HEMOLYTIC ANEMIAS

HEMOLYTIC ANEMIA

A i f i d d t ti• Anemia of increased destruction– Normochromic, normochromic anemia– Shortened RBC survival– Reticulocytosis - Response to increased

C d iRBC destruction– Increased indirect bilirubin– Increased LDH

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIATesting

• Absent haptoglobin• Hemoglobinuria• Hemoglobinuria• Hemoglobinemia

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIACauses

INTRACORPUSCULAR HEMOLYSIS• INTRACORPUSCULAR HEMOLYSIS– Membrane Abnormalities– Metabolic Abnormalities– Hemoglobinopathies

• EXTRACORPUSCULAR HEMOLYSIS– Nonimmune – Immune

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIAMembrane Defects

Mi k l t l d f t• Microskeletal defects– Hereditary spherocytosis

• Membrane permeability defects– Hereditary stomatocytosis

• Increased sensitivity to complement– Paroxysmal nocturnal hemoglobinuriaParoxysmal nocturnal hemoglobinuria

RED CELL CYTOSKELETONRED CELL CYTOSKELETON

HEREDITARY SPHEROCYTOSIS

D f ti b t t i l l• Defective or absent spectrin molecule• Leads to loss of RBC membrane,

leading to spherocytosis• Decreased deformability of celly• Increased osmotic fragility• Extravascular hemolysis in spleen• Extravascular hemolysis in spleen

SPLENIC ARCHITECTURE

HEREDITARY SPHEROCYTOSISHEREDITARY SPHEROCYTOSISOsmotic Fragility

80

100

sis

40

60

Hem

olys

0

20% H

0.3 0.4 0.5 0.6

NaCl (% of normal saline)

Normal HS

Paroxysmal Nocturnal yHemoglobinuria

• Clonal cell disorder• Clonal cell disorder• Ongoing Intra- & Extravascular hemolysis;

classically at nightclassically at night• Testing

– Acid hemolysis (Ham test)– Sucrose hemolysis– CD-59 & CD-55 negative

A i d d fi it f GPI A i t d t i• Acquired deficit of GPI-Associated proteins (including Decay Activating Factor)

GPI BRIDGE

Paroxysmal Nocturnal Hemoglobinuria

GPI Proteins

• GPI links a series of proteins to outer leaf of cell membrane via phosphatidyl inositol bridge with membrane anchor viabridge, with membrane anchor via diacylglycerol bridge

• PIG-A gene, on X-chromosome, codes for th i f thi b id lti l d f tsynthesis of this bridge; multiple defects

known to cause lack of this bridge• Absence of decay accelerating factor leads toAbsence of decay accelerating factor leads to

failure to inactivate complement & thereby to increased cell lysis

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIAMembrane abnormalities - Enzymopathies

D fi i i i H M h h t• Deficiencies in Hexose Monophosphate Shunt– Glucose 6-Phosphate Dehydrogenase

(G6PD) Deficiency

f h h• Deficiencies in the EM Pathway– Pyruvate Kinase Deficiency

G6PD DEFICIENCY – Function of G6PD

G6PDFunctions

• Regenerates NADPH allowing regeneration of• Regenerates NADPH, allowing regeneration of glutathione

• Protects against oxidative stressProtects against oxidative stress• Lack of G6PD leads to hemolysis during

oxidative stress– Infection– Medications

Fava beans– Fava beans• Oxidative stress leads to Heinz body

formation, extravascular hemolysiso a o , a as u a o ys s

Glucose 6-Phosphate Dehydrogenasep y gDifferent Isozymes

6080

100

vity

(%)

204060

PD A

ctiv

Level needed for protection vs ordinary oxidative stress

020

0 20 40 60 80 100 120

G6P

RBC Age (Days)

Normal (GdB) African Variant (GdA-)Normal (GdB) African Variant (GdA )Mediterranean (Gd Med)

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIACauses

INTRACORPUSCULAR HEMOLYSIS• INTRACORPUSCULAR HEMOLYSIS– Membrane Abnormalities– Metabolic Abnormalities– Hemoglobinopathies

• EXTRACORPUSCULAR HEMOLYSIS– Nonimmune – Immune

EXTRACORPUSCULAR HEMOLYSISEXTRACORPUSCULAR HEMOLYSISNonimmune

• Mechanical• Infectious• Chemical• Chemical• Thermal

O ti• Osmotic

Microangiopathic Hemolytic Anemiag p yCauses

• Vascular abnormalities (No coagulation• Vascular abnormalities (No coagulation abnormalities)– Thrombotic thrombocytopenic purpuray p p p– Renal lesions

• Malignant hypertension• Glomerulonephritis• Glomerulonephritis• Preeclampsia• Transplant rejection

Vasculitis– Vasculitis• Polyarteritis nodosa• Rocky mountain spotted fever• Wegener’s granulomatosis• Scleroderma renal crisis

Microangiopathic Hemolytic AnemiaMicroangiopathic Hemolytic AnemiaCauses - #2

– Vascular abnormalities• AV Fistula• Cavernous hemangioma

I t l l ti d i t• Intravascular coagulation predominant– Abruptio placentae– Disseminated intravascular coagulation

IMMUNE HEMOLYTIC ANEMIAGeneral Principles

• All require antigen antibody reactions• All require antigen-antibody reactions• Types of reactions dependent on:

– Class of AntibodyClass of Antibody– Number & Spacing of antigenic sites on cell– Availability of complement– Environmental Temperature– Functional status of reticuloendothelial system

• Manifestations• Manifestations– Intravascular hemolysis– Extravascular hemolysist a ascu a e o ys s

IMMUNE HEMOLYTIC ANEMIAGeneral Principles - 2

• Antibodies combine with RBC & either• Antibodies combine with RBC, & either1. Activate complement cascade, &/or2. Opsonize RBC for immune system2. Opsonize RBC for immune system

• If 1, if all of complement cascade is fixed to red cell, intravascular cell lysis yoccurs

• If 2, &/or if complement is only partially fixed macrophages recognizepartially fixed, macrophages recognize Fc receptor of Ig &/or C3b of complement & phagocytize RBC,complement & phagocytize RBC, causing extravascular RBC destruction

IMMUNE HEMOLYTIC ANEMIAIMMUNE HEMOLYTIC ANEMIACoombs Test - Direct

• Looks for immunoglobulin &/or complement of surface of red blood cell (normally neither f d RBC f )found on RBC surface)

• Coombs reagent - combination of anti-human immunoglobulin & anti human complementimmunoglobulin & anti-human complement

• Mixed with patient’s red cells; if immunoglobulin or complement are onimmunoglobulin or complement are on surface, Coombs reagent will link cells together and cause agglutination of RBCstogether and cause agglutination of RBCs

IMMUNE HEMOLYTIC ANEMIAIMMUNE HEMOLYTIC ANEMIACoombs Test - Indirect

• Looks for anti-red blood cell antibodies in th ti t’ i l f dthe patient’s serum, using a panel of red cells with known surface antigensC bi ti t’ ith ll f• Combine patient’s serum with cells from a panel of RBC’s with known antigensAdd C b ’ t t thi i t• Add Coombs’ reagent to this mixture

• If anti-RBC antigens are in serum, l ti tiagglutination occurs

HEMOLYTIC ANEMIA - IMMUNE

D R l t d H l i• Drug-Related Hemolysis• Alloimmune Hemolysis

– Hemolytic Transfusion Reaction – Hemolytic Disease of the Newborn

• Autoimmune Hemolysis– Warm autoimmune hemolysisWarm autoimmune hemolysis– Cold autoimmune hemolysis

IMMUNE HEMOLYSISIMMUNE HEMOLYSISDrug-Related

• Immune Complex Mechanism– Quinidine, Quinine, IsoniazidQuinidine, Quinine, Isoniazid

• “Haptenic” Immune MechanismPenicillins Cephalosporins– Penicillins, Cephalosporins

• True Autoimmune Mechanismh ld O d– Methyldopa, L-DOPA, Procaineamide,

Ibuprofen

DRUG-INDUCED HEMOLYSISDRUG INDUCED HEMOLYSISImmune Complex Mechanism

• Drug & antibody bind in the plasma• Immune complexes either• Immune complexes either

– Activate complement in the plasma, or– Sit on red blood cell

• Antigen-antibody complex recognized by RE system

d ll l d “ b d ” f• Red cells lysed as “innocent bystander” of destruction of immune complex

• REQUIRES DRUG IN SYSTEM• REQUIRES DRUG IN SYSTEM

DRUG-INDUCED HEMOLYSISDRUG INDUCED HEMOLYSISHaptenic Mechanism

• Drug binds to & reacts with red cell f t isurface proteins

• Antibodies recognize altered protein, ±drug, as foreign

• Antibodies bind to altered protein & pinitiate process leading to hemolysis

DRUG-INDUCED HEMOLYSISDRUG INDUCED HEMOLYSISTrue Autoantibody Formation

• Certain drugs appear to cause antibodies that react with antigens normally found on RBC surface, and do so even in the absence of the drug

DRUG-INDUCED HEMOLYSIS -DRUG INDUCED HEMOLYSIS Mechanisms

ALLOIMUNE HEMOLYSISALLOIMUNE HEMOLYSISHemolytic Transfusion Reaction

• Caused by recognition of foreign antigens on transfused blood cells

• Several types– Immediate Intravascular Hemolysis (Minutes) - Due to

preformed antibodies; life-threateningpreformed antibodies; life-threatening– Slow extravascular hemolysis (Days) - Usually due to

repeat exposure to a foreign antigen to which there i ll l ild twas a previous exposure; usually only mild symptoms

– Delayed sensitization - (Weeks) - Usually due to 1st exposure to foreign antigen; asymptomatic

INCOMPATIBLE RBC TRANSFUSIONRate of Hemolysis

60

80

100

ells

(%)

20

40

60

urvi

ving

Ce

0

20

0 1 2 3 4 5 6 7

S

Weeks Post-Transfusion

Normal Immediate Intravascular HemolysisSlow Extravascular Hemolysis Delayed Extravascular Hemolysis

ALLOIMMUNE HEMOLYSISALLOIMMUNE HEMOLYSISTesting Pre-transfusion

• ABO & Rh Type of both donor & i i trecipient

• Antibody Screen of Donor & Recipient, including indirect Coombs

• Major cross-match by same procedure j y p(recipient serum & donor red cells)

ALLOIMMUNE HEMOLYSISALLOIMMUNE HEMOLYSISHemolytic Disease of the Newborn

Due to incompatibility between mother• Due to incompatibility between mother negative for an antigen & fetus/father positive for that antigen Rh incompatibilitypositive for that antigen. Rh incompatibility, ABO incompatibility most common causes

• Usually occurs with 2nd or later pregnanciesUsually occurs with 2nd or later pregnancies• Requires maternal IgG antibodies vs. RBC

antigens in fetusg

ALLOIMMUNE HEMOLYSISALLOIMMUNE HEMOLYSISHemolytic Disease of the Newborn - #2

Can cause severe anemia in fetus with• Can cause severe anemia in fetus, with erythroblastosis and heart failure

• Hyperbilirubinemia can lead to severe brain• Hyperbilirubinemia can lead to severe brain damage (kernicterus) if not promptly treated

• HDN due to Rh incompatibility can be almost• HDN due to Rh incompatibility can be almost totally prevented by administration of anti-Rh D to Rh negative mothers after each gpregnancy

AUTOIMMUNE HEMOLYSIS

D t f ti f t tib di th t• Due to formation of autoantibodies that attack patient’s own RBC’s

• Type characterized by ability of autoantibodies to fix complement & site of RBC destruction

• Often associated with either lymphoproliferative disease or collagen vascular disease

AUTOIMMUNE HEMOLYSISAUTOIMMUNE HEMOLYSISWarm Type

Usually IgG antibodies• Usually IgG antibodies• Fix complement only to level of C3, if at all

I l b li bi di t ll t• Immunoglobulin binding occurs at all temps• Fc receptors/C3b recognized by

mac ophages the efo emacrophages; therefore,• Hemolysis primarily extravascular

70% i t d ith th ill• 70% associated with other illnesses• Responsive to steroids/splenectomy

AUTOIMMUNE HEMOLYSISAUTOIMMUNE HEMOLYSISCold Type

• Most commonly IgM mediated• Most commonly IgM mediated• Antibodies bind best at 30º or lower• Fix entire complement cascade• Fix entire complement cascade• Leads to formation of membrane attack

complex, which leads to RBC lysis in p , yvasculature

• Typically only complement found on cells• 90% associated with other illnesses• Poorly responsive to steroids, splenectomy;

i t l h iresponsive to plasmapheresis

HEMOLYTIC ANEMIAHEMOLYTIC ANEMIASummary

• Myriad causes of increased RBC• Myriad causes of increased RBC destruction

• Marrow function usually normal• Marrow function usually normal• Often requires extra folic acid to

maintain hematopoiesismaintain hematopoiesis• Anything that turns off the bone

marrow can result in acute lifemarrow can result in acute, life-threatening anemia