(pt 3) exam #3 - immunohematology (cls 544)

uncommon blood groups

genomic testing

allows for discovery of new blood group antigens that are either of high or low prevalence

• The corresponding antibodies are rarely encountered

• Knowledge of these least common antigens and/or antibodies better prepares laboratory personnel for when they encounter an uncommon antibody

blood group system 

• one or more antigens governed by a single gene or complex of two or more closely linked homologous genes; the genetic basis confirmed

  • Antigens where the genetic basis is unknown are placed into collections

what must happen for an antigen to form a new blood group?

antigen must be:

• Defined by a human alloantibody

• Inherited character

• Encoding gene must be known

• Gene location on chromosome must be known

• Gene must be unique from other blood group system genes

ISBT 200 series (general)

• collections are antigens that have a biochemical, serologic, or genetic relationship but do NOT meet the criteria for a system

  • Antigens classified as a collection are assigned a 200 number

ISBT 700 vs 900 series 

• All remaining RBC antigens NOT associated with a system or collection are catalogued into the ISBT 700 series (low prevalence antigens)OR the ISBT 901 series (high prevalance antigens)

  • 700 series: low prevalence antigens in occur in less than 1% of the population

  • 901 series: high-prevalence antigens occur in greater than 90% of the population

paroxysmal COLD hemoglobinuria (PCH)

acquired hemolytic anemia–seen in children w viral infections or idiopathically in adults

• IgG cold autoantibody (Autoanti-P) reacts in cold areas of the body

• Hemolysis occurs when antibody is incubated w/ cells

• antibody often demonstrates specificity towards the high-prevalence P antigen

• Antibody screen usually negative

• Positive DAT (with complement only)

  • Complement-mediated hemolysis

• Hemoglobinuria

• Intravascular and extravascular hemolysis

• Donath-Landsteiner test is diagnostic for PCH

  • Biphasic hemolysin has anti-P specificity

paroxysmal NOCTURNAL hemoglobinuria (PNH)

acquired stem cell disorder caused by a variant in the PIGA gene

• Presents with pancytopenia

• Cells deficient in glycosyl phosphatidylinositol-anchored proteins (GPI-APs)

• DRBCs in PNH patients lack DAF (CD55) & MIRL (CD59)

  • Both regulate complement → more sensitive to complement mediated hemolysis

• Leads to an immune response which triggers hemolysis of these cells

• Hemosiderin present in the urine

• Intravascular hemolysis

• Determined with flow cytometry of BM analysis

rare blood types by ethnic group

• African-American: U-, Fy(a-b-)

• Native American and Alaskan Native: RzRz

• Pacific Islander and Asian: Jk(a-b-)

• Hispanic: Di^b- (Diego B-negative)

• East European and Russian Jewish: Dr^a- (Drori A-negative, Cromer blood system)

• Caucasian: Kp^b- and Vel-

(general) diego blood group system (DI; 010)

• Named after the first antibody maker in a Venezuelan family during an investigation of HDFN (caused by anti-Dia)

• Antigens carried on Band 3, chromosome 17

• Anion exchanger (AE1 transporter)

diego blood group system antigens

• 23 antigens designated to this blood group system (expressed on newborn RBCs)

  • Antigens of interest (antithetical pairs): Di^a/Di^b and Wr^a/Wr^b

  • High prevalence antigens: Di^b and Wr^b

  • Low prevalence antigens: Di^a and Wr^a

• Wrb expression dependent on interaction of Band 3 and normal GPA (MNS)

  • Note: GPA-deficient RBCs are also Wr(a-b-)

(diego blood group) enzyme treament + antibodies

• Resistant to ficin, papain, DTT, and glycine-acid EDTA

• Antibodies are usually IgG, sometimes IgM

• Anti-Dia, anti-Dib, and anti-Wra implicated in HTRs and/or HDFN (anti-ELO)

  • Autoanti-Wra common in serum of patients diagnosed with WAIHA

(general) YT blood group system (YT; 011)

• Six antigens (Yt^a / Yt^b antithetical)

  • Yt^a is high prevalence antigen

  • Yt^b is a low prevalence antigen

  • Three phenotypes: common Yt(a+b-), Yt(a+b+), and rare Yt(a-b+)

• Represent amino acid substitution on the glycosylphosphatidylinositol (GPI)-linked RBC glycoprotein acetylcholinesterase or (AchE), chromosome 7

• Antigens are absent from RBCs of people with PNH III

(YT blood group) enzyme treatment + antibodies

• Enzyme/chemical treatment

  • Results of ficin and papain treatment varies

  • DTT destroys antigens

  • Resistant to glycine-acid EDTA

• Antibodies NOT implicated in HDFN

• Monocyte phagocytosis assays (MPA) helpful in determining clinical significance of anti-Yt^a

(general) Xg blood group system (XG; 012)

• Two antigens: Xg^a & CD99

• The gene coding for Xg^a antigen is located on the X chromosome

  • More commonly seen in females (89%) than males (66%)

• Phenotypic relationship

  • Xg^a positive ppl = high expression of CD99 on RBCs

  • Xg^a negative females = low expression of CD99 on RBCs

(XG blood group) enzyme treatment + antibodies

• Enzyme/chemical treatment

  • Sensitive to ficin and papain

  • Resistant to DTT treatment

• Antigens weakly expressed on cord RBCs and some adult females

• Anti-Xg^a usually IgG with some examples naturally occurring

  • Not implicated in HDFN or HTRs

  • Few examples of anti-CD99 reported

(general) scianna blood group system (SC; 013)

• Currently consists of eleven antigens: Sc1, Sc2, Sc3 etc 

• Found on RBC adhesion protein, erythroid membrane-associated protein (ERMAP) located on chromosome 1

• Antigens expressed on cord RBCs

(SC blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Resistant to enzymes

  • DTT treatment varies on blood group antigens

• Antibodies

  • Alloantibodies rarely encountered

  • Usually IgG (react at AHG phase)

  • May cause HTR, mild HDFN has been reported

  • Autoantibodies to Sc1 and Sc3 have been reported

(general) dombrock blood group system (DO; 014)

• 3 phenotypes: Do(a+b−), Do(a+b+), and Do(a−b+) 

• ART4 gene encodes for Dombrock GPI-linked glycoprotein, chromosome 12

  • Ten antigens

  • Do^a and Do^b are antithetical antigens

• High-prevalence antigens, Gya and Hy, have phenotypic relationship

  • Gy(a-) phenotype is null phenotype

• Antigens are found on cord RBCs but absent on PNH III RBCs (absent GPI anchor)

(DO blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Resistant to ficin, papain, and glycine-acid EDTA

  • DTT treatment weakens antigenic expression (sensitive)

• Antibodies

  • Usually IgG and react optimally with enzyme-treated RBCs

  • Anti-Do^a and anti-Do^b implicated in delayed HTRs but not HDFN

  • Difficult to identify = weakly reactive and disappear

(general) colton blood group system (CO; 015)

• The high-and low- prevalence antithetical antigens are Co^a and Co^b, respectively

  • Co3 present on all RBCs except for rare Co(a-b-) phenotype

  • Co4 seen on two individuals with rare null phenotype

• Located on integral membrane protein, aquaporin-1 (AQP1), chromosome 7

  • Accounts for 80% of water reabsorption in the kidneys

• Antigens expressed on RBCs of newborns

(CO blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Resistant to ficin, papain, chloroquine, and DTT

• Antibodies

  • Usually IgG antibodies

  • Enhanced with enzyme-treated RBCs

  • Anti-Co^a, anti-Co^b, and anti-Co3 implicated in HTRs and HDF

(general) landsteiner-wiener blood group system (LW; 016)

• 4 LW antigens, chromosome 19

• LW^a and LW^ab are the common, high-prevalence antigens

• LW^b = low-prevalence

• Resistant to enzymes and glycine-acid EDTA

• Antigens depressed during pregnancy, lymphoma, and leukemia

• Autoanti-LW common in serum from patients with WAIHA

• Anti-LW not implicated in serious HDFN or HTRs

(LW blood group) similarities bewteen Rh & LW systems

• Rh(D) positive RBCs will react strongly in the presence of anti-LW

• Rh(D) negative RBCs may be nonreactive or react only weakly with anti-LW

• Anti-LW reacts equally well with cord cells regardless of their D type

• Anti-LW never reacts with Rh_null cells

(LW blood group) how to distinguish between anti-LW & anti-D?

• test with DTT-treated D+ RBCs

  • D antigen not denatured by DTT, LW antigen destroyed by DTT

  • Positive reaction (anti-D), negative reaction (anti-LW)

(general) chido-rodgers blood group system (CH/RG; 017)

• Located on the fourth component of complement C4

• Nine antigens, chromosome 6

• Not intrinsic to RBC membrane; antigens are adsorbed onto RBCs after birth

• Crossmatch-compatible units may not be located due to high prevalence

(CH/RG blood group) enzyme treatment + antibodies 

• Enzyme/Chemical Treatment

  • Enzymes destroy these antigens

  • Resistant to DTT and glycine-acid EDTA

• Antibodies

  • Usually IgG and react weakly (clinically insignificant for transfusion)

  • Both anti-Ch and anti-Rg can be neutralized with pooled plasma

(general) gerbich blood group system (GE; 020)

• six high-prevalence Gerbich antigens

  • Ge2, Ge3, Ge4, GEPL, GEAT, and GETI

  • Seven low-prevalence antigens

• Antigens expressed at birth (located on sialoglycophorin C and D)

• RBCs of Gerbich or Leach phenotypes have weak expression of Kell blood group antigens

(GE blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Ficin destroys Ge2 and Ge4 antigens

  • Ge3 is ficin resistant

  • Resistant to DTT treatment and glycine-acid EDTA

• Antibodies

  • Some are IgM but mostly IgG

  • Clinical significance varies

  • Can be eluted from DAT+ cord RBCs

  • Most common: Anti-Ge2, anti-Ge3 causes HDFN

(general) cromer blood group system (021)

• 21 high-prevalence antigens and low-prevalence antigens, chromosome 1

  • Carried on decay accelerating factor (DAF/CD55), a complement regulatory protein

  • PNH III RBCs deficient in DAF will lack Cromer antigens (absent GPI linkage)

(cromer blood group) enzyme treatment + antibodies

• Enzyme Treatment

  • Resistant to ficin, papain, and glycine-acid EDTA

  • DTT weakens antigenic expression

  • Destroyed by chymotrypsin

• Antibodies

  • Usually IgG

  • Not implicated in HDFN

  • DAF strongly expressed on placenta and absorbs antibodies

(general) knops blood group system (022)

• 14 blood group antigens, chromosome 1

  • Kna, McCa, SI1, Yka high-prevalence antigens

• Weak at birth; later expressed on complement receptor 1 (CR1)

(knops blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Weakened by ficin and papain

  • Destroyed by DTT

  • Resistant to glycine-acid EDTA

• Antibodies

  • Primarily IgG antibodies (react at AHG)

  • Difficult to absorb and elute

  • Reactivity enhanced with longer incubation at 37°C

  • Clinically insignificant for both HTRs and HDFN

(general) indian blood group system (IN; 023)

• 6 antigens in the system

  • Mainly discuss In^a (IN1, low-prevalence) and In^b (IN2, high-prevalence)

  • Located on CD44 glycoprotein, chromosome 11

  • Weakly expressed on cord RBCs

(IN blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Sensitive to enzymes and DTT

  • Resistant to glycine-acid EDTA

• Antibodies

  • Usually IgG (react at AHG phase)

  • Does not bind complement

  • Seen in positive DATs but not implicated in HDFN

  • Rare cases of HTRs reported

OK blood group system (OK; 24)

• 3 high-prevalence antigens

• Carried on CD147, or basigin, a receptor essential for Plasmodium falciparum invasion, noted as gene BSG, chromosome 19

• Ok^a well developed on RBCs from newborns

• Enzyme/Chemical Treatment

  • Resistant to enzymes, DTT, and glycine-acid EDTA

• No reports of anti-Ok^a (IgG) implicated HDFN reported

(general) raph blood group system (025)

• only antigen is MER2; originally defined by two monoclonal antibodies, chromosome 11

• Encoded by the CD151 gene

  • Essential for the assembly of basement membranes in the skin and the kidneys

  • has been recognized by human polyclonal antibodies

  • MER2 is abundant on platelets; decreases over time with maturation of erythroid cells

• Alloanti-MER2 has been found in patients with end-stage renal disease

(raph blood group) enzyme treament + transfusion considerations

• Enzyme/Chemical Treatment

  • Resistant to ficin and papain

  • Sensitive to trypsin, a-chymotrypsin, pronase, and AET

• Transfusion Considerations

  • Little is known about the clinical significance of anti-MER2, may cause HTR

  • 8% of the population has MER2- RBCs; transfuse with crossmatch-compatible units

(general) john milton hagen blood group system (JMH; 026)

• Established after it was shown that the JMH protein is the GPI-linked glycoprotein CD108 and the gene (SEMA7A) was cloned, chromosome 15

• Consists of 8 antigens

  • JMH is a high-prevalence antigen & present on glycosylphosphatidylinositol (GPI) linked glycoprotein

• Paroxysmal nocturnal hemoglobinemia (PNH) - Lack all GPI linked glycoproteins

(JMH blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Destroyed w ficin, papain, and DTT

  • Resistant to glycine-acid EDTA

• Anti-JMH often found in elderly patients (usually IgG)

  • Antibodies are generally clinically insignificant

(general) GIL blood group system (GIL; 029)

• only one high-prevalence antigen, GIL

  • Genetically discrete from all other blood group systems

• Located on the glycerol transporter aquaporin 3 (AQP3); AQP3 gene, chromosome 9

(GIL blood group) enzyme treatment + testing considerations

• Results of enzyme treatment

  • Enhanced with ficin and papain

  • Resistant to DTT and glycine-acid

• Testing considerations

  • DAT positive noted

  • No clinical HDFN occurrences

  • One occurrence of HTR associated with anti-GIL

rh-associated glycoprotein blood group system (RHAG; 030)

• RhAG does not have Rh blood group antigens

  • Presence is essential for Rh antigen expression

• Absence of RhAG due to inactivating mutations in the RhAG gene results in the Rh null phenotype

• Partial suppression of RH gene expression caused by mutations in the RHAG gene occurs in the Rh mod phenotype

• Consists of 6 antigens: Duclos, Ola, and DSLK…, chromosome 6

• Clinical significance unknown for anti-Duclos and anti-Ola

(general) FORS blood group system (FORS; 031)

• only one antigen, FORS1, low prevalence ; originally though to be a subgroup of A

• GBGT1 gene produces glycosyltransferase causing the formation of Forssman glycosphingolipid by the addition of N-acetylgalactosamine to the P antigen, chromosome 9

• Group O RBCs expressing FORS1 antigen do not react with Dolichos biflorus or monoclonal anti-A

• Forssman glycolipid serves as pathogenic receptor for E. coli ð increased susceptibility for E. coli infection in human cells

(FORS blood group) enzyme treatment + antibodies 

• Enzyme/Chemical Treatment

  • Enhanced with ficin and papain; resistant to DTT and glycine-acid EDTA

• Mostly IgM antibodies with optimal reactivity at RT or 4°C

  • Clinical significance = unknown

(general) JR blood group system (JR; 032)

• only one antigen, Jra

  • High-prevalence in most populations

  • Located on ABCG2 gene on chromosome 4

  • Fully developed at birth

• Presents as a problem in chemotherapy due to its involvement in multidrug resistance in tumor cells

• Jr(a-) phenotype more common in Japanese population

  • Anti-Jra is usually IgG (rare antibody)

(JR blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Resistant to ficin, papain, DTT, and glycine-acid EDTA

• Documented in severe cases of HDFN

  • Some anti-Jra patients have been transfused with Jr(a+) RBC units [incompatible] without issue; others have led to HTRs

(general) LAN blood group system (LAN; 033)

• only one antigen, Lan (high-prevalence > 99% population)

  • Lan- phenotype occurs in about 1 in 20,000 people

• Lan gene, ABCB6, encodes for ATP-binding cassette transporters, chromosome 2

  • Lan null phenotype: other porphyrin transporters compensate for the ATP- dependent uptake of heme

• Cord RBCs have a stronger reaction with monoclonal anti-Lan versus adult RBCs

(LAN blood group) enzyme treatment + antibodies + transfusion considerations

• Enzyme/Chemical Treatment: resistant to ficin, papain, DTT, and glycine-acid EDTA

• Anti-Lan formed due to exposure via pregnancy or transfusion

  • IgG antibody; reacts optimally at AHG phase; some able to bind complement

• Transfusion Considerations

  • Due to clinical significance, Lan- RBCs should be used

  • Not known to cause HDFNS but has been observed in positive DAT of newborns

(general) vel blood group system (VEL; 034)

•  only one antigen (high-prevalence), Vel

  • Gene SMIM1 located on chromosome 1 → absence of gene results in null phenotype

• Weak antigen expression on cord RBCs; varies on adult RBCs

(VEL blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Antigen resistant to glycine-acid EDTA and DTT

  • Anti-Vel enhanced with enzyme-treated RBCs

    • Some examples of anti-Vel did not react with DTT treated RBCs

• Antibodies

  • Mostly IgG but can be IgM as well

  • Ability to cause severe immediate HTR and HDFN

  • In testing, anti-Vel may cause in vitro and in vivo hemolysis

  • Characterized by its ability to activate complement

(general) CD55 blood group system (035)

• Only one antigen, CD59.1; chromosome 11

• CD59 plays major role in protecting against complement-regulated hemolysis by binding to C8 and C9 → interference with MAC formation

  • PNH (acquired hemolytic anemia) caused by mutation in the GPI-linker gene

  • PNH patients are deficient in all GPI-linked proteins including CD59

    • Symptoms include hemolysis, strokes, and neuropathy

(CD55 blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Demonstrate increased reactivity with enzyme-treated RBCs, not affected by DTT-treated RBCs

• Antibodies: one example of anti-CD59.1 (IgG)

(general) augustine blood group system (AUG; 036)

• 4 antigens, AUG1, AUG2 (Ata), AUG3 (ATML) and AUG4 (ATAM)

  • AUG1, AUG2 (Ata) and AUG 4 are high-prevalence

  • At(a-) only identified in the AA population

  • AUG gene, SLC29A1 located on chromosome 6

  • Antigens are fully developed at birth

(AUG blood group) enzyme treatment + antibodies

• Enzyme/Chemical Treatment

  • Resistant to ficin, papain, DTT, and glycine-acid EDTA

• Antibodies

  • Anti-At^a usually IgG and reacts at AHG phase, cause HTR

  • Anti-AUG3 cause HDFN

• Associated with severe HTRs; one case of HDFN

  • Low prevalence antigen AHG 3 has corresponding antibody that caused severe HDFN

SID blood group system (SID; 038)

• 1 high-prevalence carbohydrate antigen, soluble form is Tamm-Horsfall glycoprotein found in urine (96%)

• B4GALNT2 gene, located on chromosome 17

• Variable in RBC expression, weakens during pregnancy, not detected at birth

• Antigen resistant to ficin, papain, DTT and glycine acid-EDTA

• Antibody usually IgM, can naturally occur, can react at AHG phase

(SID blood group) testing considerations

• Known to have characteristically shiny, refractile agglutinates under microscope

• Inhibited/neutralized with urine from Sd(a+) individuals, clinically insignificant

• Enhanced with enzyme treated RBCs

Er blood group system (ER; 044)

• High prevalence antigens Er^a and Er³

• Low-prevalence: Er^b, ERSA, and ERAMA

• 5 antigens, PIEZO1 gene, chromosome 16

• IgG antibody, anti-Er3 indicates HTR

EMM blood group system (EMM; 042)

• 1 antigen, high prevalence, PIGG gene, chromosome 4

• Located on GPI-anchored protein, deficiencies in GPI synthesis can lead to the Emm-negative phenotype, e.g. PNH III

• Resistant to enzyme treatment

• Anti-IgG or IgM, naturally occurring, clinically insignificant

which blood group structures are carbohydrates? (7)

• ABO

• P1PK

• LE ; H ; I

• GLOB ; FORS

which blood group structures are single pass proteins? (11)

• MNS ; LU ; XG ; KEL

• SC ; LW ; GE 

• KN ; IN ; OK

• Vel

which blood group structures are multi-pass proteins? (11)

• FY ; RH ; JK

• LAN ; DI ; CO

• KX ; RAPH ; GIL

• RHAG ; JR

which blood group structures are GPI-linked proteins? (5)

• YT

• DO

• CROM

• JMH

• EMM

ISBT Ii collection 207

• One antigen: i

• i antigens are found on most human and on soluble glycoproteins in body fluids

• Strongly expressed on cord cells

ISBT collection 210 + MN CHO collection 213

• Collection 210

  • Two antigens Lec and Led, precursors to Lewis antigens

• MN CHO Collection 213

  • Six polymorphic antigens

    • Hu, M1, Tm, Can, Sext, and Sj

  • Associated with the M or N antigen in the MNS system

ISBT 700 series

• Low-prevalence of less than 1% of most random populations

• Gene for these antigens is unknown

• When identified is placed into a blood group system

• Antigens currently make up the 700 series of the ISBT classification:

  • By, Chra, Bi, Bxa, Toa, Pta, Rea, Jea, Lia, Milne, RASM, JFV, JONES, HJK, HOFM, and REIT

  • May cause HDFN

ISBT 901 series

• High-prevalence antigens that represent more than 90% of most random populations

• Gene for these antigens is unknown

  • When identified is placed into a blood group system

• Antigens currently make up the 901 series of the ISBT classification:

  • ABTI: weak Vel expression I ABTI- RBCS

  • LKE

HLA antigens on RBCs (general)

• HLA class I antigens (HLA-A, -B, and -C) are present on all nucleated cells

• Mature RBCs are not nucleated and generally do not have detectable level of HLA antigens

• HLA antigens are not considered a blood group antigen

• Three antigens: Bg^a, Bg^b, and Bg^c, detectable on mature RBCs

(HLA antigens) BG antigens

• Bg^a (HLA-B7), Bg^b (HLA-B17), Bg^c (HLA-A28)

• HLA antigens on RBCs not destroyed by enzyme, removed by chloroquine

• Bg antibodies can be adsorbed by using platelet concentrate

• IgG class antibodies, react weakly in serologic tests

• Clinically insignificant, rare delayed HTR, no HDFN, significant in TRALI

uncommon blood groups applications to routine blood banking

• Identification of uncommon blood group antibodies

  • Prevalence (low or high), known ethnicity, effect of enzymes, and chemical treatments is critical

• Antibodies to low-prevalence antigens: HDFN, incompatible crossmatch

• Antibodies to high-prevalence and other uncommon antigens