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The Osler InstituteBlood Bank ID. Joe Chaffin, MDBonfils Blood Center, Denver, COThe Fun Just Never Ends A. Blood Bank I Blood GroupsB. Blood Bank II Blood Donation and Autologous Blood Pretransfusion TestingC. Blood Bank III Component TherapyD. Blood Bank IV Transfusion Complications* Noninfectious (Transfusion Reactions)* Infectious (Transfusion-transmitted Diseases)E. Blood Bank V (not discussed today but availableat www.bbguy.org) Hematopoietic Progenitor Cell TransplantationF. Blood Bank Practical Management of specific clinical situations Calculations, Antibody ID and no-pressure samplequestionsBlood Bank IBlood GroupsI. Basic Antigen-Antibody TestingA. Basic Red Cell-Antibody Interactions1. Agglutinationa. Clumping of red cells due to antibody coatingb. Main reaction we look for in Blood Bankingc. Two stages:1) Coating of cells (“sensitization”)a) Affected by antibody specificity, electrostaticRBC charge, temperature, amounts of antigenand antibodyb) Low Ionic Strength Saline (LISS) decreasesrepulsive charges between RBCs; tends toenhance cold antibodies and autoantibodiesc) Polyethylene glycol (PEG) excludes H2O, tendsto enhance warm antibodies and autoantibodies.2) Formation of bridgesa) Lattice structure formed by antibodies and RBCsb) IgG isn’t good at this; one antibody arm mustattach to one cell and other arm to the other cell.c) IgM is better because of its pentameric structure.P}Chaffin (12/28/11)Blood Bank Ipage 1

Pathology Review Course2. Hemolysisa. Direct lysis of a red cell due to antibody coatingb. Uncommon, but equal to agglutination.1) Requires complement fixation2) IgM antibodies do this better than IgG.B. Tube testing1. Immediate spin phasea. Mix serum, 2-5% RBC suspension; spin 15-30 sec.1) Most common: 2 drops serum, 1 drop RBCs.b. Antibodies reacting here are usually IgM2. 37 C phasea. Add potentiator ( /-), incubate at 37 C, spin.b. Potentiators and incubation times:1) 10-15 minutes for LISS2) 15-30 minutes for albumin or PEG3) 30-60 minutes for no potentiation3. Indirect antiglobulin (“antihuman globulin”) phasea. Wash above to remove unbound globulins.b. Add antihuman globulin, spin.C. Alternatives to tube testing1. Column agglutination technology (Gel testing)a. Add RBCs and plasma to top of tube, incubate, spin.b. Microtubes are filled with gel particles and anti-IgG1) Anti-IgG grabs onto IgG-coated RBCs and inhibitstheir migration through gel immunologically2) Gel particles separate RBC clusters physically(inhibit agglutinates from migrating through gel).c. Results:1) Negative: RBCs form button at bottom ofmicrotube.2) Positive: RBCs stopped in areas through themicrotube (more positive higher position in tube)d. Can be automated (ProVue machine)e. Similar sensitivity to PEG tube testing2. Solid-phase Red Cell Adherence Testinga. Antibody binds to lysed or intact RBC antigens thatare bound by manufacturer to the sides of microwellsb. Add patient serum, incubate, wash: If positive,antibody binds to test RBCs.c. Indicator RBCs (coated with monoclonal anti-IgG)attach to antibody on test RBCs.d. Spin and interpret1) Negative: RBCs in a button at bottom of microwell,(indicator cells didn’t bind to the test RBCs).2) Positive: RBCs spread in a “carpet” all along themicrowell (indicator cells did bind to test RBCs).e. Can be automated (Galileo, Galileo Echo, NEO)f. Similar sensitivity to PEG tube testingpage 2Blood Bank IP}Chaffin (12/28/11)

The Osler InstituteD. The Antiglobulin Test (“Coombs Test”)1. Indirect: see above; demonstrates in-vitro RBC coatingwith antibody and/or complement.2. Direct: red cells from patient washed, then mixed withantihuman globulin; demonstrates in-vivo RBC coatingwith antibody and/or complement.3. IAT variationsa. Unknown antibody check: Use RBCs with a knownantigen profile, as in an antibody screenb. Unknown RBC antigen check: Use serum with knownantibody specificity, as in RBC antigen testingc. Can be used to check for an unknown antigen ORunknown antibody, as in the crossmatch procedure4. Specificity possibilities for the antiglobulina. Anti-IgG, -C3d (“polyspecific”); most common to start1) Detect red cells coated with either of the above2) May also detect other immunoglobulins (becausethe anti-IgG detects light chains, too)b. Anti-IgG and anti-IgG (heavy chain-specific)1) Both detect IgG-coated red cells2) Anti-IgG used for PEG, gel, and solid phase testsc. Anti-C3b, -C3d1) Detects either of the above complement components2) Most useful in evaluating IgM-related hemolysis,cold agglutinin disease5. IgG-sensitized RBCs (“Coomb’s control”, “check cells”)a. Use after negative DAT or IAT tube test (not gel orsolid-phase) to ensure functioning of AHG reagentb. Add IgG-coated cells to AHG-cell mixturec. Negative bad AHG or no AHG addedd. Other errors (e.g., omitting test serum) missed.E. Dosage1. Some antibodies react more strongly with RBC antigensthat have homozygous gene expression.2. For example, imagine a hypothetical anti-Za. Patient 1 genotype: ZZ (Homozygous for Z)b. Patient 2 genotype: ZY (Heterozygous for Z)P}Chaffin (12/28/11)Blood Bank Ipage 3

Pathology Review Coursec. If anti-Z shows dosage, it will react stronger withpatient 1’s RBCs (see below).RBC GenotypeReaction with anti-ZZZ3 ZY1 3. Most common in Kidd, Duffy, Rh and MNS systemsF. Enzymes1. Proteolytic enzymes (e.g., ficin, papain) cleave RBCsurface glycoproteins and can strengthen reactions byenhancing antigen expression or allowing antibodies tobind better to previously shielded antigens2. Enzymes may also directly destroy other antigens3. Useful in antibody identification to confirm or refute aparticular antigen as target of an antibody (see table)4. The “Enzyme Classification”EnhancedABO-relatedABO, H SystemsLewis SystemI SystemP SystemRh SystemKidd SystemDecreasedMNS SystemDuffy SystemLutheran SystemUnaffectedKell SystemDiego SystemColton SystemG. Neutralization1. Certain substances, when mixed with a red cell antibody,inhibit the activity of that antibody against test red cells.2. Some of these are pretty weird! (See table below)Neutralization of AntibodiesABOLewisSdaSaliva (secretor)Saliva (secretor for Leb)Hydatid cyst fluidPigeon egg whitesHuman urineChido, RodgersSerumP1H. Lectins1. Seed/plant extracts react with certain RBC antigens2. Especially useful in polyagglutination (T, Tn, etc)3. May be commercial or homemadepage 4LectinSpecificityDolichos biflorusUlex europaeusVicia gramineaArachis hypogeaGlycine maxSalviaA1HNTT, TnTnBlood Bank IP}Chaffin (12/28/11)

The Osler InstituteII. Blood GroupsA. General characteristics1. Definitiona. Blood group antigen: Protein, glycoprotein, orglycolipid on RBCs, detected by an alloantibody1) NOTE: Antigens are not limited to RBCsb. Blood group system: Group of blood group antigensthat are genetically linked (30 total systems per ISBT)2. Significancea. “Significant” antibody causes HTRs or HDFNb. Most significant antibodies are “warm reactive”;meaning they react best at IAT (37 C).c. Most insignificant antibodies are “cold reactive”;meaning they react best below 37 C.d. Warm antibodies most often IgG, colds usually IgM.e. IgM antibodies are usually “naturally occurring” (notransfusion or pregnancy required for their formation).f. ABO is the exception; see asterisks in table below“WARM-REACTIVE”IgGRequire exposureCause HDNCause ly occurringNo HDN*No HTRs*“Insignificant”*B. ABO and H Systems1. Basic biochemistry (see figure below)a. Type 1 and 2 chains1) Type 1: Glycoproteins and glycolipids in secretionsand plasma carrying free-floating antigens2) Type 2: Glycolipids and glycoproteins carryingbound antigens on RBCs.b. Se gene (FUT2; FUT “fucosyltransferase”)1) “Secretor” gene (chrom 19); Precursor to making Aor B antigens in secretions2) FUT enzyme adds fucose to type 1 chains atterminal galactose; product is type 1 H antigen3) 80% gene frequencyc. H gene (FUT1)1) Closely linked to Se on chrom 192) FUT enzyme adds fucose to type 2 chains atterminal galactose; product is type 2 H antigen.3) Virtually 100% gene frequency (Bombay hh).d. H antigen required before A and/or B can be made onRBCs (type 2 H) or in secretions (type 1 H).1) Single sugar added to a type 1 or 2 H antigen chainmakes A or B antigens and eliminates H antigen.a) Group A sugar: N-acetylgalactosamineP}Chaffin (12/28/11)Blood Bank Ipage 5

Pathology Review Courseb) Group B sugar: Galactose2) As more A or B is made, less H remains.a) H amount: O A2 B A2B A1 A1B2. ABO antigensa. Genotype determined by three genes on long arm ofchrom 9: A, B and O (O is nonfunctional).b. A and B genes code for transferase enzymes, notdirectly for an antigen (as above)c. ABO antigens begin to appear on fetal RBCs at 6weeks gestation; reach adult levels by age 4.1) Also present on platelets, endothelium, kidney,heart, lung, bowel, pancreas tissue3. ABO antibodiesa. Antibodies clinically significant, naturally occurringb. Begin to appear at 4 months of age; reach adult levelsby age 10 and may fade with advanced agef. Three antibodies: anti-A, anti-B and anti-A,B; differby blood group1) Group A and B: Anti-A or –B is predominantlyIgM, but each reacts strongly at body temperatures.2) Group O: Anti-A and –B are predominantly IgG,and react best at body temperatures3) Group O: Anti-A,B is IgG reacting against A and/orB cells (reactivity can’t be separated into individualspecificities).TypeWhitesBlacks Asians Native %4%5% 1%4. ABO blood groupsa. Group O1) The most common blood group across racial lines2) Genotype: OO3) Antigen: Ha) Ulex europaeus lectin reacts with H antigen.page 6Blood Bank IP}Chaffin (12/28/11)

The Osler Institute4) Antibodies: Anti-A, anti-B, anti-A,B (see above)a) Because of strong IgG component to all aboveantibodies, mild HDFN is common in O momsb) Why not severe? Weak fetal ABH expression,soluble ABH antigens (neutralize antibodies)b. Group A1) Possible genotypes: AA, AO2) Antigens: A, H3) Antibody: anti-B (primarily IgM).4) A subgroupsa) A1 (80%) and A2 ( 20%) most importantb) Monoclonal anti-A agglutinates both types wellc) A1 red cells carry about 5x more A on RBCsurfaces than A2 cellsd) Qualitative differences also exist in the structureof the antigenic chains (type 3 and 4 for A2).e) 1-8% of A2 and 25% of A2B form anti-A1. Usually clinically insignificant IgM Common cause of ABO discrepancies. If reactive at 37C, avoid A1 RBC transfusion.f) Dolichos biflorus lectin agglutinates A1 but notA2 RBCs.c. Group B1) Genotypes: BB, BO2) Antigens: B, H3) Antibodies: Anti-A (primarily IgM).4) B subgroups: Usually unimportant and less frequentd. Group AB1) Least frequent ABO blood type (about 4%)2) Antigens: A and B (very little H)a) Can be further subdivided into A1B or A2Bdepending on the status of the A antigen3) Antibodies: none5. ABO testingCellAnti-A Anti-BSerumA1 cells B cells4 004 04 4 04 4 00004 4 ABOGroupABABOa. Cell grouping (“forward grouping”)1) Patient red cells agglutinated by anti-A, anti-B.b. Serum grouping (“reverse grouping”, “back typing”)1) Patient serum (or plasma) against A1 and B RBCs.c. Note the opposite reactions!1) If forward reactions are not opposite of reverse, anABO discrepancy is present.P}Chaffin (12/28/11)Blood Bank Ipage 7

Pathology Review Coursed. Both serum and cell grouping required unless testingbabies 4 months of age or reconfirming ABO testingdone on donor blood (requires cell grouping only).6. ABO discrepanciesa. Disagreement between the interpretations of cell andserum grouping (e.g., forward A, reverse O);caused by antigen and/or antibody problems ortechnical errors.b. Antigen problems1) Missing antigensa) A or B subgroupsb) Transfusion or transplantationc) Leukemia or other malignancies2) Unexpected antigensa) Transfusion/transplantation out-of-groupb) Acquired B phenotype (more below)c) Recent marrow/stem cell transplant.d) Polyagglutinationc. Antibody problems1) Missing antibodiesa) Immunodeficiencyb) Neonates, elderly, or immunocompromisedc) Transplantation or transfusiond) ABO subgroups2) Unexpected antibodiesa) Cold antibodies (auto- or allo-)b) Anti-A1c) Rouleaux/plasma expanders (false positive)d) Transfusion or transplantatione) Reagent-related antibodiesd. Technical errors1) Sample/reagent prep, mix-ups, or interpretationerrors7. Weird stuff about ABOa. Acquired B phenotype1) A1 RBC contact with enteric gram negatives: Coloncancer, intestinal obstruction, gram-negative sepsis2) AB forward (with weak anti-B reactions), A reverse3) Bacterial enzymes deacetylate group A GalNAc;remaining galactosamine looks like B and reactswith forms of monoclonal anti-B (ES-4 clone).Cell TypingSerum TypingAntiAAntiBInterpA1cellsBcellsInterp4 1-2 AB04 A4) Use monoclonal anti-B that does NOT recognizeacquired B, acidify serum (no reaction with anti-B)page 8Blood Bank IP}Chaffin (12/28/11)

The Osler Instituteb. B(A) phenotype1) Opposite of acquired B (group B patients with weakA activity); this condition is inherited, not acquired2) Cross-reaction with a specific monoclonal anti-A;test using different anti-A shows the patient as B.c. Bombay (Oh) phenotype1) Total lack of H, A and B antigens due to lack of Hand Se genes (genotype: hh, sese)2) Naturally occurring strong anti-H, anti-A, anti-B3) Testing: O forward, O reverse, but antibody screenwildly positive and all units incompatible4) “Para-Bombay” phenotypea) Like Bombays, are hh, but unlike Bombays,have at least one Se geneb) Phenotypes: Ah, Bh, ABhc) RBCs may be Bombay-like, but may also showfree or RBC A or B antigens (unless group O).d) Allo-anti-H present in serum.5) Both Bombay and Para-Bombay need H-negativeblood (from Bombay donors)8. Consequences of ABO incompatibilitya. Severe acute hemolytic transfusion reactions1) Among most common blood bank fatalities2) Clerical errorsb. Most frequent HDFN; usually mild, howeverC. Lewis System1. Biochemistry (see figure below)a. Type 1 chains onlyb. One gene: Le (FUT3)1) Second gene, le, is nonfunctionalc. FUT enzyme adds fucose to subterminal GlcNAc(left side of figure below).1) This makes Lea (Lewis A) antigen.2) Lea antigens cannot be modified to make Leb.d. In secretors, Se product (FUT2) adds fucose, then Leproduct adds fucose; this makes Leb (Lewis B).1) In secretors, Leb formation occurs preferentially.P}Chaffin (12/28/11)Blood Bank Ipage 9

Pathology Review Course2) As a result, the vast majority of the chains of thosewho carry Le and Se are Leb rather than Lea.3) In non-secretors, Lea is only possible Lewis antigen.e. Unlike ABO, antigens are not tightly bound(remember, they are made from type 1 chains); rather,they adsorb onto the surface of RBCs.1) Leb does this better than Lea; another reason thatmost adults with both Le and Se will be Le(a-b ).2) Le(a-b ) people still have Lea, just in much smallerquantities that may not show up on RBCs.f. Same chain can carry Le and ABO antigens (unlike theinverse relationship with ABO and H).2. Lewis phenotypes, antigens, and antibodiesa. Phenotypes: Le(a-b ), Le(a b-), Le(a-b-)b. 22% of blacks are Le(a-b-), vs. only 6% of whites.c. Antibodies are naturally occurring, cold-reacting IgM.1) Primarily in Le(a-b-)2) Neutralize with saliva from secretors.3) Antibodies commonly also show ABH specificity(e.g., anti-LebH reacts best with O or A2 RBCs)3. Consequences of incompatibilitya. Antibodies are generally insignificantb. Rare HTRs (more commonly with anti-Lea)c. No HDFN (antibody doesn’t cross placenta and Leantigens are not present on fetal RBCs).4. Weird stuff about Lewisa. Lewis antigens decrease during pregnancy.1) Pregnant patients may appear Le(a-b-) and havetransient, insignificant Lewis antibodies.2) Increased plasma volume dilutes the antigens andincreased plasma lipoproteins strip the antigensb. Le(a-b ) people don’t make anti-Lea.1) Still have Lea, just not visible on their RBCs.c. Children’s Lewis type may vary, as antigen chains areconverted [more Lea than Leb initially, with a transientperiod of Le(a b )]; by age 2, are Le(a-b )d. Infection associations:1) H. pylori attaches to gastric mucosa via Leb antigen.2) Norwalk virus also attaches via Leb3) Le(a-b-) are at risk for Candida and E. coli infectionD. I System1. Antigens built on type 2 chains.2. Expression is age-dependent.a. Simple chains found on neonates make i antigen.b. Branched chains in adults make I antigen.c. “Big I in big people, little i in little people”d. Occasional adults lack I; they are known as “iadult”;more common in Asianspage 10Blood Bank IP}Chaffin (12/28/11)

The Osler Institute3. Antibodies (usually autoantibodies)a. Cold reacting IgM, with auto-anti-I seen commonlyb. Naturally occurring, common, usually insignificantc. Like Lewis, antibodies commonly have H specificityas well (e.g., anti-IH reacts better against O and A2)4. Classic associationsa. Auto-anti-I1) Cold agglutinin disease2) Mycoplasma pneumoniae infectionb. Auto-anti-i1) Associated with infectious mononucleosis2) Less often a problem than auto-anti-Ic. Iadult phenotype1) Cataracts2) HEMPASE. P System (the cool one)1. Also built on ABO-related chains2. Antigensa. P1 is the only antigen1) P, Pk not officially in P system anymore2) These three antigens define the overall P phenotype.3) Most common P phenotype: P1 (P P1 Pk–).b. Very rare people lack all three and make anti-PP1Pk.1) Acute HTR and early spontaneous abortionsc. P antigen is parvovirus B19 receptor.d. Pk antigen is receptor for various bacteria and toxins3. Antibodies (anti-P1)a. Cold reacting, naturally occurring, insignificant IgM;rare anti-P1 reactive at AHG is potentially significantb. Titers elevated in those with hydatid cyst disease(Echinococcus) and bird handlers1) Bird feces contains P1-like substance.c. Neutralized by hydatid cyst fluid, pigeon egg whites4. Association with paroxysmal cold hemoglobinuriaa. Biphasic IgG with anti-P (not P1) specificity1) Binds in cold temps, hemolyzes when warmed2) “Donath-Landsteiner biphasic hemolysin”b. Historically in syphilis, now after viral infx in childrenF. Rh System1. Second most important blood group (after ABO)2. Old (incorrect) Rh antigen terminology systemsa. Fisher-Race (DCE or CDE)1) Five major antigens: D, C, E, c, ea) “Rh positive” really means “D positive.”b) Absence of D designated “d” (no d antigen)c) C/c and E/e are antithetical (e.g., can’t have bothC and c or E and e from same chromosome)2) Eight potential combinations based on presence ofgenes for above antigens (ie, “DCe”, “dce”, etc.)P}Chaffin (12/28/11)Blood Bank Ipage 11

Pathology Review Courseb. Wiener (Rh-Hr)1) Different, archaic names for the five main antigens2) Believed that main Rh genes (for presence orabsence of D, for C or c and for E or e) inherited asone genetically linked group, or “haplotype.”3) Shorthand names to the haplotypes; nomenclature isstill in use and is essential to know (though theoryof how these are inherited has been disproven).Wiener’s “Haplotypes”(with DCE Equivalents)R1: DCeR2: DcER0: DceRz: DCEr’ : dCer”: dcEr : dcery : dCEa) Rules for converting Wiener’s modifiedhaplotypes into Fisher-Race terminology: “R” D, “r” d “1” or “prime” C “2” or “double prime” E “0” or “blank” ce Any sub- or superscript letter CE4) Only four of the above combinations occur withsignificant frequency: R1, R2, R0 and r. ( 97% ofblacks and whites use only these four). R0 most common in blacks, least common inwhites. r is always second in frequency. R1 always comes before R2.“The Big Four”Whites: R1 r R2 R0Blacks: R0 r R1 R25) Asians us. D ; their order is R1 R2 r and R0.c. Current understanting of Rh genetics/structure1) Two genes, RHD and RHCE (chromosome 1) codefor two main Rh proteins (RHD and RHCE)2) D type determined by presence/absence of RHD3) One protein gives both C/c and E/e antigens;combination determined by which alleles of RHCEare present (CE, Ce, cE, or ce)3. Rh antibodiesa. Exposure-requiring, warm-reacting IgGb. D induces the most antibodies, then c and E1) Traditional: 80-85% of D negatives make anti-Dwhen exposed to one unit of D pos RBCs2) Recent data: 20-30% in hospital settingsc HTRs with extravascular hemolysispage 12Blood Bank IP}Chaffin (12/28/11)

The Osler Instituted Severe and prototypical HDFN with anti-D, severeHDFN with anti-c, mild HDFN with anti-C, -E, -e4. Weird stuff about Rha. D-negative phenotype1) Unusual because caused by mutations and deletionsrather than by synthetic actions of a gene product2) Caucasians: D-negatives have deletion of RHD gene3) African-Americans: Point mutations in RHD gene(“pseudogene”)4) Asians: Usually have inactive RHD geneb. D Variants1) Weak D (formerly “Du”)a) Usual D testing: Monoclonal IgM withpolyclonal IgG read only at immediate spinb) Almost all D test as D with these reagentsb) Some D individuals have decreased Dexpression and require IAT to detect D antigen.c) Possible reasons for weak D Mutated form of RHD Point mutation causing altered amino acidsin membrane or inner part of RHD Type 1 common in Caucasians RHCe on opposite chromosome to RHD (“Cin trans”) inhibits D expressiond) Testing requirements Weak D test for all D-negative blood donors Not required for D-negative blood recipients Previously a concern, for fear of wasting Dneg units on D patients Monoclonal antibodies mentioned abovemake this very unlikely The only patients who definitely need weakD testing are apparently D-negative babieswith D-negative moms.e) Weak D moms do not need RhIG prophylaxis2) Partial D (“D Category”, “D mosaic”)a) At one time considered a form of weak Db) Lack portions (epitopes) of D antigen.c) RHD gene mutations leading to alteration ofexterior part of RHD antigend) Antibodies form against absent parts of RHD;this antibody appears to be anti-D at first glanceP}Chaffin (12/28/11)Blood Bank Ipage 13

Pathology Review Coursee) Classic: Anti-D in a D-positive personf) Most common: DVI (D “six”) in whites Monoclonal anti-D usually types these as Dnegative (prevents D exposure as recipients)g) Note that partial C and partial e antigens exist,and can result in unusual antibodiesh) Partial D moms do need RhIG prophylaxisi) Partial D vs. weak D may be impossible withoutmolecular testing; if in doubt for prenataltesting, consider patient D-negative3) Del (“D-E-L”)a) Appear D-neg but have tiny amounts of D seenafter elution of reagent anti-D from RBCsb) Primarily seen in Asian populations (up to 1/3 ofD-negative Asians)c. These antibodies go together 1) Anti-E formation commonly accompanied by anti-c(not necessarily vice-versa)2) Think “Big 4”; R2R2 gives both E and c exposured. Compound Rh antigens1) G Antigen present when either C or D is present Anti-G reacts against (D C-), (D-C ), or(D C ) RBCs (rarely against D-C-G ) Common presentation: D-negative person formsanti-D when not obviously exposed to D Important because if D-neg mom has anti-G, sheDOES still need RhIG to prevent anti-D Can cause HTRs (give D-C- blood) See bbguy.blogspot.com/2011/08/g-whiz.html2) f Present when RHce is inherited (r and R0). Anti-f is often seen with anti-e or anti-c Can cause mild HDFN and HTRG. Kidd System1. Kidd antigensa. Jka, Jkb, Jk3 (very high frequency)b. Jka slightly more common than Jkb in AfricanAmericans but similar in whites and Asiansc. Antigens reside on a urea transport protein2. Kidd antibodiesa. Exposure requiring, warm-reacting IgG (often withIgM component as well)1) Can fix complement (with IgM component)2) Severe acute HTRs possibleb. Marked dosage effect1) Antibodies may not react at all against cells withheterozygous Kidd antigensc. Variable antibody expression1) Antibody often disappears with time/storage.page 14Blood Bank IP}Chaffin (12/28/11)

The Osler Institute3. Weird stuff about Kidda. Delayed HTRs (most famous association)1) Anamnestic response2) Intravascular and often severeb. Mild HDFN at worst1) Child can only be one antigen different from mom;remember dosage discussion above.H. MNS System1. Basic biochemistrya. Glycophorin A (GPA) carries M or N antigens.b. Glycophorin B (GPB) carries S or s, and U antigens.2. MNS antigensa. M frequency roughly equals N (each 75%)b. s ( 90%) is more frequent than S ( 50%W, 30%B)c. If S-s- (as seen in 2% of African-Americans), may alsobe U-negative (U is extremely high frequency).d. Vicea graminea lectin reacts against N antigense. Mur: Hybrid antigen seen in nearly 10% of Chinese1) Significant antibodies can form; more frequent insome areas than anything but anti-A or -B3. MNS antibodiesa. M and N antibodies are mostly opposite of S, s and Uantibodies (see below)Anti-M & anti-NAnti-S, -s and -UNaturally occurringRequire exposureCold IgMWarm IgGDosageMinimal dosageInsignificantSignificantb. Anti-M and anti-N can usually be ignored unlessreactive at 37C; not so with anti-S and anti-s1) Though anti-M is usually insignificant, it has beenrarely associated with severe HDFN.c. Effect varies by enzyme, but enzymes generallydecrease all MNS antigens except U4. Weird stuff about MNSa. N-like antigen (‘N’)1) GPB always has terminal 5 amino acid sequencethat matches GPA’s terminal sequence when it isexpressing N; this is known as ‘N’.a) Not really true N antigen, but it’s close enoughto prevent most M N- from making anti-N.2) Seen in all except those who lack glycophorin B.a) 1% of blacks lack S, s, and U; rare in whitesb) Anti-N nearly exclusive to African-Americansb. Auto-anti-N induced by hemodialysis1) Formaldehyde sterilization of machineP}Chaffin (12/28/11)Blood Bank Ipage 15

Pathology Review Course2) Modification of N leads to rare autoantibodyI. Duffy System1. Duffy antigens and genesa. Fya from Fya gene; high frequency in Asiansb. Fyb from Fyb gene; high frequency in caucasiansc. Absence of both antigens, Fy (a-b-), is most commonFy phenotype in African-Americans (68%, evenhigher in Africa).1) Due to inheritance of two copies of Fy gene, whichgives no functioning Duffy glycoprotein2) Fy is an Fyb gene variant, and gives Fyb antigen innon-RBC tissues2. Duffy antibodiesa. Anti-Fya more common and significant than anti-Fybb. Exposure requiring, warm-reactive IgGc. Marked dosage and variable expression like Kidd Abs3. Consequences of incompatibilitya. Severe HTRs, usually delayed and extravascularb. Often mild, occasionally severe HDFN4. Weird stuff about Duffya. Fy(a-b-) and malarial resistance1) Fy(a-b-) humans are resistant to Plasmodium vivaxand P. knowlesi infection.J. Kell System1. Extremely important group clinically and serologically2. Kell antigensa. Low frequency: K, also known as “KEL1” (9%whites, 2% blacks), Jsa, Kpab. High frequency: k or “KEL2” (99.8%), Jsb, Kpbc. Kx antigen: Bound to Kell glycoprotein on the red cellmembrane; required for proper Kell antigen expression1) Actually a separate blood group (Kx system)2) When Kell antigens decrease, Kx increases (as inK0, aka “Kell null”)3) When Kx decreases (as in “McLeod syndrome”, seelater), Kell antigens decrease, too.d. Kell system antigens destroyed by thiol reagents (2ME, DTT, ZZAP) but not by enzymes alone.3. Kell antibodiesa. Anti-K1) Most common non-ABO antibody after anti-D2) Exposure-requiring, warm reacting IgG13) More common from transfusion than pregnancyb. Anti-k1) Very uncommon due to high antigen frequency2) Antibody is just like anti-K4. Consequences of incompatibilitya. Severe HTRs1) May be acute or delayed; usually extravascular.page 16Blood Bank IP}Chaffin (12/28/11)

The Osler Instituteb. Severe HDFN1) Less common than ABO or RHD HDFN2) Damages EARLY RBC precursors, so tends to besuppressive rather than hemolytica) Lower bilirubin and reticulocytopenia than withanti-D HDFN5. Weird stuff about Kella. Kell null phenotype (“K0”)1) All Kell antigens decreased, Kx increased2) Significant anti-Ku (“universal”) with exposureb. McLeod phenotype1) Kx absent, all Kell antigens markedly decreased2) No anti-Ku like K0, but can form anti-Kx and antiKm (Kell “McLeod”); only compatible withMcLeod RBCs3) Phenotype is part of McLeod “syndrome”a) Hemolytic anemia with acanthocytesb) Myopathy, ataxia, peripheral neuropathy,cognitive impairment, cardiomyopathyc) Occasional association with X-linked chronicgranulomatous disease NADPH oxidase deficit Organisms phagocytized but not killed Catalase-positive organisms (Staph)K. Diego System1. Over 20 antigen system built on “band 3”a. Important RBC membrane structureb. Carries HCO3- anions out of RBCs (for CO2 removal),and anchors membrane to cytoskeleton2. Diego antigensa. Dia and Dib antithetical pair1) Dia very low frequency except in some SouthAmericans and Asians2) Dib very high frequency in all populationsb. Wra and Wrb antithetical pair1) Wr “Wright”2) Wra very low frequency, Wrb very high frequency3. Diego antibodiesa. Di antibodies are IgG, while Wr antibodies may haveIgM componentb. Both anti-Dia and –Dib can cause HDFN that may besevere but generally not HTRsc. Anti-Dib can show marked dosage effectd. Anti-Wra is common, naturally occurring, and maycause both HTRs and severe HDFN (IgG IgM)e. Anti-Wrb, on the other hand, is rarely seen as analloantibody but may be an autoantibody inautoimmune hemolytic anemia (AIHA)P}Chaffin (12/28/11)Blood Bank Ipage 17

Pathology Review CourseL. A few other systems and antigens (in brief)1. Dombrock Systema. Doa/Dob antigens; Dob more frequent1) Either antibody may cause HTRs but generallydon’t cause HDFN2) Warm-reactive IgGb. High frequency antigens Joa, Gya, Hy1) Mild HTRs or HDFN possible, but antibodies arevery rare2) Near 100% incidence for all of these3) Joa- and Hy negative exclusively in blacks4) Gya negative in Japanese and eastern Europeans2. Colton (Co) Systema. Antigens (Coa and Cob) located on water transportmembrane protein (aquaporin 1)b. Coa very high frequency (near 100%), Cob about 10%c. Both antibodies may cause significant HDFN3. Lutheran (Lu) Syst

Pathology Review Course page 6 Blood Bank I P}Chaffin (12/28/11) b) Group B sugar: Galactose 2) As more A or B is made, less H remains. a) H amount: O A 2 B A 2B A 1 A 1B 2. ABO anti