Download Antibodies Formerly Known as - Mississippi Valley Regional

Antibodies Formerly Known as
‘High-Titer, Low-Avidity (HTLA)
Antibodies’ : a review.
Jackie Ensley, MLS(ASCP)SBB
Mississippi Valley Regional Blood Center
1
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
2
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.
3
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
4
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 sure
o 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
_____ Classification
o 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 hospitals
o Note: the second arrow
shows the lab will describe
the reactivity of the
antibody
Example 5
• Antibody of
Undetermined Specificity
o Not HTLA antibodies
o 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’
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.
11
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.
12
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√
Titer:
128
13
0√
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
14
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
15
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
16
Example
D
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+
17
Antibodies with HTLA Activity
•
•
•
•
Chido/Rodgers System
JMH System
Knops System
COST Collection
18
Chido/Rodgers System
• Anti-Ch was reported in 1967 and anti-Rg was reported in
1976
o 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.
19
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
20
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.
21
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% albumin
o 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 nonreactive to confirm Chido/Rogers
22
Chido/Rodgers:
Neutralization
Original plasma
reactivity
Control plasma
reactivity
Neutralized plasma
reactivity
1+
1+
0√
1+
1+
0√
2+
2+
0√
1+
1+
0√
1+
1+
0√
1+
1+
0√
1+
1+
0√
23
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
24
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
Hagen
o Also called ‘old boys’ antibody
25
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)
26
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
27
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.
28
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.
29
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.
30
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%
31
Knops System:
Important note
• Anti-Sla is a common specificity produced by Blacks and
initially may be confused with anti-Fy3 because most Fy(ab-) RBCs are also likely to be Sl(a-).
Phenotypes (%
occurance)
Sl(a+) aka Sl1
Caucasians
98
32
Blacks
60
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
33
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-).
34
COST
• Csa Most populations
Blacks
• Csb Most populations
>98%
96%
34%
35
COST
• Reactions
Effect of enzymes
Ficin / papain
Resistant
Trypsin
Resistant
α-Chymotrypsin
Resistant
Pronase
Resistant
Sialidase
Resistant
DTT 200 mM
Variable
Acid
Resistant
36
Comparison
Ficin/
Papain
Trypsin
αChymot
rypsin
Pronase Sialidas
e
DTT 200 Acid
mM
Ch/Rg
Sensitive
Sensitive
Sensitive
Sensitive
Resistant
Resistant
Resistant
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
Variable
Resistant
37
Other
Can be
neutralized
with
pooled
plasma
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
38
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.
39
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’
40
Questions?
Thank you!
41
Good Articles for Reference
• IMMUNOHEMATOLOGY, Volume 26, Number 1, 2010. 3038. 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
42