Red Cell Disorders

Question 1

Blood cellular elements

Answer 1

Peripheral blood film (smear)
Bone marrow aspirate
Bone marrow biopsy (tissue section)

Question 2

Automated complete blood counts (CBC)

Answer 2

Red cell part
White cell part
Platelet part

Question 3

CBC: red cells

Answer 3

Hemoglobin concentration
-Measured spectrophotometrically (HiCN)
Hematocrit
-Fraction of whole blood that is red cells
RBC concentration (RBC count)
-Measured directly by particle counting
-Impedance and/or electro-optical methods
Red cell indices

Question 4

Red cell indicies

Answer 4

MCV (mean cell volume)
-Average volume per red cell
-Measured by impedance and/or electro-optically
MCH (mean cell hemoglobin)
-Average content (mass) of hemoglobin per red cell
MCHC (mean cell hemoglobin concentration)
-Average concentration of hemoglobin in the red cells
RDW (red cell distribution width)
-Coefficient of variation of red cell volume

Question 5

CBC: Red cells

Answer 5

Hemoglobin (g/dl):  measured
Hematocrit (%):  calculated (MCV x RBC)
RBC count (x106/µl):  measured
MCV (fl):  measured
MCH (pg):  calculated (Hgb/RBC)
MCHC (g/dl):  calculated (Hgb/Hct)
RDW (%):  measured (SD/MCV)

Question 6

CBC: White cells

Answer 6

WBC count (x103/µl)
Differential count [5 part] (% of total WBC)
Absolute counts (cells/µl)
Lymphocytes
Monocytes
Neutrophils
Eosinophils
Basophils

Question 7

CBC: platelets

Answer 7

Platelet count (x103/µl)
MPV (mean platelet volume) (fl)
PDW (platelet distribution width) (%)

Question 8

Reticulocyte Count

Answer 8

Manual
-Number of Reticulocytes per 100 red cells
Automated
-% Retics
*Normal: 0.5-1.5%
*% counts must be corrected for degree of anemia
-Absolute count
*Normal: 24,000-84,000/µl
3% or 150,000/µl is a useful guide for a good marrow response to moderate anemia

Question 9

Red Cells on Blood Films

Answer 9

Evaluate the size, color, and shape of the red cells
Assess by
-Direct examination of blood film (smear)
-Red cell indices
Correlates with cause of anemia
Helps in choosing logical next steps
Size: Microcytic, normocytic, or macrocytic
Color: Hypochromic or normochromic
Shape: Normal or a variety of possible abnormal forms

Question 10

Size & color

Answer 10

Normochromic
normocytic
Polychromasia
Hypochromic, microcytic
Macrocytic

Question 11

Shape

Answer 11

Spherocytes
Eliptocytes
Target cells
Schistocytes
Poikilocytosis

Question 12

Hemoglobin

Answer 12

Tetramer of globin chains
Each chain has a heme prosthetic group capable of binding oxygen
Heme is a porphyrin ring and a coordinated iron atom

Question 13

Adult blood contains 3 hemoglobins

Answer 13

HbA, α2β2, 97% of total
HbF, α2γ2, 3-5%
HbA2, α2δ2, 1.5-3.5%

Normal and variant hemoglobins can be detected and quantified by electrophoresis, HPLC, or isoelectric focusing

Question 14

Red cell disorders typically manifest as

Answer 14

Anemia
-Decreased number of red cells
Polycythemia
-Increased number of red cells

Question 15

Anemia

Answer 15

Reduction of oxygen carrying capacity of blood
-Reduction of red cell mass
-Labs
Decreased hemoglobin concentration (Hgb)
Decreased hematocrit (Hct)
Decreased red cell concentration (RBC count)

Question 16

Response to Anemia

Answer 16

Decreased tissue oxygen tension →
Increased erythropoietin production →
Hyperplasia of erythroid precursors
-May even lead to extramedullary hematopoiesis
Reticulocytosis is the hallmark of increased marrow output of red cells

Question 17

Anemia clinical

Answer 17

Clinical consequences depend upon severity, speed of onset, and mechanism
Pallor, fatigue, and lassitude are common presenting symptoms
Slow onset gives more time to adapt
Premature destruction
-Hyperbilirubinemia, jaundice, pigment gallstones
Ineffective erythropoiesis
-Increased iron absorption, iron overload
Severe congenital anemias
-Growth retardation, skeletal abnormalities, cachexia

Question 18

Anemia of Blood Loss

Answer 18

Acute blood loss

chronic blood loss

Question 19

Acute blood loss

Answer 19

Hypovolemia is the most immediate threat
-Cardiovascular collapse, shock, and death
Fluid volume is fully restored in 2-3 days
-Hemodilution reveals the extent of the anemia
-Normochromic, normocytic anemia
Red cell production increases in several days
-↑ Epo → Erythroid precursors → Red cells

Question 20

Chronic blood loss

Answer 20

Anemia develops when
Rate of blood loss exceeds production capacity
Run out of raw material, e.g. iron

Question 21

Hemolytic anemias

Answer 21

Anemias associated with accelerated destruction of red cells
Classify by cause
Intrinsic, or intracorpuscular, factors
-Most inherited
*Membrane abnormalities, enzyme deficiencies, hemoglobin synthesis disorders
Extrinsic, or extracorpuscular, factors
-Acquired
*Immune mediated, mechanical destruction, infections

Question 22

Hemolytic Anemias general features

Answer 22

Shortened red cell life span
-Premature red cell destruction
Increased rate of erythropoiesis
-Reticulocytosis
Accumulation of the products of hemoglobin catabolism
-Iron accumulation is a problem in chronic hemolytic anemias
*Iron is recycled and gut absorption is increased

Question 23

Hemolytic Anemias

Answer 23

Extravascular hemolysis

Intravascular hemolysis

Question 24

Extravascular hemolysis

Answer 24

Red cell destruction within the cells of the mononuclear phagocyte system
-More common mode of red cell destruction
Occurs largely within the spleen and liver
Removes damaged and immunologically targeted red cells from the circulation
Leads to
-Jaundice
-Pigment gallstones
-Haptoglobin decrease
-Reactive hyperplasia of the mononuclear phagocyte system i.e., splenomegaly

Question 25

Intravascular hemolysis

Answer 25

Red cell destruction within the vascular space -Less common mode Results from -Mechanical damage *Defective heart valve -Biochemical or chemical damage to the membrane *Complement fixation, clostridial toxins, or heat Leads to: Hemoglobinemia Hemoglobinuria Hemosiderinuria Unconjugated hyperbilirubinemia Jaundice Haptoglobin absence from plasma Acute tubular necrosis in kidney, if massive hemolysis

Question 26

Hereditary Spherocytosis

Answer 26

Inherited defect in the red cell membrane that renders the red cells spheroidal, less deformable, and vulnerable to splenic sequestration and destruction Autosomal dominant, most common Autosomal recessive 25% of the time, more severe form

Question 27

Hereditary Spherocytosis | defect

Answer 27

Defect is in the membrane cytoskeleton that stabilizes the red cell membrane -Ankyrin most common defective protein -Band 3 next most common -Spectrin (α and β) and band 4.2 most of remainder Reduced membrane stability → loss of membrane fragments (but retain volume) after release into the periphery → become spherical Spherical cells are have limited deformability → sequestered in splenic cords → destroyed by macrophages Spleen is critical element in causing the anemia

Question 28

Hereditary Spherocytosis | features

Answer 28

``` Splenomegaly, 500-1000 g Spherocytes in peripheral blood Note: Not all spherocytes are HS !! Reticulocytosis Erythroid hyperplasia of marrow Hemosiderosis Jaundice, mild Pigment gallstones, 30-40% of adults ```

Question 29

Hereditary Spherocytosis clinical

Answer 29

``` Anemia -Most moderate varies from subclinical to profound *20-30% virtually asymptomatic Splenomegaly Jaundice Generally stable Punctuated sometimes by -Aplastic crisis *Parvovirus B19 infection Hemolytic crisis -Increased splenic sequestration and destruction -Usually associated with an intercurrent event like infectious mononucleosis Gallstones ```

Question 30

Hereditary Spherocytosis Diagnosis

Answer 30

Family history, hematological findings, and laboratory evidence - Osmotic fragility test * Increased red cell lysis when incubated in hypotonic saline (in about 2/3 of cases) - MCHC increased

Question 31

Hereditary Spherocytosis treatment

Answer 31

No specific treatment | Splenectomy can correct the anemia but not the spherocytosis

Question 32

G6PD Deficiency epidemiology

Answer 32

``` X-linked recessive Glucose-6-phosphate dehydrogenase deficiency -G6PD A- variant -10% of black Americans Protective against Plasmodium falciparum ```

Question 33

G6PD Deficiency path

Answer 33

Intravascular hemolysis predominantly -Mild component of extravascular Decreased NADPH and glutathione -GSH neutralizes peroxide and other reactive oxygen species -Oxidized hemoglobin → precipitates as Heinz bodies *Heinz bodies damage red cell membrane → hemolysis *Heinz bodies are removed by splenic macrophages → bite cells

Question 34

G6PD A-

Answer 34

Moderately reduced half-life | Episodic hemolysis after exposure to oxidative stress

Question 35

Oxidative stresses

Answer 35

Infection—most common Drugs -Primaquine, chloroquine, sulfonamides

Question 36

G6PD Deficiency | clinical

Answer 36

Sudden onset of back pain with hemoglobinuria 2 to 3 days after an oxidant stress Hemolysis is self-limited

Question 37

G6PD Deficiency | labs

Answer 37

``` Normocytic anemia Heinz bodies -Best seen during active hemolysis -Supravital staining Bite cells RBC enzyme analysis -After hemolysis has subsided ```

Question 38

Sickle Cell Disease epidemiology

Answer 38

``` Autosomal recessive Most common hemoglobinopathy in black Americans Heterozygous (Sickle cell trait, HbAS) -No anemia -8% of black Americans Homozygous (Sickle cell disease, HbSS) -Anemia Protective against falciparum malaria ```

Question 39

Sickle Cell Disease path

Answer 39

Missense point mutation in β-globin gene -Glutamic acid to valine at 6th position of β chain HbS aggregates and polymerizes when deoxygenated -Causes red cells to change shape (sickle or boat shape) -Sickling is reversible initially with reoxygenation -Repeated sickling leads to membrane damage and eventually to irreversible sickling -Membrane damage leads to intracellular dehydration -Sickling makes the cells “sticky” Extravascular hemolysis predominantly of sickle cells

Question 40

Factors that affect rate and degree of sickling

Answer 40

Amount of HbS -HbS in HbAS is too low to produce sickling or anemia Interaction with other hemoglobins -HbS interacts weakly with HbA -HbF inhibits HbS polymerization *Infants are usually 6 months old before disease manifests -HbC (HbSC Disease) milder disease than HbSS *2-3% of black Americans heterozygous for HbC

Question 41

Factors that affect rate and degree of sickling contin

Answer 41

``` MCHC -↑ MCHC → more sickling (membrane damage) -↓ MCHC → less sickling (α-thalassemia) Acidosis → more sickling Time in low oxygen tension -Normal capillary flow → no sickling -Sluggish flow → more sickling *Spleen, bone marrow -Inflammation → more sickling -Stickiness → more sickling ```

Question 42

Sickle Cell Disease clinical

Answer 42

Key processes are chronic hemolysis and tissue injury from vascular occlusion -Extravascular hemolysis *Irreversibly sickled cells are recognized and removed by the mononuclear phagocytes *Minor intravascular hemolysis results from the mechanical fragility of sickled cells Microvascular occlusion results primarily from increased adhesiveness and inflammation

Question 43

Sickle Cell Disease Crisis

Answer 43

Vaso-occlusive crises (pain crises) Sequestration crisis Aplastic crisis

Question 44

Vaso-occlusive crises (pain crises)

Answer 44

Episodes of hypoxic tissue injury and infarction associated with severe pain

Question 45

Sequestration crisis

Answer 45

Rapid enlargement of spleen with entrapment of red cells can result in hypovolemic shock

Question 46

Aplastic crisis

Answer 46

Transient cessation of erythropoiesis Parvovirus B19 ↓ Reticulocytes, worsening anemia

Question 47

Autosplenectomy

Answer 47

Spleen is enlarged but dysfunctional by age 2 -Howell-Jolly bodies appear Chronic erythrostasis causes hypoxic tissue damage and fibrosis Spleen becomes a small piece of fibrous tissue

Question 48

Increased susceptibility to infections

Answer 48

Encapsulated bacteria -Pneumococcus, H flu -Sepsis and meningitis are most common cause of death in children Salmonella osteomyelitis

Question 49

Sickle cell disease

Answer 49

``` Acute chest syndrome -Vaso-occlusion of pulmonary capillaries -Most common cause of death in adults Dactylitis -Painful swelling of hands and feet in infants (6-9 mo) -Due to bone infarcts Stroke / Seizures Aseptic necrosis of femoral head Leg ulcers Calcium bilirubinate gallstones Renal findings in HbAS and HbSS -Microhematuria due to medulla infarcts -Lose concentrating ability ```

Question 50

Sickle Cell Disease Lab

Answer 50

``` Sickle cell screen -Sodium metabisulfite reduces O2 tension, induces sickling Hb electrophoresis -HbAS: HbA 55-60%, HbS 40-45% -HbSS: HbS 90-95%, HbF 5-10%, no HbA Peripheral Blood -HbAS: normal -HbSS: sickle cells, target cells Prenatal screening -Fetal DNA to detect point mutation ```

Question 51

Sickle Cell Disease treatment

Answer 51

``` hydroxyurea “Gentle” inhibitor of DNA synthesis ↑ HbF ↓ Leukocytes → anti-inflammatory ↑ MCV → ↓ MCHC Nitrous oxide (NO) production -Vasodilation -Inhibits platelet aggregation ```

Question 52

Thalassemias

Answer 52

A heterogeneous group of disorders characterized by diminished globin synthesis Epidemiology -Autosomal recessive -α-thalassemia in Southeast Asia, black Americans -β-thalassemia in black Americans, Greeks, Italians

Question 53

α-Thalassemia path

Answer 53

``` Decreased α-globin chain synthesis due to gene deletions -4 genes for α-globin -Lie in tandem on chromosome 16 Silent carrier: -One gene deletion Asymptomatic, normal red cells ```

Question 54

α-thalassemia trait

Answer 54

``` Two gene deletion Mild anemia, increased RBC count Black American type (α - / α - ) Asian type (- - / α α) -At risk for more severe types in offspring Labs -Decreased MCV, Hb, Hct -Increased RBC count -Normal RDW, serum ferritin, Hb electrophoresis No treatment required ```

Question 55

HbH (β4) disease

Answer 55

Three gene deletion Moderately severe hemolytic anemia -HbH is a mildly unstable hemoglobin with high oxygen affinity -Excess β chains form inclusions that are removed by macrophages and result in the destruction of the red cells *Also has some effect on erythroid precursors but erythropoiesis is effective -Average Hgb is 3 g/dl less than normal controls HbH on electrophoresis (5-30%)

Question 56

Hb Bart (γ4) disease

Answer 56

Four gene deletion Hb Bart is a high affinity hemoglobin useless for oxygen delivery to the tissues Intrauterine death -Intrauterine transfusion is possible and can save some Hb Bart on electrophoresis (>80%) -HbH and Hb Portland

Question 57

β-Thalassemia path

Answer 57

``` Mutations in β globin gene -Promoter region -Coding sequence -Splice sites Results in either -β0 No chain synthesis -β+ Diminished chain synthesis ```

Question 58

β-thalassemia minor (β/β+) [β-thal trait]

Answer 58

Mild microcytic anemia or microcytosis without anemia Mild protection against falciparum malaria Shortened red cell life span

Question 59

β-thalassemia minor (β/β+) [β-thal trait] labs

Answer 59

``` Decreased MCV, Hb, Hct Increased RBC count Normal RDW, serum ferritin Hb electrophoresis -Decreased HbA, Increased HbA2 and HbF ```

Question 60

β-thalassemia major (β0/β0) [Cooley’s anemia]

Answer 60

Severe hemolytic anemia Excess α chains precipitate -Membrane damage and extravascular hemolysis -Erythroid precursors undergo apoptosis (ineffective erythropoiesis) Extramedullary hematopoiesis

Question 61

β-thalassemia major (β0/β0) [Cooley’s anemia] | labs

Answer 61

``` Increased RDW, reticulocytes Hb electrophoresis: No HbA, Increased HbA2 and HbF Long term transfusion requirement -Systemic iron overload Bone marrow transplantation ```

Question 62

β-Thalassemia other gene combos

Answer 62

Variable and intermediate disease | Depending on amount of β-globin produced

Question 63

Paroxysmal Nocturnal Hemoglobinuria path

Answer 63

Acquired membrane defect in multipotential myeloid stem cells -Red cells, granulocytes, and platelets affected -Loss of GPI anchor that complement regulatory proteins use to attach to the red cell surface *Decay accelerating factor, CD55 *Membrane inhibitor of reactive lysis, CD59 -Cells are more susceptible to complement lysis Intravascular complement mediated lysis of red cells, neutrophils, and platelets -Nocturnal because acidosis enhances complement attachment to cells *Paroxysmal and nocturnal only 25% of the time *Chronic hemolysis without dramatic hemoglobinuria is more common

Question 64

Paroxysmal Nocturnal Hemoglobinuria clinical

Answer 64

Episodic hemoglobinuria Increased incidence of thrombosis Increased incidence of severe infections Association with aplastic anemia

Question 65

Paroxysmal Nocturnal Hemoglobinuria labs

Answer 65

``` Sucrose hemolysis test (sugar water test) -Screening test -Sugar enhances complement attachment Acidified serum test (Ham test) -Confirmatory test -Activates alternative complement pathway Peripheral Blood -Normocytic anemia with pancytopenia *Microcytic if iron deficiency develops ```

Question 66

Immune Hemolytic Anemia

Answer 66

``` Extrinsic hemolytic anemias with extravascular or intravascular hemolysis Antibodies and/or complement play a central role Classification Autoimmune -Warm type -Cold type Drug induced Alloimmune ```

Question 67

Warm Autoimmune Hemolytic Anemia

Answer 67

70% of AIHA Primary (idiopathic) 50% Secondary -Leukemia, lymphoma, other neoplastic disease, autoimmune disease (esp. SLE)

Question 68

Warm Autoimmune Hemolytic Anemia path

Answer 68

IgG antibodies that react against red cell antigens coat the red cells IgG may fix C3b to the red cells Extravascular hemolysis -IgG and/or C3b coated red cells are phagocytosed by macrophages in the spleen and liver

Question 69

Warm Autoimmune Hemolytic Anemia clinical & lab

Answer 69

Jaundice Splenomegaly Positive Direct Antiglobulin Test (DAT, Coombs’ test) -Detects RBCs sensitized with IgG and/or C3b Positive Indirect Antiglobulin Test (IAT, indirect Coombs’ test) -Detects red cell reactive antibodies in the serum Unconjugated hyperbilirubinemia Peripheral blood -Normocytic anemia, spherocytes may be present

Question 70

Cold Autoimmune Hemolytic Anemia

Answer 70

``` 30% of AIHA Primary (idiopathic) Secondary -Post-infection (mycoplasma, infectious mononucleosis) -Lymphoid neoplasms ```

Question 71

Cold Autoimmune Hemolytic Anemia oath

Answer 71

IgM antibodies bind red cells usually in cooler areas of the body IgM fixes complement to the red cell IgM dissociates at warmer temperatures in most cases May result in intravascular hemolysis if complement is activated through to the membrane attack complex Otherwise, extravascular hemolysis -C3b coated cells are phagocytosed by macrophages in the liver

Question 72

Cold Autoimmune Hemolytic Anemia clinical labs

Answer 72

Hepatosplenomegaly Raynaud’s phenomenon may result from IgM induced red cell agglutination in the cooler peripheral circulation ``` Positive DAT (for C3b) If intravascular hemolysis, -Hemoglobinuria -Hemoglobinemia -Decreased haptoglobin ```

Question 73

Drug Induced Immune Hemolytic Anemia mechanisms

Answer 73

Drug absorption Immune complex Autoantibody Membrane modification

Question 74

Drug absorption

Answer 74

Penicillin | Drug absorbs onto red cells → Antibody binds drug on cell → extravascular hemolysis

Question 75

Immune complex

Answer 75

Quinidine Drug-Antibody complexes are absorbed onto the red cells → +/- complement fixation → intravascular or extravascular hemolysis

Question 76

Autoantibody

Answer 76

α-methyldopa Drug induces a red cell autoantibody Indistinguishable from warm AIHA Antibody usually disappears in several months, if drug is discontinued

Question 77

Membrane modification

Answer 77

Cephalosporins Red cells become “sticky” Immunoglobulin and complement components adhere non-specifically

Question 78

Angiopathic Hemolytic Anemia

Answer 78

Intravascular hemolysis due to vascular lesions causing direct mechanical damage to the red cells

Question 79

Macroangiopathic | hemolytic anemia

Answer 79

Calcified stenotic aortic valve -Sheer forces due to turbulent flow and abnormal pressure gradients Artificial mechanical heart valves

Question 80

Microangiopathic hemolytic anemia

Answer 80

Microvascular lesions such as fibrin or platelet thrombi damage red cells as they pass - Disseminated intravascular coagulation (DIC) * Most common cause of microangiopathic hemolytic anemia - Thrombotic thrombocytopenic purpura (TTP) - Hemolytic uremic syndrome (HUS) - Malignant hypertension - Systemic lupus erythematosus - Disseminated cancer

Question 81

Microangiopathic hemolytic anemia peripheral blood

Answer 81

Microangiopathic hemolysis is an important diagnostic clue rather than a major problem in and of itself Peripheral Blood -Red cell fragments (schistocytes) *Helmet cells, triangle cells, burr cells