• What is immunohematology?
o Specialized branch of medical science
o concepts and clinical techniques related to modern transfusion therapy
o determine blood group and type, so blood and organs are safely transfused and transplants
o prevent materno=-fetal transplacental rxns (HDN, hemolytic dz of the newborn)
• what is the sig hx of blood transfusions?
o William harvery described circulation of blood in 1616
o 1665, Lower, dog-dog transfusion -> feasibility demonstrated
o 1667, Denis, animal-human -> banned experimentation
o 1818, Blundell, human-human -> techniques, rational indications
o 1800s, Various, frequent severe reactions -> search for blood substitute (saline)
• What dis Denis do in 1667?
o Transfuse blood from docile lamb to man accused by wife of running thru streets of Paris naked w other women, in attempt to rid “wild” blood
o As soon as blood entered veins, felt heat, HR inc, sweaty face
o Upset stomach, kidney pain, felt like choking
o After sleeping all night, really black urine (Hb, bilirubin, blood)
• What are the main sxs of acute hemolytic reaction?
o Systemic: fever, chills o Heart: inc HR o Vascular: hypotension, uncontrollable bleeding o Transfused vein: heat sensation o Chest: constricting pain o Lumbar: pain o Urinary: Hb, bilirubin
• Who first described the ABO blood groups? Significance?
o Karl Landsteiner in 1901
o Until then, all blood assumed to be the same
o Often tragic consequences of blood transfusions not previously understood
• What are the Ag’s and Ab’s found in each blood type?
o A: a-B, A Ag
o B: a-A, B Ag
o AB: no Ab’s, A and B Ag’s
o O: a-A and a-B, no Ag’s
• Where are the A-B Ag’s found? What other Ag is there? What other phenotype?
o Expressed on proteins or lipid bi-layer pf RBCs
o Also H-Ag= foundation on which both A and B are created
o H-Ag: RBC-glu-gal-NAG-gal-fru (A-B attached to oligosaccharide off RBC)
o Bombay phenotype: no H-Ag, need transfusion from other H-def, 1/10000 in India, 1/million in Europe
• What is the clinical significance of blood group Ab’s?
o Abs to system destroy RBC in vivo
o Cause HDN
o Are common
• What are the AIHAs?
o Cold: cold hemaglutinin dz, paroxysmal cold Hb-uria
• What are other types of immune hemolytic anemias?
o Alloimmune: Hemolytic dz of newborn (HDN), Hemolytic transfusion rxns
o Drug induced
• A transfusion is a tissue graft. What are the types? Blood?
o Also Abs against chemicals, drugs
o Autograft: animal has auto-Abs to its own blood
o Allograft: allo-Abs, against another individual of its own species
o Xenograft: heterologous Abs, against another species
o ABO Abs are allo-Abs; but also heterologous bc formed in response to exposure to gut and environmental bacteria, reacting w allo-Ags
• What was the first blood group system to be identified? Applications?
o ABO, most significant for transfusion practice
o Only system that reciprocal Abs are consistently and predictably present in sera of ppl who have no exposure to RBCs
o One of the first human characteristics proven to be inherited
o Used by lawyers in paternity suits, police in forensic science, anthropologists
• What are the ABO Abs?
o Occur naturally, aka natural isohemaglutinins, against A and B Ags
o Complete Abs=IgM class (u heavy chain) (st IgG, y HC)
o IgM large enough to produce visible agglutination of RBCs
o Formed w/o prior sensitization to non-self RBCs
o Form by encountering similar antigenic determinants found on micro-organisms (not present at birth)
• What are the relative amounts a-A and a-B in blood?
o Both decrease with age, highest at 10yrs
o a-A: much higher, in B and O
o a-B: about half, in A and O
• what is the difference bw sensitization and agglutination?
o S: Abs attach to a single RBC
o A: Abs attach to multiple RBCs, forming a clump of Ag-RBCs
• What is the mechaniom of immune hemolysis?
o C1,4,3 attach to sensitized RBCs
o C5,6,7,8 attach to those, signaling intravascular hemolysis
• How does complement activation occur in hemolysis (up to C1s)?
o 6 C1q subunits, 2 bind Fc of Ab to activate C’
o 1) Pentameric IgM bind Ags on cell surface, adopt “staple” form; C1q binds IgM
o OR 2) IgG bind Ags, C1q binds at least 2 IgG
o BOTH: binding of 2 C1q subunits to Ig activates C1r, cleaves and activates serine protease C1s
o IgM only requires 1 molecule, so may be better at fixing C’ and causing hemolysis
• What is the final course of C’ activation?
o C5b + C6 + C7 = complex, binds surface, basis to form membrane attack complex, binds to C8 + C9
o MAC=pore (causes lysis); C8 damages membrane, C9 polymerizes to form pore
• What is the fate of intravascular Hb after incompatible transfusion? Ssx?
o Hb Peaks in plasma at 6 hrs, after all Hpt used up
o Hpt drops to zero in first 2 hrs (bc binds all Hb, takes to liver)
o Urine Hb and serum also peak ~6 hrs, but lower levels
o All falls back to zero by 24 hrs
o Red/brown urine sign of EXTRAVASCULAR RBC destruction
o Jaundice also important sign of hemolysis
• How is ABO blood type testing done?
o Forward grouping: pt RBC added to reagent a-A and a-B antisera
o Reverse grouping: pt serum added to reagent A and B cells
o Crossmatching: last step before transfusion. Mix donor RBC w recipient plasma and test for rxn
• What are the levels of agglutination of RBCs?
o Graded on a scale of 1-4
o Negative reaction, no clumps
o +2: dispersed clumps
o +4: strongly positive, all RBCs in a single clump, can’t “de-glutinate”
• What happens if each blood type is added to a-A serum?
o A: agglutinates
o B: no rxn
o AB: agglutinates
o O: no rxn
• What happens if each blood type is added to a-B serum?
o A: no rxn
o B: agglutinates
o AB: agglutinates
o O: no rxn
• Method? Blood type? Reagent a-A binds pt RBCs, but not a-B:
o Forward grouping
o Type A
• Method? Blood type? Pt antibodies bind B cells, not A cells:
o Reverse grouping
o Type A
• What are the ABO blood compatibilities for receiving?
o A: from A or O
o B: from A or O
o AB: from A, B, AB, or O
o O: only from O
• What are the ABO blood compatibilities for donating?
o O: to O, A, B, AB
o A: to A, AB
o B: to B, AB
o AB: to AB only
• How are ABO blood groups genetic? What are the types?
o Make a punnett square of Mom and Dad types (AB x AO, etc)
o Each A, B, or O Ag is one allele from each parent
o We have two alleles
o A=AA, AO
o B=BB, BO
• What are the most common blood types? Least?
o Worldwide, O; >50% in Africans, Native Americans, Australian Aborigines o O+ very common in US o English: A and O o SE Asian and Indians: O and B o Least: AB
• What were the major developments in immunohematology?
o 1901, Landsteiner, ABO groups, explained transfusion rxns
o 1907, ottenberg, donor-recipient match, compatibility testing
o 1910, Moss; Agglutination parallels hemolysis, Testing practical and sensitive
o 1939 Levine and Stetson; discover Rh, immunization by pregnancy, Explained HDN
o 1940; Landsteiner & Weiner; Rh polymorphism, Improved safety
o 1945 Coombs, Mourant & Race; Anti-Human Globulin, Demonstrate IgG Abs
• How was the Rh factor discovered?
o 1940 Landsteiner and Weiner discovered Ab that agglutinated ~85% of ABO compatible blood
o Hypothesized it was same Ab causing HDN (discovered by Levine and Stetson)
o Abs produced by injecting blood from Rhesus monkeys into guinea pigs and rabbits
o Hence “Rh factor”
• What is hemolytic dz of the newborn, HDN?
o Aka: erythroblastosis fetalis
o Occurs secondary to transplacental transfer of maternal Abs:
o 1) ABO incompatibility: uncommon
o 2) Rh incompatibility: more common
o Occurs w Rh- mother, Rh+ father, firstborn OK, subsequent babies affected after mother sensitized
• How does woman become sensitized to Rh+ blood?
o First pregnancy: Rh- mom with RH+ fetus
o Cells from Rh+ fetus enter woman’s bloodstream, usu during birth
o Becomes senstitied, makes Abs (anti-D)
o Next Rh+ pregnancy: maternal Abs attack fetal RBCs
• How does immunization work for HDN?
o Inject mother w “RhoGam” (Rh immune globulin= anti-D)
o anti-D destroys Rh+ fetal RBCs before mother can mount an immune response
o Must do before birth of first Rh+ child
• What is the Fisher-Race theory of Rh factors?
o Each Rh gene: multiallelic, 3 sets of 2 alleles: D d, C c, E e
o Rh genes: C, c, D, E, e…but no “d” (a-d has never been identified)
o Rh+ means D-Ag positive= DD or D”d” (weak D)
o Rh- means D Ag negative
o D is the most antigenic of this group. A-D is strong IgG
• What is the significance of Rh in blood transfusions?
o Ppl receiving an Ag they don’t have MAY form Abs
o Serum of Rh- person doesn’t have anti-D unless had previous exposure to D Ag
o Unlike ABO Abs, the only way a-D is formed is through placental sensitization or transfusion
• What happens to D”d” ppl?
o It’s a weak subgroup of D Ag
o Person appears to be Rh- (D-) w a-D typing sera, so need more testing
o a-D coats weak D cells w/o agglutination
o 2-3% of “Rh-“ are actually D”d”
o All seemingly Rh- pts should be screened for D”d” variant, aka “weak D”
o Screening for weak D = add a-Ig (indirect a-Ig test)
• What is the charge on RBCs?
o Negative, they repel each other
• How is Direct coombs (direct antiglobulin test, DAT) used to detect RBC Abs introduced into pt’s circulation?
o Used in cases of blood transfusion rxns and HDN
o RBCs coated w Abs in vivo, but no visible agglutination
o Pt RBCs mixed w AHG (anti-human globulin, a-Ig)
o If RBCs were actually coated by Abs in vivo, AHG will bind, giving visible agglutination
o If neg, nothing binds to RBCs, no agglutination
• What are applications of DAT?
o Detects in vivo sensitization by Ab or complement
o AIAH, hemolytic transfusion rxn, HDN, Drug/RBC Abs, passive immunization
• What is the Indirect Coombs test used for?
o For acquired blood group Abs
o Detect IgG againsts RBC Ags. Rxns occur invitro
o Abs result of prior exposure to foreign Ag
o Usu no agglutination in vivo
o Known Ags are added to pt blood to see if they have certain Abs
o AHG added to cause visible agglutination (if Abs present)
• What is mechanisms of extravascular immune hemolysis?
o Abs bind RBCs (sensitized), complement binds
o M0s eat w or w/o C’
o M0s detect with Fcy
• What are the features of blood group IgM Abs?
o Cold reacting o Naturally occurring o Complete o Fix Complement to C’9 o Intravascular hemolysis
• What are features of IgG Abs?
o Warm reacting o Immune o Incomplete o Opsonizing o Extravascular hemolysis
• What is pre-transfusion testing? Crossmatch?
o ABO, Rh, Ab screen (AST or indirect coombs)
o C: D and R should be same ABO and Rh
o D RBCs mixed with Pt serum, look for agglutination or hemolysis
o If either occurs, they are incompatible
o If R has Abs, must determine before transfusion
o D is screened to make sure LACK Ags
• What are the 5 most common blood groups assoc w transfusion rxns?
o ABO o R(Rh+ 85%) o Kell (K k) (k 91%) o Duffy, Fya, Fyb, Fy (FyaFyb 46%) o Kidd, Jk, JKa, Jkb, Jkab (50%)
• What are the universal donors and recipients?
o Donor: O negative, reserved for emergency situations w massive blood loss
o R- AB positive
• What is the blood type distribution in the US?
o O+: 37% o A+: 35 o B+: 8 o O-: 6 o A-: 6 o AB: 3 o B-: 1 o AB-: 0.6
• Blood type? aA no rxn, aB + rxn, A cells + rxn, B cells no rxn:
• Blood type? No rxn aA or aB, + rxn both A and B cells:
• Blood type? + rxn both aA and aB, no rxn A or B cells:
• How is Rh typing done?
o Can only add aD to pt blood to see if Rh+
o Ppl don’t naturally have aD Abs if they are Rh-
o So can only do forward typing
o Positive= agglutination
• Rh type? Adding aD causes agglutination: