Hemolytic , Macrocytic, Aplastic Anemia

Dr . Yenny Dian Andayani SpPD -KHOM

Division Hematologic Oncology Medic

Dept Internal Medicine Moh Hoesin General Hospital /
Faculty of Medicine Sriwijaya University
Palembang
 Morphological classification of anemias

Anemia - Morfologi
• MCV, MCH , MCHC nilai normal ?
1. Microcytic, hypochromic anemia
( decreased MCV < 80 dan MCH 27 pg )
2. Normocytic, normochromic anemia
( normal MCV 80 -95 fl dan MCH 27-34 pg)
3. Macrocytic, normochromic anemia
( increased MCV > 95 pg )
 Basic Blood Test
• Routine blood
consist of : WBC, RBC, Hb, Hct, Red blood cell
indices , Platelet count.
• CBC ( Complete Blood Count )
consist of routine blood + MCV, MCH, MCHC,
Peripheral Blood smear, RDW, Diff count WBC.
 Etiology of anemia
• Production  : anemia aplastic
• Destruction : anemia hemolytic
• Loss of blood : anemia cause hemorrhagic
• Disturbance metabolism : anemia chronic disease
 HEMOLYTIC ANEMIA
• Anemia of increased destruction
- Normochromic, normochromic anemia
– Shortened RBC survival
– Reticulocytosis - Response to increased RBC
destruction
– Increased indirect bilirubin
– Increased LDH
 Classification of Hemolytic Anemia

• Intracorpuscular factor
• Extracorpuscular factor
 Causes of HEMOLYTIC ANEMIA

• INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
• EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune
1. Intracorpuscular factor
Red cell abnormality
A. Hereditary
1. Membrane defect (spherocytosis, elliptocytosis)
2. Metabolic defect (Glucoze-6-Phosphate-Dehydrogenaze (G6PD)
deficiency, Pyruvate kinase (PK) deficiency)
3. Hemoglobinopathies (unstable hemoglobins,
thalassemias, sickle cell anemia )

B. Acquired
1. Membrane abnormality-paroxysmal nocturnal hemoglobinuria (PNH)
Mechanisms of hemolysis /
Pathogenesis:

1. Intra vascular
2. Extra vascular
 Hemolysis - RBC destruction

Extravascular Hemolysis Intravascular Hemolysis

Ingested by RE cell ( spleen Hgb liberated in

& liver) blood vessel

 Serum
Heme Globin Hgb + haptoglobin
haptoglobin

Reutilized +
Hgb + albumin
Iron Protoporphyrin hemalbumin &
plasma Hgb
Hgb excreted +
Reutilized bilirubin in urine hemoglobinuria
& hemosidenuria
Intravascular hemolysis :

- Red cells destruction occurs in vascular space

- Clinical states associated with Intravascular hemolysis:
Acute hemolytic transfusion reactions
Severe and extensive burns
Paroxysmal nocturnal hemoglobinuria (PNH)
Severe microangiopathic hemolysis
Physical trauma
Bacterial infections and parasitic infections (sepsis)
- Laboratory signs of intravascular hemolysis:
Indirect hyperbilirubinemia
Erythroid hyperplasia
Hemoglobinemia
Methemoalbuminemia
Hemoglobinuria
Absence or reduced of free serum haptoglobin
Hemosiderinuria
 Extravascular hemolysis :
- Red cells destruction occurs in reticuloendothelial system (RES)
- Clinical states associated with extravascular hemolysis :
Autoimmune hemolysis
Delayed hemolytic transfusion reactions
Hemoglobinopathies
Hereditary spherocytosis
Hypersplenism
Hemolysis with liver disease

- Laboratory signs of extravascular hemolysis:

Indirect hyperbilirubinemia
Increased excretion of bilirubin by bile
Erythroid hyperplasia
Hemosiderosis
 Anamnesa
• Fatigue
• Pallor
• Shortness of Breath
• Bleeding/petechiae
• Joint symptoms
• Rash-eg malar
• Family History
• Medications
 Physical Exam Findings
• Tachycardia
• Tachypnea
• Jaundice
• Splenomegaly
• Signs of congestive heart failure in rapidly
progressive anemia
Laboratory features:
Hematology test
1. Laboratory features
- Normocytic/macrocytic, hyperchromic anemia
- Reticulocytosis
- Increased serum iron
- Antiglobulin Coombs’ test is positive

2. Blood smear
- Anisopoikilocytosis, spherocytes
- Erythroblasts
- Schistocytes

3. Bone marrow smear

- Erythroid hyperplasia
 Extra corpuscular Factor
Autoimmune Hemolytic Anemia (AIHA)
* warm-reactive antibodies:
I. Primary
II. Secondary
1.Acute
- viral infections
- drugs (  -Methyldopa, Penicillin, Quinine, Quinidine)
2. Chronic
- rheumatoid arthritis, systemic lupus erythematosus
- lymphoproliferative disorders
(chronic lymphocytic leukemia, lymphomas,
WaldenstrÖm’s macroglobulinemia)
- miscellaneous (thyroid disease, malignancy )
 * cold-reactive antibodies:

I. Primary cold agglutinin disease

II. Secondary hemolysis:
- Mycoplasma infections
- Viral infections
- Lymphoproliferative disorders
III. Paroxysmal cold hemoglobinuria
Diagnosis
- Positive Coombs’ test (DAT)

Treatment:
- Steroids
- Splenectomy
- Immunosupressive agents
- Transfusion If needed
 Intracorpuscular FACTOR
A. Accquired :
Paroxysmal Nocturnal Hemoglobinuria (PNH)

1. Pathogenesis

- An acquired clonal disease, arising from a somatic mutation in a

single abnormal stem cell

- Glycosyl-phosphatidyl- inositol (GPI) anchor abnormality
- Deficiency of the GPI anchored membrane proteins
(decay-accelerating factor =CD55 and a membrane inhibitor
of reactive lysis =CD59)
-Red cells are more sensitive to the lytic effect of complement
- Intravascular hemolysis

2. Symptoms
- Passage of dark brown urine in the morning
3. PNH –laboratory features:

- Pancytopenia
- Chronic urinary iron loss
- Serum iron concentration decreased
- Hemoglobinuria
- Hemosiderinuria
 Lab Tests for PNH
• Acidified serum lysis test (Ham’s test)
• Sugar water (sucrose hemolysis) test
• Flow cytometry: lack of CD59 on RBCs, or lack of
CD59 or CD55 on granulocytes
• Treatment
- Washed RBC transfusion
- Allogenic bone marrow transplantion
 Intracorpuscular Factor
B. Herediter ( defect)
• Hemoglobinopathies
– Sickle Cell Disease, Sickle cell trait, Hemoglobin SC
– Thallasemias
• Unstable RBC Membrane
– Hereditary spherocytosis
• Metabolic Machinery
– G6PD deficiency
– Pyruvate kinase deficiency
 G6PD Deficiency
• It is an X-Linked recessive inheheritance ( males
usually affected and females are carriers)
• Risk factors : black, male, or having a family
history of G6PD deficiency
• G6PD enzyme functions in the Pentose –
Monophosphate shunt and in process, catalyzes the
reduction of NADP + to NADPH required in
triggering a cascade of events that can detoxify the
harmful oxidant H2O2
 Drugs that affect it
• Drugs - can precipitate this reaction include :
Anti malaria agents, sulfonamide, aspirin
NSAIDs, nitrofurantoin, quinidine, quinine
Other :
exposure to certain chemicals such as those in mothballs
and flava beans
 What are the symptom ?
• The most common symptoms :
- Abnormal palerness or lack of color of the skin
- jaundice, or yellow of the skin, eyes, and mouth
- dark color of urine
- Fever
-weakness
-Dizziness, confution
-Intolerance to physical activity
 Sign of anemia

• Pale skin
• Rapid pulse
• Heart murmur
• Enlarged spleen and liver
 Required test
• Blood tests are taken to measure levels of :
- Red cells, assess size, shape of red cells
- Hb level
- Reticulocytes
Other Blood test :
- Combs test
- Heinz body presentation
 Treatment
• Stopping use drug
• For severe case
- corticosteroid
- Intravenous imunoglobulin
-Imunosupresive
• Change in diet
• Transfusion if needed
 MACROCYTIC ANEMIAS
• Macrocytic anemias are characterized by large
RBCs with a normal hemoglobin content.
• Macrocytic anemias  Megaloblastic or
non-Megaloblastic
– Megaloblastic anemias are associated with
defective DNA synthesis and therefore,
abnormal RBC maturation in the bone
marrow (a nuclear maturation defect).
 MEGALOBLASTIC ANEMIAS
• The biochemical basis for this is as follows:
– Megaloblastic dyspoiesis (abnormal synthesis) occurs when
the DNA synthesis in the hematopoietic system is disrupted
or slowed down.
– Other rapidly proliferating cells in the body are also
affected.
– Administration of drugs that interfere with DNA metabolism
can be the cause of a megaloblastic anemia.
– On rare occasions there is an inherited disorder that affects
DNA synthesis
DIFFERENTIAL DIAGNOSIS
WITH A HIGH MCV
 NON-MEGALOBLASTIC MACROCYTIC ANEMIA

• Note that the macrocytic RBCs are not oval, but

are round.
• There are no hypersegmented neutrophils or
Howell-Jolly bodies
NON-MEGALOBLASTIC MACROCYTIC
ANEMIA
 Megaloblastic anemia / Macrocytic
anemia
• 95% of cases of megaloblastic anemia are
- Deficiency of vitamin B12
- Deficiency of folic acid
• Macrocytic blood picture
• Megaloblastic erythropoesis.
 Vitamin B12 deficiency
• Inadequate diet (no animal products!)
• Malabsorption
• Pernicious anemia (PA)
• Partial or total gastrectomy
• Blind loop syndrome
• Fish tapeworm
• Primary hypothyroidism
VITAMIN B12 ABSORPTION

Mucosal cell
Folate/B12 DNA Synthesis
 Clinical Feature
Sympton and sign Vit B12 Def :
• Severe : anemia, neuropathy
• Other symptom : sore mouth,loss of taste, atropy
mucosa of the tongue.
• Disorder of the central nervous system :
paresthesias of the hands & feet, unsteadiness of
gait, memory loss etc.
 Folate deficiency
• Reduced intake ( nutritional & malabsorpsi)
• Increased utilisation (pregnancy, malignancy,
hyperthytoidsm)
• Defective utilisation : drugs (anticonvulsant, oral
contraceptive), alcoholism.
• Reduced hepatic stores
alcohosm, hepatoma
 Sypmtom & Sign folic acid def

• Often go undiagnosed, especially alcoholic who

have a very poor diet and maintain blood alcohol
levels above 100 mg/dl→ enteropatic cycle of
folate supply to the intestine and tissues impared.
• Diagnosis is made difficult → clinician must be
suspicious of the possibility of folate def. in the
alcoholic.
 The peripheral blood smear
Macrocytic Anemia
• oval macrocytes, anisocytosis, poikilocytosis
• Hypersegmental neutrophils (>5% with more than
five nuclear lobes)
• Platelets bizarre in shape and size (giant platelets)
• Neutropenia
• Thrombocytopenia (not as severe as in AA)
• Low reticulocytosis
• The bone marrow shows a megaloblastic
erythropoesis
 Bone marrow smear in Macrocytic
Anemi

• Megaloblastic erythropoesis
• Bone marrow rich in cells,
• Giant metamyeolcytes
• Giant bands
• Many Howell-Jolly’es bodies
• Cabot’s rings
 Biochemical findings in MA
• Serum indirect (unconjugated) bilirubin 
• Serum LDH  (principally LDH-1)
• Serum iron 
– (unless the anemia is complicated with iron
deficiency)
• Vitamin B12 concentration ↓
• Folic acid concentration ↓
 The diagnosis of megaloblastic anemia

• Serum vitamin B12 concentration

• Serum folic acid concentration
• If vitamin B12 and folate assays are within
normal limits, then the bone marrow
examination is performed (before blood
transfusion or vitaminB12, folate administration).
• Schilling test
 Diagnosis

• Establised based on laboratory test.

• DD Macrocytosis
Dysplastic anemias,
Liver disease
Hemolysis  exposure to the
chemotherapeutic agents.
 Treatment
• Folic Acid and Vit B12 ( etiology must known
well)
• Severe with anemias : PRC transfusion
 Aplastic anemia
• Aplastic anemia is a severe, life threatening syndrome in
which production of erythrocytes, WBCs, and platelets
has failed.
• Aplastic anemia may occur in all age groups and both
genders.
• The disease is characterized by peripheral pancytopenia
and accompanied by a hypocellular bone marrow.
Hypocellular bone marrow in aplastic
anemia
 Aplastic anemia
• Etiology
– Acquired
» Most cases of aplastic anemia are idiopathic and there is no history
of exposure to substances known to be causative agents of the
disease
» Exposure to ionizing radiation – hematopoietic cells are especially
susceptible to ionizing radiation. Whole body radiation of 300-
500 rads can completely wipe out the bone marrow. With
sublethal doses, the bone marrow eventually recovers.
» Chemical agents – include chemical agents with a benzene ring,
chemotherapeutic agents, and certain insecticides.
» Idiosyncratic reactions to some commonly used drugs such as
chloramphenicol or quinacrine.
 Aplastic anemia
» Infections – viral and bacterial infections such as
infectious mononucleosis, infectious hepatitis,
cytomegalovirus infections, and miliary tuberculosis
occasionally lead to aplastic anemia
» Pregnancy (rare)
» Paroxysmal nocturnal hemoglobinuria – this is a
stem cell disease in which the membranes of RBCs,
WBCs and platlets have an abnormality making
them susceptible to complement mediated lysis.
» Other diseases – preleukemia and carcinoma
 Aplastic anemia
– Congenital disorders
» Fanconi’s anemia – the disorder usually becomes
symptomatic ~ 5 years of age and is associated
with progressive bone marrow hypoplasia.
Congenital defects such as skin
hyperpigmentation and small stature are also seen
in affected individuals.
» Familial aplastic anemia – a subset of Fanconi’s
anemia in which the congenital defects are absent.
 Clinical features
• Fatique
• Heart palpitation
• Palor
• Infections
• Ptchiae
• Mucosal bleeding/gum bleeding
 Aplastic anemia
• Pathophysiology:
– The primary defect is a reduction in or depletion of
hematopoietic precursor stem cells with decreased production
of all cell lines. This is what leads to the peripheral
pancytopenia.
• This may be due to quantitative or qualitative damage to the
pluripotential stem cell.
• In rare instances it is the result of abnormal hormonal
stimulation of stem cell proliferation
• or the result of a defective bone marrow microenvironment
• or from cellular or humoral immunosuppression of
hematopoiesis.
 Aplastic anemia
Lab findings
Severe pancytopenia with relative lymphocytosis
(lymphocytes live a long time
Normochromic, normocytic RBCs (may be slightly
macrocytic)
Mild to moderate anisocytosis and poikilocytosis
Decreased reticulocyte count
Hypocellular bone marrow with > 70% yellow
marrow
 Differential Diagnosis of pancytopenia and hypoplastic
marrow

1. Aplastic anemia
2. Hypoplastic myelodysplastic syndrome or
hypoplastis AML
3. PNH
4. Hypoplastic antecedent phase of acute
lymphocytic leukemia
5.Hypoplastic antecedent of hairy cell leukemia
6. Idiopathic myelofibrosis
7. Pure red cell aplasia
8. Agranulocytosis.
 Diagnosis Criteria for Severe AA

• At least two of following

- Absolute neutrophil count < 0,5 x 10 9 /L
- Platelet count < 20 x 10 9/L
- Anemia with corrected reticulocyte count < 1 %.
• One of the following
- Bone marrow cellularity <25 %
- Bone Marrow cellularity < 50 % with fewer the
30 % the neutrophil cell
 Treatment
• Marrow tranplantation isI curative for < 40 years.
• Only one –third of patients have suitable donor.
• Immunosuppressive therapy : not curative
-ATG
-Cyclosporin
-Androgen
- Corticosteroids
 Anemia of chronic disease
=Anemia of chronic disorders
(ACD)
 Definition
ACD -- anemia that occurs in patients with
infectious, inflammatory, or neoplastic diseases
that persist for more than 1 or 2 months.
It does not include anemias caused by marrow
replacement, blood loss, hemolysis, renal
insufficiency, hepatic disease, or endocrinopathy,
even when these disorders are chronic.
Disorders Associated with the Anemia of Chronic Disease
• Chronic infections
- Pulmonary infections: abscesses, emphysema,
tuberculosis, pneumonia
- Subacute bacterial endocarditis ,Pelvic inflammatory
disease
- Chronic urinary tract infections, Chronic fungal disease
- HIVinfections, Osteomyelitis
• Chronic, noninfectious inflammations
- Rheumatoid arthritis
- LES (Systemic lupus erythematosus)
- Severe trauma, thermal injury, Vasculitis
• Malignant diseases
- Cancer
- Hodgkin’s disease and Non-Hodgkin’s Lympmhomas
- Leukemias
- Multiple myeloma
• Miscellanous
- Alcoholic liver disease
- Thrombophlebitis
- Ischemic heart disease
• Idiopathic ACD
 Pathogenesis
• Shortened red cell life span, moderately 20-30%
(from 120 to 60-90 days)
• Relative bone marrow (erythropoiesis) failure
- Cytokines released from inflammatory cells (TNF-,
IL-1, IFN-) affects erythropoiesis by inhibiting the
growth of erythroid progenitors
- Serum erythropoietin levels in patiens with ACD are
normal when compared to healthy subjects but much
lower than levels in non-ACD anemic patients
 Pathogenesis

ABNORMAL IRON METABOLISM

• Activation of the reticuloendothelial system with increased iron
retention and storage within it
• impaired release of iron from macrophages to circulating
transferrin (impaired reutilization of iron)
• Reduced concentration of transferrin
(decreased production, increase sequestration in the spleen and in
the foci of inflammation, increase loss )
 ACD

Infection and inflammation

Interleukin-1 (IL-1)
Other Cytokines Stored iron in
reticuloendothelial
system

[Leukocytes (granulocytes)]

Lactoferrin Lactoferrin iron

Plasma iron

Decreased circulating plasma

iron
 ACD
infection
Inflammation
Immune response
Macrophage activity

Increased phagocytosis
- Decreased RBC survival
Reticulo Increased ferritin synthesis
endothelial - Increased stored iron
system Increased membrane receptors
- Increased avidity for RBCs
and iron-binding proteins

IL-1

Granulocytes

Acute-phase
reactants

Lactoferrin

Circulating
plasma iron (Lactoferrin-iron complex)
Decreased plasma iron
(hypoferremia)
 Symptoms and Sign

• Symptoms of the underlying disease

( malignancy or chronic inflammatory disease) :
weight loss, anorexia, fever,chills,myalgias and
artralgia.
• Symptoms of the anemia
 Laboratory features
• The anemia is usually mild or moderate ( Hb 7-11g/dl)

• The anemia is most often normochromic and normocytic (MCHC

and MCV are normal)
- MCV 70-80 fl in 5-40% of patients with ACD
- MCHC 26-32 g/dl in 40-70%
• Erythrocyte sedimentation rate (ESR) - usually rapid
• Retikulocytes - most often normal or slightly decreased number,
increased count is rarely
 Laboratory features
Iron metabolism
1. Serum Iron - decreased (it is necessary for the diagnosis of
ACD)
2. TIBC - reduced or low-normal (N)
3. Saturation index is decreased and is often < 15 %.
4. Serum Ferritin-increased or normal
5. Serum Transferrin Receptor (sTR)-Normal
6. Sideroblasts in the bone marrow-reduced (5-20%)
 Differential diagnosis
Laboratory Iron deficiency ACD
features without iron with iron
deficiency deficiency .
sFe   

TS <10% >10% <10%

TIBC  , N N, 

sFerritin <10g/L >200g/L, N <30g/L, N

Sideroblasts <10% 10-20% <10%

sTR  N 
 Therapy
1. Treatment of the underlying disorder
2. Iron supplementation (IS)
- for patients with ACD with chronic infection
or malignancy IS should be strictly avoided
- IS benefit patients with ACD associated with
auto-immune or rheumatic disorders.
- when ACD is complicated by iron deficiency
(about 27% patients).
3. Transfusion demand -- > patients who have low Hb and are
symptomatic
4. Recombinant erythropoietin 10.000 units 3 times a week i.v.
or s.c. 2-3tg, in the absence of response 20000j, If there is
still no respose, the treatment should be discontinued. (in
40% of patients it reduces number of transfusions)
5. Sequential administration of erythropoietin and iron
(48h later)
5. Iron chelation with deferoxamine - in some patients therapy
was associated with a rise in hemoglobin level
6. In future anti-TNF-antibodies
 POYCYTHEMIA

Yenny Dian Andayani

Hematology Oncology Medic Dept.of Internal

Medicine Moh Hoesin General Hospital
/Faculty of Medicine University Sriwijaya
Palembang.
 Myeloproliferative disease

arise from precursors of the "myeloid" lineage in the bone marrow

1. Polycythemia vera (PV)

2. Essential thombocytosis (ET)

3. Myelofibrosis with myeloid metaplasia (MMM)

4. Chronic Myelomonocytic leukemia (CMML)

 Classfication of Polycythemia
• I. Primary (autonomous)
Polycythemia Vera
• II. Secondary Polycythemia
A. Physiologically appropriate (
decreased tissue oxygenation)
B. Physiologycally in appropriate
(normal tissue oxygenation ).
 Physiologycally inappropriate
EPO Overproduction

-
 Tumors ; renal , brain , hepatoma , uterine fibroid ,

pheochromocytoma

 Renal artery stenosis

 Adrenal cortical hypersecretion
 Exogenous androgens
 NS

 Bartter’s syndrome

 Renal cyst , hydronephrosis

 Physiologycally approprite (decreased tissue
oxygenation)
EPO Production secondary to hypoxia
-
 Lung disease

 High altitude

 Smoking

 Cyanotic Heart disease

 Methemoglobinemia

 High O2 affinity hemoglobin

 Cobalt
 Clinical Features

- Head ache
- Thrombosis  common cause of death
- Pruritis ( aggravated by bathing ) 50%

- Erythromelagia
- Digital ischemia ( palpable pulse )
- Joint pain
- Weight loss
- Headache , vertigo
- Visual disturbance
- Conjunctival plethora
- Palpable splenomegaly 70%
 Clinical Features

- Lab : elevated leukocyte alkaline phosphatase ( LAP ) 70%

elevated serum B12 40%

- Risk to transform to acute leukemia 1.5 %

spent phase 10-25%

( Spent phase : normalization of red cell mass asso. with

cytopenia , increasing splenomegaly (extramed.hemat.) &

collagen fibrosis of BM )
-BMP : hipersellarity
 WHO criteria for diagnosis of Polycythemia Vera

 A1. elevated RBC mass > 25% above mean normal predicted value,or Hb >
18.5g/dL ( Male ) Hb > 16.5 g/dL (female )

 A2. No cause of 2nd erythrocytosis,including:

Absence of familial erythrocytosis
No elevation of EPO from - hypoxia ( PaO2 92 % )
- high O2 affinity Hb.
- truncated EPO receptor
- inappropiated EPO production by tumor
 A3. Splenomegaly

 A4. Clonal genetic abnormality other than Ph chromosome or BCR/ABL fusion

gene in marrow cells

 A5. Endogenous erythroid colony formation in vitro

 WHO criteria for diagnosis of Polycythemia Vera

 B1. Thrombocytosis > 400,000

 B2. WBC > 12,000
 B3. BM Biopsy showing panmyelosis with prominent erythroid &
megakaryocytic proliferation
 B4. Low serum EPO levels

Diagnosis : A1+A2 and any other of cathegory. A

or A1+A2 and any 2 of cathegory. B
or > 99th percentile of method specific reference range
of age ,gender,altitude of residence
 Criteria Polycythemia Vera study
group
• Category A
1.total red cell mass Male ≥36 ml/kg
Female ≥32 ml/kg
2.Arterial oxygen saturation ≥ 92 %
3. Splenomegaly
• Category B
1. Thrombocytosis > 400 x 10 3/ul
2. Leukocytosis 12x 103/ul
3. Increased leukocyte alkaline
phosphatase (LAP)
4. Serum B12 > 900 pg/ml or B12
binding capacity > 2200 pg/ml

Pv Diagnosis : when A1+A+2+A3 and any 2 from category B are present.

 PV & Secondary polycythemia

Finding PV 2nd Polycythemia

Splenomegaly + -
+ -
Leukocytosis + -
increased normal
Thrombocytosis normal normal
RBC volume increased normal
arterial O2 sat increased normal
B12 level Panhyperplasia normal
LAP decreased normal
Bone Marrow +
-
EPO level
 Staging & Prognosis

Hct. > 45% risk to thrombosis  Death

age > 70 yrs. & previous history of thrombosis

; important predictor of recurrent thrombotic events

 Treatment
aim ; - reduce thrombotic risk & slow leukemic transformation

- based on risk of thrombosis

Low risk High risk

-age < 60yr. Intermediate risk -age > 60yr.

-no Hx thrombosis -Previous Hx. thrombosis
-Plt. < 1,500,000 -CVD risk(smoking, )
-no CVD risk
 Treatment
Treatment of choice is “ Phlebotomy “

Hct. < 45 % in men , < 42% in women

“ Hydroxyurea “ is supplemented to decreased Hct.

“ IFN –alfa “ use for cytoreduction in younger

( decreased risk to leukemic transformation of hydroxyurea )

 Treatment

Busulfan or P-32 in elderly pt. with hydroxyurea intolerated

Low dose ASA ( 40 mg ) ; alleviate of microvascular sequelae

( headache, vertigo,visual disturbance , erythromelalgia )

Anagrelide ; used in all MPD to lowering platelet count

HOW IS FOLIC ACID INVOLVED IN DNA
SYNTHESIS?
» Is involved in the following reaction:
MEGALOBLASTIC ANEMIAS
» Therefore, with decreased folic acid, the net result is the
same as that for decreased vitamin B12 – there is decreased
conversion of dUMP to dTMP, and thus, dTTP which is
required for DNA synthesis.
» Defective DNA synthesis may occur when dUTP gets used
in place of dTTP because there is a great increase in
erroneous DNA copying where dUTP is put in place of
dTTP.
» Humans need to get about 50 ug/day of folic acid from the
diet
• Causes of megaloblastic anemia
– Vitamin B12 deficiency
MEGALOBLASTIC ANEMIAS
» 85% is due to lack of intrinsic factor (IF) secondary to
gastric atrophy – this is called pernicious anemia (PA) and it
occurs most often in older adults (over 40), most commonly
in individuals of Northern European descent, and more
commonly in women. Antibodies against the parietal cells
in the G.I. tract and against IF are commonly found in these
individuals.
» After gastrectomy
» Malabsorption syndrome
» Inadequate dietary intake (rare)
» Defective or absent transport proteins (transcobalamine)
CAUSES OF B12 DEFICIENCY
MEGALOBLASTIC ANEMIAS
– Folic acid deficiency
» Inadequate diet is the major cause and this occurs most often in the
poor, elderly, and alcoholics.
» When there is increased requirement such as in pregnancy,
hemolytic anemia, leukemia, etc.
» Malabsorption – may be due to diseases of the upper small
intestine such as ileitis, tropical sprue, and gluten sensitive
enteropathy.
» When there is increased loss such as in patients undergoing
hemodialysis
» Drug inhibition – examples are oral contraceptives, anticoagulant
drugs, alcohol, and isoniazid
MEGALOBLASTIC ANEMIAS
• Clinical manifestations occur in two categories – those found in folic
acid or vitamin B12 deficiency, and those mainly found in B12
deficiency
– In both types of deficiency the symptoms include pallor,
weakness, lightheadedness, a smooth, sore tongue, and diarrhea
alternating with constipation
– In vitamin B12 deficiency, and occasionally folic acid
defefiency, there are neurological disturbances including
numbness and tingling of extremities, gait abnormalities, and
mental disturbances.
» Older theories suggest that the B12 deficiency leads to a defect in
the degradation of propionyl CoA to succinyl CoA leading to an
accumulation of propionyl CoA:
MEGALOBLASTIC ANEMIAS
» When there is a build up of propionyl CoA (3 carbons), it is used
in place of acetyl CoA (2 carbons) as a primer for fatty acid
synthesis.
» Fatty acids with odd numbers of carbons get incorporated into
neural membranes
» This leads to disruption of membrane function with subsequent
demyelination of nerve fibers.
– Newer theories suggest that the defiency of methionine leads to
decreased production of S-adenosyl-methionine (SAM), a key
intermediate in methylation reactions of myelin and this leads to
demyelination
• Lab findings
– Macrocytic, normochromic anemia (MCV=100-140, MCHC is
normal))
– MCH is increased (due to increased cell size)
MEGALOBLASTIC ANEMIAS
– Hemoglobin and RBC counts are decreased
– WBC and platlet counts are decreased
– On a peripheral smear, a triad of things is commonly seen: oval
macrocytes, Howell Jolly bodies (nuclear DNA fragments), and
hypersegmented neutrophils (5 or more lobes). In addition:
» Anisocytosis is usually moderate
» Poikilocytosis is striking with nucleated RBCs,
polychromatophilia, and cabot rings (spindle remnants).
» RBC dimorphism is seen with concomitant IDA.
» The absolute reticulocyte count is decreased because of
ineffective erythropoiesis.
 PERIPHERAL SMEAR OF MEGALOBLASTIC
ANEMIA
Oval Macrocyte

Howell Jolly Body

Cabot ring
 PERIPHERAL SMEAR OF
MEGALOBLASTIC ANEMIA

Hypersegmented neutrophil
MEGALOBLASTIC ANEMIAS
– The bone marrow will show hypercellularity, yet there are
decreased numbers of all cell types in the peripheral blood
because ineffective hematopoiesis is occurring and many cells
are dying prematurely in the bone marrow.
– In folic acid deficiency – there will be decreased serum and
RBC folate
– In B12 deficiency – there will be decreased serum vitamin B12
– Specific tests for PA
» Gastric analysis – if there is no free HCl after histamine
stimulation, this may indicate PA since the same cells that secrete
HCl, also secrete intrinsic factor (IF)
» Test for antibodies to IF
MEGALOBLASTIC ANEMIAS
» Schilling test – is the definative test for the diagnosis of PA.
( Know this test!)
» The test measures the amount of an oral dose of radioactively
labeled B12 that is absorbed in the gut and excreted in the urine.
» This is followed by an injection of unlabeled vitamin B 12 to
saturate all vitamin B12 receptors in the tissue and plasma. Thus
any amount absorbed in the gut will be in excess, and will be
filtered in the kidneys to appear in the urine.
» If there is no radioactivity in the urine, this means that there is
either malabsorption or PA.
» The test is repeated, but this time the radioactively labeled B12 is
accompanied by a dose of IF.
» If absorption is now normal, this means that the patient has PA
SCHILLING TEST
SCHILLING TEST RESULTS
NON-MEGALOBLASTIC MACROCYTIC
ANEMIA
 NON-MEGALOBLASTIC MACROCYTIC
ANEMIA
• Note that the macrocytic RBCs are not oval, but
are round.
• There are no hypersegmented neutrophils or
Howell-Jolly bodies
DIFFERENTIAL DIAGNOSIS
WITH INCREASED MCV
DIFFERENTIAL DIAGNOSIS
WITH A HIGH MCV
LAB RESULTS OF MEGALOBLASTIC VS NON-
MEGALOBLASTIC MACROCYTOSIS
Megaloblastic Anemia
Megaloblastic Anemia
 B-12 Physiology
• Normal B-12 absorption:
– Dietary B-12 binds to R factor in saliva and gastric juices.
– In duodenum, pancreatic enzymes promote dissociation from R
factor and binding to Intrinsic Factor (IF)
– IF-B12 complex taken up by ileal receptor cubilin.
– Released into plasma bound to transcobalamines TC I, II, or III.
– Enters cells through receptor mediated endocytosis and
metabolized into two coenzymes: adenosyl-Cbl and methyl-
Cbl.
Folate/B12 DNA Synthesis
 HEMOLYTIC ANEMIA
Testing

• Absent haptoglobin
• Hemoglobinuria
• Hemoglobinemia
 HEMOLYTIC ANEMIA
Causes

• INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
• EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune
 HEMOLYTIC ANEMIA
Membrane Defects

• Microskeletal defects
– Hereditary spherocytosis
• Membrane permeability defects
– Hereditary stomatocytosis
• Increased sensitivity to complement
– Paroxysmal nocturnal hemoglobinuria
 Paroxysmal Nocturnal
Hemoglobinuria
• Clonal cell disorder
• Ongoing Intra- & Extravascular hemolysis; classically at
night
• Testing
– Acid hemolysis (Ham test)
– Sucrose hemolysis
– CD-59 negative (Product of PIG-A gene)
• 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 via diacylglycerol bridge
• PIG-A gene, on X-chromosome, codes for synthesis of
this bridge; multiple defects known to cause lack of this
bridge
• Absence of decay accelerating factor leads to failure to
inactivate complement & thereby to increased cell lysis
 HEMOLYTIC ANEMIA
Membrane abnormalities - Enzymopathies

• Deficiencies in Hexose Monophosphate Shunt

– Glucose 6-Phosphate Dehydrogenase Deficiency
• Deficiencies in the EM Pathway
– Pyruvate Kinase Deficiency
 G6PD DEFICIENCY
Function of G6PD

Infections
Drugs
2 H2O H2O2 Hgb

Sulf-Hgb
GSSG 2 GSH

NADPH NADP Heinz bodies

6-PG G6PD
G6P
Hemolysis
Glucose 6-Phosphate Dehydrogenase
Functions

• Regenerates NADPH, allowing regeneration of

glutathione
• Protects against oxidative stress
• Lack of G6PD leads to hemolysis during oxidative stress
– Infection
– Medications
– Fava beans
• Oxidative stress leads to Heinz body formation, 
extravascular hemolysis
Glucose 6-Phosphate Dehydrogenase
Different Isozymes

Level needed for protection vs ordinary oxidative stress

 HEMOLYTIC ANEMIA
Causes

• INTRACORPUSCULAR HEMOLYSIS
– Membrane Abnormalities
– Metabolic Abnormalities
– Hemoglobinopathies
• EXTRACORPUSCULAR HEMOLYSIS
– Nonimmune
– Immune
 EXTRACORPUSCULAR
HEMOLYSIS
Nonimmune

• Mechanical
• Infectious
• Chemical
• Thermal
• Osmotic
 Microangiopathic Hemolytic Anemia
Causes

• Vascular abnormalities
– Thrombotic thrombocytopenic purpura
– Renal lesions
• Malignant hypertension
• Glomerulonephritis
• Preeclampsia
• Transplant rejection
– Vasculitis
• Polyarteritis nodosa
• Rocky mountain spotted fever
• Wegener’s granulomatosis
Microangiopathic Hemolytic Anemia
Causes - #2

– Vascular abnormalities
• AV Fistula
• Cavernous hemangioma
• Intravascular coagulation predominant
– Abruptio placentae
– Disseminated intravascular coagulation
 IMMUNE HEMOLYTIC ANEMIA
General Principles

• All require antigen-antibody reactions

• Types of reactions dependent on:
– Class of Antibody
– Number & Spacing of antigenic sites on cell
– Availability of complement
– Environmental Temperature
– Functional status of reticuloendothelial system
• Manifestations
– Intravascular hemolysis
– Extravascular hemolysis
 IMMUNE HEMOLYTIC ANEMIA
General Principles - 2

• Antibodies combine with RBC, & either

1. Activate complement cascade, &/or
2. Opsonize RBC for immune system
• If 1, if all of complement cascade is fixed to red
cell, intravascular cell lysis occurs
• If 2, &/or if complement is only partially fixed,
macrophages recognize Fc receptor of Ig &/or
C3b of complement & phagocytize RBC,
causing extravascular RBC destruction
 IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Direct
• Looks for immunoglobulin &/or complement of surface
of red blood cell (normally neither found on RBC
surface)
• Coombs reagent - combination of anti-human
immunoglobulin & anti-human complement
• Mixed with patient’s red cells; if immunoglobulin or
complement are on surface, Coombs reagent will link
cells together and cause agglutination of RBCs
 IMMUNE HEMOLYTIC ANEMIA
Coombs Test - Indirect

• Looks for anti-red blood cell antibodies in the patient’s

serum, using a panel of red cells with known surface
antigens
• Combine patient’s serum with cells from a panel of
RBC’s with known antigens
• Add Coombs’ reagent to this mixture
• If anti-RBC antigens are in serum, agglutination
occurs
HEMOLYTIC ANEMIA - IMMUNE
• Drug-Related Hemolysis
• Alloimmune Hemolysis
– Hemolytic Transfusion Reaction
– Hemolytic Disease of the Newborn
• Autoimmune Hemolysis
– Warm autoimmune hemolysis
– Cold autoimmune hemolysis
 IMMUNE HEMOLYSIS
Drug-Related

• Immune Complex Mechanism

– Quinidine, Quinine, Isoniazid
• “Haptenic” Immune Mechanism
– Penicillins, Cephalosporins
• True Autoimmune Mechanism
– Methyldopa, L-DOPA, Procaineamide, Ibuprofen
DRUG-INDUCED HEMOLYSIS
Immune Complex Mechanism

• Drug & antibody bind in the plasma

• Immune complexes either
– Activate complement in the plasma, or
– Sit on red blood cell
• Antigen-antibody complex recognized by RE system
• Red cells lysed as “innocent bystander” of destruction of
immune complex
• REQUIRES DRUG IN SYSTEM
 DRUG-INDUCED HEMOLYSIS
Haptenic Mechanism

• Drug binds to & reacts with red cell surface

proteins
• Antibodies recognize altered protein, ± drug, as
foreign
• Antibodies bind to altered protein & initiate
process leading to hemolysis
 DRUG-INDUCED HEMOLYSIS
True 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
 ALLOIMUNE HEMOLYSIS
Hemolytic Transfusion Reaction
• Caused by recognition of foreign antigens on transfused blood
cells
• Several types
– Immediate Intravascular Hemolysis (Minutes) - Due to preformed
antibodies; life-threatening
– Slow extravascular hemolysis (Days) - Usually due to repeat exposure
to a foreign antigen to which there was a previous exposure; usually
only mild symptoms
– Delayed sensitization - (Weeks) - Usually due to 1st exposure to
foreign antigen; asymptomatic
INCOMPATIBLE RBC
TRANSFUSION
Rate of Hemolysis
 ALLOIMMUNE HEMOLYSIS
Testing Pre-transfusion

• ABO & Rh Type of both donor & recipient

• Antibody Screen of Donor & Recipient, including
indirect Coombs
• Major cross-match by same procedure (recipient
serum & donor red cells)
 ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn
• Due to incompatibility between mother negative for an
antigen & fetus/father positive for that antigen. Rh
incompatibility, ABO incompatibility most common
causes
• Usually occurs with 2nd or later pregnancies
• Requires maternal IgG antibodies vs. RBC antigens in
fetus
 ALLOIMMUNE HEMOLYSIS
Hemolytic Disease of the Newborn - #2

• Can cause severe anemia in fetus, with erythroblastosis

and heart failure
• Hyperbilirubinemia can lead to severe brain damage
(kernicterus) if not promptly treated
• HDN due to Rh incompatibility can be almost totally
prevented by administration of anti-Rh D to Rh negative
mothers after each pregnancy
 AUTOIMMUNE HEMOLYSIS
• 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 HEMOLYSIS
Warm Type
• Usually IgG antibodies
• Fix complement only to level of C3,if at all
• Immunoglobulin binding occurs at all temps
• Fc receptors/C3b recognized by macrophages; 
• Hemolysis primarily extravascular
• 70% associated with other illnesses
• Responsive to steroids/splenectomy
 AUTOIMMUNE HEMOLYSIS
Cold Type
• Most commonly IgM mediated
• Antibodies bind best at 30º or lower
• Fix entire complement cascade
• Leads to formation of membrane attack complex, which
leads to RBC lysis in vasculature
• Typically only complement found on cells
• 90% associated with other illnesses
• Poorly responsive to steroids, splenectomy; responsive to
plasmapheresis
 HEMOLYTIC ANEMIA
Summary

• Myriad causes of increased RBC destruction

• Marrow function usually normal
• Often requires extra folic acid to maintain
hematopoiesis
• Anything that turns off the bone marrow can
result in acute, life-threatening anemia
 Week 2: Hemolytic Anemia
• Normocytic anemia • Heinz bodies
• Fanconi anemia • PK deficiency
• Diamond-Blackfan • Cytoskeleton
(PRCA) • Spherocytosis
• Types of hemolysis • Elliptocytosis
• Evidence of hemolysis • PNH
• Reticulocyte
• G6PD deficiency
 Normocytic Anemia
• Acute hemorrhage
• Hypo-proliferative
• Acquired aplastic anemia
• Constitutional aplastic anemia
• Pure red cell aplasia (PRCA)
 Acute Hemorrhage
• No changes in CBC until plasma volume
increases
• Later, reticulocytosis
 Hypoproliferative Anemia
• Pancytopenia
• < 25% BM cellularity
• Low retic count
• Bone marrow defect
• Immunologic suppression
• Growth Factor deficiency
• Abnormal stem cells
 Acquired Aplastic Anemia
• Idiopathic
• Drugs and chemicals (eg, chloramphenicol,
benzene, insecticides)
• Radiation
• Virus
• Paroxysmal nocturnal hemoglobinuria
 Constitutional Aplastic Anemia
• Fanconi’s anemia
• Chromosomal breaks
 Pure Red Cell Aplasia PRCA
• Infections
• Drugs
• Diamond-Blackfan syndrome
• No EPO deficiency
 Hemolytic Anemia
• Intravascular vs extravascular
• Bilirubin metabolism
• Types
– Membrane defect
– Metabolic defect
– Extracorpuscular
 Evidence of Hemolysis
• Low RBC survival with chromium tagging study
• Unconjugated bilirubin
• Plasma Hb
• Decreased serum haptoglobin
 Evidence of Erythropoiesis
• Polychromasia
• Increased reticulocyte
• “Shift” macrocytosis
• Hypercelluar BM
 Correcting Retic Count
Retic Index = Retic % x Patient Hct
Normal Hct

Absolute Retic = Retic % x RBC/mm3

Retic Production Index = Retic Index

Days in circulation
 Membrane Defect
• Spherocytosis
• Elliptocytosis
• PNH (sensitivity to complement lysis -- sugar
water test, Ham’s test)
• Stomatocytosis (possibly Rh null)
 Spherocytes

Elliptocytes
Osmotic Fragility
 Paroxysmal Nocturnal Hemoglobinuria

• Hematopoietic stem cell disorder

• Mutation of phosphatidylinositol glycan class A (PIG-A)
gene
• Glycosylphosphatidylinositol (GPI) anchors membrane
proteins
• Without GPI, unable to regulate completment activities
on membrane
• Hemolysis is pH dependent
• Thrombosis can occur
 Lab Tests for PNH
• Acidified serum lysis test (Ham’s test): PNH cells lyse
due to complement activation in acidified serm
• Sugar water (sucrose hemolysis) test: RBCs sensitive to
complement will lyse in sucrose and serum
• Flow cytometry: lack of CD59 on RBCs, or lack of
CD59 or CD55 on granulocytes
Stomatocytes
 Metabolic Defect
• G6PD deficiency
– Hexose monophosphate
shunt
– Most common RBC enzyme
defect, >50 variants
– X-linked
– Low glutathione due to low
NADPH
– Oxidative lysis, Heinz
bodies, spherocytic
– Primaquine, fava beans
• Pyruvate kinase deficiency
– Glycolysis
– Low RBC ATP level
– Non-spherocytic
• B12 and folate deficiency
– Macrocytic
– HJ bodies
• Hemoglobinopathies
– Poikilocytosis
– Abnormal Hb
Heinz body preparation with Crystal violet
Unstable hemoglobin
 Extracorpuscular Factors
• Antibodies • Abnormal plasma lipids
– Autoimmune – Acanthocytosis
– Isoimmune • Venom
• Drugs, antibiotics – Snake
• Fresh water – Spider
– Bee
 Extracorpuscular Factors
• Trauma • Microorganisms
– DIC
– Malaria
– Hemolytic uremic
syndrome (HUS) – Babesia
– TTP – Clostridium
– Angiopathy
– Gram negative
– Heat
endotoxin
– Heart valves
– “March” hemoglobinuira
 Schistocytes

Malaria
 HEMOLYTIC ANEMIA

OUTSIDE THE RED CELL

WITHIN THE RED CELL
1. Membrane defects AUTO-IMMUNE NON-IMMUNE
- HS 1. Warm 1. Hypersplenism
- HE 2. Cold 2. Fragmentation syndromes
- Hereditary pyropoikilocytosis - grafts / valves / AS
3. Transfusion reactions
- HTN / Pre-eclampsia
- Hereditary stomatocytosis 4. Drug associated - March hemoglobinuria
2. Enzyme defects - MAHA
-G6PD - TTP/HUS
- DIC
-Pyruvate kinase - hemangioma
3. Hemoglobin defects 2. Infections/Toxins (Malaria,
- SCA Babeosis, Bartonella, Clostridium
welchii, snakes, spiders)
- Thalassemias
3. Drugs
- Unstable hemoglobin
4. Liver dz (Spur cell)
5. PNH
 HEMOLYTIC ANEMIA
Intravascular Causes in Yellow

OUTSIDE THE RED CELL

WITHIN THE RED CELL
1. Membrane defects AUTO-IMMUNE NON-IMMUNE
- HS 1. Warm 1. Hypersplenism
- HE 2. Cold 2. Fragmentation syndromes
- Hereditary pyropoikilocytosis - grafts / valves / AS
3. Transfusion reactions
- HTN / Pre-eclampsia
- Hereditary stomatocytosis 4. Drug associated - March hemoglobinuria
2. Enzyme defects - MAHA
-G6PD - TTP/HUS
- DIC
-Pyruvate kinase - hemangioma
3. Hemoglobin defects 2. Infections/Toxins (Malaria,
- SCA Babeosis, Bartonella, Clostridium
welchii, snakes, spiders)
- Thalassemias
3. Drugs
- Unstable hemoglobin
4. Liver dz (Spur cell)
5. PNH
Alf Alving, Scientist, U.S. Army
(mid 1950s)
 RBC
Extravascular Hemolysis
RES

HBG
Fe

Transferrin CO

Storage and Lung

recycled in
marrow
Biliverdin

Uncojugated
bilirubin

Conjugation
Intravascular Hemolysis

RBC LYSIS

HBG

HAPTOGLOBIN
HBG TAKEN UP BY RENAL
REMOVED BY LIVER TUBULAR CELLS

HEMOGLOBINEMIA HEMOSIDERIN

HEMOGLOBINURIA CELLS SLOUGHED IN

URINE 1 WEEK LATER
 Classic Presentation

1. New onset pallor and anemia

2. Splenomegaly
3. Jaundice
3.  Indirect bilirubin (<5)
4.  Reticulocyte percentage
5.  LDH (esp LDH-2)
6.  RBC life span
7.  Haptoglobin
< 25
 HEMOLYTIC ANEMIA

OUTSIDE THE RED CELL

WITHIN THE RED CELL
1. Membrane defects AUTO-IMMUNE NON-IMMUNE
- HS 1. Warm 1. Hypersplenism
- HE 2. Cold 2. Fragmentation syndromes
- Hereditary pyropoikilocytosis - grafts / valves / AS
3. Transfusion reactions
- HTN / Pre-eclampsia
- Hereditary stomatocytosis 4. Drug associated - March hemoglobinuria
2. Enzyme defects - MAHA
-G6PD - TTP/HUS
- DIC
-Pyruvate kinase - hemangioma
3. Hemoglobin defects 2. Infections/Toxins (Malaria,
- SCA Babeosis, Bartonella, Clostridium
welchii, snakes, spiders)
- Thalassemias
3. Drugs
- Unstable hemoglobin
4. Liver dz (Spur cell)
5. PNH
 Hereditary Spherocytosis

- Autosomal Dominant
- ~1:3000
- Spectrin, Ankyrin, Protein 3
- Dx: osmotic frag., neg. direct Coombs
- Tx: Splenectomy, cholecystectomy, vaccine,
folate
Hereditary Eliptocytosis

- Autosomal Dominant
- ~1:4500
- Protein 4.1
- Dx: >75% elliptic RBC
- Tx: Splenectomy
Hereditary Stomatocytosis

- Autosomal Dominant
- Lack of Protein 7.2 (stomatin)
  Permeability to Na, K
- Stomatocytes, xerocytes  target cells
- Tx: Splenectomy
Stateville Penitentiary, near Joliet, IL (mid
1950s)
 Warm Immunohemolyis

- Most common in adult women (CT, SLE)

- IgG bound to RBC activate phagocytes and
complement
- Dx: + direct Coombs
- Tx:
- 1. Prednisone 1mg/kg ( RES, Ab production)
- 2. Splenectomy
- 3. Cyclophosphamide
- 4. Azathioprine
- 5. IV gamma globulin
- 6. Transfusions (cross-matching impossible)
 Cold Immunohemolyis

- monoclonal production of cold agglutinins

- response to infection (M. pneumoniae, EBV)
- IgM mediated complement attack (no Fc)
- Tx:
- 1. Underlying cause
- 2. Splenectomy, Steroids, have minimal role
 - 1931 – Nobel prize for
discovery of hexose-
monophosphate shunt and
glycolytic pathway (Otto
Meyerhof, Gustav Embden
students)

- 1931-1944 discovery of
cytochromes, flavin
adenine dinucleotide,
nicotinamide adenine
dinucleotide.

- 1944 – offered 2nd Nobel

prize, but prevented from
accepting it by Hitler
Otto Warburg (1883-1970)
 TTP

- Ab inhibits protease that normally cleaves vWF

- 1. Intravascular hemolysis
- 2. Thrombocytopenia
- 3. Non focal neurologic findings
- 4.  Renal function
- 5. Fever
- Dx
- Negative direct Coombs
- Fragmented RBC, but no spherocytes
- Normal coagulation tests
 HUS

- O157:H7 Shiga-like verotoxins that damage renal

vascular endothelial cells
- Clinically similar to TTP, no neuro manifestations
- Tx
- Plasmapheresis, dialysis, transfusions
- Role of glucocorticoids, dextran, heparin uncertain
 Liver disease: Spur Cell Anemia

  cholesterol to phospholipid ratio

- Splenic traffic jam
- Clinically similar to TTP, no neuro manifestations
- Limited Treatment
 PNH

- Somatic mut. on X-chromosome

- Gene makes GPI anchor
- Many proteins can’t attach to RBC
- No DAF and membrane inhibitor of reactive lysis
(MIRL), RBC sensitive to complement
- Hypercoaguable state
- Dx: pancytopenia,  LAP, sucrose hemolysis, Ham’s
test (lysis in acidified serum)
- Tx: Transfusion, glucocorticoids, Fe
 G6PD

- X chromosome, recessive
- 11% African American males
  sensitivity to oxidative stress
- Heinz bodies, bite cells
- Triggers: infection, drugs, met. acidosis, moth
balls, fava beans
- Dx: enzyme assay (fluorescent spot) after acute
episode
- Tx: avoid triggers
 DRUGS THAT CAUSE HEMOLYTIC ANEMIA

OXIDANT PENICILLIN TYPE IMMUNE COMPLEX AUTOIMMUNE

1. Antibiotics (Ab + Drug-Membrane) (Ab-Drug + Membrane) (AutoAb to Rh Ag)
- nitrofurantoin 1. Penicillins (Most common type) 1. methyldopa
- sulfa 2. Cephalosporins 1. quinidine
- dapsone 3. Synthetic penicillins 2. rifampin
- nalidixic acid
2. Primaquine
3. Pyridium
4. Doxorubicin
5. Methylene blue
MISCELLANEOUS
1. Vitamin K (water soluble)
 Cinchona plant, Costa Rica, containing
both quinine and quinidine, named for the
Countess of the Spanish town of Chinchon
Hematuria, Hemoglobinuria, and Myoglobinuria
Causes of Intravascular Hemolysis
1. Transfusion reactions
2. Infections
- Clostridium welchi
- Malaria, Babeosis
- Bartonella
- Mycoplasma pneumonia
3. Fragmentation syndromes
- grafts / valves / AS
- HTN / Pre-eclampsia
- March hemoglobinuria
- TTP/HUS
- DIC
- hemangioma
4. G6PD deficiency with oxidant stress
5. PNH
6. Infusion of hypotonic solutions
7. Snake and Spider venoms
8. Some autoimmune hemolytic anemias (RhoD)
 Hemolytic Anemias -
Extracorpuscular defects
What is destroying this RBC?
 Extracorpuscular defects
• Immune hemolytic anemias
– These anemias result from a shortened RBC survival
mediated by the immune response, specifically humoral
antibodies.
– There are three broad categories:
• Alloimmune in which the patient produces alloantibodies to foreign
RBC antigens introduced through transfusion or pregnancy
– Transfusion reaction
» An immediate transfusion reaction is characterized by acute
intravascular hemolysis, mostly associated with ABO IgM
isoantibodies. The patient’s antibodies destroy the donor’s cells.
RESULTS OF INTRAVASCULAR
HEMOLYSIS
 Extracorpuscular defects
» A delayed transfusion reaction occurs 2-14 days after
transfusion and usually is the result of an anamnestic
response in which IgG antibodies are made in an individual
who has been previously sensitized. Extravascular
hemolysis of IgG coated antibodies occurs in the spleen.
– Hemolytic disease of the newborn
» RBCs of the fetus are destroyed by maternal IgG antibodies
that cross the placenta.
» The baby is often born jaundiced, anemic and with
hepatosplenomegaly.
Extravascular hemolysis
 Extracorpuscular defects
• Autoimmune hemolytic anemia – This represents an abnormality in
which the immune system’s ability for self-recognition is lost and
antibodies are made to the RBC antigens (autoantibodies). They
bind to the RBCs and initiate hemolysis.
– Warm autoimmune hemolytic anemia - In this type of immune
hemolytic anemia the serologic reactivity of the IgG antibody
involved is optimal at 370 C.
» Primary, idiopathic - severe, but self-limiting anemias that may
last several weeks to years.
» Secondary – associated with some underlying disease
(lymphoproliferative, neoplastic, SLE, RA, viral or bacterial
infection, chronic inflammatory disease)
Extracorpuscular defects
» In both the primary and secondary form of the disease, most
hemolysis is extravascular and complement is not necessary
for cell destruction, though it may be involved (Ag-Ab
complexes may be pitted from the cell membrane in the
spleen or the cell itself may be ingested by phagocytic
cells).
» The anemia is moderate to severe, the RBCs are
normochromic, normocytic with polychromasia (increased
reticulocytes).
» Spherocytes, schistocytes, etc. may be seen and are
indicative of the hemolytic process.
Extracorpuscular defects
» The direct Coombs test is positive. This tests for RBCs sensitized
with IgG Ab or complement. IgM antibodies will agglutinate
RBCs in saline, but IgG antibodies are not large enough to
overcome the zeta potential of the RBCs. In the direct Coombs
test, antihuman globulin (AHG), which contains antibodies to
human antibodies and complement, is added to cells suspected of
having IgG or complement bound to them. When AHG binds to
the IgG or complement that is bound to RBCs, it bridges the
distance between the RBCs which leads to agglutination and a
positive test.
» Treatment is glucocorticoids, splenectomy, or other
immunosuppressive drugs.
Zeta potential
Warm autoimmune hemolytic anemia
 Extracorpuscular defects
– Cold autoimmune hemolytic anemia – pathologic cold
autoantibodies are usually IgM antibodies that fix complement
and are optimally reactive below 370 C. It is normal to have
benign cold autoantibodies, but their thermal amplitude and
concentration are not high enough to cause problems. The
pathologic forms can be divided into three types:
» Cold agglutinin syndrome – This is idiopathic, chronic, usually in
individuals older than 50, and usually due to an IgM monoclonal
antibody.
» Secondary, cold autoimmune hemolytic anemia – due to
polyclonal IgM antibodies that develop with Mycoplasma
pneumonia infections, infectious mononucleosis, or
lymphoproliferative disease. Is usually transient.
COLD AGGLUTININ
Extracorpuscular defects
» In both a and b the extent of the disease is related to the thermal
amplitude of the antibody – if it reacts at 30-320 C, it can cause
problems when the peripheral circulation cools to that
temperature:
» IgM binds and fixes complement, upon warming the antibody
dissociates, but complement remains bound leading to either
intravascular or extravascular hemolysis.
» The patient may experience acrocyanosis of hands, feet, ears, and
nose (with agglutination blood flow slows down, the skin turns
white and then blue; upon warming, the skin turns red).
» Blood counts are difficult to perform unless the blood is warmed.
» The Coombs test with anti-complement antibody is positive.
Extracorpuscular defects
» The cold agglutinin test is positive at 0-200 C and usually up to 300
C. The titer is usually 1:1000 or greater.
» Paroxysmal cold hemoglobinuria (the third type of cold
autoimmune hemolytic anemia) – is found in association with viral
disorders and syphilis and may be chronic.
» This is characterized by massive, intermittent, acute intravascular
hemolysis and hemoglobinuria upon exposure to cold.
» It is caused by a biphasic IgG antibody that binds at low
temperature and fixes complement.
» Upon warming, to body temperature, the intravascular hemolysis
occurs and is accompanied by fever, shaking chills, and abdominal
and back cramps.
Cold autoimmunne hemolytic anemia
 Extracorpuscular defects
• Drug induced immune hemolytic anemia – many
different drugs can cause this and 3-4 different
mechanisms may be involved.
– Immune complex mechanism
• The drug binds to plasma proteins and antibodies are made against
the drug.
• The antibodies bind to the drug to form an immune complex which
adsorbs nonspecifically to the patients RBCs, complement is fixed,
and acute intravascular hemolysis occurs.
IMMUNE COMPLEX
MECHANISM
 Extracorpuscular defects
– Drug adsorption (hapten) mechanism
• The drug binds nonspecifically to proteins on the RBC membrane,
antibodies are made (usually IgG), they bind to the drug and
extravascular hemolysis occurs.
– Membrane modification mechanism
• The drug modifies the RBC membrane so that normal plasma
proteins bind nonimmunologically.
• In rare instances a cross reacting antibody causes a hemolytic
anemia.
– Methyldopa induced mechanism
• The drug induces formation of autoantibodies causing extravascular
destruction.
• It may change autoproteins so that they are no longer recognized as
self, or it may cause a direct loss of T suppressor cells.
DRUG ADSORPTION
MEMBRANE MODIFICATION
 Extracorpuscular defects
• Nonimmune hemolytic anemias
– Caused by antagonists in blood or abnormalities in plasma
lipids.
• Chemicals and drugs
– Include drugs that cause oxidative injury
– Inhalation of arsine gas
– Lead intoxication (in addition to interfering with heme
synthesis, lead can cause membrane damage by interfering with
energy production)
– Injection of large volumes of water.
• Animal venoms – bees, wasps, spiders, scorpions in susceptible
individuals, rarely snakebites
• Infectious agents – malarial parasites, Babeiosis, Clostridium
perfringens, Bartonella bacilliformis
BABEIOSIS AND MALARIA
 Extracorpuscular defects
• Abnormal plasma lipid composition – note that these were also included
in intracorpuscular problems, because they lead to intrinsic problems with
the RBC.
– Spur cell anemia – associated with severe hepatocellular disease
which leads to increased serum lipoproteins, increased membrane
cholesterol, decreased deformability and decreased survival
– Abetalippoproteinemia – leads to an increased
cholesterol/phospholipid ratio, acanthocytes, and decreased RBC
survival.
– Caused by physical injury to RBCs
• Microangiopathic hemolytic anemia
– Caused by microcirculatory lesions that cause the RBCs to tear due
to sheer stress
» Disseminated cancer

•
Spur cell anemia
 Extracorpuscular defects
» Hemolytic uremic syndrome
» Idiopathic thrombotic thrombocytopenic purpura – microthrombi
are deposited in the microvasculature
» Malignant hypertension
» Disseminated intravascular coagulation.
• Macroangiopathic hemolytic anemia
– Due to abnormalities in the heart or large vessels causing RBC
hemolysis
» Prosthetic heart valves
• March hemoglobinemia
– Transient hemolytic anemia occurring after strenuous exercise
with contact with a hard surface (running, marching)
• Thermal injury
MICROANGIOPTHIC
HEMOLYTIC ANEMIA
THERMAL INJURY
Thermal injury