Hemolytic anemia

Fazel Elahi (MD)Oncologist &Hematologist

Production of Erythrocytes: Erythropoiesis

Erythrocytes (RBCs)

Components of Whole Blood

Withdraw blood

and place in tube

1 2 Centrifuge

Plasma

(55% of whole blood)

Formed

elements

Buffy coat:

leukocyctes and

platelets

(<1% of whole blood)

Erythrocytes

(45% of whole blood)

Erythropoietin Mechanism

Reduces O2

levels in blood

Erythropoietin

stimulates red

bone marrowEnhanced

erythropoiesis

increases RBC

count

Normal blood oxygen levels Stimulus: Hypoxia due to

decreased RBC count,

decreased availability of O2

to blood, or increased

tissue demands for O2

Start

Kidney (and liver to a

smaller extent) releases

erythropoietin

Increases

O2-carrying

ability of blood

Structure of Hemoglobin

Hemolysis:

Any condition characterized by a

significantly decreased erythrocyte life

span

RBC destrution

Hemolysis

Ineffective erythropoesis

hematoma

Haemolysis may be predominantly

extravascular (i.e. phagocytic uptake) or

intravascular (i.e. in the blood stream

DefinitionA haemolytic anaemia is an anaemia

resulting from an increased rate of red cell

destruction. This results in a shortening of

the red cell life span

Erythropoiesis in the bone marrow can

expand by up to 6-fold to compensate for

accelerated red cell destruction, but

anaemia results when red cell destruction

exceeds erythropoiesis

Increased erythropoiesis leads to a reticulocytosis

classification

Intavascular or Extravascular

Inheritance or Aquired

Extrinsic or Intrinsic

HEMOLYTIC ANEMIACauses

INTRACORPUSCULAR HEMOLYSIS

Membrane Abnormalities

Metabolic Abnormalities

Hemoglobinopathies

EXTRACORPUSCULAR HEMOLYSIS

Nonimmune

Immune

Classification

The haemolytic anaemias can be classified

as inherited or acquired

Inherited haemolytic anaemias

can be subclassified into

I. Haemoglobin defects

II. Red cell membrane defects

III. Red cell enzyme defects

HEMOLYTIC ANEMIAMembrane Defects

Microskeletal defects

Hereditary spherocytosis

Membrane permeability defects

Hereditary stomatocytosis

Increased sensitivity to complement

Paroxysmal nocturnal hemoglobinuria

Hereditary hemolyisis

Hemoglobinopathy

(alfa,beta thal, S,S beta ,C ,SC…..)

Membran disorders

( HS ,HE, HPP, STOMATOCYTOSIS …)

Enzyme defect:1-Glycolytic pathway

2-Pentose phosphate pathway

3-Nucleotid metabolism

INHERITED HAEMOLYTIC ANAEMIAS

HAEMOGLOBIN DEFECTS Thalassaemia

Sickle cell anaemia

Other haemoglobin defects

MEMBRANE DEFECTS Hereditary spherocytosis

Other membrane defects

ENZYME DEFECTS G6PD deficiency

Other enzyme defects

All Hereditary etiology of hemolysis

have intrinsic defect

Only nonhereditary intrinsic defect

(PNH)

In some of intrinsic defect hemolysis one extrinsic

factor cause hemolysis or aggrevated hemolysis

(G6PD)

Acquired haemolytic anaemias

immune or non-immune

The entire classification encompasses a

very large number of different

haemolytic disorders

ACQUIRED HAEMOLYTIC

ANAEMIAS

IMMUNE Auto-immune haemolytic anaemia

Allo-immune haemolysis

NON-IMMUNE Microangiopathic haemolytic anaemias

Infections

Drugs and toxins

Acquired membrane disorders

Mechanical and physical agents

Aquired Immunohemolytic

Microangiopathic

Traumatic

Infectious agent

Chemical agent

Physical agent

Spur cell anemia

PNH

hypophosphATEMIA

Vit .E deficency

EXTRACORPUSCULAR

HEMOLYSISNonimmune

Mechanical

Infectious

Chemical

Thermal

Osmotic

Microangiopathic Hemolytic

AnemiaCauses

Vascular abnormalities Thrombotic thrombocytopenic purpura

Renal lesionsMalignant hypertension

Glomerulonephritis

Preeclampsia

Transplant rejection

VasculitisPolyarteritis nodosa

Rocky mountain spotted fever

Wegener’s granulomatosis

Microangiopathic

Hemolytic Anemia

Vascular abnormalities AV Fistula

Cavernous hemangioma

Intravascular coagulation predominantAbruptio placentae

Disseminated intravascular coagulation

Hemolysis

Acute

Chronic

Acute on chronic

Clinical finding

Acute haemolysis presents with :

Fatigue, Pallor, Jaundice, Fever, Chills, Low

back pain, Splenomegaly and Congestive

cardiac failure

Haemoglobinuria results if the haemolysis is

predominantly intravascular

Clinical finding

Chronic haemolysis :

(e.g. thalassemia, sickle cell anaemia etc)

Present with pallor, jaundice, splenomegaly

and congestive cardiac failure; but in addition there

may be gallstones, bony deformities and

pathological fractures due to marrow erythroid

expansion with thinning of the bone cortex

Causes of Intravascular Haemolysis

Infection :Malaria, clostridium perfringen sepsis G6PD deficiency

Severe auto-immune haemolytic anaemia

Paroxysmal nocturnal haemoglobinuria

Paroxysmal cold haemoglobinuria

Transfusion :

ABO mismatched blood, infected blood

Burns

Cardiac prosthesis

Intravascular haemolysis

Elevated plasma haemoglobin (haemoglobinaemia) urinary haemoglobin (haemoglobinuria)

serum LDH

urinary haemosiderin

Elevated unconjugated bilirubin and reduced haptoglobins are found in both intra- and extravascular haemolysis

Chronic intravascular haemolysis

Results in iron deficiency due to the loss of haemoglobin, haemosiderin and ferritin in

the urine

Extravascular haemolysis

Red cells are destroyed by

phagocytosis by macrophages in

the spleen, liver and bone marrow

This uptake system is often referred as

the reticulo-endothelial system (R.E.S).

In this situation, free haemoglobin is

not liberated into the bloodstream

Detecting reasons of hemolysis

Duration and severity

Jundice (acholuric )

Splenomegaly

Cholelithiasis (black stone)

Leg ulcer

Skeletal abnormality

Fever , shaking chills

Pain ( limb, back,abdominal,headache )

Malaise ,vomiting ,hypotention

Shock ,oliguria, aneuria

History ( famalial , personal , drugs )

Physical exam

lab test :

1- Sign of accelerated RBC destruction

2- Sign of accelerated erythropoesis

Normal blood film: There is a slight degree of anisocytosis (variation in

cell size). The width of central pallor is less than one-

third of the diameter of the cell.

Laboratory finding

Peripheral blood film

Increased red cell destruction e.g. jaundice, unconjugated hyperbilirubinaemia, elevated serum lactate dehydrogenase (LDH), reduced haptoglobins

Increased erythropoiesis e.g. reticulocytosis, polychromasia, radiological changes of erythroid hyperplasia in chronic haemolysis

The biochemical profile is characteristic of a pre-hepatic jaundice with unconjugated hyperbilirubinaemia and elevated LDH, but normal hepatocellular enzymes

There are a relatively small number of causes of intravascular haemolysis

Therefore, tests for intravascular haemolysis are useful because the differential diagnosis for haemolysis can be considerably simplified if it can be shown that predominantly intravascular haemolysis is taking place

RBC MORPHOLOGY HAEMOLYTIC ANAEMIA

Spherocytes hereditary spherocytosis, immune haemolysis, sepsis

Fragmentation DIC, artificial heart valves, malignancy, TTP, HUS

Agglutination immune haemolysis

Target cells liver disease, thalassaemia, haemoglobin C

Sickle cells S/S, S/C, S/Thal

Blister cells G6PD deficiency

Acanthocytes liver disease, pancreatitis, abetalipoproteinaemia

Prickle cells pyruvate kinase deficiency

post-splenectomy blood film ,nucleated red cells, Howell-Jolly bodies, acanthocytes, target cells, mild to moderate leukocytosis and thrombocytosis

This film is from a splenectomised patient who was in chronic renal failure, and therefore also shows burr cells.

Reticulocytes are larger than mature red cells and stain purplish with Romanowsky stains (e.g. May Grunwald Giemsa). Reticulocytosis leads to a rise in the MCV (mean cell volume).

The reticulocyte count is a simple test

that provides a rough guide to the rate of red

cell production

The reticulocyte count does not neccessarily

reflect true erythroid activity because of

premature or delayed release of reticulocytes

from the marrow and variable rates of

reticulocyte maturation in response to different

conditions

Reticulocyte count

(reference range 0. 2 - 2.0%)

Number of reticulocytes per litre

(10-100 x 109/L)

Reticulocyte count: methylene blue stain

BMA

Erythroid Hyperplasia

(haemoglobinaemia):

Reddish plasma in severe intravascular haemolysis

Urinary haemosiderin May persist for several weeks after an

acute haemolytic episode, and may

therefore be useful in detecting a bout

of recent haemolysis

Urinary haemosiderin: Perl's iron stain of

urine cellular debris

Sign of RBC destruction

Uncongugated bilirubin increased

Urobilinogen “ “ “

LDH LDH2>LDH1 “ “ “

Haptoglobin deceased

GHb glycosylated hemoglobin “

RBC life span (t ½ Cr ) “

Carbon menoxide increased

Free haemoglobin binds with high affinity to a serum glycoprotein, haptoglobin and the complex is cleared by the reticulo-endothelial system (R.E.S.), resulting in a low or absent serum haptoglobin.

Albumin combines with haem to form methaemalbumin which gives a brown colour to the plasma. Methaemalbumin can be biochemically detected by Schumm's test

(LDH) is released from red cells during haemolysis and this contributes to a rise in serum LDH, which is a useful marker for intravascular haemolysis.

Massive deposition of free haemoglobin in the renal tubules can cause acute oliguric renal shutdown, a medical emergency, as seen in the haemolytic transfusion reaction.

Intravascular hemolysis

HEMOGLOBINEMIA

HEMOGLOBINURIA

HEMISIDRINURIA

METHEMOGLOBINEMIA

METHEMALBUMINEMIA

HEMOPEXIN decreased

Urine hemosiderrin

Spacific Lab test Antiglobulin (coomb”s ) test

Osmotic fragility test

Autohemolysis

Heat stability test (denaturation procedure )

Heinz body formation

Isopropanol precipitation test

Sickling test

Retic with incubation (H.inclusion)

Identifying Enzyme deficency

Hemoglobin & globin chain study

PNH Test HAM& sucrose lysis test ,flucytometry

Glutathion stability test

Ascorbate - cyanid test

Sign of accelerated erythropoesis

Retic

NRBC (erythroblastosis)

Polychromatophilia

Macrocytosis

Basophilic stipling

Leukocytosis (PMN rise), leukopenia

Thrombocytosis ,thrombocytopenia, large Plt

Erythrophagocytosis

Autoagglutination

BM erythroid hyperplasia , megaloblastic change

Effective erythropoesis (PITR ,EITR )

Macrocyte

Tin-macrocyte ,leptocyte ,wafer cell

Target cell .codocyte

Spherocyte , microspherocyte

Elliptocyte ovalocyte

Stomatocyte I,II,III, spertostomatocyte spheroechinocyte IV (dense crenated spher ,prickle cell )

Acanthocyte.spur cell

Echinocyte .Burr cell,Crenated cell,berry cell type I,II,III

Schistocyte ,helmet ,fragmented RBC

Sickle cell ,Drepantocyte, boat shape RBC

Keratocyte ,Horn cell

Tear drop ,tennis racket, dacrocyte

Basophilic stippling

Heinz body ,howell jolly body

Agglutination ,heophagocytosis

Cabot Ring

Reticulocyte Manual Count by

Supravital Stain: Normal Count

Reticulocytes: Elevated Count

Hemolytic crisis

Aplastic crisis

Megaloblastic crisis

Compensated hemolytic state

A state of hemolysis in which the

resulting increased erythrocyte

production is able to keep up

with accelerated RBC

destruction, thus averting any

anemia

Hemolytic anemiaA state of hemolysis in which increased erythrocyte

production is insufficient to keep up with

accelerated RBC destruction, thus producing

anemia

This anemia is characterized as

normochromic/normocytic, except when sufficient

outpouring of the larger reticulocytes produces a

resulting elevation of the MCV

Diagnosis of hemolytic anemia is

performed in four steps

Step -1

1- Establish that anemia exists

Step - 2Marrow response

Hemolysis is demonstration of an

attempted marrow response to erythrocyte

destruction

Reticulocyte count

Absolute retic count (in cells/µL)

Reticulocyte production index (RPI)

Step - 3

Look for erythrocyte detritus/their

component catabolites, such as free

hemoglobin, methemoglobin,

methemalbumin, bilirubin, and

urobilinogen, as well as the specific

binding proteins for these catabolites,

such as haptoglobin and hemopexin.

Step - 4

The first distinction to make is to determine whether the hemolysis is (extravascular hemolysis) or(intravascular hemolysis)

Both types produce indirect hyperbilirubinemia, urobilinogen in stool and urine, decreased serum haptoglobin, and reticulocytosis

intravascular hemolysis produces hemosiderin in the urine sediment, free hemoglobin in the serum (which may be grossly visible), and free denatured hemoglobin in the urine. helmet-shaped schizocytes (or "schistocytes"), which can be seen on the routine peripheral blood film

Extravascular hemolytic anemias may produce spherocytes,

Leptocyte (hypochromic microcytic

red cell), Blood smear

Poikilocyte (DIC), Blood smear,

Tear Drop Cell, Blood smear

Basophilic Stippling, Blood smear

Pappenheimer's body, Blood smear

Howell-Jolly's body, Blood smear

Rouleaux Formation, Blood smear

Malaria, Blood smear

Thalassemia, Bone marrow

Schizocytes

FREQUENCY

Internationally: Hemolytic anemia represents

approximately 5% of all anemias.

RACE

Sickle cell disorders are found primarily in Africans, African Americans, some Arabic peoples, and Aborigines in southern India

Several variants of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency exist. The A variant generally is found in West Africans and African Americans. Approximately 10% of African Americans have at least 1 gene for this variant

The Mediterranean variant occurs in individuals of Mediterranean descent and in some Asians

SEX

Most cases of hemolytic anemia are not specific to any gender.

Autoimmune hemolytic anemia (AIHA) is slightly more likely to occur in females than in males.

G-6-PD deficiency is a X-linked recessive disorder. Males usually are affected, and females are carriers

AGE

Hemolytic anemia can occur at any age.

Hereditary disorders usually are evident

early in life

AIHA is more likely to occur in middle-aged

and older individuals

Peripheral smear and morphological examination

Identifies polychromasia, indicating RBC immaturity

reticulocytosis

Demonstrates spherocytes, suggesting congenital

spherocytosis or AIHA

Can identify schistocytes (fragmented red cells),

suggesting TTP, HUS, or mechanical damage

Can help diagnose a concomitant underlying

hematological malignancy associated with hemolysis

(ie, CLL)

Reticulocyte count

An increased reticulocyte count is a criterion for

hemolysis but is not specific for hemolysis

An increase may be caused by blood loss or a bone

marrow response to iron, vitamin B-12, or folate

deficiencies

The reticulocyte count may be normal or low in

patients with bone marrow suppression despite

ongoing severe hemolysis

Lactic acid dehydrogenase

Serum LDH is a criterion for hemolysis. LDH is not specific because it is ubiquitous and can be released from the neoplastic cells of the liver or other damaged organs.

Although an increase in LDH isozyme 1 and 2 is more specific for RBC destruction, these enzymes also are increased in patients with myocardial infarction.

Serum haptoglobin

A low serum haptoglobin is a criterion for moderate-to-severe hemolysis

A decrease is more likely in intravascular hemolysis than in extravascular hemolysis, but it is an acute phase reactant

The presence of concomitant infection, other reactive states, or chronic hemolysis may mask the diagnosis by raising haptoglobin levels

Indirect bilirubin

Unconjugated bilirubin is a criterion for hemolysis, but it is not specific because an elevated bilirubin also may indicate Gilbert disease

With hemolysis, the level of indirect bilirubin usually is less than 4 mg/dL

Higher levels of indirect bilirubin indicate compromised hepatic function or cholelithiasis and hemolysis

Changes in the LDH and serum

haptoglobin levels are the most

sensitive general tests because the

indirect bilirubin is not always

increased

Imaging Studies:

Use ultrasound to estimate spleen size. The

physical examination occasionally does not

detect significant splenomegaly.

Chest radiograph is used to evaluate

cardiopulmonary status

Other Tests:

ECG and other studies are used to evaluate

cardiopulmonary status

Prognosis:

Depends upon the underlying cause for hemolysis

Patient Education:

The patient must be able to identify symptoms and

signs of hemolysis recurrence and seek prompt

medical attention if they occur.

There are numerous fragmented RBC's seen here. Some of the irregular shapes appear as "helmet" cells. Such fragmented RBC's are known as "schistocytes" and they are indicative of a microangiopathic hemolytic anemia (MAHA) or other cause for intravascular hemolysis. This finding is typical for disseminated intravascular coagulopathy (DIC)

The CBC of a patient with microangiopathic hemolytic anemia (MAHA) demonstrates a markedly increased RDW (red cell distribution width) due to the marked variation in size and shape of the RBC population.

Erythrocyte Inclusions with Wright’s Stain

Inclusion Composition Appearance Condition

Basophilic Precipitated Evenly dispersed Lead poisoning

stippling ribosomes fine or coarse granules thalassemia

other anemia

Howell-Jolly Nuclear Dense, round Post splenectomy

bodies fragment blue granule

Pappenheimer Iron-containing Small blue granules Anemiasbodies granules in clusters

Organism Small blue inclusion Malaria

Babesiosis

Basophilic Stippling

Howell-Jolly Body

Malaria

RBC Inclusions: Composite

Erythrocyte Distribution Abnormalities

Rouleaux formation Stacking of RBCs due to

increased plasma proteins

coating RBCs

Agglutination Antibody-mediated

clumping;

temperature dependent

Rouleaux Formation

Agglutination Reaction

Variations in RBC Size and

Shape

Anisocytosis Variations in size (e.g. microcytes)

Poikilocytosis Variations in shape (e.g. target cells)

Hypochromia Increased central pallor due to

decrease in hemoglobin

Variations in RBC Size and Shape

Anisocytosis Variations in size (e.g.

microcytes)

Poikilocytosis Variations in shape (e.g. target

cells)

Hypochromia Increased central pallor due to

decrease in hemoglobin

Hypochromic Microcytic RBC

Normal Hypochromic

microcytic

Hypochromia without Anisocytosis:

Thalassemia Trait

Severe Hypochromia: Iron

Deficiency Anemia

Mixed Population: Treated Iron

Deficiency Anemia

Microcytic Hypochromia: Alpha

Thalassemia (a-/--)

Microcytic Hypochromia: Beta

Thalassemia Major

Microcytic Hypochromia: Beta

Thalassemia Major

Macrocytic Anemia: Macro-

Ovalocytes

Shape Abnormalities of Erythrocytes

Terminology Description Condition

Target cells Central hemoglobin; target-shaped Liver disease;

thalassemia:

Abnormal Hgb; iron deficiency

Echinocyte Short spicules, equally-spaced Uremia, hypokalemia,

artifact

Acanthocyte Spiculated, irregular Liver disease (alcohol),

Post-splenectomy

Spherocyte Spherical, no central pallor HS, Immune hemolytic

anemia

Schistocyte Fragmented RBC, helmet cells MAHA, burns

Ovalocyte Oval/elliptical shaped Hereditary elliptocytosis,

Megaloblastic anemia

Sickle cell bipolar spiculated shape Hgb S-containing

“banana” shaped hemoglobinopathy

Teardrop cell single elongated extremity Myelophthistic changes

Bite cells Irregular gap in membrane G6PD deficiency

Target Cells

Diagnostic possibilities

Liver disease

Hemoglobinopathy

Thalassemia

Iron deficiency

Post-splenectomy

Lipid disorders

Echinocytes (Burr Cells)

Acanthocytes (Spur Cells)

Target

CellsSpur Cells

Morphologic Changes in Liver

Disease

Hepatorenal Syndrome:

Burr + Spur Cells

Spherocytes

Spherocytes: Autoimmune Hemolytic

Anemia

Spherocytes:

Hereditary Spherocytosis

Schistocytes: Microangiopathic Hemolytic Anemia

Elliptocytes: Hereditary

Elliptocytosis

Sickle Cell Anemia: Hgb SS

Hemoglobin SC Disease

Hemoglobin S-Beta Thalassemia

Teardrop Cells

Bite Cells

Heinz Bodies