10. AIHA Flashcards
the first clue to an autoAb
DAT+ with plasma Abs reactive with ALL cells tested
important to obtain patient’s … (3) in autoAb investigation
- diagnosis
- transfusion hx
- medications
clinical s/s that point to AIHA (6)
- Normocytic or macrocytic anemia
- Reticulocytosis
- ↓ haptoglobin
- ↑ lactate dehydrogenase
- ↑ bilirubin
- DAT+
cold AIHA often associated with… (4)
- Pneumonia (Mycoplasma pneumoniae)
- Anemia
- Lymphoma
- Viral infection
DAT+ specificity
trxn
IgG with specific Ab
DAT+ specificity
WAIHA
IgG and/or C3
DAT+ specificity
CAIHA
C3
DAT+ specificity
drug interaction
IgG
serum may be nonreactive
DAT+ specificity
Clot tube stored at 4°
C3
no serum Ab
DAT+ specificity
HDFN
IgG
ABO or alloAb from mom
CAIHA phase of rxn
IS
strong reactions may carry over to AHG
may cause an ABO discrepancy
cold autoAb
IgM found in normal healthy individuals against I or H antigens
benign cold auto Ab
anti-I more common
benign cold auto titers
<64 at 4°
benign cold max temp
RT
benigns autos are NR with…
I= (cord and adult i) cells at RT
anti-H most common in —– type people
A1 type
least amount of H antigen present
occasional clinical manifestation of high-titer anti-HI benign cold autos
group O cells will have decreased red cell survival after transfusion
O cells have the most H antigen
3 pathologic cold autoAb conditions
- CAIHA
- Cold Agglutinin Syndrome (CAS)
- Paroxysmal Cold Hemoglobinuria (PCH)
characterized by agglutination, at room temp, of red cells in an EDTA specimen
CAS
CAS etiology
underlying disorder
infection (eg, Mycoplasma pneumoniae or Epstein-Barr virus)
autoimmune disorder
lymphoid malignancy
CAS type of anemia
extravascular
investigation of CAS is difficult because agglutinins are active at refrigerator temp
how do we cirumvent?
The blood tubes need to be prewarmed and samples must be transported at 37C.
CAS resolution
usually self-limiting when underlying condition is resolved
—- anti-H is CS (Bombay)
allo
CAS
DAT
eluate
titer
specificity
C3 only
eluate NR
>1000 at 4°
anti-I
treatment for severe CAS cases targets…
C1
PCH population
children
acute transient condition secondary to infection
biphasic hemolysin
definition
PCH IgG
binding to RBCs occurs at low temps, but hemolysis does not occur until C3-coated cells are warmed to 37°
affects the extremities
PCH
diagnostic test for PCH
Donath-Landsteiner test
PCH
DAT
eluate
IAT
specificity
DAT+ with C3 only
eluate NR
IAT=
anti-P
PCH sx
anemia, frequently marked
hemoglobinurea
resolve ABO discrepancy once cold autoAb is IDd
prewarm technique
steps of prewarm technique
- incubate plasma and reagent cells at 37° for 15 min
- two drops plasma added to reagent cells
- incubate for 30 mins w/o potentiator
- if AHG, warm saline must be used for wash
methods to eliminate cold autoAb after ID
sulfhydryl reagents (DTT and 2-ME) denature IgM pentamer
rarely used to resolve cold autoAb
adsorption at 4°
transfusions for benign cold autoAb pts
XM compatible by prewarm
and/or
use of IgG AHG reagent
transfusions for CAS pts
transfusion should not be withheld due to serologic incompatibility
transfuse smallest volume possible
transfusions for PCH pts
randomly selected units of blood
if pt does not response adequately, use P= units
WAIHA lab findings (6)
- ↓ H&H
- ↑ MCV
- ↓ haptoglobin
- ↑ bilirubin
- ↑LDH
- reticulocytes, spherocytes, nRBCs, fragmented RBCs
polychromasia
symptom of WAIHA
bluish tint to RBCs
WAIHA etiology
secondary to disease state (Hodgkins; CLL; SLE; viral infection; immune deficiencies; GI autoimmune disease)
idiopathic
hallmark of WAIHA
DAT+
mostly IgG and C3
WAIHA with —- + are more hemolytic and harder to tx
C3d
WAIHA pts may have a +/= ——, because…
ABS
at low titer, all autoAb is adsorbed
DAT+ before ABS+
warm autoAb is detectable in plasma after…
titer rises and it spills into plasma from saturated RBCs
panagglutinin in AHG
warm autoAb
main problem warm autoAb poses to BB
panagglutinins “cover” underlying alloAb
overview of BB investiation into warm autoAb (8)
- pt hx check (dx may establish link between serological findings and primary disease)
- TS
- ABID
- DAT
- elution
- techniques to remove Ab
- Ag type
- XM
ABO grouping usually not affected by…
warm autoAb
if ABO is affected, it’s with VERY strong DAT+
cannot interpret ABO if…
autocontrol is +
DAT+ may cause false pos Rh results when…
anti-D reagent has a high protein content
contains molecular additives
“slide and modified tube anti-D”
most BBs use a low protein anti-D
using a slide and modified tube anti-D requires…
Rh control with every test
contains all additives, but no anti-D
most BBs use ——— anti-D for Rh typing
monoclonal blend
IgG and IgM, from multiple clones for partial Ds
monoclonal blend anti-D only requires control if…
pt is AB+
control is by manufacturer, or 6% albumin
weak-D testing is invalid when DAT+ because…
weak-D control is +
why must specimens for DAT be collected in EDTA tubes?
EDTA binds Ca2+
C3 requires calcium
C3 would otherwise bind normal cold agglutinins in the fridge
2 causes of rare AIHA with DAT=
- low levels of IgG
- IgM or IgA cause
next step after DAT+ with IgG+
eluate
eluates should always be performed if…
pt transfused in last 3 weeks
alloAb may ONLY be present on transfused cells in first 3 weeks
eluate pattern with WAIHA, DIIHA, and HTR
WAIHA —panagglutination
DIIHA —strong DAT, but no ABID rxns
HTR —specific alloantibody pattern
if a warm autoAb has a specificity at ABID stage…
run a selected cell panel for autoAb
How do we know that the Ab identified at ABID is an allo or auto Ab?
Ag typing
If pt =, it’s an allo
If pt +, it’s an auto
cannot Ag type on patients…
transfused in last 3 months
blood for patients with specific autoAbs
evaluated for need for Ag= blood on a case by case basis
technique of choice if patient has panagglutinin, assuming…
autoadsorption
not transfused w/i last 3 months
purpose of autoadsorption
remove autoAb to reveal underlying alloAb
autoadsorption procedure
- mix equal volumes cells and plasma, incubate at 37° (may use enzymes, PEG, ZZAP)
- mix autoadsorbed plasma with additional aliquots of pt cells until all autoAb removed
- test adsorped plasma for alloAb
usually takes the same number of repititions as the rxn strength
ficin and papain are good for autoadsorption when…
strength is W+ or 1+
ZZAP
DTT + papain
elutes IgG from cells, opening up more space for adsorption
volume PEG in autoadsorption
same as volume of patient plasma
drawbacks of autoadsorption (4)
- cannot be performed on patient transfused in last 3 months
- residual saline from RBC washes dilutes weak Ab
- ZZAP destroys Kell (anti-K not removed from plasma)
- requires large volume of RBCs, and patients are usually anemic
indications for allogeneic adsorption
- pt transfused in last 3 months
- pt cells in short supply
principle of allogeneic adsorption
donor or reagent RBCs of known phenotypes are used to remove autoantibody and selective alloantibodies
allogeneic adsorption procedure
- select 3 donor cells of known phenotypes
- wash them
- mix with patient plasma in equal volumes, adsorb at 37°
- each adsorped plasma is added to more aliquots of cells until all autoAb reactivity is gone
- underlying alloAb remains in at least one of the plasmas
use same enhancement techniques as with autoadsorption
antibody against —— are difficult to find using allogeneic adsorption
high freqs
ex) anti-U
drawbacks of allogeneic adsorption (3)
- hard to find high freqs
- saline from washings dilutes weak alloAb
- differential cells difficult to find
cells may be available from reference lab
problem with Ag typing in patients with warm autoAb
IgG coating cells may interfere with Ag typing
false positives occur during Ag typing when…
resolution
pt has DAT+
elution to remove IgG from cells prior to Ag type
delay Ag type until time when pt becomes DAT=
cannot use —— to render cells DAT= for Ag typing
appropriate methods (3)
acid elution—denatures RBC antigens
- EGA
- gentle heat elution
- chloroquine disphosphate treatment
how to remove IgM (cold auto) from cells for Ag typing
warm saline washes
sulfhydryl reagents
WAIHA general tx
- corticosteroids (prednisone)
- immunosuppressive drugs
- splenectomy (rare)
transfusions should be avoided for ——- because… (4)
WAIHA patients
- compatible blood usually not available
- transfused blood will not survive
- transfusions stimulate alloAb production
- transfusions may actaully increase anemia over time (↓ signal for erythropoiesis)
transfusion criteria for WAIHA pts (4)
- Ag= for corresponding alloAb
- compatible blood if autoAb has a simple specificity (ie. anti-e)
- units compatible with adsorbed plasma (incompatible with neat plasma)
- phenotypically matched RBCs (very hard to find)
mixed type AIHA
prevalence
IgM and IgG present
7-10% cases
severe hemolysis, very low H&H, responds well to steroid tx
mixed type AIHA
ABS/panel reactive at IS and AHG
mixed type AIHA
mixed type AIHA associated with…
SLE
CLL
DIIHA
hemolytic anemia occuring when drugs induce formation of Ab against drug or RBC antigens
tx for DIIHA
d/c drug
3 categories of DIIHA
- WAIHA-like DIIHA —Ab to RBC membrane components
- Drug adsorption DIIHA —Ab to drug bound to RBC
- Immune complex DIIHA —Ab to part drug and part RBC membrane
drugs act as ——, inducing an immune response by binding to RBC
haptens
HTN drug for pregnant women causing DIIHA
methyldopa
drug adsorption DIIHA causes —– hemolysis
extravascular
drug adsorption
DAT
ABS
eluate
DAT strongly + due to IgG, rarely C3
ABS=
eluate NR
requires RBCs to react
drug adsorption DIIHA lab confirmation procedure
- incubate O cells with drug
- wash
- test pt plasma or eluate with drug-coated RBCs
- observe for agglutintion or hemolysis
rarely done; usually only in reference lab
immune complex DIIHA causes —— hemolysis
severe intravascular
DIIHA with hemoglobinuria/emia
immune complex
immune complex
DAT
eluate
ABS
DAT+ due to C3, rarely to IgG
eluate NR
ABS=
reactivity only in presence of drug
immune complex DIIHA lab confirmation procedure
- mix pt serum + normal serum + drug
- mix control — pt serum + normal serum + saline
- test O RBCs with mixtures
- observe for agglutination
purpose of normal plasma included in mixtures for immune complex DIIHA workup
provides complement
drug that demonstrates multiple DIIHA mechanisms
cefotetan
drug-dependent
DIIHA where drug must be present in test system to detect
nonimmunological protein adsorption
drugs may cause DAT+ by altering RBC membrane so plasma proteins bind nonspecifically
all proteins —Ig, complement, albumins etc
multiple myeloma drug that can cause DAT+
why?
how to resolve?
darzalex (daratumumab)
anti-CD38, which is present on RBCs (but more so on myeloma cells)
DTT destroys CD38 on RBCs
you probably have autoAb if… (3)
- DAT+
- autocontrol +
- rxns with all cells showing similar strength
you probably have alloAb if… (4)
- DAT +/=
- autocontrol +/= (recent transfusion)
- prior immunization
- variation in rxn strength
you may have multiple specificities if… (3)
- all cells reactive
- variation in rxn strength
- variation in phase
you may have a trxn sample if… (3)
- DAT+ mf
- eluate exhibits Ab with specificity
- previous specimen with ABS=
frequencies of WAIHA, CAS, and PCH
70-80%
18%
<2%
peak age for WAIHA, CAS, PCH, and benign
60s
60s
children
adults
primary Ig class for WAIHA, CAS, PCH, and benign
IgG
IgM
IgG
IgM
Ab specificities for WAIHA, CAS, PCH, and benign
Rh, Kell
Ii
P1
Ii, H
disease associations for WAIHA, CAS, PCH, and benign
malignancy, autoimmune, HIV
epstein-barr, M. pneumo, lymphoproliferative
viral infection, syphilis
none
tx for WAIHA, CAS, PCH, and benign
block spleen, steroids, drugs, surgery
tx sx
supportive
none
drug-dependent DIIHA
immune complex
drug-adsorption
drug-independent DIIHA
autoantibody formation
drugs causing immune complex DIIHA
piperacillin
ceftriaxone
drugs causing drug-adsorption DIIHA
penicillin
1st gen ceph (Keflex, Keflin)
drug causing nonimmune protein adsorption
Keflin
drugs causing autoAb formation
methyldopa
2nd and 3rd gen ceph
fludarabine
requirements for 3 cells used to allogeneically adsorb
- at least one must be negative for all CS Ags, so the alloAb is left in at least one plasma
- R1R1, R2R2, and rr
