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Warm Auto Immune HA Flashcards

1
Q

What is immune HA

A
  • shortened RBC survival mediated through the immune response
    -when RBC are destroyed prematurely
    -hemolysis occurs due to AB or complement or both
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2
Q

What is the role of complement

A

-serum proteins that interact with each other
to initiate complement dependent cell mediated lysis.

-involvement of complement can be confirmed with a positive direct antiglobulin test C3d

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3
Q

What is intravascular hemolysis

A

-hemolysis that occurs in the vascular system because of the complement activation

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4
Q

What is Igm

A

-pentameric structure
-single molecule can initiate complement from the classical pathway

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5
Q

What is IgG

A

-activate complement based on factors like subclass, number of locations of IgG molecules , physical location of Red cell AG and avidity

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6
Q

What is extravascular hemolysis

A

-phagocytosis of red cells within the mononuclear phagocyte system (MPS)
-involves the spleen and the liver
-red cells that are coated with AB interact with the Fc receptors lining the splenic cord causing partial or complete phagocytosis

-if the cell cant repair itself = partial spherocytic are formed
-unlike RBCs. spherocytes lack deformability and get trapped in the spleen and those that escape are seen in the PBS

-Cells sensitized by IgG and C3b/C3d are removed in the liver and spleen

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7
Q

How does Autoimmune Hemolytic Anemia occur

A

-when pt immune system does not recognize its own red cell AG as “self”
-mechanisms to control autoreactive AB are lost so the AB become directed against pt red cells which causes the pt to produce autoAB that bind to their own red cells.

can be primary or secondary to another disease

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8
Q

What are the major types of AIHA

A
  1. Warm Autoimmune Hemolytic Anemia
    (In 70% of cases, autoantibodies have an optimal temp of 37˚C)
  2. Cold Agglutinin Syndrome or Cold Hemagglutinin Disease (autoantibodies have an optimal temp of 4˚C and 25˚C to 31˚C)
  3. Paroxysmal Cold Hemoglobinuria (PCH)
  4. Drug-Induced Hemolytic Anemia (12% cases)
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9
Q

What is Warm Autoimmune Hemolytic Anemia

A

-can be idiopathic without any underlying disease
-can be due to condition or medication (secondary)
-mild to severe
-most are IgG AB
-extravascular hemolysis occurring in the spleen
-gradual onset , signs and symptoms will not appear unti anemia has set in
-Rh specificity (Anti-e)

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10
Q

Disorders associated with WAIHA

A

-Lymphoproliferative disorders such as CLL, Hodgkin’s disease, Non-Hodgkin’s Lymphoma, MM, and Waldenstrom’s Macroglobulinemia.
* Autoimmune disorders such as Lupus and Rheumatoid arthritis.
* Neoplastic Disorders include solid ovary, breast, and pancreas tumours.
* Viral infections such as Hep A and Hep B
* Chronic inflammatory disorders

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11
Q

What will you see in WAIHA when in the lab

A

-lyses RBC with HGB released
-hemoglobinuria in the urine

need to tell the difference between hemoglobinuria and hematuria where the RBCs are intact and lysis has not occured

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12
Q

CBC Results in AIHA

Cold AIHA

A
  • Decreased RBC
  • Increased MCV
  • Rule of 3 violation
  • Very increased MCH
  • Very increased MCHC
  • PBS: agglutination
    present
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13
Q

CBC Results in AIHA

Warm AIHA

A
  • Normal MCV
  • Rule of 3 acceptable
  • Very increased MCHC
  • PBS: spherocytes and increased polychromasia
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14
Q

What are the serological problems of WAIHA

A

-AutoAb in serum can mask alloAB because it reacts with all the cells and is usually directed against an AG that is found on all the cells
-PANAGGLUTINATION
-can be specific for an high incidence AG (can get hard to find units for pts)
-the pts Red cells are coated with the autoAB which can interfere will testing like phenotyping

if your auto is positive (do DAT see why its positive and if DAT is positive then it means that AB is attached to red cells which is why you would have to do a differential test)

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15
Q

how do you Investigate and Detect a WARM Autoantibody

A

Antibody Screen is positive in MTS and IAT tube method
* Possible Reverse grouping anomaly
* If you suspect a Warm, Set up a panel with an Auto Control and DAT testing

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16
Q

What is Antiglobulin testing

A

-AB are gamma globulins
-An antibody to gamma globulin can form bridges between red cells sensitized with the antibody and cause them to agglutinate
-. Because some IgG and IgM antibodies can also cause C3 to attach to the red cells, Polyspecific AHG serum also contains Anti-C3

Reagent will be
IgG (Anti-IgG) and Complement (Anti-C3d, Anti-C3b)

-this test is important because it can detect IgG AB and complement proteins that are attached to red cells in vitRO or viVO

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17
Q

The Antiglobulin Test (Coombs)
DAT vs IAT

A

DAT-Detects Antibody or Complement that has attached to the Red Cell membrane in VIVO

IAT-Detects Antibody or Complement that has
attached to the Red Cell membrane in VITRO
plasma + screening cells

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18
Q

What is Direct Antiglobulin Testing

A

-Detection of IgG or complement that is bound to RBCs in VIVO
-AHG added after RBCs are washed
-NO INITIAL INCUBATION
-Agglutination = IgG or complement attached to RBC

In Normal circumstances, red cells are not sensitized with IgG/C3.

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19
Q

What is Indirect Antiglobulin Testing

A

-Detection of IgG or complement that is bound to RBCs in VITRO
-2 steps in procedure
-ABs (in serum/plasma) are incubated at 37˚C with RBC
Antigens in vitro.
* RBC suspension is washed
* Combined with AHG reagent to detect agglutination

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20
Q

Steps of DAT just know difference between 2

A

Wash 5% cells, add Polyspecific AHG after washing, centrifuge, and read macroscopically.
* Confirm all negative reactions under the microscope
-Sit for 5 minutes, read macroscopically and under the microscope. read again because complement like C3 gets enhanced after 5 mins
* Add Coombs Control to negative tubes only.

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21
Q

Steps of IAT

A

Add patient plasma/serum to known cells, read at IS, 37˚C, and AHG.
* Centrifuge and read macroscopically at IS.
* Incubation required
* Incubate at 37˚C and read macroscopically.
* Wash cells, Add Polyspecific reagent read macroscopically and under the microscope.
* Add Coombs control to negative tubes only.

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22
Q

What are IgG sensitized cells

A

-control system to check when the AHG results are negative (no agglutination)
-added after coombs - AGGLUTINATION in order to validate testing
-commercial reagents are O red Cells

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23
Q

What are false negatives with IgG caused by

A

1) Failure to add the AHG reagent
2) Failure of the AHG reagent to react
3) Failure to wash the RBCs adequately

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24
Q

What does AB screening do

A

AB detection that have specificity to red cell ag

25
What is AB identification
Identifies specificity of red cell AB
26
What is Cross match
-determines serologic compatibility between donor and patient before transfusion
27
What antigen typing
identifies specific red cell AG in patient or donor
28
How can the following cause a positive DAT Transfusion reaction HDFN AIHA Drug related mechanism
Transfusion reaction- donor cells coated with IgG + pt AB HDFN - fetal red cells coated with IgG +Maternal AB crossing the placenta AIHA - IgG or C3 on pt red cells + Patient AutAB Drug related mechanism: IgG-drug complex attached to RBC + Immune complex formed with drug
29
What is a differential DAT SLIDE 29 listen
-done when the initial DAT with a polyspecific AHG is positive -Reagents are Monospecific Anti-IgG and Monospecific Anti-Complement (-C3b, -C3d, -C4b or- C4d) -C3d, C3b, C4b or C4d are the complement components that remain on the Red Blood cell if the complement cascade has been activated. For Testing Complement in a Differential DAT, negative reactions must be let sit for 5 minutes RT to enhance the complement reactions.
30
AIHA vs. Transfusion Reaction -positive DAT SLIDE 31
-diagnostic of AIHA -hallmark of transfusion reaction - when the pt makes as AB against the transfused donor cells -this is why a transfusion history is important when investigating a positive DAT because IF there is no recent transfusion then it can be due to a AIHA
31
If IgG is already on the patient’s cells, what happens when you add the AHG?
-will give a false positive in tests that use the IAT method with AHG -tests like Auto Control, Weak D testing, and Phenotyping IAT method
32
What happens when you have a positive DAT and weak D testing needs to be done
- weak D would be positive with the presence of an autoantibody since it is already attached to the red cells -ANTI D IS NOT attached to the cells =FALSE POSITIVE -Rh control would be positive as well
33
OBG antigen phenotyping that requires IAT method: when dat is positive
- add anti sera and cells for IAT testing -cells are already coated with IgG -= false positive so you cant tell if the pt has the AG or not -if blood products are needed then give blood LACKING that AG. -if there is previous info use that or genotype pt at CBS
34
What is neutralization of the AHG reagent
-washing of red cells is very important in AHG testing -if cells are improperly washed any unbound AB and complement can bind to the AHG reagent and inhibit its reaction with the AB or C3d molecules attached to the red cells to detect this error- add CCC to a negative reaction -if there is agglutination then proper washing has occured
35
Sources of false positives in Antiglobulin testing
Red cells are agglutinated before washing step and addition of AHG = Potent cold reactive AB from pt Use of dirty glassware = particles or contaminants improper centrifugation or over centrifugation = red cells packed very tightly on centrifugation so that non specific clumping cant disperse
36
Sources of false negative error in Antiglobulin testing failure to wash cells adequately during testing before added AHG testing is interrupted or delayed; AHG reagent is not added immediately after washing failure to ID weak positives loss of reagent activity
1- unbound human serum globulins neutralize AHG 2- bound IgG or complement can detach from coated red cells 3-technicial error in testing 4-improper reagent storage, bacterial contamination
37
Sources of false negative error in Antiglobulin testing failure to add AHG reagent improper centrifugation - under centrifugation inappropriate red cell concentrations- outside the 2-5%
1-technical error in testing 2-conditions for promoting agglutination are not optimal 3-concentration of red cells influences the agglutination reaction
38
What are the expected reactions of WAIHA
* Antibody Screen is positive * Full Panel is positive * Auto Positive * DAT Positive * Differential DAT usually indicates IgG and/or Complement on cells * Perform Eluate to determine if autoantibody is present * Autoabsorption may be performed to determine if an underlying antibody is present.
39
What type of further testing will you have to do for AIHA
-because the autoAB can mask the alloantibody produced before the AIHA was developed you need to test both cells and serum/plasma (eluate/panels) -if blood is needed the PC would be "Least Compatible" and the Dr would be notified . Consent would be required -the packed cells would be super phenotyped and need to match exactly so alloAB do not develop
40
What is needed for a WIAHA transfusion
Full IAT Crossmatch (patient plasma and donor cells) * “Best compatible” or “Least Incompatible” units are provided
41
how do you crossmatch patients with WAIHA
-need full phenotyped matching units to prevent an immune response to develop AB -if an alloAB is produced then it can be masked and undetected because of the pts panagglutination -need to issue antigen negative red cells with comments in LIS after authorization from DR
42
how would you conduct a WAIHA investigation Testing the patient red cells and patient’s plasma
ABsorption - removing AB from plasma. Run the plasma with normal panel ADsorption - adding AG so that AB can attach to it and be removed from the plasma The ag adsorbs the AB Elution -removing an AB bound to CELLS (autoAB or donor cells covered with pT AB ) Elution can only be done if pt HAS BEEN transfused in teh last 3 months
43
What is Elution used for
- to eluate AB coating Redcells Unabsorbed AB in patient serum are washed and absorbed with selected cells * The antigen and antibody complex is then disassociated (eluded) by the addition of a low pH solution * The eluted solution is then used to perform an antibody investigation * Last Wash must be tested along with the eluate
44
What are the limitations of Elution Activity of the eluate depends on:
Activity of the eluate depends on: 1) The number of Antibodies bound to cells 2) Dissociation of Antibody during last wash 3) Use of Patient cells older than 72 hours may yield weaker reactions (alters final pH) 4) Failure to adjust pH to proper range can result in hemolysis of red cells
45
What are the methods to eluate
Glycine - removes AB by lowering pH - quick Heat/freeze/thaw - removed AB physically - quick, good for ABO AB- only good for ABO AB What we will do in lab Ether /organic solvents - removes AB by organic solvent - sensitive - hazardous or flammable. Turns solution blue add drop by drop
46
how do you test the eluate
- test against a panel of cells -reaction can indicate specific AB (Anti e) OR the AB can be non specific and react will all the cells
47
What is the method for making an eluate
LAST WASH MOST IMPORTANT Last wash is saved - treat like eluate solution- should be AB free -run last wash with panel as well -wash cells 4-6x before you perform the elution -Dont decant the last saline supernatant; save it and label it “Last Wash.” -do the elution on the packed cells after the last wash - * Run the “Last Wash” parallel to the eluate with the same panel.
48
What could the results of an Elution reaction be
-if your washing was successful then the LW should be AB-free =-A positive eluate with a negative LW indicates that the antibody present was taken off from the red cells and not the plasma -if there was a warm AB present then the elute would react with everything (all cells in panel are positive due to non specificity -if the eluate doesnt react it can mean that the pt could have a drug induced AIHA -* Eluates are not useful if Anti-C3d is attached to red cells.
49
Describe the adsorption technique and its limitations Rabbit Erythrocyte stroma
-removes cold (IgM) Ab like Anti I limitation can adsorb antiB and other IgM AB
50
Describe the adsorption technique and its limitations cold autoadsorption
pt red cells are used to remove cold AutoAB to determine if alloAB are present limitation dont use if recently transfused
51
Describe the adsorption technique and its limitations warm autoadsorption
pt red cells are used to remove warm AuAB to determine if alloAB are present limitation dont use if recently transfused
52
Describe the adsorption technique and its limitations differential (allogeneic) adsorption
- uses known phenotyped red cells to separate specificities: Warm auto AB from alloAB AlloAB with various specificities limitation can adsorb alloAB to a high frequency ag
53
What is the procedure for Autoadsorption of Autoantibody
using pts own red cells to remove AB -with heat or chemicals remove AutoAB from pt cells -incubate pt SERUM with AutoAB with ELUTED PT CELLS -AutoAB from serum will attach to Pt cells -repeat until the AutoAB is taken out -then alloAB will be exposed
54
slide 55
screening cells to absorb out the auto plasma is run with the panel to see if there is an underlying AB enzyme treated cells increase uptake of AB
55
What causes Drug Independent Auto Immune Hemolytic Anemia
* α–Methyldopa or Aldomet (Antihypertensive drug) * Antibodies are produced by a drug * Considered True Autoantibody because they react to RBC antigen * Not all patients are affected * Mild-moderate-severe * May persist up to 2 years after the drug discontinued -hard to diagnose because it mimics WAIHA Positive DAT * IgG attached to RBC * Possible for DAT to be positive up to 2 years after the drug is discontinued. * Positive Antibody Screen, Panel, and Auto. * Eluate reacts with ALL cells * Possible Hemolysis (extravascular)
56
What is drug dependent AIHA LISTEN
Non-Specific uptake of immunoglobulins (normal plasma proteins) by the red cell membrane. * Drug modifies the RBC membrane; plasma proteins absorb onto the patient red cells. * NOT an actual antigen antibody reaction, Antibody formed against the drug/protein complex * Cephalothin (Cephalosporin) * DAT is positive to C3d * Antibody Screen is negative * Eluate is negative Drug covalently binds to red cell membrane protein and remains firmly attached. AB FORMED AGAINST THE DRUG/PROTEIN COMPLEX * Reacts ONLY with the drug-treated cells * DAT is Positive with IgG, not Anti-C3 ***********
57
What is the most common drug that causes drug dependent AIHA and how do you test for it
Penicillin 1) Coat the red cells with penicillin in vitro. 2) Perform antibody screen, the Penicillin coated cells are to run in parallel with regular screening cells. 3) Only drug coated cells will react.
58
Quinidine
Drugs + plasma proteins and become immunogenic * Drug and Anti-Drug complex are formed, and binds complement. * Complement non-specifically binds to red cell * DAT is positive for Complement (C3d) and occasionally IgG * Antibody Screen is negative * Eluate is negative * common drug Quinidine
59
ANTI CD38

Transfusion Yr 2 Sem 2 (10 decks)

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