells, regardless of Rh type
17. Lewis The Le gene codes for enzyme L-fucosyltransferase to attach fucose to the subterminal to the GLcNAc on the ABH precursor substance to form Leª Ag The Se gene codes for enzyme, α-2-L-fucosyltransferase to attach 2ndfucose to the terminal Gal on the ABH precursor substance to form Le b Ag Genes: Le and le. The are co-dominant. Solubleantigens produced by tissues and found in body fluids (plasma) Adsorbed on the RBC Examples: Lese Le(a+b-) LeSe Le(a-b+) leSe Le(a-b-) lese  Le(a-b-)
18. Lewis Antibodies Primarily IgM Frequently detected at RT Can activate complement and cause hemolysis in invivo and invitro testing Enzymes enhance reactivity Lewis antibodies can be neutralized by Lewis positive plasma or saliva Often times a pregnant Mom will develop Lewis antibodies and phenotype will appear Le(a-b-) Doesn’t cause HDN [doesn’t cross placenta] and cord cells Le(a-b-) Can cause HTR
19. P Antigens 3 antigens: P, P1 and P(k) give rise to 5 phenotypes P1 phenotype: P1, P and P(k) antigens on red cells P2 phenotype: P and P(k) antigens on red cells p phenotype: Null phenotype[no P antigens] (rare) Common Precursor Substance is Lactosylceramide Deteriorates in storage Variable antigen expression on red cells Found in secretions like plasma and Hydatid cyst fluid Binding site for Parvovirus B19
20. P Antibodies P1 produces no antibody Has all 3 antigens on red cells [P1, P, P(k)] P2 produces Anti-P1 P and P(k) antigens IgM, naturally occurring Reacts best at RT or 4°C Rarely activates Complement Enhanced by enzymes Can be neutralized (inhibited) by P1 substance p produces Anti-PP1P(k) [antibody used to be called Anti-Tjª] Reacts with all red cells except null p type IgM and IgG antibody Wide thermal range Activates Complement Causes severe HTR and HDN Associated with spontaneous abortions in early pregnancy
21. P Antibodies Autoanti-P IgG [autoantibody] Causes Paroxysmal Cold Hemoglobinuria [PCH] Biphasic hemolysin Found in children <14 yr following a viral infection PerformDonath–Landsteinertest
22. M and N Antigens Glycophorin A: carries M and N antigens Rich in sialicacid Enª: found on all cells that are M+ or N+ If cells lack Glycophorin A, they will have no: Enª, M or N antigens M and N are easily Destroyed by enzymes and by ZZAP M and N antigens are well developed at birth M(k) NULL gp Silent (amorph) gene RBCs lack M, N, S, s, and U antigens Normal rbc survival
23. S,s,U Antigens Glycophorin B: carries the S, s and Uantigens The U antigen is ALWAYSpresent when S & s are inherited [less than 1% of S=s= are U= Less sensitive to destruction by enzymes If cells lack Glycophorin B, they will have no: S, s, and U antigens and are only found in theBlack population S, s and U antigens are well developed at birth Glycophorin B associates with the Rh protein [explains why S, s expression is reduced on Rh null cells]
24. M, N, S, s, U Antibodies S,s and U IgG and react at 37°C / AHG Shows minimal dosage effect **May bind Complement May not react with enzyme-treated cells Can cause HTR and HDN Anti-U: Found mostly in African Blacks [35% are U=] M and N Usually reacts at room temperature [saline reactivity] or below IgM and IgG component Do not bind Complement **Shows Dosage: antibodies react better with homozygous cells [M/M and N/N] rather than heterozygous [M/N]
25. Kell Antigens K is rated second in Immunogenicity after D antigen K antigen is detected early in fetal life, at 10 weeks are well developed at birth 3 antithetical partners: Co-dominant K [Kell] and k [Cellano] Kpa [Penny] and Kpb[Rautenberg] Jsa [Sutter] and Jsb [Matthews] K, Kpa and Jsa are low incidence k, Kpb and Jsb are high incidence
26. Kell Antigens sensitive to sulfhydryl reagents Not denatured by enzymes Kothe Null phenotype [no Kell antigens expressed] K0 individuals can develop anti-Ku Kx antigens are increased in those who are K0 Kxis produced by a gene XK1on the X chromosome [not part of the Kell system] McLeod Phenotype When the XK1 gene is not inherited, Kx is absent Associated with Chronic Granulomatous Disease
27. Kell Antibodies Anti-K is most common IgG(react well at AHG) 20% bind Complement Clinically significant Can cause HDN and HTR Fetus should be carefully monitored Reactivity may be increased with PeG
28. Duffy Antigens 3 main alleles: Fya and Fyb and Fy [silent allele] 4 high incidence antigens: Fy3, Fy4, Fy5 and Fy6 Fy3 is a precursor common to Fya and Fyb All normal red cells positive for Fy3, Fy4, Fy5 and Fy6 Fygene is Null phenotype [lack all Duffy antigens] If Rh null, you lack Fy5 antigen Fya and Fybantigens do not store well Fya, Fyb, and Fy6 are destroyed by enzymes and ZZAP Fy3, Fy4 and Fy5 are not destroyed by enzymes
29. Duffy Antibodies IgGin nature React best at AHG phase Enhanced by LiSS Do not bind complement showdosage Clinically significant Do cause acute and delayed HTR Do cause mild to severe HDN To distinguish Anti-Fy3 from Anti-Fy5: Rh null cells
30. Kidd Antigens Jk3 is a high incidence antigen found on all Jka+ or Jkb+ cells Kidd antigens are involved in urea transport in red cell Kidd Antigens are well developed at birth NULL phenotype: JKJK Jk(a-b-) Found in Polynesians: Filipinos, Japanese and Chinese Resistant to lysis in 2M urea [normal red cells lyse < 1 min] Jk(a-b-) lyse in 30 minutes Unable to concentrate urine
31. Kidd Antibodies IgG Clinically significant: can cause HDN The Kidd antibodies are notorious for causing delayed hemolytic transfusion reactions Show dosage Titers rise quickly and fall just as fast [DHTR] Enhanced by enzymes, LiSS and PeG Anti-Jk3: Found in some individuals who are Jk(a-b-)
32. Pretransfusion Testing ID patient with name and date of birth Complete label with 2 patient identifiers such as First and Last name and Medical ID number, date and time of collection, Phleb ID Remember Type and Screens are good for 72 hrs ABO and Rh testing Antibody Screen Antibody Identification [if necessary] Antigen testing [if necessary] Crossmatch Immediate spin detects ABO compatibility AHG detects clinically significant alloantibodies and autoantibodies
33. Antibody Identification
34. Antibody Identification “Ruling out” means crossing out antigens that did not react on HOMOZYGOUS cells Is everything else ruled out? Circle the antigens that are not crossed out Look for a matching pattern The rule of three must be met to confirm the presence of the antibody. A p-value ≤ 0.05 must be observed Phenotype patient if the patient has NOT been recently transfused
35. Antiglobulin Testing
36. Adsorptions Useful for detecting alloantibodies masked by an autoantibody Differential Adsorption Used in patients who have been recently transfused or positive DAT 3 cells: R1R1, R2R2 and rr Among the 3 cells: 1 cell must be negative for K, another negative Jka and third one negative for Jkb Autologous adsorption Uses washed patient cells Incubate patient’s plasma with patient’s cells for up to 1 hr to remove autoantibody
37. Antigen Frequency Calculation used to estimate number of units to screen vs. ordering from reference lab Uses the antigen negative frequency [100-frequency] Y = x number of units needed # combined antigen neg frequencies
38. HDFN Categories ABO Antibodies Mild HDN due to Anti-A,B DAT: 1-2+ Can affect every pregnancy Mom is Group O, child is A or B Bilirubin peaks in 1-3 days IgG Antibodies Anti-D: most severe Anti-K: 2nd most common DAT: 2-4+ Affects 2nd and future babies Bilirubin levels higher and longer Mother has an antibody capable of crossing the placental barrier that is specific to an antigen present on the red blood cells of the fetus. Fetal red cells become coated with the IgGalloantibody and undergo accelerated destruction both before and after birth. Kernicterus: Bilirubinemiadue to increased red cell destruction
39. Testing Mom Type and Screen Antibody ID [if necessary] Antibody Titers Amniocentesis [if necessary] RhIG Evaluation on Rh Neg moms Fetal Screen [Rosette test] KleihauerBetke PCR Infant Cord Blood: Type and DAT Elution [if necessary] Bilirubin levels [if necessary]
40. Neonatal Transfusions Intrauterine and Exchange Mosby 2009
41. Donors
42. Donor Testing
43. Donor Testing
44. Donor Deferrals
45. Components
46. Components
47. Quality Control RBC ≤ 80% HCT Leukoreduced: RBC or SDP: 95% ≤ 5.0 x 106wbcs Random plt: 95% ≤ 8.3 x 105wbcs Maintains 85% of rbcs Platelets: Single: ≥ 5.5 x 1010 platelets Pheresis: ≥ 3.0 x 1011 platelets pH ≥ 6.2 Cryo: 1 unit: ≥ 80 IU of F8 and ≥ 150 mg fibrinogen Granulocytes: ≥ 1.0 x 1010wbcs
48. Transfusion Reactions Acute Intravascular Tends to present immediately or within 24 hours after transfusion Symptoms: Fever, chills, chest pain or hypotension. Transfused patients develop oliguria, hemoglobinuriaand hemoglobinaemia. Causes: clerical errors (ABO antibodies) Delayed Extravascular A type of transfusion reaction occurs 3 to 10 days later Symptoms: unexplained fever Anemia [no evidence of bleeding] The patient may also have jaundice, high bilirubin, positive DAT. Causes: patient develops an IgG antibody to a red cell antigen that they lack.
49. Transfusion Reactions cont.. TRALI Adverse reaction with hypotension and pulmonary edema. Cause: Occur when human leucocyte antigen (HLA) or human neutrophil antigen (HNA) Symptoms: Acute onset of fever, chills, dyspnea,respiratory failure hypotension. TACO Adverse reaction with hypertension and pulmonary edema. Cause: This is usually due to rapid or massive transfusion of blood- cardiac problems Symptom: Dyspnea, hypertension and pulmonary edema It’s in the Bank! April 2014
50. Transfusion Reactions cont.. Allergic Urticarial Allergic reaction Cause: Antibody to plasma protein Symptoms: hives Anaphylaxis Anaphylaxis is a life-threatening allergic reaction that can occur after only a few milliliters of blood have been transfused Cause: Because they lack IgA, their immune systems develops anti-IgA Symptoms: mild upper respiratory symptoms. Bacterial Contamination Severe reaction to component Cause: Due to gram neg endotoxin Symptoms: Shock, fever, low BP, chills TRALI Adverse reaction 1° after plasma products, within 6 hrs Cause: HLA or WBC antibodies Symptoms: Acute onset of fever, chills, dyspnea, respiratory failure hypotension.
51. Transfusion Reaction Workup Stop transfusion Keep saline lines open Return product to blood bank for inspection Check for clerical errors Test the post-transfusion sample: Check for visual hemolysis Perform DAT if positive, compare to pre-transfusion DAT Repeat ABO and RH if necessary Perform Elution and Antibody ID if necessary Additional testing per pathologist
52. Let the Games Begin….. An example of a technical error that can result in an ABO discrepancy is: Acquired B phenomenon Missing antibodies or antigens Leaving out the plasma in the reverse cells Washing the cells 4 times instead of 3 after the 37 C incubation Excluding ABO, this antibody is rated second only to D in immunogenicity: Anti-Fya Anti-K Anti-Jka Anti-S
53. Review Questions A red cell unit that has AS1 preservative added to it, has an expiration date of: 5 days 21 days 42 days 1 year Symptoms of dyspnea, cough, hypoxemia, and pulmonary edema within 6 hrs of transfusion is most likely which type of reaction? Febrile Non-hemolytic TRALI Hemolytic Anaphylactic
54. Review Questions A patient has an Anti-E antibody. What kind of crossmatch, if any, should the technologist perform? An immediate spin crossmatch only An immediate spin and an AHG crossmatch No crossmatch is necessary, just give out E negative units No crossmatch is required, the antibody is not clinically significant  Which of the following tests can distinguish a patient who has had Hepatitis B from one that has received the Heptavax [Hep B] immunization series? Hbs Ag Hbs Ab Hbcore Ab HCV Ab
55. Review Questions Extravascular destruction of the red cells in a delayed transfusion reaction is characterized by: Increased bilirubin, decreased haptoglobin, increased hemoglobin Increased bilirubin, falling hemoglobin, positive DAT Decreased bilirubin, falling hemoglobin, negative DAT Decreased bilirubin, increased haptoglobin, falling hemoglobin The H antigen is found in highest concentration on what blood group type? A B O AB
56. Review Questions With Warm Autoimmune Hemolytic Anemia, the DAT is results are usually: Positive for IgG only Positive for C3 only Positive for either IgG and/or C3 Negative most of the time  The most serious hemolytic transfusion reactions are due to antibodies from which blood group system? Rh Kell Duffy ABO
57. Review Questions Which of the following constitutes permanent rejection for a blood donor? Had a tattoo 5 months ago Received 2 units of blood 3 years ago Had a confirmed positive test for Hepatitis B 13 months ago Received an immunization for Rubella 2 months ago  A moderate type of post-donation reaction that is characterized by a decreased pulse, decreased blood pressure and loss of consciousness is called: Hyperventilation Shock Vasovagel Hematoma
58. Review Questions Fresh Frozen Plasma is the product of choice for treating all the following disorders except: Bleeding caused by DIC Bleeding due to deficiency of coagulation factors Bleeding due to decreased platelets Bleeding due to Coumadin overdose An individual that is identified as Rh null lacks which of the following antigens? M,N,S,s ABO Rh and Fy5 Kidd
59. Review Questions What is the minimal interval between 2 1-unit red cell donations? 2 days 2 weeks 4 weeks 8 weeks What is the main purpose of performing an antibody screen? To detect mostly IgM antibodies To detect clinically significant IgG antibodies To detect ABO antibodies To detect all antibodies
60. Review Questions What is the terminal sugar on the B antigen? L-fucose N-acetylglucosamine N-acetylgalactosamine D-galactose In an emergency situation and you have no blood type for the patient, which type of red cells should you give? AB Negative A Positive O Negative O Positive
61. Review Questions Which of the following statements about Rh antibodies is true? They are predominately IgG They activate Complement They best react at RT They are predominately are IgM What is the terminal sugar on the H antigen? L-fucose N-acetylglucosamine N-acetylgalactosamine D-galactose
62. Review Questions Which of the following is the correct Wiener notation for DCe/DcE? R1R1 R2R2 R1R2 rr What is the purpose of the immediate spin crossmatch? To detect clinically significant antibodies To detect a low frequency antibody To detect ABO incompatibility To detect an ABO discrepancy