WAHIA, DIHA, and CAD Flashcards
Why is DAT positive in WAIHA?
Patients auto antibody is coated RBC’s
Usually IgG is strong positive
C3 is present in 1/2 of patients
Why is a control done with positive DAT?
To ensure that spontaneous agglutination of antibody coated cells is not occurring.
Control can be saline, PBS, or 6% albumin
What enhances warm auto antibody reactivity?
PEG, solid phase, Gel, enzyme treated cells
Usually reacts in IAT phase after 37C incubation
What is primary or idiopathic WAIHA?
Occurs as primary condition with no underlying or associated disorder
What are secondary causes of WAIHA?
Lymphoma, lupus, CLL, and other auto immune disorders and chronic inflammatory conditions
What to drugs cause WAIHA>
methyldopa and procainamide
Why is elution done in WAIHA?
to remove antibody attached to RBC’s
Attempt to ID specificy to reactions > almost always panreactive but if some cells are negative could indicate an alloantibody
If eluate is negative = drug induced
Elution doesn’t need to be preformed on subsequent samples since is unlikely to change
How do you resolve abo discrepancies due to spontaneous agglutination?
Patients cells are heavily coated with IgG and aggluntinate with centrifugation.
Use glycine acid EDTA (EGA) or chloroquine to remove antibody
What is the difference between relative, broad, or apparent specificity
Broad = negative rh null cells Relative = c like or e like (strong with e positive cells) Apparent = single specificity (e, c, D, E, C, f) (patient will be positive for antigen with positive DAT)
What are common WAIHA specificities?
Usually Rh group
LW, U, Kell system, Ena, and Wrb have been reported
How do you remove autoantibody to detect allo antibodies?
Auto adsorption - not recently transfused
Homologous - phenotypically matched cell
differential - 3 sets of adsorptions are preformed to rule out alloantibodies
How do you preform auto adsorption?
Need large volume of patients RBC’s
Use ZZAP, WARM, or enzymes to remove bound antibody so that RBC;s can bind more antibody
Often 3-4 adsorptions are required to remove all antibody
What is ZZAP?
Ficin or papain (enzyme) + DTT
prepared in house
What is WARM?
commercial form of ZZAP
Why are enzymes generally not used?
Not as effective as WARM or ZZAP?
What are the common reasons auto adsorptions can fail?
Chemical treatment did not remove all of antibody
Chemical treatment altered binding sites so antibody can’t bind
Antibody may be alloantibody
What is the risk of performing allogenic adsorption?
Remove antibody to high prevalence antigen
How are allogenic adsorptions preformed?
3 sets of cells are used (R1R1, R2R2, rr) that are negative for K-, Fya-Fyb-, Jka-Jkb-, S-s-
Can enzyme treat to remove Fy(a-b-)
ZZAP removes K-
2 parts of RBC to 1 part of Plasma (PEG can be added)
What if adsorbed plasma is reactive?
May need additional adsorptions
Underlying allo if pattern exist
Enzyme or ZZAP removed autoantibody binding site - test raw serum against treated cells; no reactivity means repeat adsorption with untreated cells
What is DTT?
breaks sulfhydryl bonds
- 01M dispererse IgM cold autoagglutinins on RBC and differentiates IgM from IgG
- 2M denatures Kell, LU, DO antigens and used in ZZAP
What is in ZZAP?
Ficin and DTT
Commercial version is WARM
How does ZZAP work in adsorption?
DTT removes IgG bound antibody
Enzyme enhance binding of autoantibody
What else can ZZAP be used for?
Can be used in place of enzymes or DTT for ABID
Ex) Denature Kell antigens when high frequency is suspected
What is AET (2-aminoethylisothiouronium bromide?
Like DTT it destroys disulfide bonds and can be used to create Ko cells
What does EGA (EDTA glycine-acid) do?
Dissociates bound IgG so that cells can antigen typed
If transfused recently - cell separation 1st
Perform DAT on treated cells to verify
Inactivates Kell, Bg, and Era
Does not denature MNS or Duffy
What is CDP used for?
Dissociate cell bound IgG to phenotype cells
Removes Bg antigens
What is RESt used for?
Rabbit Erythrocyte stroma - rabbit RBC’s have I and B
Can be used in recently transfused to adsorb cold agglutinins. Can’t use plasma for abo typing.
Removes I, H, B and P1 antibodies
What chemicals can be used to remove autoantibody from autologous RBC’s?
gentle heat elution or partial heat elution
ZZAP = papain and DTT
EGA = EDTA Gylcine Acid
Chloroquine diphosphate - CPD
Protylotic enzymes such as ficin can increase antibody bind for adsorption
What antigens are destroyed by enzymes?
Duffys MNS Ena Ge JMH Ch/Rg Inb Ex) auto anti-Ge2 will not be adsorbed out
How do you QC enzyme treated cells?
enzyme treated cells should be positive with G. max var soja and untreated cells should be non reactive
What are some characteristics of a benign cold auto agglutinin?
reacts < 25C Titer at 4C < 64 titer at 22C < 16 Autoantibody rarely hemolyzes enzyme-treated red cells in vitro using acidified serum Auto antibody is polyclonal DAT is weak positive or negative for C'
What are the characteristics of pathological CAD?
titer at 4C is often > 1000
Antibody reacts at 4C but also at 25-34C
DAT positive due C’
What test is used to diagnosis pathological CAD?
Thermal amplitude test
Serial dilutions of patient’s serum/plasma in saline (see notes for special collection procedures) are tested at (37 C, 30 C, RT, 4 C) against I+ adult cells, cord cells, and autologous cells.
What may be done of patient has hemolytic anemia but titer at 4C is not signicficant?
Repeat the titer at 30 C using 30% albumin as the diluent instead of saline. Agglutination reactions at 30 C in albumin seem to correlate best with the presence or absence of CHD.
What is the pathology in CAD?
Auto antibody is IgM that binds to RBC’s at colder temps; C’ is activated and intravascular or extravascular hemolysis can occur
What are symptoms of CAD?
Weakness, pallor, weight loss, jaundice, Raynaud phenomenon and hemoglobinuria
What are the characteristics of idiopathic cold AIHA?
People usually experience symptoms in the winter that resolve in the summer
Mono clonal IgM
Usually anti-I specificity or anti-Pr
Rarely assoc. with severe anemia
What diseases may cause CAD?
Chronic lymphocytic leukemia
Lymphoma
B-cell neoplasms
Histiocytic lymphoma
What disease may cause secondary CAD?
Following infections
most common is Mycoplasma pneumonia and infectious mononucleosis
In children= after mumps, measles or chicken-pox
What are the characteristics of CAD following mycoplasma pneumonia?
Auto anti-I that is polycolonal (unlike idiopathic CAD)
Rarely causes hemolysis and resolves in 2-3 weeks
Thought that M. Pnuemonia alters I antigen on RBC’s