CLS 3311 Advanced Clinical Immunohematology

CLS 3311Advanced Clinical Immunohematology Other Blood Groups Kidd, Kell, Duffy and MNSs

KiddBlood Group SystemGenetics • Three alleles: Jka, Jkb, Jk • Codominant Inheritance • Jkis a silent allele (amorph) • Looking at the Kidd phenotype frequencies on the next slide (Page 186 in your text), what is the frequency of the Jka antigen in the White population? • Did you came up with 77%? Combine the frequencies of the first two rows of the table, both of which are Jka positive.

Kidd PhenotypeFrequenciesTable 8-11 Pg. 186 Jka Frequency: 77%, 91% and 73% respectivelyJkb Frequency: 72%, 43% and 77% respectively

Phenotype Frequencies What is the purpose of learning the phenotype frequencies of each blood group antigen? • Gives faculty ghoulish pleasure! • When crossmatches are required it helps the Tech know how many units to crossmatch or antigen type to find compatible blood. If a patient has anti-Jka antibody how many RBC units need to be antigen typed to find 2 compatible units? 77% of the population is positive for the antigen therefore 23% are NEGATIVE for the antigen. Approximately 2 out of 10 units are compatible. Need to antigen type 10 units.

Phenotype Frequencies • What if there is more than one antibody present in the patients serum? How do you determine how many units to antigen type or crossmatch? • Multiply the frequency of donors negative for one antigen by the frequency of donors negative for each of the other antigens. • For example if a patient has anti-c, -Fya and -S. Among donors c neg = 18%, Fy(a-) = 34% and S neg = 45%. The frequency of compatible units would be: .18 x .34 x .45 = .0275 which is 2.8% of the donor population is compatible.

Kidd Antigens • Biochemistry:Single protein band that is part of the RBC Urea transport system. • People who are Jk (a–b–) resist lysis in 2M Urea for 15 to 30 minutes. Jk (a+ and/or b+) cells swell and burst rapidly. Testing cells in 2M Urea is used as a screening process to detect Jk null red cells. • Well developed at birth: HDN • Enzymes ENHANCE antigen expression • Ficin or Papain treatment of red blood cells enhances anti-Kidd reaction strength (makes stronger).

Kidd Antigens • Approximately 14,000 antigens on RBC • Not very accessible on the RBC membrane • DemonstrateDosage • Requires a Homozygous Cell to rule out on Panel • Found only on RBCs • Jk3 Ag • Present with Jka and/or Jkb antigens • Known as a Common Antigen: Whenever you have either Jka or Jkb antigens on the RBC you also have Jk3 antigens.

Jk (a-b-): Jk Null Phenotype Jka, Jkb, Jk3 antigens NOT on the RBC Two Mechanisms • Inheritance of Homozygous Jk/Jk genotype • No detectable Jk antigens • Form anti-Jk3 antibody: reacts with Jka, Jkb and Jk3 antigens. • Dominant Inhibitor Gene: In(Jk) • Weak expression of Kidd antigens • Does not make anti-Jk3

Jk (a-b-)Jk Null Phenotype Anti-Jk3 antibody Most of the antibody reactivity is directed against the Jk3 antigen. Reacts with virtually ALLcells Transfuse with Jk (a-b-) red cells See a need to screen large #’s of RBC units with 2M urea? This is a quick way to screen a lot of units in a short amount of time. The other option is to antigen type each unit for both Jka and Jkb.

Kidd Antibodies • Good complement binding IgG antibodies • Polyspecific AHG may be the only way to detect these antibodies since the antibody often quickly falls below detectable levels and only the complement on the red cell is evidence of their presence. • Cause Hemolytic Transfusion Reactions (often Delayed) • Often found in combination with other antibodies: i.e. Multiple antibodies

Kidd Antibodies • Show (Demonstrate) dosage • Homozygous expression results in stronger reaction with antibody than heterozygous. • Rbc Immune: Result from exposure to foreign red blood cells – transfusion or pregnancy. • Can rule out ONLY with homozygous cells

Kidd Antibodies NOTORIOUS REPUTATION Delayed Hemolytic Transfusion Reaction • 7-10 days following transfusion a patient begins demonstrating the signs of a hemolytic transfusion reaction: anemia, weakness, dizziness, pallor, etc. • The antibody had, indeed, dropped below detectable levels. NEED to check previous records. • PEG and Enzymes enhance the reactivity of Kidd antibodies.

Kell Blood Group SystemGenetics • KEL gene codes for both Kell and Km proteins on the RBC membrane. AllKell system antigens are found on these two proteins. • Specific genetic base pair mutationsaccount forKell system Low Frequency antigens (K, Kpa, Jsa, etc.). • Typically, a KEL gene variant codes for only one low frequency antigen at a time. • K0(null)is a Silentallele: No Kell antigens present on RBC

Xk Gene KELgene codes for the entire Kell system glycoprotein, some 720 amino acids.It also codes for the Km antigen. KEL Gene RBC Kell system glycoprotein: Kell Ag’s reside here. Kx Km Kx is NOT a Kell system antigen -but there is interaction between the Kell protein and the Kx antigen.

Kell Null (K0) Phenotype 1.K0is a silent Kell allele 2. When homozygous K0K0 inherited no Kell system antigens are expressed. 3. Kx antigen expression is enhanced 4. Very rare Kx

Kell Blood Group SystemAntigens • Phenotype Frequencies: Harmening Pg. 179 • Typical Kell System Phenotype would include all of the High Frequency (k, Jsb, Kpb, etc.) antigen’sPLUSONELow Frequency Antigen: K or Jsa or Kpa. • i.e. Typical Kell System Phenotype: K, k, Jsb, Kpb or k, Jsa, Jsb, Kpb, etc. • There are between 2500 – 6000 antigen sites on the RBC membrane

K+ = 9% and 3.5%Kpa = 2.3% and Rare Jsa = Rare & 20%k + = 99.8% and 100% Kpb = 100% and 100% Jsb = 100% & 80%

Kell Antigens • Found ONLY on RBCs • NOT on WBCs, Epi’s, etc. • K (K1) and k (K2) antigens are very immunogenic • Second only to the D antigen in immunogenicity • Anti-K is a very common antibody identified in the Transfusion service • NOTdenatured by enzymes: papain, ficin, etc. • Well developed at birth. • HDN: Antigen is present on fetal cells as early as 10 weeks gestation

Kell Antigens • Sulfhydral Reagents destroy Kell system antigens. • 2ME, DTT, AET, ZZAP (papain & DTT) are all sulfhydral reagents (destroy disulfide bonds) • Kell glycoprotein has much folding that is held together by disulfide bonds • You need to look up each reagent’s full name. • Treatment of red blood cells with Sulfhydral Reagents results inKell null (K0) cells: • Destroys Kell system antigens. No longer detectable on the red blood cell membrane.

Red cell immune Dosage Complement LISS Can cause HTR (hemolytic transfusion reaction), HDN: Reacts best AHG phase Variable: occasional Only up to C3, so no lysis due to complement There are documented cases of LISS NOT enhancing anti-K antibodies Kell System AntibodiesNext to ABO and Rh, anti-K is the most common antibody encountered in the Transfusion Service

Kell System Antibodies • There areexamples of IgG anti-K that are room temperature reactive. • Non-RBC immune IgM anti-K • This is rare. Associated with bacterial infections • Antibodies to Kpa, Jsa • HDN, HTR: Rare • Reactivity is similar to anti-K • Antibodies to k, Kpb, Jsb: Very rare. Why?

McLeod Phenotype • Lack Kx and Km antigens from RBC membrane • Weakened expressionof Kell system antigens • Found only in males: X-linked transmission • Associated with Chronic Granulomatous Disease (CGD): unable to kill ingested bacteria • Hemolytic Picture:compensated hemolytic anemia with acanthocytes, reticulocytes, etc.

McLeod Phenotype k, Kpb, Jsb Weakened expression of Kell system antigens - Loss of Kx and Km

Duffy Blood Group System:Genetics • The Duffy gene codes for a glycoprotein also found in other tissues: brain, kidney, spleen, heart and lung. • Alleles at Duffy locus: Fya, Fyb, Fyx, Fy3, Fy4 & Fy (Silent Allele), ETC. Codominant Alleles. Syntenic with Rh gene. • Duffy silent allele Fy can make for some interesting inheritance patterns as seen on the next slide.

Father:HeterozygousFyaFyb Mother: Appears Homozygous FyaFya Scenario 1: Expected inheritance pattern Scenario 2: Unexpected inheritance pattern Fya Fyb Fya Fyb Fya FyaFya FyaFyb Fya FyaFyb FyaFya FyaFya FyaFyb Fya Fy FybFy FyaFy

Duffy Antigens • Phenotype Frequencies Pg. 183

Fya and Fyb Antigen’s • Well developed at birth: HDN • Do not store well: antigen elutes off the red blood cell during storage - expression becomes weaker • Use in-date reagent red cells: IAT and Panels should be within expiration date. • If expired reagent red cells are used the strength of the Duffy antigen expression needs to be tested with reagent anti-sera (anti-Fya, etc.)

Duffy Antigen’s • Fya, Fyb and Fy6 antigens are Destroyed by enzymes (Ficin, Papain, Bromelin, Chymotrypsin but NOT by neuraminidase): Fy3 and Fy5 antigens are NOT. • Moderately immunogenic • There are approximately 17,000 antigens per red blood cell.

Duffy Antigen’s • Fyx gene codes for a Weakened Expression of Fyb and Fy3 antigens • Fyx person may type Fy(b–) but will adsorb and elute Fyb antigens • No Fyx antibodies • Fy3 Antigen is NOT destroyed by enzymes • Common antigen: present when Fya and/or Fyb antigens are present. • Anti-Fy3 antibody seen in Fy (a–b–) phenotype. Reacts with all Duffy Positive cells. Acts like an inseparable anti-Fya, -Fyb.

Fya and Fyb Antibodies • RBC Immune: Usually IgG, React best at AHG. Cause HTR and HDN • DO NOT agglutinate enzyme treated cells • Can rule out ONLYwith homozygous cells • Anti-Fyb • Most commonly present with other unexpected antibodies: Multiple antibodies.

Duffy Antibodies Anti-Fy5 • Similar to anti-Fy3 EXCEPT: • Anti-Fy5 fails to react with Rh Null red cells that express Fy3 and is non reactive with red cells from Fy(a–b–) Blacks. • Anti-Fy5 DOES agglutinate red cells from Fy(a–b–) Whites • Theory: Fy5 formed by the interaction of the Rh and Duffy gene products.

Duffy and Malaria • Black people with the Duffy phenotype of Fy(a–b–) appear to have resistance to Plasmodium vivax, a causative agent of Malaria. • Duffy antigens appear to be a receptor for the P. vivax organism and when the antigen is not present on the red blood cell membrane P. vivax is unable to access the red blood cell • Some area’s of West Africa are 100% Fy(a–b–). What is the percentage of the American Black population that are Fy(a–b–) ?

MNSs Blood Group System: Genetics • M & N locus and the S & s locus are linked and found on chromosome #4. • Genes produce two distinct glycophorins or sialyglycoproteins (SGP) on the RBC membrane.

MN Genetics • MN Locus genes produce Glycophorin A (GPA) • M-GPA’s 1st five aa’s = Ser-Ser-Thr-Thr-Gly • N-GPA’s 1st five aa’s = Leu-Ser-Thr-Thr-Glu • Amino acids (aa) 2, 3 & 4 are the same for both • Harmening: Page 166, Figure 8-1. • Glycophorin A (GPA) is a glycoprotein also known as MN-sialoglycoprotein or -sialoglycoprotein

MN Antigens • Well developedat birth • Antigen sites:2.5 x 105 / red cell • Destroyed by enzymes: ficin, papain and bromelin • Enzyme snips off the end of the glycophorin • RBC antigens: found only on RBCs • Demonstrate Dosage: Must us homozygous cell to rule out on panel

Ss Genetics Ss genes code for the production of Glycophorin B(GPB) • S glycophorin B has Met at aa position 29 • s glycophorin B has Thr at aa position 29 • Glycophorin B(GPB) is a glycoprotein also known as Ss-sialoglyprotein and -sialoglycoprotein) • First five aa of GPB are same as ‘N’ GPA: • Leu-Ser-Thr-Thr-Glu • Harmening Page 166 Figure 8-1

Ss Antigens • Less easily destroyed by enzymes • S is more susceptible than s. • Antigen Sites: GPB - 1.0 x 106 / red cell • Demonstrate dosage • REQUIRE a homozygous cell to rule out on a panel • Well developed at birth: HDN

AABB Technical Manual, 14th Edition, Page 320

Anti-M Antibodies • NON RBC immune • Generally thought to be predominantly IgM room temperature or saline reacting antibodies • Some also have IgGin addition to IgM • Most are NOT clinically significant • Do NOT react with enzyme treated red cells: enzymes destroy M antigens

Anti-M Antibodies • Reactivity enhanced with increased serum:cell ratio • Some are pH dependent: react best at pH 6.5 • Rarely causes HTR or HDN, BUT… • Remember, some do have an IgG component that enables them to cause HDN and/or HTR. If anti-M reacts at 37oC or AHG phases need to treat as clinically significant. Rule out with Homozygous cells is necessary!

Anti-N Antibodies • Non RBC Immune: Cold reacting IgM or IgG, saline agglutinin • Does NOTbind complement • DoesNOT react with enzyme treated cells • Demonstrates dosage: rule out with homozygous cells on a panel • NOT implicated in HDN or HTR

Ss Antibodies Anti-S and anti-s • IgG reactive at 37oC and AHG phase • CLINICALLY SIGNIFICANT • Anti-S usually doesNOT react with enzyme treated reagent red blood cells • Anti-s may or may not react with enzyme treated reagent red blood cells - variable • RBC immune - Cause HTR and HDN

U Antigen and Antibody • U antigen is found on Glycophorin B at the very base next to the red cell membrane • High frequency antigen • RBC Immune antibodies • Can cause HTR and HDN

SENSORY OVERLOAD!!!! • You have just been bombarded with the particulars of four different blood group systems! How to organize all this!?!?!?!? Categorize Each Member of Each Blood Group as to: • RBC Immune • Destroyed by enzymes • Dosage • HDN, HTR • Complement Activation, etc.

CLS 3311 Advanced Clinical Immunohematology