Hematology - Hemolytic Anemia

hemolytic anemia

a state of accelerated erythrocyte destruction (loss of survival) characterized by premature removal of circulating red blood cells and increased bone marrow production of replacement cells

classification:

• acute versus chronic

• inherited versus acquired

• intrinsic versus extrinsic

• intravascular versus extravascular

• fragmentation versus macrophage-mediated

anemia of increased destruction

hemolytic anemia -

decreased RBC survival

normocytic, normochromic anemia

polychromasia

reticulocytotic - increased in response to increased RBC destruction

haptoglobin - decreased

bilirubin - increased indirect (unconjugated)

increased LD

urine hemosiderin - increased

signs of hemolysis

hemoglobinuria

hemoglobinemia

→ hemoglobin in the urine

→ bilirubin is increased in plasma and haptoglobin is decreased

causes of accelerated RBC destruction

two main categories -

1. hereditary causes

   1. intrinsic defects

2. acquired causes

   1. primarily extrinsic defects

   2. exception: paroxysmal nocturnal hemoglobinuria - intrinsic

diseases associated with accelerated RBC turnover

RBC life span - 120 days

three phases -

bone marrow production phase

circulating phase

removal phase

initial lab tests

CBC - Hgb, HCt, MCV, MCH, MCHC, RDW,

RBC morphology

reticulocyte count, polychromasia

serum haptoglobin

serum LD

serum bilirubin (direct and indirect)

direct antiglobulin test

automated hemogram

anemia - varies with the cause and rate of hemolysis

RBC indices - MCV clues:

• slight macrocytosis <= 110 may be due to increased reticulocytosis

• > 115 - think macrocytic anemia

• < 70 with normal MCHC, think hemoglobinopathy i.e B-thalassemia

peripheral blood smear clues

reticulocytosis - causes polychromasia and macrocytosis

spherocytes - hereditary spherocytosis, autoimmune H.A, burns, ABO-HDN

target cells - hemoglobinopathies, jaundice, post-splenectomy

cell fragments - DIC, HUS, TTP, mechanical hemolysis

increased RBC production response

Increased renal erythropoietin (EPO) secretion due to anemia

marrow erythroid hyperplasia with increased storage Fe on biopsy

reticulocytosis - increased raw and corrected counts

circulating nRBCs if marrow stress is severe

chemistry tests

elevated LDH (LD) - directly released from RBCs

haptoglobin - decreased/depleted

elevated bilirubin

hemoglobinuria/hemoglobinemia

direct antiglobulin test

a positive DAT (firect anti-globulin test) indicates in-vivo coating of RBCs with immunoglobulins (IgG, IgM, + or C3)

suggests that the hemolysis is due to an immune mechanism

• weak +DAT may or may not indicate immune hemolysis

• DAT doesn’t separate autoimmune from alloimmune type hemolytic anemia

urine studies

hemoglobinuria gives pink/red to “smoky” or “cola-colored” urine

• free urine Hgb can be measured

urine hemosiderin test - cytospin sediment stained with prussian blue, stains sloughed renal tubular cells containing Hgb breakdown Fe blue

• develops late (days after the hemoglobinuria event)

RBC survival assay

Takes patient blood and mixes it with radioactive label

blood is given back to patient intravenously

measurements taken periodically to measure the circulating RBC

normal half life is 25-32 days

• mild hemolysis - 20-25 days

• moderate hemolysis - 15-20 days

• severe hemolysis < 15 days

tempo and site of hemolysis

intravascular hemolysis - rapid tempo

• free plasma hemoglobin, hemoglobinuria, unconjugated bilirubin, depleted haptoglobin

extravascular hemolysis - slower tempo, mainly spleen

• conjugated bilirubin

hemolytic anemia - causes

intrinsic hemolysis

• membrane abnormalities

• metabolic abnormalities

• hemoglobinopathies

extrinsic hemolysis

• nonimmune

• immune

hereditary shape abnormalities

all result from disturbances in either the make-up or function of the RBC membrane, resulting in abnormal shapes of the RBC and in decreased survival of erythrocytes

• hereditary spherocytosis

• hereditary elliptocytosis

• hereditary pyropoikilocytosis

• hereditary stomatocytosis/hydrocytosis

• hereditary xerocytosis

• acanthocytosis

structure of RBC membrane

1. Lipid bilayer

2. central cytoskeleton

key normal functions:

• flexibility - to squeeze through tiny capillaries

• maintenance of biconcave shape - for max gas exchange and other functions

categories of RBC membrane defects

1. lipid content - e.g Acanthocytosis

2. cytoskeleton - e.g Hereditary spherocytosis, hereditary ellipotcytosis, hereditary pyropoikilocytosis

3. permeability of the membrane - e.g hereditary stomatocytosis, hereditary xerocytosis

acanthocytosis

cause - decreased plasma and membrane lipid balance, causing irregularly formed RBCs

genetic cause - abetalipoproteinemia, McLeod syndrome

acquired cause - alcoholic cirrhosis (Burr cell anemia), myelodysplasia, hypothryroidism, malnutrition

additional tests - decreased triglycerides and cholesterol, sphingomyelin increased, peripheral blood smear - marked acanthocytosis, retic count - normal to increased

hereditary spherocytosis

most common membrane defect

autosomal dominant

cause - decreased or dysfunctional cytoskeleton protein - spectrin, ankyrin, protein 4.2 or Band 3

results - loss of biconcavity and deformability of RBCs and increased turnover of cells

• splenic environment degenerates cells leading to early lysis

Lab evaluation:
 Hgb & Hct: Normal to moderate decrease
 MCHC: usually increased (36 or greater)
 Peripheral smear: Spherocytes and anisocytosis
 Reticulocyte count: Increased
 Osmotic fragility: increased (more fragile)

 Treatment: Splenectomy for severe cases
 Decreases hemolysis, increases RBC survival
 Howell-Jolly Bodies, Target Cells & Pappenheimer
bodies usually seen on peripheral blood smear

osmotic fragility test

test ability of RBCs to swell in a hypotonic solution

• normal RBC can swell to 1.8 x resting volume before lysis will occur

• spherocytes have less redundant membrane and lyse earlier

osmotic fragility

Increased Fragility:
 Hereditary Spherocytosis- the classic example
 Hereditary Stomatocytosis
 Warm AIHA
 Hereditary Pyropoikilocytosis
 Decreased Fragility
 Hereditary Xerocytosis
 Thalassemia
 Hereditary Elliptocytosis-variable

hereditary elliptocytosis

Elliptical RBCs
 Cause: Defective spectrin
 Severity varies: 90% of patients are
asymptomatic and do not experience
significant hemolysis
 Labs:
 H & H: normal – decreased
 Peripheral smear: Elliptocytes
 Retic count: elevated
 Osmotic fragility: variable

hereditary pyropoikilocytosis

Autosomal recessive, rare
Subset of HE
 Cause: Defective spectrin
causes variable RBC sizes and
shapes
 Labs:
 MCV: decreased
 RBC morph shows budding,
fragmentation,
microspherocytes, elliptocytes
 Osmotic fragility: increased
 Positive heat sensitivity test:
RBCs fragment when warmed to
45 C

permeability problems

H. Stomatocytosis:
 Cause: Membrane defect
 allows water to enter the cell
 MCV increased
 MCHC decreased
 Osmotic fragility: Increased
 H. Xerocytosis:
 Membrane defect
 allows water to leave the cell
 Target cell morph
 MCV increased, MCHC increased
 Osmotic fragility: Decreased

Rh-null disease

Lack of all Rh-Hr antigens on red cells
 Defect in membrane with increased
permeability to K
 Mild anemia with stomatocytes and
elliptocytes

clinical findings in hereditary membrane defects

highly variable, many have well=compensated hemolytic anemia

• trends: the more severe the disease, the earlier in life it’s detected

• high incidence of bilirubin gallstones

• hereditary spherocytosis can be clinically relieved by splenectomy

hereditary RBC disorders due to deficiencies of the glycolytic pathway

glycolytic pathway - Embden-meyerhoff pathway

Embden-Meyerhoff pathway

RBCs have no mitochondria, therefore no oxidative metabolism

energy created via E-M pathway in which 1 molecule glucose generates a net 2 ATPS

multiple enzymes involved, among which there may be inherited deficiencies, leading to hemolytic anemia

as a group, these are called “hereditary non-spherocytic hemolytic anemias”

metabolic defect

G6PD deficiency
 Hexose monophosphate
shunt
 Most common RBC
enzyme defect, >50
variants
 X-linked
 Low glutathione due to
low NADPH
 Oxidative hemolysis
 Heinz bodies,
Spherocytic
 Primaquine, fava beans

 Pyruvate kinase
deficiency
 E-M Glycolysis
 Low RBC ATP level
 Non-spherocytic

hereditary non-spherocytic hemolytic anemia - general characteristics

early onset (infancy), with jaundice, splenomegaly, pigment gallstones

no associated with drug ingestion

autosomal recessive inheritance

hexose monophosphate shunt deficiencies

10% of energy created here, but critically generates NADPH, a reducing agent essential to prevent oxidative damage to RBC membrane

key enzyme - GLucose-6-phosphate-dehydrogenase (G-6-PD)

G-6-PD deficiency

inheritance - X:linked

• fully expressed in males, females are heterozygous and have 2 populations of RBCs (those deficient and those note)

• cause:

  • decreased G6PD prevents synthesis of glutathione which is required to prevent hydrogen peroxide buildup in RBC during oxidant stress due to infections, chemicals, and food. Increased H2O2 levels irreversibly denature hemoglobin resulting in Heinze bodies and hemolysis.

G6PD deficiency - lab results

reticulocytes counts - increased during hemolytic event

supravital stain for Heinz bodies = +

fluorescent spot test - negative

quantitative G6PD assay - decreased

clinical findings

Key: Episodic hemolysis
 Differential narrowed to G-6-PD deficiency
 OR PNH, Malaria, or some unstable Hgb
 What brings on an episode of
hemolysis?
 Oxidative stress : certain drugs, infections,
foods (fava beans)

drugs/chemicals/foods associated with hemolytic events in G6PD deficiency

anti-malarial agent primaquine

sulfonamides

naphthalene - moth balls

fava beans

lab findings

Variable degree of anemia, reticulocytosis
 RBC Morphology:
 HEINZ BODIES- clumps of denatured Hgb
 “BITE” cells- where spleen has removed a Heinz
body, leaving a dent or bite out of the RBC
 Enzyme screens for G-6-PD, also for
Glutathione Reductase activity

PK deficiency - lab approach

Inheritance: Autosomal recessive
 Cause:
 Missing Pyruvate kinase: Needed to convert
Phosphoenolpyruvate to pyruvate
 A decrease of PK in the EMP prevents synthesis of ATP
needed for cell membrane function
 Lab Evalulation:
 Specific enzyme screening for PK
 Fluorescent spot test for PK: Decreased
 PEP + ADP ---PK ----> Pyruvate + ATP
 Pyruvate + NADH + H -LDH----> Lactate + NAD
 Quantitative PK: Decreased
 Osmotic fragility: Normal

Methemoglobin reductase deficiency

Glycolytic pathway enzyme needed to
reduce ferric (Fe3+) to ferrous (Fe2+) iron
in hemoglobin, allowing O2 binding
 Autosomal recessive inheritance
 Labs: Increased methemoglobin on
assay
 Decreased NADH- methemoglobin
reductase activity on enzyme screen

Paroxysmal Nocturnal Hemoglobinuria

A rare Acquired Stem Cell disorder that results in RBC
membrane defect
 Missing PIG-A gene
 Deficiency of phosphatidyl inositol glycan
 Membrane defect makes the RBC have increased
sensitivity to complement-mediated cell lysis
 As a result, red blood cells hemolyze too early
 The red cells leak hemoglobin into the blood, which can
pass into the urine
 This can happen at any time, but is more likely to occur
during the night or early morning
 RBC precursors lack CD 55 and CD59 (complement
inhibiting)

block complement from binding to RBCs

PNH clinical features

Occurs most often in adults
 Irregular Episodes of acute intravascular
hemolysis
 Worse during sleep when pH is lower,
enhancing complement binding
 Hemoglobinuria
 Moderate thrombocytopenia → platelets may be affected
 Venous thrombosis, infections

PNH - lab findings

Peripheral blood:
 Normocytic to macrocytic → increased prod of reticulocytes
 NRBCs
 Neutropenia & thrombocytopenia → WBC and platelets increased
 Indices: normal
 BM: Normoblastic
 Retic count: increased 5% - 10%
 Additional tests:
 Immunophenotyping: Decreased CD 55 & 59
 Sucrose Hemolysis test positive
 Acid Hemolysis Test (HAM’S test)
 Decreased haptoglobin
 Urine hemosiderin nearly always positive