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Antibodies Formerly Known as ‘High-Titer, Low-Avidity (HTLA) Antibodies’ :

a review.

Jackie Ensley, MLS(ASCP)SBBMississippi Valley Regional Blood Center

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Objectives• Describe serology observed with these antibodies• Review the blood group systems/collections associated

with this reactivity• Discuss how MVRBC handles and reports out these

antibodies• Outline how to approach identification of these antibodies

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Terminology• What do we call this reactivity?

o Currently, the term ‘HTLA antibody’ is not considered the best terminology. HTLA stood for High Titer, Low Avidity antibody and is a description, not an identification of the antibody

o There has been no official decision in the blood bank community on what to call these antibodies and many facilities are struggling to come up with appropriate terminology

o On April 24th Medical Director Dr. Bruce Marshall is attending two seminars regarding this ever-evolving issue and more united decisions may come from these.

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MVRBC terminology• MVRBC has started using ‘Antibody of Undetermined

Specificity’ if the actual identity of the antibody can not be determined.

• In attempting to identify the antibody techs will use the chemicals and reagent cells available in the lab to define the serological reactions. o Example: Antibody reactivity decreased with ficin, decreased with

.2M DTT• This indicates anti-JMH but we may not have the antigen negative

cells available to completely identify as these cells are rare and hard to find. May be able to say ‘Probable anti-JMH’ or leave as antibody of undetermined specificity

MVRBC terminology• Moving forward, the MVRBC Reference Labs have come

up with specific terminology to help better define these antibodies, and to enhance the communication and understanding with our hospitals

• The following slides show specific examples that may be encountered

Example 1• The specificity of the

antibody is determined for sureo NOTE: With the 2nd arrow

we will be attempting to inform the hospital whether this unusual antibody is clinically significant or not.

Example 2• Probable Antibody of

_____ Classificationo The lab can classify the

antibody but can not put a single specificity to

the antibody

Example 3• Antibody of

Undetermined Specificity (formerly called HTLA)o NOTE: The 2nd arrow

shows the lab will describe the reactivity of the antibody

Example 4• HTLA-like Antibody of

Undetermined Specificity o Will be used for clarity with

our hospitalso Note: the second arrow

shows the lab will describe the reactivity of the antibody

Example 5• Antibody of

Undetermined Specificityo Not HTLA antibodieso Truly unknown antibody,

everyone comes across these

o May follow with phrase ‘future testing may allow ID’

o May include phrase such as ‘Antibody to a low

frequency antigen’

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What distinguishes this category?

• Reacts with majority of panel cells• Frequently described as “reactive weakly by the

antiglobulin test.” o Reactions are very weak and will break apart very readily due to

the weak attraction between the antigens and antibodies (low avidity).

o Repeated reactions may be irreproducible and not give the same strength or reactivity

• However, reactions of 1-2+ have also been seen using gel technique.

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What distinguishes this category?

• These antibodies are not clinically significant• Weak reactivity with high titers >64 are characteristic, but

titers below 64 have also been seen. A high titer does not give us a specificity, but can help the lab determine if they are on the right track with identification.o MVRBC has stopped using titers on a regular basis to identify

these antibodies. We find it is more conclusive to rely on serological reactions than the titer value. However, for MVRBC the titer is still available to use as an identification aid.

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Titer Reactivity Example

1:1 1:2 1:4 1:8 1:16

1:32

1:64

1:128

1:256

1:512

1:1024

1:2048

W+

W+

W+

+/- +/- +/- +/- +/- 0√ 0√ 0√ 0√

Titer:128

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How do I know when I have one of these antibodies?

• Most times is a process of elimination, all other possible antibodies or combination of antibodies is ruled out.

• Reactivity is not explained and doesn’t seem to follow any pattern

• Need to run enough cells so exclude possibility of any other alloantibodies being present

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Suspecting these antibodies

• Usually negative DAT, but each patient is different with unique history, diagnosis and treatment plans

• Reaction pattern suggests an antibody to a high-frequency antigen, and all or most cells are reactive at IgG.

• Reactions do not seem to fit a specific antibody or combination of antibodies pattern

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Suspecting these antibodies

• 1-2 phenotypically similar cells are run and are reactive• Theoretically should not adsorb out of the plasma because

of the low-avidity of the antibody

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ExampleD C c E e K k Fya Fyb Jka Jkb Lea Leb P1 M N S s Gel PeG

1 0 0 + 0 + + + + + + 0 0 + + + 0 0 + 1+ W+

2 0 0 + 0 + 0 + + + 0 + 0 + 0 0 + 0 + 2+ W+

3 0 0 + 0 + + + 0 + + 0 0 + + + 0 + 0 0 0√

4 + + 0 0 + 0 + + 0 + 0 + 0 + + + 0 + 1+ W+

5 + 0 + + 0 0 + 0 + + 0 + 0 + + 0 0 + 1+ +/-

6 + 0 + + 0 0 + 0 + 0 + 0 + + + 0 + 0 1+ 1+

7 + 0 + + 0 0 + + 0 + + 0 + + + + + + W+ W+

8 0 + + 0 + 0 + + 0 0 + 0 + + + 0 + + 1+ 1+

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Antibodies with HTLA Activity

• Chido/Rodgers System• JMH System• Knops System• COST Collection

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Chido/Rodgers System• Anti-Ch was reported in 1967 and anti-Rg was reported in

1976o Named after the first antibody producers, Chido and Rodgers.

• Function: Part of the complement cascade. Ch and Rg are antigen determinants located on the fourth component of complement (C4), which becomes bound to RBCs from the plasma.

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Chido/Rodgers System: Phenotypes

• Chido/Rodgers describes the cluster of antigen determinants on C4. o Individuals can form antibodies to the epitopes they lack

Chido Ags % occurrence Rodgers Ags % occurrence

CH/RG:1, 2, 3 88.2% CH/RG:11, 12 95%

CH/RG:1, -2, 3 4.9% CH/RG:11, -12 3%

CH/RG: 1, 2, -3 3.1% CH/RG:-11, 12 2%

CH/RG:-1, -2, -3 3.8%

CH/RG:-1, 2, -3 rare

CH/RG:1, -2, -3 rare

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Chido/Rodgers:Disease Associations

• Disease Associations:o Ch- (Lack of C4b) gives increased susceptibility to bacterial

meningitis in children. o Rg- individuals (lack of C4a) have a much greater susceptibility for

SLE.

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Chido/Rodgers:Neutralization

• Antibodies are neutralized by plasma, which has Ch/Rg. This helps in identification:o Control plasma

• Equal volumes of patient plasma and 6% albumino Neutralized plasma

• Equal volumes of patient plasma and pooled plasma• Test the control and neutralized plasmas with known

incompatible panel cells at same method where original incompatibility was seen

• Control needs to be reactive, Neutralized will be non-reactive to confirm Chido/Rogers

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Chido/Rodgers:Neutralization

Original plasmareactivity

Control plasma reactivity

Neutralized plasmareactivity

1+ 1+ 0√

1+ 1+ 0√

2+ 2+ 0√

1+ 1+ 0√

1+ 1+ 0√

1+ 1+ 0√

1+ 1+ 0√

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Chido/Rodgers:Chemical Effects

Effect of Enzymes

Ficin / papain Sensitive

Trypsin Sensitive

α-Chymotrypsin Sensitive

Pronase Sensitive

Sialidase Resistant

DTT 200 mM Resistant

Acid Resistant

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JMH System• JMH became a system in 2000 after it was shown that the

JMH antigen is carried on the GPI-linked CD108 glycoprotein.

• Named after the first antibody producer, John Milton Hageno Also called ‘old boys’ antibody

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JMH System: GPI-Linked

• Glycosylphosphatidylinositol (GPI anchor) is a glycolipid that anchors proteins to the cell membrane.o Defects in the GPI anchors synthesis occur in diseases such as

paroxysmal nocturnal hemoglobinuria (PNH)

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JMH System: Chemical effects

Effect of Enzymes

Ficin / papain Sensitive

Trypsin Sensitive

α-Chymotrypsin Sensitive

Pronase Sensitive

Sialidase Resistant

DTT 200 mM Sensitive

Acid Resistant

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Knops System• Reported in 1970, Knops was established as a system in

1992 when the antigens were found to be located to CR1.• What is CR1?

o Complement receptor 1 (CR1) is a membrane-bound glycoprotein found on most blood cells, except platelets.

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Knops System:What is CR1?

• CR1 binds C3b and C4b and has an inhibitory effect on complement activation by classical and alternative pathways, protecting RBCs from autohemolysis.

• Erythrocyte CR1 is important in processing immune complexes by binding them for transport to the liver and spleen for removal from the circulation.

• As CR1 binds particles coated with C3b and C4b, it mediates phagocytosis by neutrophils and monocytes. The presence of CR1 on other blood cells and tissues suggests it has multiple roles in the immune response, for example, activation of B lymphocytes.

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Knops System: Disease Associations

• Low levels of CR1 on RBCs may result in deposition of immune complexes on blood vessel walls with subsequent damage to the walls.

• Knops antigens (CR1 copy number) depressed in SLE, PNH, hemolytic anemia, insulin-dependent diabetes mellitus, AIDS, some malignant tumors, any condition with increased clearance of immune complexes.

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Knops System:Important Phenotypes

Phenotypes (% occurance)

Caucasians Blacks

Kn(a+b-) 94.5 99.9

Kn(a-b+) 1 0

Kn(a+b+) 4.5 0.1

McC(a+) 98 94

Sl(a+) aka Sl1 98 60

Yk(a+) 92 98

Null: Some RBCs (e.g. Helgeson) type as Kn(a-b-), McC(a-), Sl(a-) and Yk(a-) because these RBCs have low copy numbers of CR1 (approximately 10% of normal).

Most recent antigen added 2005: KCAM+ causcasians:98%, West Africans 20%

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Knops System:Important note

• Anti-Sla is a common specificity produced by Blacks and initially may be confused with anti-Fy3 because most Fy(a-b-) RBCs are also likely to be Sl(a-).

Phenotypes (% occurance)

Caucasians Blacks

Sl(a+) aka Sl1 98 60

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Knops System:Reactivity

• Reactions

Effect of enzymes

Ficin / papain Weakened (especially ficin)

Trypsin Sensitive

α-Chymotrypsin Sensitive

Pronase Resistant

Sialidase Resistant

DTT 200 mM Sensitive

Acid Resistant

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COST• Also known as Cost-Stirling• Five of the original antigens from this collection are in the

Knops system because they are carried on CR1.• Csa has variable expression on RBCs from different people.

RBCs of approximately 12% Caucasians and 15% Blacks with the Yk(a-) phenotype are also Cs(a-).

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COST• Csa Most populations >98%

Blacks 96%• Csb Most populations 34%

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COST• Reactions

Effect of enzymes

Ficin / papain Resistant

Trypsin Resistant

α-Chymotrypsin Resistant

Pronase Resistant

Sialidase Resistant

DTT 200 mM Variable

Acid Resistant

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ComparisonFicin/Papain

Trypsin α-Chymotrypsin

Pronase

Sialidase

DTT 200 mM

Acid Other

Ch/RgSensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant

Can be neutralized with pooled plasma

JMH Sensitive Sensitive Sensitive Sensitive Resistant Sensitive Resistant

Knops Weakened (especially ficin)

Sensitive Sensitive

Resistant/weakened

Resistant Sensitive Resistant

COST Resistant

Resistant

Resistant

Resistant

Resistant

VariableResistant

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DTT Treatment• .2M DTT treatment denatures Knops and JMH, but also

many other antibodies not associated with high titer, low avidity reactivity. Be Careful!

• Other systems denatured:o Cromer, Dombrock, Ge3, Indian, Kell, Lutheran, Scianna, Yta

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Compatibility• Most red cell units will be incompatible• MVRBC recommends least incompatible red blood cells as

providing antigen negative blood is not feasible• These antibodies are not clinically significant. Present data

indicates these antibodies do not cause increased red cell destruction when incompatible blood is transfused or hemolytic disease of the newborn. o Transfusion. 2007 Jul;47(7):1290-5. Do patients with autoantibodies or clinically

insignificant alloantibodies require an indirect antiglobulin test crossmatch? By Lee, E. et al.

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Summary• They don’t always act like we thought they do, especially

that gel is used so often. And we don’t call them what we used to.

• Chemical treatments are usually more helpful than titration studies

• Identification relies heavily on exclusion of the other ‘usual suspects’

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Questions?Thank you!

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Good Articles for Reference• IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010. 30-

38. A review of the Chido/Rodgers Blood Group. By R. Mougey

• IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010. 2-7. The Knops blood group system: a review. By J.M.Moulds