W7 - Hemolytic Anemia, Maternal Allo, G Antigen, Serological

What is polyagglutination?

A group of cells that agglutinate regardless of blood group

• Cryptic antigen on rbc membrane is exposed and polyagglutinin are naturally occurring

• detected when preforming crossmatch if donner cells are polyagglunatble

What are the categories of polyagglutination?

1) Acquired —> due to bacterial enzyme on rbc membrane

2) Inherited —> due to somatic mutation of hemopoitic tissues

What is the dolichos biflorus used for?

To differentiate between A1 and A2 cells

Which plant lectin is used to differentiate between A1 and A2 cells?

Dolichos Biflorus

Which plant lectin is used to screen for anti-H ?

Ulex europaeus

What is Ulex europaeus used for?

To screen for bombay pt aka pt for anti-H

What is HTLA?

High Titer; Low avidity

aka the description of the antibodies, NOT an antibody itself

What are the criteria for HTLA?

High titer: >64 and low avidity: weak to 1+

What does the serology screen look for HTLA?

• Screen & panel cells reacting w+ to 1+

• Autocontrol: Nonreactive

• 1:10 dilution: w+ to 1+

• Double titration: Tubes 1 to 7: 1+

What are some examples of aquired polyagglutination?

T, Tk, Acquired-B

What are some example of inherited polyagglutination?

Tn

Which antibodies exhibits HTLA characteristics?

• Anti-Chido

• ANti-Rodgers

• Anti-York

• ANti-CsA

• Anti-Knops

Which antigen has two name depending on where it’s loctated? What are the name?

Bg

RBC —> Bg antigen

Platelets —> HLA class I

What is the notation for Bennet-Goodspeed antigen?

Bg

What is the signficance of Bg antigen?

Can cause immediate and delayed hemolytic transfusion rxn

What would you use to remove Bg antigen?

Chloroquine diphosphate and EGA

Which HLA antibodies are found on RBC?

Bg antigens

What are some characteristics of Anti-CD38?

• monoclonal antibody used to treat multiple myeloma

• normal rbc contains weak CD 38

• anti-CD38 interferes w/ antibody screen, ABID and cross match

What should you use to remove CD38 from RBC?

DTT

Why is it important to treat Anti-CD38?

Pt w/ multiple myeloma has a bunch of CD38 and so they need to be washed with DTT or else they cannot tell which antigen is on the cell : (

What is some characteristics of Anti-CD47?

• CD47 present on RBC and platelets

• Might cause ABO discrepancy

• Not denatured by DTT

• correlates to hematologic and solid malignancies

What type of antibody is Anti-CD47? Why might it cause a problem?

anti-CD47 is IgG4 —> not detected by Gamma-clone anti-IgG

How to resolve anti-CD47?

Allogeneic adsorption

What is IVIg used to treat?

WAIHA, humoral immunodeficiencies, chronic immune thrombocytopenia purapura

So when youre doing panel cells and you see weak reactions; what should you think of?

Bg antibodies,Anti-CD 38, Anti-CD47 or IVIg

What is the definition of hemolytic anemia?

Shortened RBC survival mediated through the immune response specifically by humoral antibody

What are some classification of Immune HA?

1) AIHA

2) Alloimmune HA

3) Drug induced HA

What are some categories of AIHA?

• Warm AIHA

• CAS

• Mixed type

• PCH

What are some categories of Alloimmune HA?

• Hemolytic Transfusion Reaction

• HDFN

What is the % of C3 only in the warm?

13%

What is Cold agglutinin syndrom?

When IgM autoagglutinin binds to RBC in peripheral circulation and then binds/activates complement

• IgM dissociates when RBC returns to warmer parts of circulation but RBC remains coated w/ complements

What are the types of Drug-Induced Hemolytic anemia?

1) Drug-adsorption

2) Autoantibody

3) Drug-dependent

What are the 2 forms of HTR?

1) Alloantibody —> Patient makes antibody against donor’s antigen

2) Passive: Donor’s antibody against recipient’s antigens

What is the binding site of antigens to RBC?

Fab

What is the binding site of antigen to complement?

Fc

What is the main antibody in Warm IgM AIHA?

IgM agglutinins reactive @ 37C

What would you expect from DAT and IAT in Warm IgM AIHA?

DAT Control +; IAT: w= @37C and v weak @AHG

What is the eluate of Warm IgM AIHA?

Hemolysis at 37C

What is the antibody in WAIHA?

Usually IgG

What is the IAT / DAT of WAIHA?

IAT: Panagglutination

DAT: IgG only

What should you see in the Eluate and Adsorption in WAIHA?

Pannagglutination; Adsorbed plasma non-reacive if no alloantibody is present

What is the specificity of WAIHA?

Usually anti-e

Why is Warm IgM AIHA more fatal?

Because it involves IgM which can bind complement thus resulting in intravascular hemolysis and severe hemolysis

What is antibody responsible in Mixed Type? What temps is it usually reactive at?

IgM and IgG; cold reactive @ 4C and >30C

What is the expected DAT of mixed type?

IgG and C3 or C3 only

Which temperature do we do adsorption for mixed type? Why?

37C first and then 4C ; Because we have both IgG and IgM :^]

How can you get a negative DAT AIHA?

When there is not enough IgG /IgM or IgA that is undetectable

What happens when you have a DAT negative AIHA?

Send to LARC fo super Coombs :^]]]]]]

What are the criteria to do phenotyping?

• Pt has no transfusion in the last 8 weeks

• Clean the cells with EGA or Chloroquine diphosphate

What are some treatment of WAIHA?

• Corticosteroid

• IVIg

• Splenectomy

• Immunosuppressive drug

What are some characteristics of Cold Reactive Antibodies?

• Reactive @ IS

• Enhanced reactivity @RT, 18C, 0-4C

• ABO discrepancy in reverse typing

• Positive antibody screen

What are the usually suspect for cold reactive antibodies?

anti-IH, anti-I, anti-i

What is the DAT for Cold Reactive Antibodies?

DAT positive for complement only

What is the difference in elution and adsorption?

Elution is testing the pt RBC and getting the antibody that was covering their cells

Adsorption is testing the pt plasma and checking which cells adsorbs it

What are the 4 mechanisms that leads to drug-related antibodies?

1) Immune complex/ innocent bystander

2) Drug adsorption

3) Membrane modification

4) Methyldopa induced

What is immune complex?

When the drug binds with the antigen to form a neo antigen on the RBC which complement will bind to the cell and cause acute intravascular hemolysis

What are the drugs involved with immune complex?

• Quinidine

• Phenacetin

What is drug adsorption?

When you take the drug and then your body makes an antibody to the drug

What are the drugs involved in drug adsorption?

• Penicillin

• streptomycin

• cephalosporin

What is membrane modificaiton?

When the drug changes the RBC membrane that causes protein to attach to the membrane

What are the drugs involved in membrane modification?

Cephalosporins

What is Methyldopa induced drug-related antibodies?

When the drug forms antibodies that mimic autoantibodies found in WAIHA

Which drug is involved in Methyldopa induced?

Methyldopa

What are the 2 disease associated with Maternal alloimmunization?

1) Hemolytic Disease of the Fetus and Newborn (HDFN)

2) Fetal/Neonatal alloimmune thrombocytopenia (FNAIT)

What is Fetal Maternal hemorhage?

When there is an injury to the mother —> resulting in maternal IgG crossing the placenta and causing UgG RBC to be removed from circulation —> resulting fetal anemia —> immature RBCs are released in response —> enlarged spleen and liver —> liver dmg due to plasma protein —> cardiac failure will cause edma —> hydrops fetalis —> fetal demise

What is the bio-marker that we check for to see if there is extra vascular hemolysis?

Increase bilirubin

What are the 2 different ways to monitor the progression of anemia?

1) Amniocentesis

2) Doppler

What is HDN?

Hemolytic Disease of the New Born

What is the path of HDN?

Hemolysis —> Increase unconjugated bilirirubin —> Kernicterus —> permanent brain dmg

What is Kernicterus?

High level of bilirubin —> causing dmg in the brain

What is some treatment of HDN?

• Neonatal exchange transfusion

• Phototherapy

What are some antibodies that cause HDFN?

• Anti-A,B

• Rh antibodies (D>E>C>c)

• Anti-K, -Kpa, -Jsa, -Jsb

• Anti-Jka, -Jkb, Jk3

• Anti-Fya, -Fyb

• Anti-S

What can increase the titer of ABO antibodies?

Yeast-Based food (yogurt), tetanus injection

What is the pathogenesis of Fetomaternal hemorrhage?

Baby Rh+ antigen yeets into the mother —> the mom body is like oh hell naw and make anti-Rh antibodies —> shoves the anti-Rh back into the baby (or the next one) and murders the RBC

What is para?

number of birth

What is gravita?

Number of pregnancy

What is included in Prenatal Testing?

Antibody screen to evaluate the risk of HDFN and to see if mom would be a good candid for Rogam (or Rh-immune globulin prophylaxis (RhIG)

What is the significant titer for anti-D?

32

What is the significant titer for other anti-bodies?

16

What are the procedures during labor and delivery?

Collect cord blood in EDTA tube (both the mom + baby) —> ABO/Rh and DAT are performed on the cord blood —> collect mom postpartum specimen an hour after delivery

Why do you need to test Rh on the new born after it’s been popped out?

Because we need to know if its Rh pos or neg so that if it is a weak D then mom needs to recieve rogam to prevent making anti-D

What happens when the mom and baby is Rh Neg but DAT is positive?

• Cancel Rosette and order Kleihauer-Betke test —> Baby’s Rh is invalid due to positive DAT —> Calculate how many vials of RhIg needed

What happens when the mom is Rh neg and the baby is Rh neg and the DAT is also neg?

Perform Weak D testing on cord blood —> RhIg is given within 72 hours after delivery

What is the protocol when Mom is Rh Neg and baby is Rh Pos w/ DAT neg

1) Perform Rosette testing on the mom

• Neg Rosette —> Give 1 vial of RhIg

• Pos. Rosette —> Send to hematology for Kleihauer-Betke test

2) Calculate how much RhIg is needed based on KB results

What is the Rosette test?

D positive fetal RBC that is coated with Anti-D and clings to a D positive indicator cells

What is methodology of Kleihauer-Betke Stain?

Hgb F is resistant to acid elution —> fetal cell will appear pink and adult cells will be ghost

How much blood can 1 vial of RhIg neutralize?

30 ml of whole blood or 15ml of packed cells

How much is in 1 vial of RhiG?

300 ug

What is the formula to calculate how much RhiG to give a pt?

# of RhiG = (% of feta; cells x maternal blood volume) / 30

What is the example of RhIG calculation?

Fetal Cells: 6

Total cells counted: 2000

6/2000 = .003 × 5000 = 15/30 =.5 —> give 2 vials

What are some situations we should give RhiG?

• Fetal death

• Abortion

• Amniocentesis

• Antepartium hemorrhage

• Ectopic pregnancy

Is RhoGam given intramuscularly or intravenously?

Intramuscularly

Is WinRho given intramuscularly or intravenously?

Intravenously

Where is the G antigen found?

Mostly D+ or C+ cells

Which haplotypes would you find G antigen?

R1: DCe

R2: DcE

R0: Dce

Rz: DCE

r’: dCe

ry: dCe

Which haplotypes do NOT have the G antigen?

r’’: dcE

r:dce

What are the exception phenotype for G antigen?

rGr (G+D-C-)

DCe (G-D+C+)

What are the characteristics of Anti-G?

• Appears as anti-D plus anti-C

• Made by:

  • D- person transfused w/ D-C+ cells

  • D-C- person transfused w/ rGr cells

  • Pregnant women who delivered a D-C+ child

How can you tell the difference between anti-D, anti-C, anti-G?

Distinguished by adsorption and elution studies