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9 ABO System RELATIONSHIP OF ABO, H, SE, AND LE 1. Lack of His genetically hh (Bombay ANTIG...

9 ABO System RELATIONSHIP OF ABO, H, SE, AND LE 1. Lack of His genetically hh (Bombay ANTIGENS OF ABO SYSTEM phenotype) a. H (HH orHh) is needed for the 1. ABO antigens defined by attachment of A and/or B sugars; H immunodominant sugar on cell surface is converted into A and/or B 2. Genetic pathway b. Group O has the gr eatest amount of H and AlB has the least amount of H. A and H 0>A2>B>Al>AlB 1/ c. Bomhays who are hh will have no A or B antigens and the type as group H Substance ~ B and H PRECURSOR / 0 ; Oh_phenotype d. Anti-H lectin ( Ulex europaeus) will NOT agglutinate Bombay cells (hh) but will agglutinate O cells (HH or ~ 44 Precursor ABO Hh) Substance -----.. Oh (Bombay) 2. Se (secretor) gene allows expression of genes A, B, H , and Leh in body fluids 3. Le antigens are plasma antigens which 3. S uhgroups of A adsorb onto r ed cells as individual a. A1 and A2 matures ❖ Principal subgroups of A a. Individual with H and Le (h ut not ❖ Serological difference based on Se gene) genes will have Hand Lea reactivity with anti-A1 (Dolichos on r ed cells and Lea only in saliva biflorus* or human anti-A1). (Se not needed for presen ce of L e8 (*lectin - plant or seed extract in saliva, but it is for HJ diluted to agglutinate speciflc b. Individual who has H, Se and Le human blood group antigens) genes will have Hand Leh on the ~ A1 cells are ~lutinated red cells and H , Leh , and decreased I& A2 cells are NOT agglutinated amounts of Lea in the body fluids CELL ANTI-A1 CELL TYPING (FORWARD/ FRONT TYPING) BY A1 pos TUBE METHOD see page 20 for gel and neg solid phase testing A2 neg 1. Reagent anti-A and -B are designed so A3 testing is p erformed at room temperature 2. Unknown cells+ antisera = NO agglutination (cells lack antigen to which antisera [reagent antibodyJ corresponds) Fundamental 3. Unknown cells + antisera = Characteristics ofLeet.ms agglutination (cells possess antigen to which antisera corresponds) REAGENT REAGENT INTERPRETATION b. Other subgroups (A3, Ax, etc) ANTI-A ANTI-B contain less A antigen and more H antigen + 0 Group A 0 + Group B + + Grouo AB 0 0 Group 0 IO SERUM TYPING (REVERSE TYPING ) A1 B A NTIBODY INTERPRETATION 1. P erformed at room temper ature with C E LL CELL IN SERUM saline suspended known gr oup A 1 and Bred cells; optimum r eactivity of 0 + Anti B Group A serum anti-A and anti-B is 4C + 0 Anti-A Group B a. Unknown serum + r eagen t r ed cells 0 0 Group AB None = NO agglutination (serum lack s antibody to antigen on red cell) + + Both Group0 b. Unknown serum + reagent red cells = agglutination (serum h as antibod y to antigen on red cells) Discrepancies in ABO Grouping Problems with Red Cells: Resolution Techniques. Rouleaux - Failure to Wash Cells Repeat with Saline Washed Cells Mixture of Cell Types- Example: A or BTransfused with 0 Check Transfusion History Cells. Subgroups (Example: A2 with or without anti-A 1) Test with Anti-A1 for A Subgroups. Unusual Genotypes (Example: Bombay) Test with Anti-H for Bombay (Bombay Lacks H Antigen and Cells will not Agglutinate with Anti-H; Bombay Serum will Agglutinate A1 & 8 Cells as well as Croup 0 Screening Cells). Disease Processes - Example: Leukemia or Bacteria (Acquired Check Patient Diagnosis; ID Beyond Scope of This Review B Phenomenon) Problems with Serum:. Rouleaux - Due to Increased Serum Proteins (Example: Saline Replacement Waldenstrom's or Multiple Myeloma). Rcx,m Temperature or Cold Reacting Antibody (H, I, M, N, Pl, or Lewis, or anti-A I in an A2 or A28 Individual) Reacting with "Mini" Cold Screen or Panel (Test at Lower Temperature) their Corresponding Antigens on Reverse Cells Age - Elderly iAntibody Production has Decreased) or Check Patient Age; "Mini" Cold Panel (May Enhance Serum Newborn {Antibody Production Has Not Reached Optimum Anti-A or Anti-8 so Interpretation will Agree with Cell Levels); Missing Anlibodies Crouping). Compromised Immune System- Example: Check Patient Diagnosis; "Mini" Cold Panel (See below) AIHypogammaglobulinemia With any ABO discrepancy, must transfose group 0 cells until the discrepancy is resolved ll@i" "Mini" Co ld Pane l Principle: Used to (1) Enhance serum anti-A and anti-B r eactions when they are expected but are not demonstrable using 1·oom temperature r eadings, and (2) Identify "cold" antibodies r eacting with other antigen s on A1 and B r eagent r ed cells. ---- - - - PATIENT SERUM TESTED WITH: INTERPRETATION A/B Cells OCells 0 Cord Cell (if available) Autocontrol + + 0 + Anti-I Unexpected antibody reacti ng at + or 0 + + or 0 0 colder temperatures (anti·H, -M~ - N, -P I and Lewis antibodies) + 0 0 0 Anti-A or Anti-B  11 1& Saline Replacement Principle: Saline replacement can differentiate rouleaux (aggregation) from agglutination. Rouleaux is typically described as having a "stack of coins". ate groupmg- appearance when observed with a oups & IJiscrepancies microscope. When the serum in the test mixture is replaced with saline, the cells dissociate. In assessing rouleaux Select Reagen.ts & Methods formation, knowledge of the patient's to Handle Rouleawc clinical diagnosis and bis/her serum protein content and proportions is negative at immediate spin, can helpful. Rouleaux is associated with receive D negative blood products. multiple myeloma and W aldenstrom' s d. Test all infants of D negative macroglobulinemia mothers e. Record weak-D pos as "D positive" Rh System ANTIGENS 4. Monoclonal/ Polyclonal Anti-D a. Separate D control not needed for A, B or O positive cells b. A negative reaction with anti-A and/or anti-Bin patient is the negative control (patient cells are not spontaneously agglutinating) c. D control is needed for any AB positive and for any ABO type negative at immediate spin for the D antigen; carry testing through to AHG for weak D typing d. If patient is AB positive, use a 6-8% albumin control, autocontrol or DAT (patient A and B cells typed with reagent anti-A and anti-B will be positive in an AB positive Testing and lnteipretation patient, i.e., no negative control) ofWeakD e. Most common cause of a positive D control is a positive DAT Evaluate Positive D - Control " anti-B anti-D D cont anti-D AHG D cont AHG l D TYPING !o + -·- i+ + - 1. Most immunogenic of all blood group antigens 0- - · + ~ i+ + 0 2. Test at immediate spin or AHG. 3. Weak D a. Negative at immediate spin but 1 10 + 0 0 0 0 -~-- - 0 0 0 0 0/+.~ OL+ 0/+ 0 0 - 0 0 positive at AHG. Must have a negative D control for the D testing 5. High protein anti-D to be considered positive at AHG. a. replaced by monoclonal or b. DONORS MUST he tested through monoclonal/polycolonal blend AHG or with sensitive weak D reagents method. b. if used, MUST use D control c. PATIENTS do NOT have to be produced by same manufacturer as tested for weak D; consider D the anti-D reagent 12 IgM Antibodies IgG Antibodies anti-I, -H anti-0 anti-K, -k REMEMBER! anti-M, -N anti-P1 anti-C, -c anti-E, -e anti-Fya, Fyb anti-Jka, -Jkb anti-Lea, -Leb anti-M (some) UNUSUAL PHENOTYPES ❖ Reacts with all adult cells (except 1. Rh null - no D, C, E, core antigens; rare i adult) cells have associated hemolytic anemia ❖ May mask clinically significant since Rh structure is integral part of alloantibody rbc membrane ❖ Remove anti-I to detect underlying antibodies by: 2. Deleted cells (D--) - missing one or llW an autoadsorption (if not more of normal Rh alleles recently transfused) or allogeneic adsorption RH ANTIBODIES llW RESt adsorption ~ using IgG AHG instead of 1. IgG clinically significant polyspecific 2. May agglutinate at 37C as well as AIIG ~ prewarming - use with caution; can result in 3. Anti-C, -c, -E, -c react stronger with decreased activity of some enzyme-treated cells clinically significant antibodies or cause weak Other Blood Group Systems antibodies to be missed LEWIS p 1. Plasma antigens t~t adsorb onto 1. P1 antigen strength deteriorates upon RBCs; Lea and Le are not alleles storage 2. Not on cord cells 2. Antibodies a. Anti-P1 3. Antibodies ❖ IgM cold antibody a. Do NOT cause HDFN (Lewis ❖ Anti-P1 (NOT anti-P) can be antigens are 110t on fetal cells and neutralized to reveal other Lewis antibodies usually Jgj'I) clinically significant alloantibodies b. IgM antibody (P1 substance in hydatid cyst ❖ Can be hemolytic fluid) c. Usually only seen in Le(a-b-) b. Anti-P persons ❖ Autoantibody- IgG; Donatl1 - d. Often seen in pregnant women who Landsteiner biphasic antibody may temporarily become Le(a-b -) fou11d in Paroxysymal Cold Hemoglobinuria I, i ❖ Reacts with all P or P1 positive 1. I - absent or weak on cord cells cells 2. i converts to I as infant matures due to MNSs branching of carbohydrate chains; i and I are not alleles 1. MIN and S/s are both codominant alleles a. Infants - i positive, I negative b. Adults - I positive, i negative 2. Antibodies a. Anti-M and -N 3. Antibodies ❖ Usually cold IgM; no HDFN a. Anti-I ❖ Often show dosage (property ❖ IgM cold antibody whereby antibody reacts strongest with cells having a homozygous  13 expression of antige11 as opposed 2. Antibodies to heterozygous cells) a. IgG ❖ Will NOT react with enzyme- b. React STRONGE R with enzym e treated cells (Mand N antigens t reated cells are destroyed by enzymes) c. Titer s rise and fall r a pidly b. Anti-M d. Associated with delayed transfusion ❖ Many examples are IgG and can r eactions cause HDFN e. Often sh ow dosage ❖ May require acidification of serum to identify DUFFY c. Anti-S and anti-s - IgG 1. Fy 3 and Fyh are codominant alleles d. Anti-U - IgG a. Antigens destroyed by enzymes ❖ Formed b y African A merican b. 68% African American s are F y(a-b-) individuals who lack S, s and U or FyFy c. Antigen typing- Fy(a+b -) KELL ❖ Caucasians - h omozygous for Fya 1. K and k are codominant alleles (FyaFya) a. 91% are K negative ❖ African Americans - probably b. Antigen s inactivated with 2-ME, heterozygous for· Fya (FyaFy); can DTT, or AET cause dosage problems 2. Antibodies - IgG 2. Antibodies a. IgG KIDD b. Weak examples may show dosage. 1. Jka and J kh are codominant alleles Negative reaction with en zyme treated cells Relationship Testing (Parentage Testing) DIRECT AND INDIRECT TESTING Indirect Example: 1. RBC blood gr oups with codominant a anti- alleles can b e u sed for parentage testing 0 along with HLA system and DNA + analysis + 2. Direct exclusion Question? a. marke r present in child b. marker absent from father and Ls the alleged father, tested b elow, excluded? mother (most common) Testing: F a ther: Fya F ya c. father has two differ ent marker s in Mother: Fyh F yb the sam e system and child lack s Baby: Fyh Fyb either marker (a group AB father with a group O child) Answer: The alleged fath er appears to be excluded. Direct Example: Must now d etermine if father is homozygous for Fya or if he carries a anti- silent allele. Father could be FyaFy in 0 which case the baby could be FybFy 0 which would not exclude the alleged + father 4. Indirect exclusion a. child lack s a ma rker that the alleged father must transmit b. father apear s to be homozygous for allele child lacks c. father could have silent allele or an valuate Serologic Reactions allele not tested for Used in. Paternity Testing 14 4. Notes ABOUT PATERNITY TESTING... a. Enzyme treated ( ficin , papain, trypsin and bromelin) cells are available to compare with panel results of untreated cells Maternity is Assumed b. Panel results ❖ Compare enzyme-treated and In paternity testing, there is a chain untz-eated cells - enhanced: Kidd, of sample custody that must be I, Pl , Lewis and Rb; destroyed: Dully, M, N, S ands adhered to in legal cases ❖ Dosage - Rh, M, N, Kidd (]k8 and Jkh) and Duffy (Fr and Fyh) Molecular techniques are replacing ❖ Strength of reaction may separate serological methods multiple antibodies (Panel with 1 + and 3+ reactions may mean two different antibodies) Antibody Screening & Identification ❖ Phase of reactivity (see chart: Antibody Characteristics) SCREENING CELLS AND PANELS ❖ Autocontl·ol - if p ositive, may 1. Commercially prepared group O red indicate a delayed transfu sion cells with a specific distribution of reaction ; if positive along with all blood group antigens (screening cells panel cells, autoantibody may he contain 2-3 different cells; panels vary indicated from approximately 10 - 20 different c. Prewarmecl technique - After cells) proving no clinically significant antibodies also present, eliminate 2. Screening cells mixed with pt. serum r eactions due to co]d antibodies a. Serum (or plasma)-cell mixture is ❖ Warm serum and cells separately tested at various temperatures, with to 37°C before mixing together different enhancement media, and ❖ Wa sh with warm saline prior to with antiglobulin r eagent (i.J1direct furtl1er testing (crossmatcb , antiglo.b ulin test {IAT)) panel, etc.) b. Patient serum ( or plasma) may also be tested against his own cells 5. Antigen type the patient; patient must (autocontrol) to determine the be negative for antigen to which the presence of an autoantihody antibody is directed 3. IAT (Indirect antiglohulin tes t) a. Antibody attaches to corresponding Enhancement Media antigen on red cells at 37C (may see albumin (bovine): hemolysis) t net negative surface charge; only ♦ antibody b. Excess serwn/antihody removed by uptake if under low ionic conditions; Rh saline washes (inadequately washing antibodies may show at 37°C may cause a false negative AHG as Low Ionic StrenJ?:th Saline (LISS): residual antibodies and other globulins neutralize the reagent) + t antibody uptake wnich allows time in incubation c. Antiglobulin is added and binds to the antibody on the cells Enzymes (bromelin, ficin, ~ n , ~in): d. Positive reaction is indicated by Removes sialic acid to t negative su rface charge and P.romotes cel l agglutination, ♦ reactivity of t agglutination or in size of button Rh, Kidd and Lewis antibodies; usually ♦ warm due to hemolysi s at 37C and cold autoantibodies; destroys M, N, S, Fya, e. Check cells (lgG sensitized cells) are and Fyb antigens added to any negative test; should give a positive reaction (AHG was Polyethylene glycol (PEG): ♦ antibody uptal