Transfusion Science 2

Rh Antibodies in vivo

Mostly IgG Ab ]→ enhanced in vitro by enzyme treated RBCs and with other potentiators - no complement activation (intravascular hemoloysis - extravascular hemolysis removes sensitized RBCs) *Clinical significance - transfusion reactions/HDFN

Rh Blood group Genetics

No allele to the D gene → locus RHD gene (presence of RhD protein) and locus for RHCE gene (RhCe/RhcE/Rhce/RhCE

Rh Blood Group System Nomenclature: Rosenfield vs. ISBT vs. Fisher-race vs. Wiener

Rosenfield: numbered antigen → serological PHENOTYPES - D=Rh1, C=Rh2, E=Rh3, c=Rh4, e=Rh5 (add a negative if missing) vs. ISBT: numeric genetics - Rh = 004, D = 001, C = 002… (Rosenfield for phenotype) vs. Fisher-Race: D for Rh+ and d/nothing for Rh- → C/E/c/e ]→ haplotype combinations vs. Wiener: shorthand notation - R = D and r = d, 0/nothing = ce, 1/’ = Ce, 2/” = cE, Z/y = CE

Common Haplotypes of Rh blood group system

R1r for Rh Positive vs. rr for Rh negative

D Antigen Strength

R2R2 has stronger reactions compared to R1R1 (C in cis to D)

Other Rh Antigen: Cw vs. f vs. G vs. Lw

Cw: Rh8 → Cc locus inherited - RhCE*Ce associated with R1 haplotypes ]- low frequency - anti-Cw natural occurrence and shows dosage (increase amount to increase strength) vs. f: Rh6 - compound Ag with ce in cis - Dce or dce → anti-f develops with negative c or e - receive either c- or e-negative blood (=f-negative) vs. G: Rh12 - D or C inherited code for G → anti-G mimics anti-D and anti-C (cannot separate activity of anti-G) - receive D- and C- RBCs vs. Lw: similar to Rh phenotype differ genetically → rhesus monkeys and human RBCs → anti-Lw reacts more strongly with D positive than D negative ]- clinical significance (rare)

Very Rare Unusual Phenotypes: Rh null vs. Rh mod vs. D-depletion vs. Partial Deleted

Rh null: No Rh on RBC due to amorphic (RHD absent → no RHCE expression ]- parents give one haplotype of Rh deletion each) OR regulatroy (gene inherited and normal but not expressed - RHAG missing for Rh Ag expression) vs. Rhmod: partial suppression of RH gene expression → RHAG mutations vs. D-Deletion: inherit RHD gene but nothing at RHCE locus → more D Ag than normal ]- transfuse only other D-deletion RBCs vs. Partial Deleted: inherit RHD gene with only part of RHCE locus → more D Ag than normal and reacts more strongly with anti-D ]- caused by inbreeding

Other Blood Group Systems: Kell vs. Duffy vs. Kidd vs. Lewis vs. Ii vs. P vs. MNS vs. Lutheran

Kell: K <9% and immunogenic vs. k >99% (b = high frequency)]- codominant and antithetical → large protein with disulfide bonds - sulfhydryl sensitivity - destroys Kell Ag/IgGdosage (stronger reactions with homozygous over heterozygous)/enzyme treated RBCs wont be reacted

Duffy: Fya and Fyb → codominant (born with the genes) → enzymes denature the genes ]- Malaria parasites use Duffy Ag as receptors (if negative for Duffy - they resist malaria) ]→ IgG/significant in transfusion/37C/AHG/dosage/enzymes render the Ag nonreactive

Kidd: Jka and Jkb codominant - dosage/weak/combine Ab/difficult to detect/common cause of hemolytic transfusion reactions (delayed)/enhanced reaction with enzyme ]→ Clinically significant/IgG/37C/AHG/does do dosage/Enzymes enhances

Lewis: Lea and Leb (not alleles) - Ag in secretion and plasma (glycoproteins/lipis) → absorbed onto red cells - develop after 6years ( cord cells are Lea-b-) ]- depends on H gene/Se/Le gene (h and se are amorphs) → inheritence of Le gene = Lea produced vs. Le/H/Se gene altogether to convert Lea to Leb (transferase converts H to Leb Ag - only trace amounts of Lea present but undetectable) - rare Lea+b+ ]- alleles Le and le vs. H and h vs. Se and se → natural occurring Ab in Lea-b-/not clinically significant due to Ag falling off within recent transfusion (nothing for Ab to attach to)/Not crossing Placenta or causing HDFN/IgM/RT/ISS, 37C, AHG phases/enhanced by enzymes

Ii: i Ag on cord cells → 2years i → I adult Ag]→ autoanti-I (most people - alloanti-I is rare) → pre-warm to decrease anti-I reactions → IgM/RT or colder/IS/enhanced by enzymes → strong autoanti-I → CHD → clinically significant hemolytic anemia - infection triggers (anti-i rare due to infectious mononucleosis)

P: P1 (P1 and P Ag) vs. P2 (only P Ag) - P2 can make anti-P1 (poorly developed at birth → strength varies in adults) → anti-P1 neutralized by P1 substance Ag from pigeon egg white or hydatid cyst fluid (dog tape worms) → P1PK not clinically significant/IgM usually/RT/IS, 4C, RT phases/enhanced by enzymes ]→ autoanti-P PCH (attach complement - biphasic) - binds P+ RBCs at low temps in extremities → hemolysis at 37C

MNS: M and N are alleles, S and s are alleles and U has 99.9% high frequency (whenever S or s is inherited)]- Ag are destroyed by enzymes → anti-M and -N are clinically insignificant unless AHG reaction after prewarming (non-red cell stimulating)/IgM usually/RT/RT or AHG/shows dosage/no agglutination with enzymes vs. Anti-S and -s are always clinically significant (red cell stimulating)/IgG/37C/AHG/shows dosage/enzymes have variable effects vs. anti-U reacting with S and s cells - occurs with S-s- (rare - needs U-negative blood units transferred)

Lutheran: 2 antithetical Lua (low frequency) and Lub (high frequency) Ag ]- most people are Lua-b+ → clinically significant/IgM (Lua) and IgG (Lua and b)/37C Lub vs. RT Lua/AHG Lub vs. RT Lua phase/no dosage and no effect from enzymes

Sentences to remember… Dosage vs. Enzyme activity vs. Cold Reacting Ab (IgM)

Dosage: KIDDs and DUFFY the Monkey (Rh) eat M+NSs

Enzyme activity: Enhanced - Rhea’s Kidds, Issac and Lewis like Pokemon vs. Destroyed - DufFY eNzyMSs

Cold Reacting IgM: LIiPMAbhN

Antibody Investigation Steps

Positive Ab Screen: react with any screen cells → positive (phase reactions/history/pattern) → Initial Ab Panel: known red cells with patient plasma with autocontrol - same method as potentiators in screen

Interpreting a panel

Auto control: patient cells and patient Ab → negative = alloantibody vs. positive (DAT pos) = autoAb or drug or delayed transfusion reaction (if DAT is negative → potentiator false positive - repeat with different potentiator or no enhanced solution) → Exclusion (highlight pos → mark out the negative rows containing positive Ag → exclusion chart) → Pattern matching (positive reaction match pattern) → RO3 (statistical measure probability events happen by random chance) - use screen antigram if CRO or need more RO3 → Phase of reaction (same as screen cells (IgG or IgM) → Reaction Strength (variable or missing can indicate showing dosage or multiple Abs) → Phenotype (don’t make alloAbs to Ag they possess → phenotype negative to Ag they developed Ab → confirm Ab ID) - dual populations when transfused (incorrect results) - Pos phenotype = misidentification or false pos

Screen Interpretations

1. AlloAb IgG

2. AlloAb IgG - multiple specificity

3. AlloAb IgM

4. AutoAb IgM - cold autoAb

5. AutoAb IgG - warm autoAb

Further Ab Investigation: Selected cells

Selected cells: pos Ab screen → clues before Ab panel → initial Ab panel interpretation → Ab screen exclusion or satisfy RO3 ]- select particular cells to continue → list on scrap paper the selected cells still required for exclusion or RO3 + = homo vs. - = hetero is needed to rule out → antigram specific Ag ± → grade and record → exclusion → record

Ab to high frequency Ag vs. HTLA Ab vs. Ab to low frequency Ag vs. Weak IgG Ab vs. Cold AlloAb IgM vs. HLA-related Ab vs. AutoAb

>98% incidence → k/Kpb/Jsb/Lub → all cells are pos/same phase/same strength/autocontrol neg → cannot ID Ab or any hidden Ab and exclusion is difficult ]- If Kell Ab → glycoprotein disulfide-bonded regions sensitive to sulfhydryl reagents → Ab not detected on treated cells → no Anti-K reaction → detect any other alloAb present (2-ME vs. DTT vs. AET)

HTLA: high frequency → high titre (not easily diluted out) and low avidity (weak reactions even when undiluted - IAT) → not usually clinically significant → 25% patients make other Ab → removed to ID alloAbs

Low frequency: Ab to multiple Ag → usually negative Ab screen → auto is negative on panel → full IAT crossmatch → compatible blood (negative screen with positive crossmatch)

Weak IgG Ab: waek IAT → new Ab or old → might be clinically significant → increase serum to cell ratio/repeat with different enhancements/check dosage/select different cells (fresher)/wash panel 1x (interference?)/incubate longer for more sensitization/phenotype if no recent transfusion

Cold AlloAb IgM: clinically insignificant → interfere or mask Ab → IS or RT phase reaction best (or carryover) → -P1, -M, -N, -Lea, -Leb, and -Lua (LIiPMAbhN) → incubate at RT or lower to enhance or eliminate reactivity (-M or -N treated cells vs. -P1/-Lea/-Leb neutralized with P1 or Lewis substance) → Prewarm technique

HLA-related AB: Human leukocyte Ag - expressed to variable degrees on RBCs → encoding expression of MHC (non-self, graft rejection) → not usually clinically significant - mask other Ab/interfere/confusing reactions/varying strength → PLT = clinically significant - refractory response (HLA+ cells)

AutoAbs: old patients → all cells pos → autocontrol pos → DAT pos → extravascular hemolysis → IgM (cold → auto-anti-I → negative test with cord cells) - IS react (carryover) → autocontrol IS pos → DAT pos only C3 (+ poly) → complement coated cells cause ABO grouping discrepancies → drug related (multiple myeloma) or mild anemia/mononucleosis/pneumonia or autoimmune or AIHA → use anti-IgG reagent (not poly) or eliminate IS or prewarm or use 2ME/DTT to denature IgM or Adsorption at 4C (patient’s own cells or rabbit cells adsorb out autoAb at cold temp → pretreat with enzymes and DTT → incubate 4C → remove adsorbed plasma → repeat) vs. IgG (Warm) - screen pos → panel pos → autocontrol pos at IAT → DAT pos for IgG (and possible also C3) → Eluate (reactive with all cells) → use BSA or saline tubes (LISS/PEG/Enzymes enhances reaction) → WAIHA (underlying alloAb?) → least incompatible crossmatch → adsorption (no recent transfusions) → pretreat patient cells with ZZAP (DTT+ficin) → removes attached autoAb and increases capacity for more adsorption → incubate patient serum with red cells → remove nonspecific warm autoAb → remove serum → retest panel (if recent transfusion → differential attach Ab in plasma to known Ag matching phenotype)

Red cell Phenotyping: Patient’s phenotype vs. Genotyping ]→ Application

Patient’s pheno: confirm ID (mixed field reaction if recently transfused → cell separation/genotype molecular) ]- Neg control has no Ag and Pos control is heterozygous expression of Ag (false pos if DAT IgG is pos and phenotype with IAT)vs. Genotype: PCR hybridization (advantage where testing is impossible or inconclusive)

Applications: blood group typing patients with AIHA/typing donors for alloimmunized patients (prevents further immunization)/fetal DNA type and zygosity for fathers at risk for HDFN (positive screen for mother) - chorionic villus sampling (placenta)/screening rare blood groups among donors/determining frequency polymorphism

Typing Donor Units → Full AHG XM

donor units negative for Ag corresponding to IDed Ab → Full AHG/IAT XM if Ab screen is positive or patient has irregular Ab history - patient plasma and phenotyped Ag negative donor units → IS/37/AHG (AHG reaction = clinically significant) ]→ Limits - Ab can be missed (absent Ag in screening/too weak so only detects homozygous expressions/only detected in not routine method/unknown history) and doesn’t guarantee successful transfusion