MMSC 420 Exam 2

How many IgG and IgM, respectively, would be required to activate the classical pathway for the complement cascade?

2, 1

CAS/CAD Specimens collected and maintained at ____C •Alternatively, EDTA tubes collected and warmed to 37C 15 min (mixing regularly) •Separate serum/plasma from RBCs while warm •Helps release autoadsorbed antibody back into plasma for titering to determine strength of autoagglutinin

37

Which of the following conditions is not a form of autoimmune hemolytic anemia? a)Hemolytic disease of fetus & newborn b)Cold agglutinin disease c)Paroxysmal cold hemoglobinuria d)Warm autoimmune hemolytic anemia

A

Common procedure now Wash RBCs - add glycine acid solution (pH 3.0) Dissociates AB-AG bonds, releases AB attached Harvest supernatant and buffer for ABID

Acid elution

What is the most common drug implicated in drug-induced hemolytic anemia caused by autoimmunity?

Aldomet

A pregnant patient has a positive result for DAT anti-PS and positive result for anti-IgG. When performing an adsorption, what kind of cells do we want to use?

Allogeneic adsorption

Pt 3+ AHG throughout whole panel and auto control, DAT PS, and DAT IgG!!!!!, but 0 for DAT C3bC3d panagglutination Autoantibody is caused by what medication?

Alphamethyldopa

The Autoantibody Mechanism for DIHA is implicated most frequently with which medication(s)?

Alphamethyldopa

treatment for solid organ tumors and other hematologic malignancies •Promotes phagocytic properties - attacks tumor •CD47 is present on all cells •Get positive reactions at all phases, all cells, including reverse typing for ABO

Anti-CD47 (Only multiple allo-adsorptions will remove this activity, OR Use of monoclonal Gamma-clone anti-IgG for AHG testing)

CAS/CAD 2 forms

Aute (secondary to lymphoma or M. pneumoniae infection, or infectious mononucleosis) and Chronic (common in elderly - Raynaud's-like phenomenon)

Which of the following would NOT be a patient that we would perform a DAT on? A: A patient with low Hgb and HCT levels but with no known bleeding B: A mother who has just given birth to a type O neg baby C: A patient exhibiting signs of an acute hemolytic transfusion reaction after receiving A pos RBCs from a donor D: A baby born to a mother who is type O

B

What test should be done as a reflex after a positive anti-PS DAT is observed in a 27 year old male?

Both anti-IgG and anti-C3bC3d should be run

What is the order of complement activation in the classical pathway of the complement cascade?

C1-C4-C2-C3-C5-C9

Rabbit erythrocyte stroma (RESt) is a commercial reagent rich in I-antigen and is used to facilitate antibody identification with which type of AIHA?

CAD

DAT result for ___________ Polyspecific +, IgG NEG, C3bC3d +

CAS/CAD

•Completely IgM antibody; binds complement•Some have specificity:•Auto anti-I (most)•Auto anti-i (some)•Anti-Pr (rare)

CAS/CAD

Check Cells (IgG coated RBCs) Complement Control Cells Anti-C3bC3d Polyspecific AHG Anti-IgG ALL used in a...

DAT

a test to detect in vivo coating of RBCs with globulin Test utilizes three reagents: Polyspecific AHG Anti-IgG Anti-C3bC3d

DAT (direct antiglobulin test aka direct coombs test, tests patient RBCs for coating of IgG or complement)

If COLD Autoantibody -may need to 1) wash patient RBCs with warm saline ... still POS DAT, can treat cells with ________or___________ to remove attached cold autoantibody

DTT or 2ME (if warm autoab, do same but use W.A.R.M. reagent here)

- treatment for Multiple Myeloma •Anti-CD38 antibody-based medication•Monoclonal antibody targeting CD38 on plasma cells •CD38 also on RBCs (some) •THIS attaches - will get all cells pos on antibody screening, antibody identification - looks like AIHA •DAT variable - may be negative •Adsorptions fail (insufficient CD38 on RBCs for adsorp)

Daratumumab

Type of elution most commonly used now

Digitonin Acid

DIHA _________________: •Drug becomes covalently linked to RBCs •Patient develops AB specific to drug •Anti-Drug ABs bind to the drug (bound to RBCs) •RBCs become coated with AB •Extravascular hemolysis occurs

Drug Adsorption Mechanism (esp for penicillin IV)

What do we do differently when transfusing patients with sickle cell disease?

Give sickle neg RBC products (C, E, and K neg too if patient is ag neg for these)

What is a distinguishing characteristic in intravascular hemolysis?

Hemoglobinuria

IS DAT or IAT more complicated

IAT (No need to try to attach antibody to red cells If positive, RBCs are already sensitized in the patient's specimen.)

THE FIRST EXAMPLE OF A CIMETIDINE ANTIBODY DAT positive with anti-C3•A strong antibody to cimetidine was detected- drug induced

IHA (IMMUNE HEMOLYTIC ANEMIA)

Penicillin IV has DAT strongly pos with _____ only •Hemolysis seen in patients receiving very large doses of medication •Usually resolves when drug administration is discontinued Antibody screen negative

IgG

Which drug induced antibody reaction involves the drug binding to antibody prior to binding to the red blood cell?

Immune complex

Activated by the previously activated component or complex. Membrane Attack complex is the same for both - the binding of C3b is the pivotal stage of the pathway C3b is cleaved to C3d - which is detected in Anti - Complement reagents

Lectin pathway

How is the alternate pathway of the complement cascade activated?

Liposaccharide on E. coli binds to C3

What test can be performed to identify ABO antibodies coating a baby's cells?

Lui Freeze Elution

The end result is the destruction of the membrane at the site of the activation... CELL LYSIS

MAC (C5 splits to C5a and C5b, MAC is C56789

Cold autoimmune hemolytic anemia is associated with which pathogen?

Mycoplasma pneumoniae

•DAT POS (IgG and/or C3bC3d) •Serum: non-reactive •Eluate: non-reactive •No documented cases of hemolysis occurring by this mechanism - purely theoretical

Non-Immunologic Protein Adsorption Mechanism

Cephalosporins are thought to be associated with which DIHA mechanism?

Non-immunogenic protein adsorption

Biphasic hemolysin occurs by binding to RBCs at cold temperatures and only causes complement-mediated lysis when warmed to 37C. Which AIHA is this condition associated with?

PCH

_____________ is associated with: POS DAT Polyspecific AHG and Anti-C3bC3d and IgG antibody

PCH (Paroxysmal Cold Hemoglobinuria)

Immune Complex or "Innocent Bystander" Mechanism for DIHA IgG OR IgM antibody

Patient develops AB to drug (AB-Drug complexes are formed, which adsorb onto RBCs-Cell bound complexes activate complement)

Which drug is commonly associated with the drug adsorption mechanism in DIHA?

Penicillin IV

broad spectrum = polyspecific AHG - Contains anti-IgG and anti-C3bC3d Will detect IgG Ab or C3bC3d bound to RBC

Polyspecific reagent (used in DAT)

Anti-CD47 is used to treat organ tumors and other hematologic malignancies by promoting phagocytosis. What complications can arise from pre-transfusion testing for patients receiving this treatment?

Positive reactions occur at all phases of testing, including reverse ABO typing

What kind of specimen tube should we use for a DAT?

Purple top, EDTA

Anti-C, -D, -e, -Jka CAN be found in what cells?

R1R1 (CDe/CDe)

K neg Jka pos Jkb neg

R1R1 (Cells are also now "M, N, S, s, Fya, Fybneg - enzyme treatment with papain")

K neg Jka neg Jkb pos

R2R2 (Cells are also now "M, N, S, s, Fya, Fybneg - enzyme treatment with papain")

Anti-c, -D, -E, -Jkb CAN be found in what cells?

R2R2 (cDE/cDE)

In Cold Agglutinin Disease, for a patient who has been recently transfused, what adsorption is appropriate?

Rabbit Erythrocyte Stroma Test

What is the major concern with the Immune Complex (Innocent Bystander) mechanism of DIHA?

Small amount of medication can cause significanthemolysis

A patient suffering from a hemolytic anemia will most likely have an increase in what two results?

Tbil & LDH

If the DAT is positive but the IAT is negative, what describes the situation accurately?

There are more RBC antigen sites than antibodies

What is the major concern with the Autoantibody Mechanism of DIHA?

This mimics Warm Autoimmune Hemolytic Anemia - it is important to quickly recognize drug interaction

•Symptoms- anemia, weakness, dizziness, dyspnea, jaundice, unexplained fever •Hemolysis (Acute at onset, Extravascular)

WAIHA (Typically IgG antibodies)

Jack Frost has Cold Agglutinin Syndrome secondary to a viral infection. His DAT is positive with Polyspecific AHG reagent and Anti-C3bC3d. No recent sensitizing event has occurred. Which of the following would be most helpful and appropriate to resolve his forward and reverse typing discrepancy

Wash patient RBCs with warm saline, repeat forward group testing with prewarmed patient RBCs; Prewarm Pt. Plasma and A1 Cells and B Cells, test with warmed reagents and plasma.

dissociates antibody attached to RBCs•Supernatant is removed, buffered to pH for use in identification procedure•Prompt removal from stroma so the antibody does not re-attach

acid elution (for WAIHA)

Warm Autoimmune Hemolytic Anemia is typically demonstrated by a warm-reacting panagglutinin. How this appears when the patient's plasma is tested against a 10-cell antibody identification panel is: reactivity (positive agglutination reactions) at the AHG phase on how many cells on the panel?

all

An in vivo phenomenon associated with a POS DAT is a new __________ detected in recently transfused patient

alloantibody

Warm Autoimmune Hemolytic Anemia typically requires some type of adsorption procedure. If the patient HAS had a recent sensitizing event, ____________&______________ adsorptions are options.

allogeneic and differential (diff= If we DO NOT know the patient's extended phenotype and cannot perform it reliably due to recent transfusion and/or strong antibody coating RBCs)

Contact with a surface such as the polysaccharide coating of a bacteria (C3-C5->C9 )

alternate pathway

A DAT would be done when you have a patient with unexplained...

anemia

Which antibody is associated with paroxysmal cold hemoglobinuria (PCH)?

anti-P

Performing eluate, identifying antibody -> now transfuse ___________________ blood even though antibody screening is negative

antigen NEG

Babies born to group O mothers Babies born to mothers with significant AB Babies showing signs/symptoms of HDFN

at risk for HDFN (DAT is done)

EDTA whole blood is used to reduce interference of the DAT caused by...

complement (EDTA binds calcium, prevents complement activation.)

There are 4 types of ______________ antibody reactions I.Drug adsorption II.Immune complex III.Membrane modification IV.Autoantibody formation

drug-induced

Autoantibodies may demonstrate specificity. Which is most commonly found in WAIHA (warm autoimmune hemolytic anemia)? auto anti-________

e

Cytolysis or hemolysis Cell activation Opsonization (makes cells easier to phagocytize)

effects of complement activation

Transfusion 6 weeks ago/POS DAT requires...

elution

A: Newborn with O- mother B: Recently transfused patient with a positive DAT and a 1+ reaction in all antibody screening red cells including autocontrol C: Recently transfused patient with a hemolytic transfusion reaction ALL indicate

elution needed

C2-C4-> C4b2a + C3

enzymatic activation (classical pathway)

Using a refrigerated clotted (red top) sample is associated with what DAT result?

false pos

If complement is activated, could cause _____________ result due to complement binding to the red cells, especially if the specimen was refrigerated

false positive DAT

1. True or False: DIIHA is one of the most common hematological abnormalities? 2. True or False: Many drugs can cause DIIHA.

false, true

A positive DAT does not mean decreased red cell lifespan; ________________&________________ are needed to determine the significance of positive DAT

history and physical exam

Purpose of elution: To detect and identify IgG antibodies that are coating the patient's cells causing _____________

in vivo lysis (also to help determine the cause of decreasing Hgb levels)

Immune Complex or "Innocent Bystander" Mechanism for DIHA: Can initiate _________________ or Cells become coated with complement proteins, which initiates ______________

intravascular hemolysis, extravascular hemolysis

(reflex testing - POS DAT) Anti-IgG Anti-Complement (anti-C3bC3d)

monospecific reagents

Recently transfused patients showing signs of anemia, acute hemolytic transfusion reaction and/or a delayed transfusion reaction HTR/DHTR

need to do a DAT

Does WAIHA involve IgM?

no

Does a positive DAT result means the patient's RBCs have a decreased lifespan?

no

Presence of polyagglutination- is an elution needed?

no

DAT - detects IgG or complement coating

patients red blood cells

The Drug Adsorption Mechanism for DIHA is implicated most frequently with which medication(s)?

penicillin

DAT POS - PS and IgG (may be C3bC3d Pos also) Unexplained decrease in Hemoglobin/Hct recent transfusion - suspected DHTR

perform elution (also to ID causative antibody(ies) in HDFN)

Mrs. Marlene Maple is scheduled for surgery tomorrow. She has no history of recent sensitizing events. We have confirmed (so far) that an anti-e is present WHAT do we do next?

phenotype (phenotype her RBCs for the e antigen, if her RBCs and Pos control are pos, it is an autoantibody)

may indicate immune-mediated hemolysis is present

pos DAT

•Autoantibodies to intrinsic red cell antigens •Circulating alloantibodies bound to transfused RBCs •Alloantibodies in donor plasma-containing products reacting with recipient's RBCs •Antibodies against drugs attached to RBCs •Maternal Antibodies that cross the placenta and bind to fetal RBCs •Non-red cell immunoglobulins bound to red cells (e.g., IVIG)

pos dat

A: Alloantibodies bound to transfused RBCs B: Autoantibodies to intrinsic red cell antigens C: Maternal antibodies that bind to fetal RBCs ALL cause

positive DAT

Patients with Cold Agglutinin Syndrome have very strong cold agglutinins present. What is the recommended treatment option if the patient needs to be transfused RBCs?

ransfuse the blood through a blood warming device

Agglutination with _________________ applies to drug-induced hemolytic anemia caused by immune complexes

reagent RBCs and drug

C1 = C1q - C1r - C1s (activated C1s is also called protease)

recognition (classical pathway, Fc portion attaches to complement)

Which of the following is/are usually INCREASED in WAIHA?

retics (>3%)

Direct lysis of cells, bacteria and enveloped viruses Assist with Opsonization to facilitate phagocytosis Produce peptide fragment split products which can mediate inflammatory and immune responses

roles of the complement system

K pos Jka po sJkb neg

rr (Cells are also now "M, N, S, s, Fya, Fybneg - enzyme treatment with papain")

Anti-c, -e, -K, -Jka CAN be found in what cells?

rr (dce/dce)

Elution of IgG molecules can be performed to identify possible...

specificity to RBC antigens

Non-Immunologic Protein Adsorption Mechanism of DIHA is a __________ caused by cephalosporins

theory

Reasons for decreased Hgb levels (use elution to find out) include: Adverse _______________________ Autoimmune process occurring - autoimmune hemolytic anemia Drug-induced hemolytic anemia HDFN

transfusion reaction

ELUTION NEEDED WHEN: Patient has a POS DAT with anti-IgG Patient has had a recent* sensitizing event: Transfusion Pregnancy Transplant (HSCT, BMT) what does "recent" mean?

within the past 3 months

Does the classical pathway require calcium?

yes (Fresh source of complement - Red Top tube Calcium - EDTA chelates Ca++)