Pathology

Foundations

Two numbers organize the entire lecture

Background on the cell itself: RBCs are biconcave to maximize surface area for rapid gas exchange, live 120 days, are anucleate, and run on glucose. Anemia is decreased RBC mass, most commonly assessed by hemoglobin (RBC count and hematocrit also measure mass), and it arises from either diminished production or increased destruction. Fewer RBCs → less hemoglobin → less oxygen, giving fatigue, dyspnea, lightheadedness and angina from hypoxia, plus pale skin and mucous membranes from the low hemoglobin itself.

Reticulocyte index — is the marrow trying?

• RI >3 = appropriate marrow response — the marrow is working, so suspect destruction or loss.
• RI <2 = inadequate response — a production problem, which is this entire lecture.

The reticulocyte production index (RPI) is more accurate when the patient is severely anemic or the reticulocyte percentage is very high, because it corrects for the extra maturation time of reticulocytes released prematurely into blood.

MCV — average RBC size, in fL

Microcytic · framework

Microcytic anemias are hemoglobin-synthesis failures

MCV <80 fL, characterized by decreased Hb production and therefore hypochromasia — the cells are pale as well as small.

A shortfall in any of those components gives a microcytic anemia, which is exactly why the list looks the way it does:

• Iron deficiency anemia — not enough iron (the classic)
• Anemia of chronic disease, late — iron is present but unavailable
• Sideroblastic anemias — protoporphyrin synthesis fails
• Thalassemias — globin chains fail

Microcytic · IDA

Iron deficiency anemia

The most common anemia, and iron is the most common nutritional deficiency. Beyond the typical signs of anemia, look for koilonychia (spoon nails) and pica. Plummer-Vinson syndrome is the named triad: iron deficiency anemia + esophageal webs (dysphagia) + atrophic glossitis (beefy red tongue).

Iron in vivo

Consumed as heme (meat) and non-heme (vegetable) forms; enterocytes absorb the heme form more readily. Absorbed in the duodenum and transferred into blood via ferroportin. Transferrin carries it through the blood to liver and marrow for storage, and stored intracellular iron is bound to ferritin, which also prevents free radical formation.

Causes

Chronic bleeding, malnutrition, absorption disorders, and increased demand (pregnancy, which raises TIBC).

• Breast-fed infants and toddlers — human milk is low in iron
• Menorrhagia or gastric ulcers; colon cancer or polyps
• Gastrectomy or duodenectomy — stomach acid normally keeps iron in the reduced ferrous Fe²⁺ state, which is better absorbed than oxidized ferric Fe³⁺; lose the acid or the duodenum and you lose absorption
• Hookworm infection

Stages, in order

TIBC is a measure of transferrin molecules in the blood; % saturation is the percentage of transferrin molecules carrying iron.

Labs

Microcytic hypochromic anemia · ↑ RDW · ↓ ferritin · ↑ TIBC · ↓ serum iron · ↓ % saturation · ↑ free erythrocyte protoporphyrin.

Treatment: replete iron, typically oral ferrous sulfate.

Microcytic · ACD

Anemia of chronic disease

Anemia arising in chronic inflammatory states — chronic infections, cancer, autoimmune disorders. It is the most common anemia in hospitalized patients.

Because ferroportin is the only exit door for iron, blocking it traps iron inside enterocytes and macrophages. Absorption and recycling continue, but nothing reaches the blood — so serum iron falls while stored iron piles up. Anemia starts normocytic, normochromic and becomes microcytic with time.

Labs

↑ ferritin · ↓ TIBC · ↓ serum iron · ↓/– % saturation · ↑ free erythrocyte protoporphyrin.

Treatment: treat the underlying condition; can consider exogenous EPO.

Microcytic · sideroblastic

Sideroblastic anemias

Here the defect is in the protoporphyrin half of heme, so iron arrives normally and then has nowhere to go — which makes this the microcytic anemia with an iron overload profile.

Causes

• Congenital: X-linked, mutation in ALA synthase (ALAS, the rate-limiting step)
• Alcoholism — toxic effect on mitochondria
• Lead poisoning — inhibits ALA dehydratase and ferrochelatase
• Vitamin B6 (pyridoxine) deficiency — B6 is the cofactor for ALAS. A side effect of isoniazid, and treatable with B6
• Myelodysplastic neoplasms/syndromes

Morphology

• Ringed sideroblasts — iron-laden mitochondria visible in immature erythroid precursors, in the marrow
• Basophilic stippling — in circulating red cells, from aggregates of damaged ribosomal RNA

Labs

Typically microcytic, though the alcoholism and MDS-related forms can be normocytic or macrocytic. ↑ ferritin · ↓ TIBC · ↑ serum iron · ↑ % saturation — all four together mark an iron overload state.

Macrocytic

Macrocytic anemias

MCV >100 fL. The most common cause is megaloblastic anemia from folate or B12 deficiency, both of which cause impaired DNA synthesis. Other causes: alcoholism, liver disease, some medications, myelodysplastic neoplasms, and congenital causes such as Diamond-Blackfan anemia.

Why "megaloblastic" produces the cells it does

Impaired DNA synthesis delays nuclear maturation relative to the cytoplasm. The cytoplasm keeps growing while the nucleus lags, so the cells come out too big and immature-looking:

• In erythroid precursors → megaloblastic anemia
• In granulocytic precursors → hypersegmented neutrophils

Folate deficiency

Causes: decreased dietary intake (green vegetables, some fruit) — and critically, body stores are minimal, so deficiency develops quickly, in weeks to months. Alcohol use disorder contributes through decreased dietary variety. Also increased demand (pregnancy, or high cell turnover as in cancer or hemolytic anemias), and folate-antagonist medications.

Labs: hypersegmented neutrophils · glossitis · ↓ serum folate · ↑ homocysteine, raising thrombosis risk · normal methylmalonic acid.

B12 deficiency

Dietary deficiency is less common than folate deficiency because hepatic stores are large — so it takes years, and vegans are the dietary exception. The most common cause is pernicious anemia: intrinsic factor, made by gastric parietal cells, is required for B12 absorption in the small bowel, and PA is autoimmune destruction of parietal cells → intrinsic factor deficiency. Other causes: pancreatic insufficiency, terminal ileum damage or loss (Crohn disease), and Diphyllobothrium latum, the fish tapeworm.

Findings: hypersegmented neutrophils · glossitis · ↑ homocysteine · ↑ methylmalonic acid · neurologic symptoms, because B12 is required for fatty acid and myelin metabolism — dorsal column and lateral corticospinal tract degeneration, giving decreased proprioception and vibration sense and spastic paresis respectively.

Normocytic

Normocytic anemias of diminished production

Normocytic anemias are classically split into hemolytic and non-hemolytic. Acute blood loss is a common normocytic cause, and early IDA and early ACD live here too before they turn microcytic. The production failures:

• Renal failure → decreased EPO production
• Aplastic anemia — see below
• Parvovirus B19 — infects erythroid precursors and inhibits erythropoiesis. Especially important in patients with underlying marrow stress, such as sickle cell anemia or post-HSCT patients, where losing production for even a short time causes an abrupt crash
• Myelophthisic processes — replacement of the marrow space by something else, usually metastatic cancer. Often pancytopenia, and the peripheral smear can show marrow elements not normally in blood: nucleated red blood cells and neutrophil precursors

Aplastic anemia

Damage to stem cells by medications, infections, or autoimmune/abnormal T-cell activity; some cases idiopathic. The marrow space is void of hematopoietic elements — replaced by fat — producing pancytopenia. Treatment: discontinue causative medications, supportive care, and HSCT in some cases.

Key points

The three tables to have memorized

Iron studies

Read it as two questions. Ferritin splits IDA (low stores) from everything else (adequate or high stores). Then serum iron and % saturation split ACD (iron trapped, so low) from sideroblastic (iron unusable, so it accumulates — high).

Folate versus B12

The MCV map

Grey entries are listed for completeness but belong to another lecture.

Highest yield

Six things to carry out of this lecture

1. RI <2 means the marrow is the problem; RI >3 means destruction or loss. That single number decides which half of anemia you are in.
2. Ferritin separates IDA from ACD. Both have low serum iron; only IDA has low ferritin and high TIBC.
3. Sideroblastic anemia is the microcytic anemia with iron overload — high ferritin, high serum iron, high % saturation — because the block is in protoporphyrin, not iron.
4. Lead inhibits ALA dehydratase and ferrochelatase; B6 is the cofactor for ALA synthase (so isoniazid can cause it, and B6 treats it). Look for basophilic stippling on smear and ringed sideroblasts in marrow.
5. MMA and neurologic findings are the only things separating B12 from folate. Both raise homocysteine; only B12 raises MMA and hits the dorsal columns and lateral corticospinal tracts.
6. Pancytopenia: empty marrow is aplastic, crowded marrow is myelophthisic — and nucleated RBCs with granulocyte precursors on the smear point to the latter.

Form follows function

What the normal red cell has to do

The core job is to enclose and transport hemoglobin — delivering O₂ to tissues and carrying CO₂ to the lungs. Enclosure matters on its own, because free hemoglobin is relatively toxic, particularly to the kidneys. Secondary roles: maintaining extracellular pH and electrolyte balance, delivering toxic compounds to liver and kidney, transporting hormones, and participating in immune responses.

The cell is deliberately simple — no nucleus, no mitochondria, no lysosomes, little enzyme activity. Energy comes from breaking glucose down to lactate or pyruvate through the glycolytic (Embden-Meyerhof) pathway, and that ATP is what maintains membrane integrity. ATP-depleted red cells are more susceptible to hemolysis.

Four design requirements

• Volume — enclose the maximum amount of oxygen-binding hemoglobin
• Shape — the biconcave disc is a compromise between storage volume and surface area for gas exchange
• Deformability — to traverse small tortuous routes and heart valves, then recover shape
• Durability — to survive 120 days

Where red cells come from, and the transfusion aside

Erythropoiesis is driven by EPO, moving through yolk sac (aorta-gonad-mesonephros) → liver and spleen → bone marrow. Under stress, extramedullary hematopoiesis can restart in spleen, liver, and lymph nodes. Transfused units are stored in glucose solutions, refrigerated, for up to 42 days — and undergo some hemolysis with storage as ATP is depleted, which is the same ATP-membrane link again.

Hemoglobin, briefly

2 pairs of globins (2 alpha, 2 beta in adults) plus 4 heme groups, each one atom of ferrous (2+) iron held by a porphyrin ring. Oxyhemoglobin is one ferrous iron plus one O₂. Two terms to keep separate: a hemoglobinopathy is a mutation producing an abnormal globin (sickle cell), while a thalassemia is a mutation producing decreased synthesis of a normal globin.

Classifying hemolysis

Hemolysis and its three axes

Definition: destruction of red cells resulting in release of hemoglobin. Note what is not in that definition — anemia. Hemolysis may or may not cause anemia: if increased marrow production keeps up with the loss, it is compensated.

• Rapid (acute) versus slow (chronic) — chronic hemolysis is easier to tolerate, because the marrow has time to compensate
• Hereditary (intrinsic defects) versus acquired (antibodies)
• Intravascular versus extravascular

Intravascular versus extravascular

Immune versus non-immune

The mnemonic split worth holding: allo = an antibody against a foreign antigen; auto = an antibody against your own antigens.

Laboratory detection

Proving hemolysis, then localizing it

Present in any hemolysis

• Decreased hemoglobin / hematocrit
• Increased reticulocyte count, with polychromasia on the smear — the marrow responding appropriately
• Unconjugated (indirect) hyperbilirubinemia — heme is being catabolized faster than the liver conjugates it to glucuronide
• Increased LDH

Added when hemolysis is intravascular

• Decreased plasma haptoglobin — it binds free hemoglobin released from RBCs, and is consumed doing so
• Increased free hemoglobin — hemoglobinemia and hemoglobinuria

The marrow side of the picture: erythroid hyperplasia, with increased erythroid precursors stimulated by EPO. Normoblasts are nucleated RBCs.

Infections that hemolyze red cells directly

• Babesia species (e.g. B. microti) — dangerous in asplenic patients
• Plasmodium species — especially falciparum malaria
• Clostridium welchii — produces a hemolysin

Membrane defects

Hereditary spherocytosis

Red cells are fragile and less deformable, get trapped in the spleen, and survive only 10–20 days instead of 120. It is the common inherited anemia in northern Europeans (1 in 5,000), usually autosomal dominant (75%), with heterogeneous molecular defects.

Molecular defects

• Many defects are private — confined to a single family
• Reading frame shifts and introduction of stop codons are the most common mutation types
• Defects in ankyrin, spectrin, and band 4.2; ankyrin is most common
• Protein electrophoresis shows reduced spectrin and reduced ankyrin

The CBC, and why the indices look the way they do

From her worked example: MCV 82.6 (normal), MCHC 36.5 high, RDW 16.5 high.

• MCV: low to normal range — not frankly microcytic
• MCHC increased from relative cell dehydration — the cell loses K⁺ and water, so the same amount of hemoglobin sits in a smaller volume

Diagnosis and treatment

• Spherocytes on smear — they lack central pallor, and are seen in HS and WAHA, so the finding alone does not establish a hereditary cause
• Osmotic fragility test: incubate RBCs in NaCl solutions of decreasing concentration. Spherocytes are more sensitive to osmotic lysis and lyse sooner than normal RBCs — because a sphere has no spare membrane to expand into as water enters
• Splenectomy is often helpful, increasing the lifespan of the abnormal RBCs. Afterward, expect Howell-Jolly bodies (remnants of DNA) on the smear, since no spleen remains to pit them out

Membrane defects, continued

Hereditary elliptocytosis and pyropoikilocytosis

• Hereditary elliptocytosis — most commonly mutations in α-spectrin genes (SPTA1, SPTB), autosomal dominant, common in equatorial Africa because it confers resistance to malaria. Critically, the majority of patients are NOT anemic — elliptocytes are an incidental smear finding, and treatment is not necessary, since HE is uncommonly associated with hemolysis.
• Hereditary pyropoikilocytosis — the severe form of HE, autosomal recessive, and it does cause hemolysis and anemia.

Enzyme defects

G6PD deficiency

An enzyme deficiency that renders the red cell vulnerable to oxidative injury. X-linked recessive, so clinically it appears in males. The abnormal G6PD protein misfolds and is degraded, which is why the deficiency worsens as cells age. Clinically relevant variants: G6PD A− in the African American population and G6PD Mediterranean in the Middle East. Like elliptocytosis, it may protect against Plasmodium falciparum.

Removal of H₂O₂ requires reduced glutathione and glutathione peroxidase, and the hexose monophosphate shunt is the red cell's only source of NADPH to regenerate that glutathione — hence a single enzyme gap collapses the whole antioxidant defense.

Triggers — hemolysis is intermittent and acute, not chronic

• Drugs: antimalarials (primaquine, chloroquine), sulfonamides, nitrofurantoins
• Foods: fava beans — “favism”
• Viral and bacterial infections — free radicals generated by WBCs

Smear after oxidant exposure

• Heinz bodies — precipitates of denatured globin, requiring supravital staining to see
• Bite cells — splenic macrophages pick out these inclusions, taking a bite of membrane with them

Treatment is preventive: prevent exposure to oxidants. There is nothing to replace.

Hemoglobinopathy

Sickle cell disease

A hereditary hemoglobinopathy from production of an abnormal hemoglobin, and it is common — 8% of African Americans carry the sickle cell mutation.

• Sickle cell DISEASE = homozygous; sickle cell TRAIT = heterozygous
• Other β-globin chain disorders include HbC and HbE
• HbS = α₂β^S₂ = α₂β₂^Glu→Val. The change in protein charge alters electrophoretic mobility, which is what makes hemoglobin electrophoresis diagnostic — SS = disease, AS = trait, AA = normal

Pathophysiology — a self-amplifying cycle

Diagnosis

• Clinical presentation and family history
• Blood smear: DISEASE shows sickle cells; TRAIT shows normal-appearing red cells. Also polychromatic RBCs, target cells, anisocytosis (variation in size), poikilocytosis (variation in shape), and Howell-Jolly bodies with irreversibly sickled cells
• Sickling test — mix blood with an O₂-consuming reagent to promote sickling
• Hemoglobin electrophoresis or HPLC; DNA testing for prenatal diagnosis; newborn screening in all states

The spleen, in two stages

• Splenic sequestration crisis — a potentially life-threatening complication: sudden enlargement of the spleen from trapping of RBCs, with a rapid drop in Hb/Hct, hypovolemia and circulatory collapse. The spleen is congested and sinuses dilated with RBCs
• Autoinfarction / autosplenectomy — the spleen becomes small, shrunken, fibrotic, non-functional, leaving the patient susceptible to encapsulated organisms: pneumococci and Haemophilus influenzae

Cerebral infarction is monitored by transcranial Doppler measurement of cerebral blood flow, with a treatment goal of reducing HbS below 30% by transfusion.

Treatment — every goal reduces sickling

HbF works because it contains no β chains at all, so it cannot participate in HbS polymer — it dilutes the polymerizing species rather than blocking it.

Shared complications

What chronic hemolysis costs, regardless of cause

These apply across hemolytic diseases, HS and SCD included:

• Marrow expansion — to keep up with RBC demand
• Iron overload — especially in chronically transfused patients; oral iron chelators are used in sickle cell disease and severe thalassemia
• Gallstones — bilirubin (pigment) stones, from the chronic unconjugated bilirubin load
• Aplastic crisis — from viral infection, specifically Parvovirus B19 (fifth disease), causing a rapid and life-threatening drop in Hb/Hct. Marrow shows giant pronormoblasts and pure red cell aplasia

Thalassemia · framework

Thalassemia syndromes

Heterogeneous genetic disorders resulting in decreased synthesis of adult hemoglobin (HbA, α₂β₂) — either the α chains (α-thal) or the β chains (β-thal). Two consequences follow, and both matter:

• Hypochromia from reduced HbA — there simply isn't enough hemoglobin
• Relative excess of unpaired globin chains can harm RBCs — and this is the arm that actually drives the disease severity

Gene dosage explains why the two behave differently: a pair of α-globin genes on chromosome 16 gives 4 genes total, while a single β-globin gene on chromosome 11 gives 2 genes total.

Laboratory testing — alpha and beta alike

• RBC count increased — “lots of small cells”
• MCV decreased — think thalassemia if MCV <67 fL
• MCH and MCHC decreased
• Normal RDW
• Smear: hypochromic microcytic RBCs and target cells
• Electrophoresis: increased HbA2 in β-thal trait and β-thal intermedia, but NOT in α-thal

Thalassemia · beta

β-thalassemias

Molecular basis: mutations — usually point mutations, in contrast to the deletions of α-thal — that decrease β-globin synthesis.

Clinical severity is heterogeneous, and it tracks the amount of β chain made: cells are hypochromic and microcytic with reduced O₂ carrying capacity, and free α chains precipitate, causing RBC membrane damage and extravascular hemolysis.

Pathogenesis of β-thalassemia major (Cooley's anemia)

Both arms converge on anemia, which then branches again:

• Anemia → tissue hypoxia → ↑ erythropoietin → marrow expansion → skeletal deformities — classically a skull radiograph with new bone formation on the outer table producing perpendicular radiations, the “crewcut” appearance
• Ineffective erythropoiesis → increased dietary iron absorption → iron deposits in heart, liver, pancreas → systemic iron overload (secondary hemochromatosis), demonstrated on a liver Prussian blue iron stain. Iron chelation therapy minimizes it
• Blood transfusions reduce the anemia and therefore the marrow drive — but add their own iron burden

Clinical spectrum

Thalassemia · alpha

α-thalassemias

Reduced or absent α-globin chains, most commonly due to gene deletions — the key contrast with β-thal's point mutations. Because there are four α genes, severity is graded by how many are affected.

Hemoglobin electrophoresis summary

Note the one apparent oddity: HbA2 is trace in β-thal major but elevated in minor. HbA2 needs α chains too, and in major the massive HbF production dominates the percentages.

Acquired · clonal

Paroxysmal nocturnal hemoglobinuria

A rare acquired hematopoietic stem cell disease — a clonal disorder from somatic mutations affecting the RBC membrane. The specific defect is in the phosphatidylinositol glycan class A (PIG-A) gene, which is required to form the red cell anchor. The result: cells are more sensitive to complement-mediated lysis.

The two that matter functionally: CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis, which inhibits C3 convertase). Both are complement brakes, so losing them leaves the cell defenseless against ongoing low-level complement activation.

Clinical triad

• Chronic hemolytic anemia
• Thrombosis — the most common cause of mortality
• Pancytopenia — bone marrow failure

Diagnosis

• Flow cytometry is the modern test: both RBCs and granulocytes normally express GPI-anchored proteins, and PNH cells show abnormal or absent CD55 and CD59. Alternatively, FLAER (fluorescent aerolysin), which binds the glycan portion of the GPI anchor itself rather than using antibodies
• Historical tests — both work by making complement attack easier, and both are less specific than flow: the acidified serum (Ham) test, 1939, where acidified serum activates the alternative complement pathway (can be positive in congenital dyserythropoietic anemia), and the sucrose hemolysis test, 1970, where 10% sucrose provides low ionic strength promoting complement binding (can be positive in megaloblastic anemia and AIHA)

Marrow shows erythroid hyperplasia with numerous erythroid precursors. Treatment: marrow transplant, or anti-complement (C5) antibody — eculizumab — to reduce hemolysis. Median survival 10 years.

Acquired · immune

Immune hemolytic anemias

The unifying mechanism: red cells coated with antibody and/or complement are removed from the circulation, in the spleen.

The Direct Antiglobulin Test (DAT / Coombs)

Principle: detect antibody (IgG) and/or complement (C3) on the RBC surface by adding anti-human antibody and looking for agglutination. Its job is to distinguish immune from non-immune anemias — anemia plus a positive DAT points to an immune hemolytic anemia.

Warm autoimmune hemolytic anemia (WAHA)

Defined as hemolytic anemia plus an RBC autoantibody, usually idiopathic.

• Positive Coombs test
• Spherocytes in peripheral blood — her slide calls them “microspherocytes”
• The autoantibodies react with a wide range of self-antigens, so they destroy both the patient's own cells and any cells that are transfused — which is what makes transfusion support difficult
• Treatment: corticosteroids (prednisone), rituximab (anti-CD20 B cell antibody), splenectomy

Spherocytes form here for the same mechanical reason as in HS: splenic macrophages remove part of the antibody-coated membrane, and the cell rounds up. Same shape, different cause — and the DAT is what separates them.

Cold agglutinin syndrome (CAS)

• May cause a chronic autoimmune hemolytic anemia
• Thermal amplitude of the antibody determines severity — symptoms appear in fingers, toes, and cold-exposed areas, where blood is coolest
• Usually IgM, often directed against I or i antigens. The DAT detects complement on the RBCs — not IgG, because IgM elutes as blood rewarms while the complement it deposited stays behind
• Infection-associated: Mycoplasma pneumoniae, infectious mononucleosis (EBV), CMV, influenza, HIV — this form is usually self-limited
• Older adults with monoclonal gammopathies — plasma cell myeloma, lymphomas

Paroxysmal cold hemoglobinuria (PCH)

• Rare autoimmune syndrome causing massive intravascular hemolysis on cold exposure
• IgG autoantibody against the P antigen — historically associated with syphilis, and now typically following viral infection in children
• Biphasic hemolysin — the Donath-Landsteiner antibody: antibody and early complement components bind at low temperature, then terminal complement components form on warming and the cells lyse intravascularly
• Donath-Landsteiner test: patient serum plus normal (P-positive) RBCs and fresh serum as a complement source; antibody binds cold, complement lyses on warming — giving red serum in the tube (hemolysis) and cola-colored urine (hemoglobinuria)
• Treatment: self-limited; avoid cold; corticosteroids are not helpful — a deliberate contrast with WAHA

Drug-related hemolytic anemia — three mechanisms

Treatment includes identifying and stopping the offending drug — and note her emphasis that drugs can cause severe intravascular hemolysis. The distinction that matters: in the hapten and ternary-complex mechanisms the antibody needs the drug present, so hemolysis stops when the drug is withdrawn; in the autoantibody mechanism a true self-directed antibody has been created, which can persist.

Acquired · mechanical

Microangiopathic and mechanical hemolysis

Microangiopathic hemolytic anemia (MAHA)

Red cells are damaged on contact with dense fibrin strands, producing schistocytes — red cell fragments. Causes:

• Disseminated intravascular coagulation (DIC)
• Thrombotic thrombocytopenic purpura (TTP)
• Hemolytic uremic syndrome (HUS)

Mechanical hemolysis (RBC trauma)

• Artificial heart valves — tilting valve, ball-and-cage valve
• March hematuria — hematuria after repetitive impact on the body, classically the feet

Highest yield

Eight things to carry out of the hemolysis lectures

1. Hemolysis is proven by ↑retic, ↑indirect bilirubin and ↑LDH; it is localized by haptoglobin. Low haptoglobin plus hemoglobinuria means intravascular.
2. HS: ↑MCHC from cell dehydration, ↑RDW, MCV low-to-normal. Spherocytes lack central pallor and appear in both HS and WAHA — the DAT separates them.
3. G6PD is episodic and X-linked. ↓NADPH → ↓reduced glutathione → Heinz bodies → bite cells. Triggers: primaquine, sulfonamides, fava beans, infection. Treatment is avoidance.
4. Sickle cell is a point mutation at the 6th codon of β-globin, glutamate → valine. Deoxygenation polymerizes HbS; dehydration raises MCHC and makes it worse. Goals: ↑HbF (hydroxyurea), ↓HbS to <30%, avoid dehydration and acidosis.
5. Thalassemia versus IDA: normal RDW and an increased RBC count mean thalassemia, along with MCV <67. HbA2 is raised in β-thal trait but not in α-thal.
6. Chain terminator → β⁰; splicing → β⁺. Free α chains drive ineffective erythropoiesis, which drives both the marrow expansion (crewcut skull) and the increased iron absorption behind secondary hemochromatosis.
7. α-thal is graded by gene count: 3 genes deleted = HbH (β₄), 4 genes = Hb Bart (γ₄, high O₂ affinity) and hydrops fetalis.
8. PNH: PIGA mutation → no GPI anchor → no CD55/CD59 → complement lysis. Triad of hemolysis, thrombosis (leading cause of death), and pancytopenia. Diagnose by flow cytometry; treat with eculizumab.

Objective 1 · vessel wall

Vessel wall abnormalities associated with bleeding

These are the “non-thrombocytopenic purpuras” — the vessel itself is leaky or fragile, so the patient bleeds while every screening test comes back normal.

Bleeding is variable and usually mild. Causes fall into recognizable buckets:

• Infections — meningococcemia, Rickettsia
• Drug reactions — vasculitis
• Collagen disorders — scurvy, Ehlers-Danlos
• Henoch-Schönlein purpura — immune complexes
• Hereditary hemorrhagic telangiectasia
• Amyloid infiltration of blood vessels

Objective 2 · platelet basics

What platelets are, and what happens as the count falls

Platelets are small cell fragments derived from megakaryocytes, which expand and differentiate over several days in response to thrombopoietin (TPO). Their two jobs:

• Provide the surface on which coagulation factors assemble — this is why platelets and the cascade are not really separable systems
• Prevent bleeding

Platelet count and clinical consequence

Normal count is 150–450 K/µL.

Objective 2 · non-immune causes

Splenic sequestration and dilutional thrombocytopenia

Splenic sequestration (splenomegaly)

Very common. The spleen enlarges from increased cells/tissue or vascular engorgement, and pools platelets out of the circulation. Associated with:

• Infiltration by leukemia or lymphoma
• Chronic liver disease
• Congestive heart failure

Dilutional thrombocytopenia

Trauma and complicated surgery. Platelets are simply diluted by the saline, red cells, and plasma being infused. Treatment is to transfuse platelets if the count is low enough to affect hemostasis.

Objective 3 · ITP

Immune thrombocytopenic purpura — adult chronic

An autoimmune disorder manifesting as immune-mediated thrombocytopenia. Autoantibodies (IgG) recognize one or more platelet surface glycoproteins — GPIIb-IIIa, GPIb-IX, others — and are detectable in 50–80% of patients.

She points at the analogy directly: this is the same situation as warm autoimmune hemolytic anemia (WAHA), just with platelets as the target instead of red cells.

Clinical features

• Most common cause of isolated thrombocytopenia in an otherwise healthy patient
• Any age or sex, but typically young women under 40
• Splenomegaly and intracerebral bleeds are NOT common
• Signs track the count — petechiae (pinpoint hemorrhages) and ecchymoses (bruises)

Laboratory features

ITP is a diagnosis of exclusion.

• CBC and smear: isolated thrombocytopenia with large platelets
• PT and aPTT are normal — clotting factor activity is intact
• No sensitive and specific test exists. Antiplatelet-antibody assays exist but do not make the diagnosis
• Check for HIV and hepatitis C — both cause thrombocytopenia
• Marrow, when examined, shows increased megakaryocytes, and mainly serves to exclude infiltration, leukemia, lymphoma, and MDS

Objective 3 · treatment

Treating chronic ITP in adults

Options scale with severity, and they sort cleanly by mechanism:

Acute immune thrombocytopenia in children

Clinically a different disease from the adult chronic form:

• Abrupt onset, usually following a viral illness
• Usually does NOT require steroids — observation
• Spontaneous and permanent remission, in contrast with adults, who often have chronic disease with relapses

Objective 4 · HIT

Heparin-induced thrombocytopenia

A life-threatening complication of heparin exposure, and common — 1% to 5% of exposed patients. Caused by an antibody against a complex of heparin and platelet factor 4 (PF4).

Pathophysiology — her four-step sequence

Type I versus Type II HIT

The “4 Ts” score

A clinical pretest probability score. Each of four features earns 2, 1, or 0 points:

Laboratory testing — two categories

• Immunoassays detect the antibody — they use antibodies to find the anti-heparin-PF4 antibodies in the patient's blood
• Functional tests detect platelet activation — the serotonin release assay is the classic

Treatment

Objective 4 · other drugs

Other drug-induced thrombocytopenias

• Immune mechanism — antibodies react with platelet membrane glycoproteins only when the drug is present, leading to platelet removal. Quinine, quinidine, sulfonamide antibiotics.
• Non-immune mechanism — marrow failure from a direct toxic effect on the bone marrow. Many drugs; the most common are chemotherapeutic agents.

Objective 5 · HIV and pregnancy

HIV-associated and pregnancy-associated thrombocytopenia

HIV

Common in HIV disease, and multifactorial:

• Decreased production — HIV infects megakaryocytes directly
• HIV-associated ITP — increased destruction through immune-mediated thrombocytopenia, with GPIIb-IIIa autoantibodies
• Responds to antiretroviral therapy — treat the virus and the count often recovers

Pregnancy

• Gestational thrombocytopenia — incidental, mild, no specific cause, resolves after delivery
• Other causes: severe preeclampsia / HELLP, DIC, thrombotic microangiopathies
• ITP in pregnancy — roughly a 10-fold greater risk than the general population. Occurs in any trimester, severity varies, and counts change over the pregnancy. Transplacental passage of maternal autoantibody can cause fetal/neonatal thrombocytopenia in ~25%. Most pregnancies are uneventful.

Objective 5 · NAIT

Neonatal alloimmune thrombocytopenia (NAT)

Caused by platelet alloantibodies the mother develops against foreign fetal platelet antigens — the HPA-1a antibody is the classic.

• Detected in the lab by ELISA
• The mother has a normal platelet count — the antibody is against an antigen she does not have
• The FIRST child is often affected — contrast Rh disease of the newborn, where it is the second pregnancy
• May present as newborn bleeding or intracranial bleed
• Treatment: IVIG ± corticosteroids, and platelet transfusion
• Subsequent pregnancies are high-risk

Objective 6 · TTP

Thrombotic thrombocytopenic purpura

A thrombotic microangiopathy — excessive platelet activation forming thrombi in small blood vessels. TTP is a medical EMERGENCY, and the defining paradox is that patients CLOT despite being thrombocytopenic.

Pathogenesis

• ADAMTS13 is the metalloprotease that cleaves vWF. Without its activity, sticky ultra-large vWF multimers persist
• Acquired TTP — an IgG autoantibody against ADAMTS13. This is the usual form
• Rare inherited patients exist with a mutation that inactivates ADAMTS13

Laboratory findings

• Thrombocytopenia — counts are variable
• MAHA — anemia with schistocytes, reticulocytosis ± nucleated RBCs, increased LDH, unconjugated hyperbilirubinemia
• Renal dysfunction — usually only a mild creatinine rise
• PT and aPTT are usually NORMAL unless DIC develops
• ADAMTS13 activity assay

Schistocytes are RBC fragments formed when red cells come into contact with dense fibrin meshworks.

Treatment

• Corticosteroids and rituximab (anti-CD20) — decrease autoantibody production
• Caplacizumab — an anti-vWF immunoglobulin
• Splenectomy — increases platelet survival
• Platelet transfusion may be contraindicated

Drug-induced thrombotic microangiopathy is immune-mediated like TTP — quinine, ticlopidine, clopidogrel.

Objective 6 · HUS

Hemolytic uremic syndrome

“Typical” HUS

• MAHA and thrombocytopenia — same microangiopathic picture as TTP
• Prominent acute renal failure — this is the discriminator
• Neurologic symptoms less common than in TTP
• More frequent in children, who often recover
• NORMAL levels of ADAMTS13
• Most often from food contaminated with enterohemorrhagic E. coli producing Shiga-like toxins that disrupt endothelium → platelet activation
• NOT treated by plasma exchange — no specific therapy

“Atypical” HUS (aHUS)

• Non-infective cause of HUS
• Inherited (mutations in complement component genes) or sporadic
• Chronic and recurring, with >50% developing end-stage renal failure; early mortality 10–25%
• Caused by excessive complement activation
• Responds to complement inhibitors — eculizumab

Highest yield

Six things to carry out of this lecture

1. Thrombocytopenia is either decreased production or decreased survival, and decreased survival happens by immune and non-immune mechanisms. Every disease in this lecture slots into that grid.
2. Normal platelet count + normal PT/aPTT + purpura = vessel wall disease. Nothing in the routine panel tests the vessel.
3. ITP is a diagnosis of exclusion — isolated thrombocytopenia, large platelets, increased marrow megakaryocytes, and no prophylactic platelet transfusion even at zero platelets.
4. HIT clots. Antibody against heparin-PF4, onset days 5–10, platelets are activated before they are destroyed. Stopping heparin is not enough — add a direct thrombin inhibitor, and avoid platelet transfusion.
5. NAIT: first pregnancy, normal maternal platelet count. Rh disease is the second pregnancy. That contrast is the exam question.
6. TTP has normal PT and aPTT; DIC does not. TTP is ADAMTS13 deficiency treated by plasma exchange; typical HUS has normal ADAMTS13, prominent renal failure, and is not treated by exchange.

Approach

The hemostatic history, then the lab

She opens with the point that history is an important part of hemostatic evaluation, and the questions are targeted:

• Personal or family history of bleeding?
• Spontaneous bleeding, or bleeding only under hemostatic challenge — surgery, dental work, childbirth?
• Medications, vitamins, health foods?
• Type of bleed?

Laboratory tests of hemostasis

• Routine screening — platelet count, PT, aPTT, plus fibrinogen, D-dimer, thrombin time
• Specialized — bleeding time, platelet function tests, vWF testing, factor assays (F8, F9, F11)

Objective 1 · acquired

Acquired platelet function defects — the platelets don't work

Drugs and vitamins — very common:

• Cyclooxygenase inhibitors (aspirin and other NSAIDs) — inhibit thromboxane A2 production, and TxA2 stimulates platelets
• P2Y12 receptor inhibitors (clopidogrel, ticlopidine) — the P2Y12 receptor binds ADP, which is crucial for platelet activation and aggregation

Other conditions affecting platelet function:

• Uremia — multiple effects on platelet function
• Cardiac surgery — platelet activation and destruction in the bypass circuit

Tests of platelet function

• Bleeding time — standard incision, blot until bleeding stops. Difficult to perform, not reproducible, and NOT DONE ANYMORE
• Automated platelet function analyzer — platelets exposed to agonists (collagen, epinephrine, ADP). Good at detecting antiplatelet medications, vWD, and inherited function disorders

Platelet agonists are substances that activate platelets: thrombin, TxA2, ADP, collagen, epinephrine, arachidonic acid, ristocetin. Historically tested by optical platelet aggregometry; today rapid assays can determine whether a patient is on an antiplatelet drug and whether it is working.

Objective 1 · hereditary

Hereditary platelet function defects

Two receptors, two diseases, and the receptor's job tells you which step fails:

Deficiency of GPIIb-IIIa gives Glanzmann thrombasthenia; deficiency of GP1b gives Bernard-Soulier syndrome; deficiency of vWF itself gives von Willebrand disease — three defects along one pathway.

Platelet granule disorders — “storage pool disease”

Rare disorders of platelet secretion, with variable reduction in the number and contents of:

• Alpha granules (α-SPD) — store fibrinogen and vWF
• Dense granules (δ-SPD) — store Ca²⁺, serotonin, ATP/ADP
• Rarely both (αδ-SPD)

The granule contents needed for clot formation (fibrinogen, vWF) and for further platelet activation (ADP) are simply not available. Hermansky-Pudlak syndrome = albinism + δ-SPD, with impaired release of granule-bound ADP. Molecular mechanisms are not well understood, bleeding is variable, and electron microscopy can detect the decreased granules.

Objective 2 · cascade recap

The classic cascade, as she frames it for the lab

Her caveat on the slide: this is only partly true in vivo. The two-pathway model is a description of the test tube, not of physiology — in a real vessel, tissue factor initiates and thrombin feeds back to amplify through F8 and F5.

Objective 2 · acquired

Vitamin K deficiency and liver disease

Vitamin K deficiency

• Usually hospitalized patients
• Cofactor for gamma-carboxylation of the vitamin K-dependent clotting proteins — 2, 7, 9, 10
• Malnutrition and decreased gut flora are usually responsible
• PT increases FIRST, because of the short half-life of factor 7
• Treatment: vitamin K replacement, with response in 24–48 hours

Urgent Coumadin reversal in a bleeding patient:

• Fresh frozen plasma (FFP) — contains ALL clotting factors
• Prothrombin complex concentrates — contain the 4 vitamin K-dependent factors

Liver disease

• Decreased synthesis of vitamin K-dependent factors
• Increased fibrinolysis, secondary to decreased antiplasmin
• Synthesis of abnormal fibrinogen
• Labs: increased PT and aPTT; decreased platelets with hypersplenism
• Treatment: FFP replacement when bleeding

Objective 2 · vWD

von Willebrand disease

The most common autosomally inherited bleeding disorder — estimated frequency around 1%, with no geographical or ethnic predilection. The VWF gene is on chromosome 12, cloned in 1985, and the protein undergoes extensive post-translational modification. Usually autosomal dominant transmission (types 1 and 2).

Bleeding is variable: epistaxis, spontaneous mucous membrane bleeding, excessive bleeding from wounds. Menorrhagia should prompt testing for vWD.

The factor VIII–vWF complex

• F8 is produced by endothelial cells in the liver and elsewhere — NOT by hepatocytes
• F8 binds vWF with high affinity, so if vWF levels are normal, all F8 circulates bound to vWF
• Since vWF stabilizes F8, vWF deficiency causes a secondary decrease in F8

Quantitative variants

• Type 1 — most common (65–85%). Autosomal dominant with variable expressivity, heterogeneous mechanisms. Decreased levels of normally functioning vWF (5–40%). Usually mild but heterogeneous in bleeding
• Type 3 — very rare but severe, with extremely low vWF. Can have hemarthrosis like severe hemophilia. Deletions or frameshift mutations produce either a null phenotype or a vWF that is not secreted

Qualitative variant

• Type 2 — 20–35%. Mostly missense substitutions causing defective multimer assembly. Produces structurally abnormal vWF that cannot interact normally with platelet GP1bα and F8. Mild to moderate bleeding. Many variants; Type 2A is most common

Laboratory evaluation

• aPTT — not elevated unless F8 is significantly decreased
• Platelet count normal, except low in type 2B
• vWF activity (ristocetin cofactor activity) — low in all types
• vWF antigen — corresponds to activity in types 1 and 3, but type 2 can have antigen higher than activity
• F8 activity — decreased
• vWF multimer studies — a gel study that can be helpful
• Genetics — useful in Type 3 for counselling; molecular tests exist for Type 2

Therapeutic management

• Replace vWF — VWF:F8 concentrate or recombinant vWF. Useful in all types, especially Types 3 and 2
• Stimulate release of vWF — DDAVP (desmopressin) releases vWF from endothelial Weibel-Palade bodies by agonist effect on vasopressin V2 receptors. Useful in Type 1, but check that it works in the individual patient
• Antifibrinolytics — Amicar, tranexamic acid. Inhibit conversion of plasminogen to plasmin, inhibiting fibrinolysis and thus stabilizing clots

Objective 2 · hemophilia

Hemophilia A and B

• Sex-linked (X-linked) recessive
• Hemophilia A (factor 8) — 1 in 5,000 males
• Hemophilia B (factor 9, “Christmas disease”) — 1 in 30,000 males
• Clinically indistinguishable from each other
• aPTT elevated, and a mixing study CORRECTS it — mixing normal plasma with patient plasma normalizes the aPTT
• Confirm with specific factor assays for F8 or F9
• Multiple genetic defects — >700 described for F8, >800 for F9

Severity tracks factor activity level

Factor products

Prepared from pooled human plasma or by recombinant techniques — F8 concentrates for hemophilia A, F9 concentrates for hemophilia B, and plasma-derived or recombinant VWF-F8 concentrates for vWD. Dosing depends on the situation, and the products are very expensive.

Factor 8 inhibitors

• Hemophilia A patients who have received F8 concentrates can develop an alloantibody that destroys F8
• Older patients can develop an autoantibody against F8 without having hemophilia
• Both bleed severely
• The mixing study does NOT correct — the inhibiting antibody rapidly binds and inactivates the F8 in the normal plasma you just added
• Inhibitor strength is estimated by the Bethesda titer

Treatment of an inhibitor:

• Large doses of F8 to overwhelm the inhibitor
• Recombinant 7a (NovoSeven) for high-titer inhibitors — activates X to Xa on the platelet surface, producing a “thrombin burst”
• Activated prothrombin complex concentrates — non-activated factors II, IX, X plus activated factor VII

Objective 3 · DIC

Disseminated intravascular coagulation

A “consumption coagulopathy” — consumption of platelets and clotting factors, with fibrin formation and subsequent lysis.

It can develop rapidly or slowly, and is classified as acute (uncompensated, with decreased clotting factors) versus chronic (compensated, with normal clotting factors).

Major associated disorders

• Obstetric complications — placental abruption, retained dead fetus, septic abortion, amniotic fluid embolism
• Infections — gram-negative sepsis, meningococcemia, Rocky Mountain spotted fever
• Neoplasms — widespread cancer, acute promyelocytic leukemia
• Massive tissue injury — trauma, burns
• Other — acute intravascular hemolysis, snakebites

Clinical manifestations

Signs are often masked by the underlying disorder.

• Bleeding is most common — ecchymoses, petechiae, purpura, trauma-related oozing, post-surgical hemorrhage (obstetric, trauma)
• Vascular thrombosis is less common, due to fibrin formation — renal insufficiency, necrotic skin lesions. More often seen when cancer is the underlying cause

Pathologic microvascular fibrin deposition — platelet-fibrin thrombi — causes the end-organ damage in DIC and in the other microangiopathies.

Laboratory manifestations

Management

Treat the underlying disease.

• Supportive care — vital signs, watch for bleeding and thrombosis, correct hypovolemia
• Follow labs — platelet count, PT, aPTT, D-dimer — to monitor the patient
• If bleeding, consider transfusing red cells, plasma, platelets, cryoprecipitate

Synthesis

Reading the panel — both bleeding lectures at once

Read it in three questions. Is the platelet count low? That splits quantitative from qualitative and vascular. Which clotting time is prolonged? Isolated PT points to F7 / vitamin K / warfarin; isolated aPTT points to F8, F9, F11, heparin, or an inhibitor; both point to the common pathway or consumption. Is fibrinogen low and D-dimer high? That is what makes it DIC rather than TTP or liver disease.

Highest yield

Seven things to carry out of this lecture

1. Petechiae mean platelets; hemarthrosis means factors. The bleeding pattern narrows the differential before any lab returns.
2. Qualitative platelet defects have a completely normal screening panel. Aspirin, Glanzmann, Bernard-Soulier, storage pool disease — normal count, normal PT, normal aPTT.
3. Glanzmann = GPIIb-IIIa = aggregation, no response to ADP/collagen/epinephrine. Bernard-Soulier = GP1b = adhesion, no response to ristocetin, with giant platelets.
4. PT rises first in vitamin K deficiency because F7 has the shortest half-life of factors 2, 7, 9, 10.
5. F8 is made by endothelial cells, not hepatocytes, and vWF stabilizes it — which is why vWF deficiency secondarily lowers F8 and can prolong the aPTT.
6. vWD antigen versus activity: they fall together in Types 1 and 3 (quantity), but antigen exceeds activity in Type 2 (quality). DDAVP for Type 1; concentrate for Types 2 and 3.
7. Mixing study corrects = factor deficiency; does not correct = inhibitor. And DIC is the one that moves every number — low platelets, low fibrinogen, prolonged PT and aPTT, high D-dimer.