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
What are the characteristics of CAD following infectious mononucleosis?
Auto anti-i, remains up to 12 weeks
Hemolysis not likely
What are the treatments for CAD?
treat underlying condition
Steriods and spleen removal not useful
Can try plasmapheresis but not for chronic conditions
What are the problems with using plasmapheresis to removal cold antibody.
IgM is primary intravacular so 80% will be removed but will be replaced in 5 days
must do in heated room, fluids warmed, and keep patient warm. Difficult and expensive.
Which clinically significant alloantibodies have been reported to be removed by RESt?
Anti-D, -E, Vel and IgM antibodies regardless of blood group specificity
What antibody causes PCH?
auto anti-P IgG biphasic hemolysin
What patients is PCH commonly seen in?
Commonly seen in children following febrile illness; diseases such as measles, mumps, chicken pox, infectious mono and illnesses with flu-like symptoms and upper respiratory infections
What are the symptoms of PCH?
Fever, chills, shaking, malaise, abdominal cramps, back pain
Intravascular hemolysis: Hemoglobinemia, Hemoglobinuria, Bilirubinemia, Severe anemia
What is the DAT result of PCH?
Positive with C’
IgG binds with complement in colder areas of the body, but dissociates from red cells as it circulates to warmer areas. Complement is irreversibly bound, so it remains attached.
What is the antibody screen in PCH?
Usually negative unless high titer
What test diagnosis PCH?
DL or Donath Landsteiner test
What is the principle of the Donath Landsteinter test?
Red cell destruction is due to a cold IgG autohemolysin which binds to patient cells at low temperatures and fixes complement. Hemolysis occurs when the body temperature increases to 37 C
How is DL performed?
Two tubes of blood are collected without anticoagulant; one is placed immediately at 37 C and kept there (control) and the other is placed 1st in a melting ice bath for 30 minutes and then placed at 37 C for 30 minutes. Both tubes are then centrifuged and checked for hemolysis. Hemolysis in the biphasic tube and absence of hemolysis in tube maintained at 37 is a positive D-L test.
What is the alt. method for DL test?
This test method calls for the addition of pooled normal sera as a source of complement in the test system. Active complement is essential for demonstration of the D-L antibody. Because patients with PCH may have low levels of complement, fresh normal serum may be needed in the test system as a source of complement. The patient’s blood should be allowed to clot at 37 C before using to avoid the loss of antibody by autoadsorption before testing.
What is the treatment of PCH?
Supportive care - treat underlying condition
If transfusion required, use blood warmer and transfuse slowly
In severe chronic cases may use rare P negative units
What is the cause of Paroxysmal Nocturnal Hemoglobinuria (PNH)?
Acquired hemolytic anemia caused by a red cell membrane abnormality
Membrane defect due to deficiency of complement regulator proteins:
1) DAF (decay accelerating factor) which accelerates the spontaneous decay of C3 convertase enzyme for both complement pathways
2) HRF (homologous restriction factor) which regulates the protein that inhibits complement mediated lysis by c5b-9 complex
What are the clinical features of PNH?
Classically presents with hemoglobinuria, most noticeable in first morning urine
Hemolytic episodes can be triggered by infections, surgery, administration of iron drugs
Intravascular thrombi a major complication
Pancytopenia and marrow hypoplasia
Urine hemosiderin
What is the screening test for PNH?
sugar-water test
Sucrose provides a medium of low ionic strength that promotes the binding of complement, especially C3, to rbc membranes
PNH cells will lyse; normal cells unaffected
What is the confirmatory test for PNH?
Ham’s test (acidified serum lysis test)
Complement will affix to rbc’s at a slightly acidic pH; this activates complement via alternative pathway to enhance binding of C3 to cell membranes resulting in cell lysis
What is the treatment for PNH?
Chronic disease – survival average 10 years after diagnosis Treat symptoms Iron for deficiency Corticosteroids may be helpful Anticoagulant therapy Androgens for bone marrow hypoplasia
Does a positive DAT with non reactive eluate indicate a DIHA?
If an eluate from DAT positive red cells is nonreactive, it is more likely to be due to reasons other than drug antibody - 80% of DAT positive samples yield nonreactive eluates
What drugs are known to cause DIHA?
Cephalothin/Cephalosporin/Penicillin Quinidine/Piperacillin Keflin/Tazobactim Methyldopa Cefotetan/Cefriaxone
What is the theory behind DIHA?
drugs (haptens) bind to cell membranes and antibodies may be made to:
1) the drug itself, producing in-vitro reactions typical of a drug adsorption reaction (penicillin type);
2) membrane components, producing in-vitro reactions typical of autoantibody production
3) part-drug, part-membrane components, producing an in-vitro reaction typical of the so-called immune complex mechanism.
What are the two categories of Drug induced antibodies
Drug dependent and drug independent (autoantibody like)
What are the two categories of drug dependent antibodies?
Reacts with drug treated cells and reacts in presence of drug in test system
What is the mechanism of penicillin and second and third generation cephalosporins, including cefotetan DIHA?
Drug (hapten) is adsorbed onto RBC’s and antibody is formed to penicillin. Positive eluate and antibody panel only seen with drug treated cells.
Commonly caused by large dose of IV penicillin
What is the mechanism of quinidine, piperacillin, and cefotetean DIHA?
Immune complex - antibody reacts in presence of drug in test system
Drug + antibody = complex +RBC =complex on RBC+ complement = RBC destruction
What are the serological findings of drug adsorption?
DAT+ (IgG only)
Eluate negative
IAT negative
Hemolysis develops gradually and if cell destruction occurs, it is extravascular
Must coat some cells with drug and test eluate or plasma with drug coated cells to confirm
What are the serological findings of immune complex DIHA?
DAT+ Complement only
Elution not indicated
IAT negative – antibody binds to drug, not rbcs
How would you prove it is the drug?
Antibody forms a complex with the drug
Obtain sample of the drug, grind it up or get in to some kind of solution, add patient’s serum, incubate.
Add screening cells or other known DAT negative cells. Let it sit, spin and look for hemolysis.
If there is hemolysis, then it can be due to the drug
What is the mechanism of Kefline, tazobactium, and Cephalosporins/ cephalothin DIHA?
Membrane modification - drugs cross react with penicillin. That is they will be positive with penicillin coated cells. This means there is non-immune adsorption of proteins onto the RBC. This is the now called NIPA = non immunologic protein adsorption
What are the serological findings in membrane modification?
DAT+ (can be IgG, C’, IgA, Albumin or any protein)
Eluate positive with drug coated cells
IAT negative
What is the theory of drug independent DIHA?
Drug forms a neoantigen on RBC. Antibody formed has cross reactivity with RBC and drug. Will react in absence of drug.
What drugs have been implicated in drug independent autoantibody production?
Methldopa (aldomet) - BP drug rarely used L-dopa (levodopa) Procainamide Fludarabine (fludara) - used in CLL Mefenamic aid (ponstel)
What is the serological picture for drug independent autoantibody ?
DAT will have IgG only some with C3.
IAT will be positive.
Antibodies similar to WAIHA.
Stop drugs, hemolysis stops in a few days but the DAT can be + for up to 2 years. IAT negative in a few weeks.
What can’t Piperacillin-treated (PT) RBCs be used for detection of antibody?
90% of donors and 49% of random patients will agglutinate Piperacillin-treated (PT) RBCs. They have a naturally occurring IgM antibody
How can you detect piperacillin antibodies?
Serum + piperacillin + untreated/enzyme-treated RBCs
Only anti-piperacillin in patients with immune hemolytic anemia will react
How sensitive is the routine DAT test?
DAT can detect 100 to 500 molecules of IgG/red cell and 400 to 1100 molecules of C3d/red cell.
What are causes of positive DAT?
HDFN
Autoantibodies
Hemolytic transfusion reaction
Passive antibodies from donor plasma, IVIG, or other derivatives
Nonspecific adsorb proteins (drugs or hypergammaglobilinemia)
C’ activation due to infection or autoantibodies
Donor lymphocytes from organ or bone marrow transplant produce antibodies
Why is EDTA sample preffered for DAT testing?
EDTA chelates Ca needed for C’ activation
What kind of hemolysis is caused by immune complex DIHA?
Intravascular; only small amount of drug required for secondary exposure. Can cause DIC or renal failure.
What kind of hemolysis is caused by drug adsorption DIHA?
Extravascular; usually gradual
What is principle of DAT test?
Detect in-vivo attachment of IgG or C
Anti-IgG or anti-C3d 2 Fab sites bind to FC portion that is attached to RBC’s
What happens if you delay adding anti-IgG reagent after washing?
False negative due IgG eluting off
Why must a negative control be performed with positive DAT?
To rule out spontaneous agglutination
How soon after transfusion may a patient be immunized?
Primary immunization may occur within 7-10 days after transfusion while secondary response is within 2-7 days
What is clinical significance of antilymphocyte globulin (ALG) or antithymocyte globulin (ATG)?
High titer heterophile antibody in these products will give you a positive DAT within days of injection. These were previously used for kidney transplant patients.
- Horse serum will neutralize the heterophile antibodies in the AHG
- Low ionic polycation (LIP) technique is negative when this is present.
- Panel can look like anti-Lub
What kind of hemolysis is seen in WAIHA?
Extravascular primarily in the spleen
What IgG subclasses causes the most RBC destruction in WAIHA?
IgG1 and IgG3
What are the risk associated with transfusing pateints with WAIHA>
- Stimulate alloantibody
- Increase Autoantibody hemolysis
- Depress compensation erythropoiesis
- Destruction of transfused cells may increase hemoglobinemia and hemoglobinuria
- Transfused cells may be destroyed more rapidly than the patient’s own
What are the treatments for WAIHA?
Corticosteriods - Prednisone, danazol, glucocorticoid steroids
Splenectomy
Immunosuppresion drugs
- Cyclophosphamide or Imuran (azathioprine)
Plasma exchange - Removes antibody
Filtration of plasma - Plasma filtration with immunadsoption columns containing protein A will temporarily relieve the level of plasma IgG.
What are the most common cause of a negative DAT In AIHA?
Red cell bound IgG below the threshold of the DAT
IgM and/or IgA not detectable by DAT
Low affinity IgG was washed offAT
What are some reasons to perform adsorption?
Rule out allo’s if high incidence antibody
Adsorb out anti-A/B for rare antisera
Confirm weak antigen by absorb/eluting
Confirm anti-G
What are some strategies to phenotype a recently transfused patient?
Obtain a pretransfusion sample
Retic typing - microhematocrit centrifugation
Molecular typing
If not Rh negative examine 3-4 Rh antigens for MF
How is PEG useful in adsorptions?
- Reduce number of adsorption = less dilution of alloantibody
- Enhances adsorption and reactions with adsorbed plasma
- Remember that PeG-adsorbed plasma can only be tested using anti-IgG
What are the controls used for adsorptions?
BEFORE adsorption, test the cell selected for adsorption with the unadsorbed serum/plasma to ensure reaction
AFTER adsorption, ensure that the serum/plasma is NOT reactive with the adsorbing cell (known as the “test-back”).
Test by same technique that will be used for testing the adsorbed plasma.
Perform DAT on EGA or CPD treated cells
What is a heat elution?
56C or 45C - Use existing waterbath with temperature adjustment.
Best for removal of ABO antibodies (56C).
Can be used to remove some of coating antibody in WAIHA patient to allow for limited phenotyping (45C).
What is the Lui Freeze thaw?
-20C; best for detecting ABO antibodies
What is the ultrasound elution?
Uses an ultrasound waterbath – often found where glassware is cleaned.
High-frequency sound waves cause generation of minute gas bubbles. As they grow, they cause shock waves that “shake” antibodies off the cell.
Best for removal of ABO antibodies
What are some examples of chemical elutions?
Chloroform or Chloroform/Trichloroethylene Carcinogenic, toxic Xylene Carcinogenic, flammable Ether Highly flammable, toxic Dichloromethane (Methylene Chloride)- DCM Non-flammable, relatively non-toxic
How does current commercially available kits work?
Uses a low-ionic strength wash solution to keep antibody on the cells.
Uses a Glycine solution (pH 2.0) followed by a buffer to return pH to normal.
How do you test the last wash?
IAT or Test using AHG + IgG-coated red cells (Check cells)
- Positive reaction = wash good
- Negative reaction = insufficient wash; IgG was present to neutralize
