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Clinical Laboratory Hematology, Third Edition

Shirlyn B. McKenzie | J. Lynne Williams

Immune Hemolytic Anemia



Introduction

• Immune hemolytic anemia (IHA)

–RBCs destroyed prematurely by immune process mediated by antibody and/or complement

–Presence and severity of anemia depends on:

Severity of hemolysis

Ability of BM to compensate for RBC loss

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Introduction

• Immune hemolytic anemia (IHA)

– Initial confirmation of immune mechanism

Demonstration of attachment of Ab or complement to RBCs of patient

–Diagnosis of anemia:

↓ Hb and Hct, ↑ reticulocytes and/or unconjugated bilirubin,

↓ haptoglobin



Classification of IHAs

• Based on stimulus for antibody production

–Autoimmune hemolytic anemia

–Drug-induced hemolytic anemia

–Alloimmune hemolytic anemia

• Important to determine process because each type requires specific treatment



Classification

• Autoimmune hemolytic anemia (AIHA)

–Shortened RBC survival

–Caused by production of autoantibodies against RBC antigens

–Ab-induced reactions include

Sensitization, agglutination, hemolysis




Classification

• Autoimmune hemolytic anemia (AIHA)

–Further classified

Warm-antibody autoimmune HA (WAIHA)

Cold-antibody AIHA (cold agglutinin disease/CAD)

Mixed-type AIHA (both warm-reacting and cold-reacting autoantibodies)



Table 19-1 Classification of Immune Hemolytic

Anemias



Table 19-2 Characteristics of Agglutinins in Hemolytic Anemia



Classification

• Drug-induced hemolytic anemia

–Drugs attach to RBC membrane or alter it.

–Classified based on reactions of patient's RBCs and drug in vitro test system

Drug-dependent

Drug-independent



Classification

• Alloimmune hemolytic anemia

–Antibody (Ab) development to RBC antigen (Ag) that individual lacks

Do not react with individual's own RBCs

HDFN-mother makes Abs against Ags on fetal RBCs

Transfusion reactions where recipient makes Abs to Ags on transfused (donor cells)



Sites and Factors that Affect Hemolysis

• Intravascular or extravascular hemolysis

–Depends on:

Class of Ab

Ability to fully activate complement cascade






–Extravascular hemolysis

Most common

RBC sensitized with Ab or complement

Sensitized cells phagocytized by macrophages in spleen or liver






– Intravascular hemolysis

Complement cascade activated → C9 (MAC) → RBC lysis



Figure 19-1 Immune-mediated extravascular hemolysis. Erythrocytes sensitized with antibody or complement (C3b) attach to macrophages via specific cell receptors for these immune proteins.



Table 19-3 Factors Affecting the Rate of Hemolysis in Immune Hemolytic Anemias



Mechanisms of Hemolysis

• Based on whether IgM, IgG, and/or complement are present on RBC

• Three types:

– IgG-mediated

–Complement-mediated

– IgM-mediated



IgG-Mediated Hemolysis

• IgG attaches to RBC membrane Ags via Fab region.

• Fc receptors

–FcγR-I, -II, -III on macrophages of spleen

–Bind to Fc portion of Ab attached to RBC

–Macrophage pits the Ag/Ab complex





• Fc receptors

–Damages RBC membrane

–RBC membrane reseals itself

–Repeated splenic passage—continues to lose membrane, forms spherocyte

Phagocytized by splenic macrophages




• Ab-sensitized RBCs can be entirely engulfed by:

–Macrophages

–PMN (FcγR-I, -III)

• NK cells (FcγR-III)—results in ADCC (Ab dependent cell-mediated cytotoxicity)




• Spleen

– Lightly opsonized cells more efficiently removed

• Liver

–Removes heavily sensitized cells

• Splenomegaly is common



Complement-Mediated Hemolysis

• Role of complement

–Sensitization

Only portion of complement cascade activated and deposited on RBC membrane

– Lysis of RBCs

Entire complement system activated and deposited on RBC membrane




• Initiation of complement activation

–Classic, alternate, lectin mechanisms

• Classic pathway

– Initiated by Ag/Ab reaction

IgM

–Activates complement more efficiently

–Only requires one IgM molecule

IgG (IgG1 and IgG3; occasional IgG2)

–Activation requires two IgG molecules




• Cascade initiated

–C1 binds to the Fc region of IgG or IgM.

–Activates C4, C2, C3

–Activates the terminal components C5 to C9

Membrane attack complex (MAC)

–Lytic attack to RBC membrane

–Intravascular hemolysis when complement activation C1→C9 is complete





• Cascade initiated

–Activations through C3

RBC sensitized with C3b

–Totally or partially engulfed by macrophages with receptors for C3b

C3b on RBC cleaved by plasma C3b inactivator

C3c dissociates from membrane.

C3d no receptors on macrophages

–Normal RBC survival



Figure 19-2 The complement cascade. The central event in complement activation is the activation of C3 by C3 convertases. This can occur by two separate but interrelated mechanisms, the classic and alternate pathways. The classic complement pathway is initiated by an antigen–antibody reaction. The antigen–antibody complex activates the C1q, r, s complex, which in turn activates C4 by proteolytic cleavage to C4a and C4b. C2 binds to C4b and is proteolytically cleaved by C1s to form C2a and C2b. The C4b2a complex serves as C3 convertase. In the alternate pathway, C3b serves as the cofactor of the C3-cleaving enzyme complex (C3b, P, Bb), also known as C3 convertase. Thus, C3b serves to prime its own activation. The C3b formed through the classic pathway can directly initiate the assembly of the alternate pathway C3 convertase. C3 can also be activated by spontaneous hydrolysis. The C3b complexes formed by the classic and alternate pathways activate C5 to C5a and C5b. Membrane damage is initiated by the assembly of C5b with C6, 7, 8, 9.



IgM-Mediated Hemolysis

• Macrophages do not have receptors for Fc portion of IgM.

– IgM is efficient activator of complement

Intravascular

–Complement activation through C9 and RBC hemolyzes




IgM-Mediated Hemolysis

• Macrophages do not have receptors for Fc portion of IgM.

– IgM is efficient activator of complement.

Extravascular

–Activation incomplete

–C3b coats RBCs and sensitized cells destroyed extravascularly via CR1 and CR3 receptors on macrophages.

– IgM can agglutinate cells in addition to activating complement.



Laboratory ID of Sensitized RBCs

• Two agglutination techniques

–Saline—detects IgM antibodies

–AHG test—detects IgG and/or complement

Direct AHG (DAT)

–Detects RBCs coated in vivo

–Required to differentiate AIHA from other types of HA





• Two agglutination techniques

–AHG test—detects IgG and/or complement

Indirect AHG (IAT)

–Detects antigens in plasma or serum (in vitro)

–Indicates alloimmunization or free autoAbs in patient's serum



Figure 19-3 The zeta potential of erythrocytes keeps the cells about 25 nm apart when suspended in saline. IgG antibodies have a span of about 14 nm, not enough to bridge the gap between cells and cause agglutination. IgM antibodies, however, are pentamers with a span of about 35 nm, a distance sufficient to bridge the space between cells and cause agglutination.




• Negative DAT in AIHA

–Can result from

Insufficient number IgG molecules on RBCs

Autoantibodies of IgA or IgM class

Autoantibodies with low affinity for RBCs





• Negative DAT in AIHA

–Newer techniques more sensitive

Enzyme linked DAT

Gel tests

Flow cytometry

Polybrene tests




• Positive DAT in normal individual

–Healthy blood donors and hospitalized patients

Positive DAT—No shortened RBC survival






–Possible causes:

Inefficient macrophage removal of sensitized RBCs

Insufficient quantity of Ab on cell surface

Subclass of Ab not recognized by macrophage






–Possible causes:

Thermal amplitude of antibody (< 37°C)

+ DAT due to presence of complement on RBCs

Patients with hypergammaglobulinemia or receiving high-dose IV gamma globulin exhibit nonspecific binding.



Autoimmune Hemolytic Anemias (AIHA)



AIHA

• Immune tolerance normally prevents formation of autoantibodies

–Autoimmune diseases occur because:

Genetic predisposition

Exposure to infectious agents (molecular mimicry)

Defects in regulation of immune tolerance




AIHA

• Immune tolerance normally prevents formation of autoantibodies

–Subcategories

Warm vs cold autoantibodies

–Further categorized as

• Primary vs secondary AIHA



Warm AIHA

• Accounts for ~ 70% of cases AIHA

–Optimal reactivity at 37°C

–Usually IgG (rarely IgM, IgA)

–Most Abs react with "Rh protein" complex

Do not react with Rh null or Rh deleted cells

Occasionally have single specificity within Rh system (e.g., anti-e)




Warm AIHA

• Accounts for ~ 70% of cases AIHA

–Most hemolysis is extravascular via splenic macrophages.



Warm AIHA

• Idiopathic WAIHA

–Accounts for about 60% of cases of warm AIHA

–Acute idiopathic WAIHA

Severe anemia

Developing over two to three days

Hemolysis is self limited

Several weeks → several years duration




Warm AIHA

• Idiopathic WAIHA

–Chronic idiopathic WAIHS

Hemolysis is unabating.



Warm AIHA

• Secondary WAIHA associated with:

– Lymphoproliferative disease

CLL, HD

–Neoplastic diseases

–Autoimmune disorders

SLE, RA, Crohn's disease, and so on

–Certain viral and bacterial infections

–Vaccines



Warm AIHA

• Clinical findings

–Can occur at any age

Incidence ↑ after age 40

–Common presentation

Symptoms of anemia




Warm AIHA


–Secondary AIHA

Signs and symptoms of underlying disorder

–Mild to moderate splenomegaly > 50% patients, hepatomegaly in ⅓ patients



Warm AIHA

• Laboratory findings

– Immune-mediated hemolysis

+ DAT

Autoantibody in the serum

Presence of spherocytes




Warm AIHA


–Peripheral blood

Moderate to severe normocytic/normochromic anemia

Reticulocytosis

Polychromasia, NRBCs, spherocytes, schistocytes



Figure 19-4 BA blood smear from a patient with warm autoimmune hemolytic anemia (WAIHA). The marked anisocytosis is due to the presence of spherocytes and large polychromatophilic erythrocytes. The nucleated cells are erythroblasts (Wright stain, 1000× original magnification).



Warm AIHA


–Bone marrow

Erythroid hyperplasia, erythrophagocytosis

–Other lab tests

+ DAT

–+ polyspecific AHG and anti-IgG monospecific AHG

–30% + anti-C3




Warm AIHA


–Differential diagnosis from HS

+ DAT

Autohemolysis test not corrected by glucose

Non-homogeneous population of spherocytes



Table 19-4 Laboratory Findings Associated with WAIHA



Warm AIHA

• Therapy

–Self-limited hemolytic disorders

Do not require transfusions.

–Patients needing transfusions

Difficulty finding compatible donor cells




Warm AIHA

• Therapy

–Other therapies

Immunosuppressive drugs, cytotoxic drugs

Splenectomy

Rituximab

IVIG—high dose intravenous immunoglobulin

Plasma exchange and plasmapheresis



Cold AIHA

• Accounts for ~ 16–30% of cases of AIHA

–Optimal reactivity < 37°C

–Usually IgM, with complement activation

Rarely IgA or IgG

Most Abs react with I/i Ags or Pr Ags




Cold AIHA

• Accounts for ~ 16–30% of cases of AIHA

–Severity of disease

Related to thermal range of the Ab

Most hemolysis due to complement-mediated lysis



Cold AIHA

• Idiopathic or secondary

– Idiopathic CAS (cold agglutinin syndrome)

Usually chronic, occurring after age 50

Ab involved is usually

–Monoclonal IgM/kappa with autoanti-I specificity




Cold AIHA


–Secondary CAS

Associated with infectious disease

–Usually acute, self-limiting

–Polyclonal autoAbs with specificity for Ii antigens

• anti-I—M. pneumoniae

• anti-I—Infectious mononucleosis

• anti-Pr—varicella, rubella




Cold AIHA


–Secondary CAS

Associated with lymphoproliferative disorders

–Usually chronic, found in older individuals

–Monoclonal IgMκ Ab



Table 19-5 Autoimmune Hemolytic Anemia Caused by Cold-Reacting Antibodies



Cold AIHA


–Chronic or episodic hemolytic anemia

–RBC agglutination

Areas of the body that cool to the Ab thermal range

Sludging of blood flow within capillaries




Cold AIHA


–Vascular changes

Acrocyanosis

Raynaud's phenomenon

–Pain with color change patterns in skin

–Hemoglobinuria and splenomegaly



Cold AIHA


–Automated blood counts

Falsely ↓ RBC

Falsely ↑ MCV

Must warm blood and diluting reagents




Cold AIHA


–Mild to moderate anemia

Normocytic/normochromic

Polychromasia, spherocytes, clumps of RBCs, NRBCs, erythrophagocytosis

–Bone marrow

Normoblastic hyperplasia



Table 19-6 Criteria for Clinical Diagnosis of Cold Agglutinin Syndrome (CAS)



Figure 19-5 Cold autoimmune hemolytic anemia from a patient with chronic lymphocytic leukemia. Some of the erythrocytes are in small clumps. Spherocytes are also present (peripheral blood, Wright stain, 1000 original magnification).



Cold AIHA


–Differential diagnosis

Benign cold autoagglutinins vs pathologic cold agglutinins

–Pathologic cold agglutinins

• + DAT for polyspecific AHG and monospecific anticomplement antiserum

• Cold agglutinin test—agglutinates RBCs at 0–20°C in saline, reversible at 37°C

• Titer usually > 1:1000 (normal 1:64)



Table 19-7 Comparison of Characteristics of Pathologic Cold Agglutinins Found in CAS with Those of Benign Cold Agglutinins Found in Normal Individuals



Cold AIHA

• Therapy

–Keeping extremities warm is most effective

–Secondary to lymphoproliferative disorder

Chemotherapy

–Plasma exchange for acute hemolytic episodes

Effective for short time



Paroxysmal Cold Hemoglobinuria (PCH)

• Rare autoimmune hemolytic disorder

–Can occur at any age

–Massive intermittent acute hemolysis and hemoglobinuria

–Accounts for 30–40% of all AIHA in children

Most frequent < age 5

Associated with viral and bacterial infections





• Rare autoimmune hemolytic disorder

–Usually transient disorder

Resolves spontaneously

–Transfusions may be needed in severe cases.




• Pathophysiology

–Bi-phasic complement fixing IgG antibody

Donath Landsteiner antibody

Binds RBCs at low temps (< 20°C), activates complement





• Pathophysiology

–Bi-phasic complement fixing IgG antibody

Upon warming to 37°

–Ab detaches

–RBC lysed by complement activation through C9 (MAC)

Usual reactivity—autoanti-P antibody





–Hemoglobinuria most common clinical symptom

– Jaundice, pallor, hepatosplenomegaly

–Raynaud's phenomenon can occur





–Anemia depends on frequency and severity of attack.

–Hb drops sharply—Can ↓ as low as 5

g/dL.

–Hemoglobinemia, methemalbuminemia, hemoglobinuria






–Neutropenia, neutrophil shift to left

–Reticulocytopenia, spherocytes

– ↑ serum bilirubin, BUN, LD

– ↓ serum complement, haptoglobin

–Erythrophagocytosis






–DAT usually negative for antibodies

–DAT weakly + for complement

– IAT + if performed in cold

–Donath-Landsteiner antibodies

Present in low titers < 1:32

Verified by D-L test



Table 19-8 Donath-Landsteiner (D-L) Test for Detecting the Presence of D-L Antibodiesa




• Therapy

–PCH terminates with recovery from infection.

–Transfusion if hemolysis is severe

–Plasmapheresis if hemolysis persists

–Rituximab

–Avoid exposure to cold



Table 19-9 Comparison of Cold Agglutinin Syndrome (CAS) and Paroxysmal Cold Hemoglobinuria (PCH)



Mixed-Type AIHA

• Due to presence of warm-reacting IgG autoAb and cold-reacting IgM autoAb

–Both have high titer and ↑ thermal

amplitude

–50% of cases are idiopathic.

–Remainder associated with lymphoproliferative diseases, autoimmune diseases (e.g., SLE), or HIV




Mixed-Type AIHA

• Due to presence of warm-reacting IgG autoAb and cold-reacting IgM autoAb

–Mixture of both intravascular (IgM) and extravascular (IgG) hemolysis

–Most patients respond to corticosteroids without transfusions.



Drug-Induced Hemolytic Anemias



Drug-Induced HA

• Acquired cause of hemolytic anemia

–Not all individuals taking the same drug develop HA.

–> 125 drugs identified

– Immune response to drug-induced alteration of RBC

–Must differentiate from:

Drug-induced, nonimmune hemolysis

Spontaneous autoimmune disorders



Drug-Induced HA

• Uncommon acquired cause of HA

–Resolution is withdrawal of drug.

–Classic mechanisms

Drug absorption, immune complex formation, autoantibody induction, membrane modification

–New "unifying" hypothesis




Drug-Induced HA


–New "unified" hypothesis

Drug binds to RBC membrane.

–Abs produced to react with epitopes specific to drug

–Combination of drug and RBC proteins

–Epitopes primarily on RBC membrane

Explains how patients develop more than one type of drug-induced Ab




Drug-Induced HA


–New "unified" hypothesis

–Two types:

Drug dependent—requires presence of drug during testing

Drug independent—reacts without presence of drug

Sensitized RBCs have shortened life span

+ DAT



Table 19-10 Summary of Classic Mechanisms of Drug-Induced Immune Hemolytic Anemia



Alloimmune Hemolytic Anemia



Alloimmune HA

• Ab develops to a RBC Ag that the individual lacks.

– Individual exposed to transfused RBCs from another person

–Ags on transfused cells are lacking on RBCs of recipient.

–Stimulate production of Ab (alloAb)




Alloimmune HA


–Abs react only with cells that possess the Ag.

Not the individual's own RBC




Alloimmune HA


–Detected by Ab screen (indirect AHg test)

–Seen in transfusion reactions and Hemolytic disease of the fetus or newborn (HDFN)



Hemolytic Transfusion Reactions

• Result of:

– Interaction of foreign (nonself) Ags on transfused RBCs and patient's plasma Abs

– Immunologic destruction of donor cells





• Result of:

–Two types of transfusion reactions

Immediate (IgM)

–Occurring within 24 hr, intravascular hemolysis

Delayed (IgG)

–Occurring 2–14 days after transfusion, extravascular hemolysis



Table 19-11 Comparison of Acute and Delayed Hemolytic Transfusion Reactions




• Therapy

–Acute

Terminate transfusion

Supportive care

–Delayed

No treatment



Hemolytic Disease of the Fetus and Newborn (HDFN)

• Feto-maternal blood group incompatibility

–Mother forms alloantibodies against fetal RBC antigens.

– IgG antibodies cross placenta and destroy fetal RBCs in utero.





• Feto-maternal blood group incompatibility

–Three categories:

1. Rh(D) caused by anti-D (more severe disease)

2. ABO caused by anti-A and/or anti-B (more common)

3. “Other” caused by Abs to other blood group system Ags



Table 19-12 Comparison of Hemolytic Disease of the Fetus and Newborn Caused by ABO and Rh(D)




• Pathophysiology

–Four conditions must be met for HDFN to occur:

Mother must be sensitized to RBC Ag that she lacks.

Fetus must possess Ag to which mother has been sensitized.

Mother must produce Abs to foreign Ags.

Mother's Ab must cross placenta, enter fetal circulation.




• Laboratory testing

–Mother

ABO and Rh typing, antibody screen (IAT)

–Baby

ABO and Rh typing, DAT (elution if necessary to identify Ab)





–Baby's peripheral blood:

Macrocytic/normochromic, ↑ reticulocytes, leukocytosis with left shift, ↑ NRBCs

Rh HDFN

–Marked polychromasia, mild or absent poikilocytosis, few (if any) spherocytes, ↑

bilirubin, + DAT






–Baby's peripheral blood:

ABO HDFN

–NRBCs, schistocytes, spherocytes, polychromasia, sl ↑ bilirubin, weakly +

DAT




• Therapy

–Prevent hyperbilirubinemia and anemia

– Intrauterine transfusion

Viability of fetus affected

–Phototherapy (after birth) to reduce bilirubin

–Exchange transfusion if bilirubin is rising

> 1 mg/dL/hour or significant anemia




• Rh immune globulin (RhIG)

–Passive injection that contains anti-D that prevents maternal immunization

Given at 28 weeks gestation and following birth of Rh+ infant

Dose determined on number of fetal cells in maternal circulation

–Kleihauer-Betke Test

–Rossette Test

–Flow cytometry