Master intrinsic RBC defects โ€” membrane, enzyme, hemoglobin โ€” causing hemolysis for the ASCP hematology exam.

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Chapter 13 โ€” INTRINSIC DEFECTS LEADING TO INCREASED ERYTHROCYTE DESTRUCTION

ASCP Board Exam ยท Lab-Focused Study Guide
Membrane Structure ยท Hereditary Spherocytosis ยท Osmotic Fragility & EMA ยท Elliptocytosis/HPP/SAO ยท Stomatocytosis ยท Acanthocytosis ยท PNH & Flow Cytometry ยท G6PD Deficiency ยท PK Deficiency ยท Other Enzymopathies ยท Practice Questions

1 ยท Priority Table & Quick Foundation

Topic Priority Why It’s Tested
Hereditary spherocytosis (HS): vertical defect, โ†‘ MCHC, spherocytes, DAT negative HIGH The most-tested membrane disorder; Rodak’s case study and review Q1โ€“Q3
Osmotic fragility test (OFT) and EMA binding by flow cytometry HIGH Procedure, interpretation, sensitivity, false results
Vertical vs horizontal membrane defects (HS vs HE/HPP) HIGH Mechanism questions โ€” which protein interaction is broken
G6PD deficiency: X-linked, oxidant drugs, Heinz bodies, bite cells, testing after crisis HIGH Most common RBC enzyme defect; false-normal assay with reticulocytosis
PNH: PIGA, GPI anchor, CD55/CD59, FLAER flow cytometry, thrombosis HIGH Acquired intrinsic defect; flow cytometry is the diagnostic test
Pyruvate kinase deficiency: โ†“ ATP, โ†‘ 2,3-BPG, burr cells, autosomal recessive HIGH Most common glycolytic enzyme defect causing HNSHA
Hereditary elliptocytosis, HPP (microcytes, thermal sensitivity), SAO HIGH Film-to-disorder matching
Acanthocytosis: abetalipoproteinemia, McLeod, spur cell anemia MEDIUM Acanthocyte vs burr cell and associated conditions
Stomatocytosis: OHS vs DHS/HX (MCV, MCHC, osmotic fragility, splenectomy contraindicated) MEDIUM Opposite hydration patterns
WHO classes of G6PD deficiency MEDIUM Variant (Aโˆ’, Mediterranean) to class matching
Pyrimidine 5โ€ฒ-nucleotidase deficiency: coarse basophilic stippling MEDIUM Morphology clue
Autohemolysis, hypertonic cryohemolysis, acidified glycerol lysis, SDS-PAGE, ektacytometry LOW Specialized/second-line tests
Rh deficiency syndrome, cryohydrocytosis, familial pseudohyperkalemia LOW Rare variants
๐ŸŽฏ KEY POINT โ€” Where to spend your time

Focus: HS lab picture (โ†‘ MCHC, spherocytes, DAT โˆ’, โ†‘ osmotic fragility, โ†“ EMA), vertical vs horizontal defects, G6PD triggers and testing pitfalls, PNH flow cytometry (CD55/CD59, FLAER), PK deficiency (โ†‘ 2,3-BPG, burr cells). Skim: gene frequencies by country, rare stomatocytic variants, neuroacanthocytosis genetics.

Quick Foundation โ€” from zero

  • Intrinsic defects are problems inside the RBC itself: its membrane, its enzymes or its hemoglobin (hemoglobin disorders are covered in later chapters).
  • A normal RBC must bend to squeeze through 1โ€“3 ยตm splenic slits. Bending depends on shape (surface-to-volume ratio), a flexible membrane and fluid cytoplasm (normal MCHC).
  • The membrane skeleton is held together two ways: vertical links tie the lipid bilayer down to spectrin (via ankyrin, band 3, protein 4.2, 4.1), and horizontal links join spectrin molecules to one another.
  • Break a vertical link โ†’ the bilayer sheds bits of membrane โ†’ the cell loses surface โ†’ spherocyte (HS). Break a horizontal link โ†’ the skeleton can’t snap back after stretching โ†’ elliptocyte or fragments (HE, HPP).
  • Enzyme defects either leave the cell unable to fight oxidation (G6PD โ€” hexose monophosphate shunt) or starve it of energy (PK โ€” glycolysis, low ATP).
  • PNH is the odd one out: an acquired stem cell mutation that removes GPI-anchored complement-protecting proteins, so complement lyses the cells.
๐Ÿ’ก Analogy

Picture the RBC as a tent. The fabric (lipid bilayer) is pegged to the frame (spectrin). Vertical defect = missing tent pegs โ†’ pieces of fabric blow away โ†’ the tent shrinks into a ball (spherocyte). Horizontal defect = broken joints in the frame poles โ†’ the tent gets pulled out of shape and stays stretched (elliptocyte). G6PD deficiency = no fire extinguisher; fine until a fire (oxidant drug). PK deficiency = generator failure; the pumps stop. PNH = the guard (CD55/CD59) never shows up, so intruders (complement) walk right in.

๐Ÿฅ Why this matters clinically

Getting the category right changes management: splenectomy helps HS, PK deficiency and severe HE, but is contraindicated in overhydrated and dehydrated stomatocytosis because of thrombosis. G6PD deficiency is managed by avoiding oxidant drugs. PNH is treated with the complement inhibitor eculizumab, which requires meningococcal vaccination.

Concept Plain-English Explanation
Deformability Ability of the RBC to bend and recover shape through tiny spaces
Surface-to-volume ratio How much membrane the cell has relative to its contents; โ†“ in spherocytes
Vertical interaction Links between the lipid bilayer and the skeleton (band 3โ€“ankyrinโ€“spectrin, 4.1 complex)
Horizontal interaction Links within the skeleton (spectrin dimerโ€“dimer, junctional complex)
Splenic conditioning Stress in the spleen (low glucose, low ATP, acid pH, macrophages) that makes defective cells even more spherical
Osmotic fragility How easily RBCs burst in dilute (hypotonic) saline
GPI anchor Lipid ‘hook’ that attaches certain proteins (CD55, CD59) to the membrane
HNSHA Hereditary nonspherocytic hemolytic anemia โ€” chronic hemolysis without spherocytes, usually from an enzyme defect

2 ยท Core Content Tables

2.1 Membrane Structure & Deformability

Feature Details
Three determinants of deformability (1) Cell geometry / surface-to-volume ratio ยท (2) membrane elasticity ยท (3) cytoplasmic viscosity (mainly MCHC)
Splenic filter Cells pass through 1โ€“3 ยตm slits; nondeformable cells are trapped and removed
Vertical โ€” ankyrin complex Band 3, RhAG, ankyrin, protein 4.2 (and glycophorin A) link the bilayer to spectrin
Vertical โ€” 4.1 (junctional) complex Glycophorin C, XK, Rh, Duffy, adducin, dematin, glucose transporter 1, protein 4.1 attach the bilayer to the spectrinโ€“actin junction
Horizontal interactions ฮฑ- and ฮฒ-spectrin chains form dimers, which join head-to-head into tetramers; tails meet at the junctional complex (actin, 4.1)
Cation transport Pumps and channels keep intracellular Kโบ high and Naโบ low; a leak changes cell water โ†’ overhydration (stomatocyte) or dehydration (xerocyte)

2.2 Hereditary RBC Membrane Disorders (Rodak Box 24-1)

Group Disorders Main Defect
Structural (skeletal) defects Hereditary spherocytosis (HS) Vertical interactions
Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) Horizontal interactions
Southeast Asian ovalocytosis (SAO) Band 3 deletion โ†’ rigid membrane
Cation permeability (transport) defects Overhydrated hereditary stomatocytosis (OHS) Naโบ and Kโบ leak โ†’ cell gains water
Dehydrated hereditary stomatocytosis / hereditary xerocytosis (DHS/HX) Kโบ loss โ†’ cell loses water

2.3 Hereditary Spherocytosis โ€” Genetics & Pathophysiology

Feature Details
Frequency Most common inherited membrane defect in people of northern European ancestry: about 1 in 2000โ€“3000
Inheritance ~75% autosomal dominant; ~25% nondominant (recessive or de novo). Fewer than 10% are de novo mutations; ~10% have no identifiable mutation
Genes (Table 24-1) ANK1 (ankyrin) 40โ€“65% (5โ€“10% in Japan) ยท SLC4A1 (band 3) 20โ€“35% ยท SPTB (ฮฒ-spectrin) 15โ€“30% ยท SPTA1 (ฮฑ-spectrin) <5%, recessive ยท EPB42 (4.2) <5% (45โ€“50% in Japan), recessive
Mechanism Vertical defect โ†’ lipid bilayer loosens โ†’ microvesicles are shed โ†’ loss of membrane surface โ†’ โ†“ surface-to-volume ratio โ†’ spherocyte
Splenic conditioning Spleen is hostile: low glucose, low ATP, low pH, macrophage contact โ†’ further membrane loss and dehydration โ†’ โ†‘ MCHC โ†’ trapping and removal (extravascular hemolysis)
Ion effect Cells lose Kโบ and water โ†’ dehydrated, hyperdense cells

2.4 HS Severity (Rodak Table 24-2 summary)

Grade % of Patients Hemoglobin Bilirubin Reticulocytes Notes
Mild 20โ€“30% Normal/near normal (compensated) 1โ€“2 mg/dL Up to 6% May be diagnosed late, during a crisis or family study
Moderate ~60% >8 g/dL >2 mg/dL 6โ€“10% Most common form
Moderately severe 5โ€“10% 6โ€“8 g/dL 2โ€“3 mg/dL >10% May need occasional transfusion
Severe 3โ€“5% <6 g/dL >3 mg/dL >10% Usually recessive/homozygous; transfusion dependent

2.5 HS โ€” Clinical Features

Feature Details
Classic triad Anemia, jaundice, splenomegaly
Splenomegaly About half of young children; 75โ€“95% of older children and adults
Gallstones Pigment (bilirubin) gallstones in about half of patients
Crises Hemolytic (usually with viral infection) ยท aplastic (parvovirus B19 infects erythroid precursors โ€” most serious) ยท megaloblastic (folate deficiency from high demand, e.g., pregnancy)
Other Neonatal jaundice; rare leg ulcers and extramedullary hematopoiesis

2.6 HS โ€” Laboratory Findings

Feature Details
CBC Hb normal to low ยท MCV normal or slightly โ†“ ยท MCHC โ†‘ (35โ€“38 g/dL) ยท RDW โ†‘ (>14%)
Hyperdense cells Analyzer measure of cells with MCHC >41 g/dL; >4% suggests HS
Film Spherocytes (dense, round, no central pallor), polychromasia; number varies
Hemolysis markers โ†‘ Reticulocytes, โ†‘ indirect bilirubin, โ†‘ LDH, โ†“ haptoglobin, โ†‘ urine urobilinogen
DAT Negative โ€” separates HS from warm autoimmune hemolytic anemia (also spherocytes)
Confirmation โ†‘ Osmotic fragility (esp. incubated), โ†“ EMA binding. Molecular testing not usually needed

2.7 Osmotic Fragility Test (OFT)

Feature Details
Principle RBCs placed in a series of hypotonic NaCl solutions take up water and swell; spherocytes (less spare surface) burst in less hypotonic saline
Specimen Fresh heparinized whole blood (or defibrinated). Test within 2 h at room temperature or 6 h refrigerated
Procedure Blood added to NaCl from 0.85% (isotonic) down to 0% ยท 30 min at room temperature ยท centrifuge ยท read supernatant absorbance at 540 nm
Calculation % hemolysis = (Atube โˆ’ A0.85%) รท (A0.0% โˆ’ A0.85%) ร— 100
Normal Hemolysis begins ~0.45% NaCl; complete at 0.35โ€“0.30%
HS Hemolysis begins above 0.5% NaCl โ†’ curve shifts left (โ†‘ fragility). A ‘tail’ of splenic-conditioned cells lyses at even higher concentrations
Incubated OFT Blood incubated 24 h at 37 ยฐC before testing โ€” exaggerates the defect; more sensitive (for mild cases)
Performance Sensitivity 68% (81% incubated). Compensated, unsplenectomized HS: 53% (64% incubated). Normal result does not exclude HS
Other โ†‘ fragility Immune (warm AIHA) spherocytes, HE/HPP, OHS
โ†“ Fragility Target cells: thalassemia, liver disease; also DHS/HX (dehydrated cells)

2.8 EMA Binding Test (Flow Cytometry)

Feature Details
Principle Eosin-5โ€ฒ-maleimide dye binds band 3 (mainly) and Rh, RhAG, CD47. Fewer membrane proteins โ†’ โ†“ mean fluorescence intensity (MFI)
HS result โ†“ MFI compared with normal controls
Performance Sensitivity 93โ€“96%; specificity 94โ€“99%
Practical Small sample; specimen stable up to 7 days; result in ~3 h
Also โ†“ in HPP (even lower than HS), SAO, congenital dyserythropoietic anemia type II

2.9 Other HS Tests

Feature Details
Acidified glycerol lysis test Time to 50% lysis in glycerol; ~95% sensitive, not specific
Autohemolysis Sterile incubation 48 h at 37 ยฐC*. Normal <5% (<1% with glucose). HS 10โ€“50%, corrected by glucose
Hypertonic cryohemolysis Cooling in hypertonic medium. Normal 3โ€“15%; HS >20%
SDS-PAGE Quantifies membrane proteins (spectrin, ankyrin, band 3, 4.2)
Ektacytometry (osmotic gradient) Laser diffraction measuring deformability across osmolality; research/reference labs
Molecular Not usually required; useful in atypical or severe cases

*48 h incubation is the standard autohemolysis protocol from general laboratory references; confirm against your text.

2.10 HS โ€” Treatment

Feature Details
Splenectomy For moderate to severe HS โ€” removes the site of destruction; corrects anemia in most. Delay until after age 6 if possible; laparoscopic
Before splenectomy Vaccinate against pneumococcus, meningococcus, Haemophilus influenzae type b
Post-splenectomy film Spherocytes persist (defect not fixed) + Howell-Jolly bodies, Pappenheimer bodies, target cells, โ†‘ platelets*
Relapse Anemia returns โ†’ look for an accessory spleen (Howell-Jolly bodies disappear)
Supportive Folic acid; transfusion in crises

*Post-splenectomy thrombocytosis is a general finding, not stated in this chapter.

2.11 Hereditary Elliptocytosis (HE)

Feature Details
Frequency 1 in 2000โ€“4000 worldwide; much higher in malaria regions (West Africa, Mediterranean) โ€” elliptocytes resist malaria
Inheritance / genes Mainly autosomal dominant. SPTA1 ~65%, SPTB ~30%, EPB41 ~5%; rarely GYPC
Defect Horizontal โ€” impaired spectrin dimerโ€“dimer (tetramer) association or junctional complex
Clinical ~90% asymptomatic (no anemia, normal or slightly โ†‘ retics); ~10% moderate to severe anemia
Film Elliptocytes from a few to 100% โ€” number does not correlate with severity
Leach phenotype Glycophorin C absent โ†’ Gerbich-negative blood group, elliptocytes
Other causes of elliptocytes Iron deficiency, thalassemia, megaloblastic anemia, MDS (acquired 4.1 defect), myelofibrosis โ€” usually <โ…“ of cells
Treatment Usually none; splenectomy for significant anemia

2.12 Hereditary Pyropoikilocytosis (HPP)

Feature Details
Nature Severe form of HE; autosomal recessive (homozygous or compound heterozygous spectrin defects)
Film Bizarre poikilocytes, fragments, microspherocytes, elliptocytes โ€” looks like a burn patient’s film
Indices MCV very low (50โ€“65 fL), โ†‘ RDW
Thermal sensitivity Cells fragment at 41โ€“45 ยฐC (normal cells at 49 ยฐC)
Other tests โ†‘ Osmotic fragility; EMA binding โ†“ (lower than HS)
Treatment Splenectomy improves anemia

2.13 Southeast Asian Ovalocytosis (SAO)

Feature Details
Genetics 27-bp deletion in SLC4A1 (band 3); autosomal dominant; all known cases heterozygous (homozygous likely lethal)
Where Malaysia, Indonesia, Philippines, Papua New Guinea โ€” prevalence up to 30%; protects against malaria
Cells Rigid membrane (not unstable); ~30% ovalocytes (stomatocytic), some with 1โ€“2 transverse bars/ridges
Clinical Mild or no hemolysis; no treatment needed
Tests EMA binding โ†“; DNA test for the deletion

2.14 Hereditary Stomatocytosis

Feature Overhydrated (OHS) Dehydrated (DHS) / Hereditary Xerocytosis (HX)
Frequency Rare Most common stomatocytosis
Gene / inheritance RHAG; autosomal dominant; secondary stomatin deficiency PIEZO1 (also KCNN4); autosomal dominant
Cation defect Large Naโบ influx > Kโบ loss โ†’ cell gains water Kโบ loss โ†’ cell loses water
Stomatocytes 5โ€“50% <10%; also target cells, burr cells, cells with Hb puddled at the periphery
MCV โ†‘โ†‘ 110โ€“150 fL Normal to slightly โ†‘
MCHC โ†“ 24โ€“30 g/dL โ†‘
Osmotic fragility โ†‘ โ†“
Other Moderate to severe hemolysis Mild; fetal loss, hydrops fetalis; pseudohyperkalemia; iron overload*
Splenectomy Contraindicated โ€” thrombosis Contraindicated โ€” thrombosis

*Iron overload in DHS is widely reported; verify against your edition if needed.

2.15 Other Stomatocytic Conditions

Feature Details
Familial pseudohyperkalemia Kโบ leaks out of cells stored at room temperature or below โ†’ falsely โ†‘ serum Kโบ; no or minimal hemolysis
Cryohydrocytosis Cells swell and lyse when stored at 4 ยฐC for 24โ€“48 h; band 3 or stomatin-related defects
Rh deficiency syndrome Rhnull (amorph or regulator type) or Rhmod; stomatocytes and a few spherocytes; mild hemolysis; splenectomy helps
Acquired stomatocytosis Most often a drying artifact of thick film areas; normal blood has 3โ€“5%; also alcoholism, liver disease, some drugs; seen in wet preparations

2.16 Acanthocytes vs Burr Cells (Echinocytes)

Feature Acanthocyte (spur cell) Burr cell (echinocyte)
Projections 2โ€“20 irregular spicules, uneven length and spacing 10โ€“30 short, evenly spaced projections
Central pallor Absent (dense cell) May be present
Cause Altered lipid composition of the membrane Artifact (drying, glass, old EDTA) or real
Associated with Abetalipoproteinemia, McLeod syndrome, chorea-acanthocytosis, severe liver disease (spur cell anemia), post-splenectomy* Uremia, PK deficiency, runners after a race*; <3% may be seen normally

*From general hematology references.

2.17 Neuroacanthocytosis & Acquired Acanthocytosis

Condition Genetics Key Features Acanthocytes
Abetalipoproteinemia Autosomal recessive; MTP gene (microsomal triglyceride transfer protein) No chylomicrons, VLDL or LDL โ†’ very low cholesterol; โ†‘ sphingomyelin in membrane; fat malabsorption (steatorrhea), ataxia, retinitis pigmentosa; treat with vitamins A and E 50โ€“90%
McLeod syndrome X-linked; XK gene โ†’ no Kx protein โ†“ Kell antigens; myopathy, neuropsychiatric signs; onset 40โ€“60 years Up to 85%
Chorea-acanthocytosis Autosomal recessive; VPS13A (chorein) Chorea, seizures; mean onset ~35 years 5โ€“50%
Spur cell anemia (acquired) โ€” Severe liver disease (alcoholic cirrhosis): excess free cholesterol loads the membrane โ†’ acanthocytes โ†’ splenic removal; splenomegaly; improves with liver transplant Variable, often many

2.18 Paroxysmal Nocturnal Hemoglobinuria (PNH) โ€” Pathophysiology

Feature Details
Defect Acquired mutation in a hematopoietic stem cell of PIGA (X chromosome) โ†’ cannot make the GPI anchor (first step)
Why one mutation suffices PIGA is X-linked โ€” males have one X; in females one X is inactivated
Affected cells All lineages from the clone: RBCs, granulocytes, monocytes, platelets, lymphocytes
Missing proteins ~16 GPI-anchored proteins, notably CD55 (decay-accelerating factor, DAF โ€” inhibits C3 and C5 convertases) and CD59 (membrane inhibitor of reactive lysis, MIRL โ€” blocks the membrane attack complex)
Result Complement-mediated intravascular hemolysis
RBC populations Type I normal ยท Type II partial deficiency ยท Type III complete deficiency (most sensitive). Most common mix: I + III
Epidemiology 2โ€“5 per million; peak 3rdโ€“4th decade; closely linked to aplastic anemia

2.19 PNH โ€” Clinical Features (Rodak Box 24-2)

Feature Details
Hemolysis Chronic intravascular hemolysis with acute episodes; hemoglobinuria at presentation in only ~25%; the ‘nocturnal’ pattern is uncommon
Thrombosis Leading cause of death; unusual sites โ€” hepatic veins (Budd-Chiari), abdominal, cerebral veins
Marrow failure Cytopenias; overlap with aplastic anemia and MDS
Smooth muscle dystonia Free Hb scavenges nitric oxide โ†’ esophageal spasm, abdominal pain, erectile dysfunction
Other Renal damage, fatigue, pulmonary hypertension

2.20 PNH โ€” Laboratory

Feature Details
Routine Anemia (normocytic or microcytic from iron loss), retics only mildly โ†‘ (marrow failure), โ†“ haptoglobin, โ†‘ LDH, hemoglobinuria, hemosiderinuria, DAT negative, โ†“ neutrophils/platelets possible
Other findings Iron and folate deficiency; abnormal karyotype in ~20%
Flow cytometry โ€” RBCs CD59 on RBCs shows type I/II/III populations; underestimates the clone (type III cells are destroyed; transfusion dilutes them). High-sensitivity: CD59 + CD235a (glycophorin A) โ€” detects 0.002%
Flow cytometry โ€” WBCs (preferred) Granulocytes and monocytes: absence of โ‰ฅ2 GPI-linked markers in โ‰ฅ2 lineages. FLAER (fluorescent aerolysin โ€” binds the GPI anchor itself) + CD24/CD15/CD45 (granulocytes) and CD14/CD64/CD45 (monocytes); detects ~0.01% and 0.04%
Obsolete Sucrose hemolysis (screen) and Ham (acidified serum) tests

2.21 PNH Classification (Rodak Table 24-3)

Category Hemolysis Marrow GPI-Deficient Neutrophils
Classic PNH Clinical intravascular hemolysis Cellular, erythroid hyperplasia Large clone (>50%)
PNH with another marrow disorder (aplastic anemia, MDS) Mild or subclinical Evidence of the other disorder Usually <30%
Subclinical PNH None Marrow failure Small clone (<1%), found only by high-sensitivity flow

2.22 PNH โ€” Treatment

Feature Details
Eculizumab (approved 2007) Humanized monoclonal anti-C5 โ†’ blocks the membrane attack complex โ†’ stops intravascular hemolysis, โ†“ thrombosis, โ†“ transfusion need
Required precaution Neisseria meningitidis vaccination (terminal complement needed against Neisseria)
Residual problem Extravascular hemolysis continues โ€” C3 fragments coat cells (DAT may become C3-positive)*
Cure Allogeneic HSCT โ€” the only cure; 50โ€“60% survival; reserved for severe cases
Prognosis Median survival before eculizumab ~10 years
Supportive Iron, folate, transfusion, anticoagulation

*DAT C3 positivity on eculizumab is a general clinical observation.

2.23 G6PD Deficiency โ€” Genetics & Epidemiology

Feature Details
Inheritance X-linked โ€” males hemizygous (fully affected); females heterozygous (mosaic, variable, lyonization); homozygous females rare
Frequency Most common RBC enzyme defect: ~400 million people (~5% worldwide); ~10% of African American males
By region Sub-Saharan Africa 7.5% ยท Middle East 6.0% ยท Asia 4.7% ยท Europe 3.9% ยท Americas 3.4%
Selection Protects against falciparum malaria
Variants Normal wild type G6PD-B. G6PD Aโˆ’ (Africa, class III), G6PD Mediterranean (class II). 186+ mutations known

2.24 WHO Classification of G6PD Variants

Class Enzyme Activity Clinical Example
I <1% (severe) Chronic HNSHA Serres, Madrid (rare sporadic)
II <10% Severe episodic hemolysis (drugs, fava beans) Mediterranean
III 10โ€“60% Moderate, self-limited episodic hemolysis Aโˆ’
IV 60โ€“150% None (normal) B (wild type), A+
V >150% None Rare

2.25 G6PD Deficiency โ€” Pathophysiology

Feature Details
Role of G6PD First enzyme of the hexose monophosphate (pentose phosphate) shunt; the RBC’s only source of NADPH
NADPH use Keeps glutathione reduced (GSH) โ†’ GSH detoxifies hydrogen peroxide via glutathione peroxidase
Oxidant stress No NADPH โ†’ no GSH โ†’ Hb oxidized โ†’ denatured Hb precipitates as Heinz bodies attached to the membrane
Destruction Heinz bodies pitted by splenic macrophages โ†’ bite cells; severe damage โ†’ intravascular hemolysis too (both routes)
Age effect Enzyme declines as the cell ages; reticulocytes have ~5ร— the G6PD of the oldest cells โ†’ old cells lyse first; episode is self-limited as young cells replace them (Aโˆ’ type)

2.26 G6PD โ€” Clinical Syndromes

Feature Details
Drug-induced acute hemolysis Starts hours to 1โ€“3 days after the drug; Hb falls, hemoglobinuria, back pain; retics rise in 4โ€“6 days
Drugs to avoid (Box 24-3) Dapsone, methylene blue, nitrofurantoin, phenazopyridine, primaquine, rasburicase, toluidine blue; possibly co-trimoxazole, quinolones, sulfadiazine; chemical: naphthalene (mothballs)
Infection-induced Most common trigger (pneumonia, hepatitis, typhoid*) โ€” Hb falls 3โ€“4 g/dL
Favism Fava beans โ†’ severe acute hemolysis with hemoglobinuria; mainly Mediterranean variant; children most affected
Neonatal hyperbilirubinemia Jaundice on days 2โ€“3; worse with coexisting UGT1A1 (Gilbert) mutation; risk of kernicterus; anemia usually mild
Chronic HNSHA Class I variants โ€” chronic hemolysis without triggers

2.27 G6PD โ€” Laboratory Diagnosis

Feature Details
During crisis โ†“ Hb, โ†‘ retics (up to 30%), โ†‘ bilirubin, โ†“ haptoglobin, โ†‘ LDH, hemoglobinuria. DAT negative
Film Bite cells (not specific), blister cells, polychromasia, some spherocytes
Heinz bodies Seen only with supravital stain (crystal violet / new methylene blue) โ€” not on Wright stain; removed quickly by spleen
Quantitative assay (reference) G6PD + G6P + NADPโบ โ†’ NADPH; rate of NADPH formation measured at 340 nm; reported as IU/g Hb. Deficient: <20% of normal (e.g., <4.0 IU/g Hb)
Qualitative screens Fluorescent spot test โ€” NADPH fluoresces under UV; deficient = no fluorescence ยท Dye reduction: BinaxNOW (formazan, 98% sensitive at <4.0 U/g Hb), WST-8 (orange formazan)
False normal Testing during/after a crisis โ€” old deficient cells gone, young retics have more enzyme; recent transfusion; WBC/platelet contamination (remove buffy coat)
Timing Retest 2 weeks to 2 months after the hemolytic episode (or test family)
Heterozygous females Screens often miss them (mixed populations)
DNA testing Works after transfusion, during hemolysis and in heterozygotes

2.28 G6PD โ€” Treatment

Feature Details
Prevention Avoid oxidant drugs, fava beans and naphthalene; screen before primaquine/rasburicase/dapsone
Acute Stop the drug, treat infection; transfusion if severe (Hb <7 g/dL or <9 g/dL with continuing hemoglobinuria*)
Newborns Phototherapy; exchange transfusion for severe hyperbilirubinemia
Chronic HNSHA Folic acid; transfusion; splenectomy rarely helpful

*Transfusion thresholds vary by source; use your lab/clinical guidelines.

2.29 Pyruvate Kinase (PK) Deficiency

Feature Details
Inheritance Autosomal recessive; PKLR gene (chromosome 1q21)*; homozygous or compound heterozygous
Frequency ~1 in 20,000 (white population); most common glycolytic enzyme defect causing HNSHA; Amish founder mutation (1436G>A)
Mechanism Last ATP-generating step of glycolysis (PEP โ†’ pyruvate) blocked โ†’ โ†“ ATP โ†’ Naโบ/Kโบ pumps fail โ†’ Kโบ and water loss โ†’ rigid, dehydrated cells โ†’ splenic removal
2,3-BPG Upstream intermediates accumulate โ†’ โ†‘ 2,3-BPG โ†’ right-shifted Oโ‚‚ curve โ†’ anemia better tolerated
Clinical Mild to severe (hydrops to compensated); neonatal jaundice; gallstones; splenomegaly; iron overload even without transfusion
Film Normocytic; polychromasia; burr cells (echinocytes) 3โ€“30%, more after splenectomy; no spherocytes
Reticulocytes Not proportional to anemia (retics are sequestered in spleen); can rise to 40โ€“70%* after splenectomy (5-fold)
Other tests Osmotic fragility normal; DAT negative; autohemolysis โ†‘, not corrected by glucose (corrected by ATP)*
PK assay PK: PEP + ADP โ†’ pyruvate + ATP; coupled with LDH: pyruvate + NADH โ†’ lactate + NADโบ. Decrease in absorbance at 340 nm (NADH consumed). Screening: loss of fluorescence
False normal Contaminating leukocytes (high PK, different isozyme) โ€” remove buffy coat; transfusion; high retics
Treatment Transfusion; splenectomy improves anemia; chelation; HSCT in severe cases

*Specific details (chromosome, post-splenectomy retic %, autohemolysis pattern) are from general references and marked for verification.

2.30 Other RBC Enzymopathies

Enzyme Deficiency Inheritance Key Point
Pyrimidine 5โ€ฒ-nucleotidase type 1 (P5โ€ฒNT-1) Autosomal recessive (NT5C3A) Third most common RBC enzyme defect; pyrimidine nucleotides accumulate โ†’ coarse basophilic stippling (resembles lead poisoning, which inhibits the same enzyme*); some patients have mental retardation
Glucose phosphate isomerase (GPI) Autosomal recessive Second most common glycolytic defect*; HNSHA
Hexokinase, aldolase, triosephosphate isomerase (TPI) Autosomal recessive HNSHA; TPI with severe neuromuscular disease*
Phosphoglycerate kinase (PGK) X-linked Hemolysis ยฑ myopathy, neurologic disease
GAPDH, LDH โ€” No hemolysis
Bisphosphoglycerate mutase โ€” โ†“ 2,3-BPG โ†’ left shift โ†’ erythrocytosis (not hemolysis)
Hemoglobin/redox enzymes (GSH synthesis, glutathione reductase) โ€” Oxidant-induced hemolysis like G6PD*

*Not stated in Rodak Ch. 24; from general hematology references.

3 ยท Visual Memory Hooks

๐Ÿง  Vertical vs horizontal
โ€œVertical = Very Spherical (HS); Horizontal = Hello Elliptocyte (HE/HPP)โ€.
๐Ÿง  HS genes
โ€œA Big Spectacleโ€ โ†’ Ankyrin (most common), Band 3, Spectrin-ฮฒ.
๐Ÿง  HS signature
โ€œHigh MCHC + Spheres + Negative DAT = HSโ€.
๐Ÿง  HS crises
โ€œHemolytic, Aplastic (Parvo), Megaloblastic (folate) โ€” HAMโ€.
๐Ÿง  Osmotic fragility
โ€œSpheres Swell and Split Soonerโ€ (โ†‘ fragility); โ€œTargets Tolerateโ€ (โ†“ fragility โ€” thalassemia).
๐Ÿง  EMA
โ€œEMA Eyes band 3โ€ โ€” low fluorescence in HS; lowest in HPP.
๐Ÿง  HPP
โ€œPyro = fireโ€ โ†’ cells fragment at 41โ€“45 ยฐC, look burned, MCV 50sโ€“60s.
๐Ÿง  SAO
โ€œStiff Asian Ovalsโ€ โ€” rigid, 27-bp band 3 deletion, no treatment.
๐Ÿง  Stomatocytosis
โ€œOver-hydrated = Oversized (MCV โ†‘), Dehydrated = Dense (MCHC โ†‘)โ€ โ€” both: no splenectomy.
๐Ÿง  Abetalipoproteinemia
โ€œA-beta = Acanthocytes, Ataxia, fAt malabsorption, reTinA pigmentosaโ€.
๐Ÿง  McLeod
โ€œMcLeod Kills Kellโ€ โ€” X-linked, no Kx, weak Kell, acanthocytes.
๐Ÿง  PNH
โ€œPIG-A takes the Anchorโ€ โ†’ no GPI โ†’ no CD55/CD59 โ†’ complement lyses.
๐Ÿง  CD55 vs CD59
โ€œ55 = Decay (DAF) early; 59 = MIRL at the end (MAC)โ€.
๐Ÿง  PNH killers
โ€œClots, Cytopenias, Complementโ€ โ€” thrombosis is the #1 cause of death.
๐Ÿง  FLAER
โ€œFLAER Finds the Anchorโ€ โ€” binds GPI directly; use on granulocytes/monocytes.
๐Ÿง  G6PD triggers
โ€œInfection > Drugs > Favaโ€ โ€” infection most common.
๐Ÿง  G6PD bodies
โ€œHeinz needs a supravital; Bites come afterโ€.
๐Ÿง  G6PD false normal
โ€œYoung cells lieโ€ โ€” test 2 weeks to 2 months after the crisis.
๐Ÿง  PK
โ€œPK = Poor Kick (โ†“ ATP), Plenty of 2,3-BPG, Prickly burr cellsโ€.
๐Ÿง  P5โ€ฒNT
โ€œPyrimidine = Punctate stipplingโ€ (coarse basophilic stippling).

Pattern Triggers

If you seeโ€ฆ Thinkโ€ฆ Becauseโ€ฆ
Spherocytes, โ†‘ MCHC, โ†‘ RDW, DAT negative, family history, splenomegaly Hereditary spherocytosis Vertical defect, membrane loss
Spherocytes, DAT positive Warm autoimmune hemolytic anemia Antibody-coated cells partially eaten
Curve shifted left on OFT (lysis at 0.5โ€“0.6% NaCl) Increased fragility โ€” spherocytes Less spare surface area
Decreased osmotic fragility Target cells โ€” thalassemia, liver disease; DHS More surface relative to volume / dehydrated
Many elliptocytes, no anemia, patient of African descent Hereditary elliptocytosis (common HE) ~90% asymptomatic
MCV 55 fL, bizarre fragments, microspherocytes in an infant Hereditary pyropoikilocytosis Severe horizontal defect, thermal instability
Rigid oval cells with transverse bars, Malaysian patient, no anemia Southeast Asian ovalocytosis Band 3 27-bp deletion
MCV 130, MCHC 26, many stomatocytes Overhydrated hereditary stomatocytosis Naโบ gain โ†’ water gain
High MCHC, target cells, puddled Hb, high Kโบ in stored sample Dehydrated stomatocytosis (xerocytosis) PIEZO1 โ†’ Kโบ loss
Acanthocytes + ataxia + fat malabsorption + retinitis pigmentosa Abetalipoproteinemia No apolipoprotein B lipoproteins
Acanthocytes + weak Kell antigens in an adult male McLeod syndrome Absent Kx protein
Acanthocytes in alcoholic cirrhosis with splenomegaly Spur cell anemia Excess membrane cholesterol
Hemoglobinuria, pancytopenia, Budd-Chiari, DAT negative PNH GPI-anchor loss
Bite cells after primaquine/dapsone in an African American male G6PD deficiency (Aโˆ’) Oxidant stress
Normal G6PD level during acute hemolysis with high retics False normal โ€” retest later Young cells rich in enzyme
Burr cells, no spherocytes, normal OFT, chronic hemolysis, AR inheritance Pyruvate kinase deficiency Low ATP
Coarse basophilic stippling, chronic hemolysis, lead level normal Pyrimidine 5โ€ฒ-nucleotidase deficiency Pyrimidine nucleotides accumulate

Commonly Confused Pairs

Feature A B
HS vs warm AIHA DAT negative; family history DAT positive; acquired
Vertical vs horizontal defect HS โ€” membrane loss (spherocytes) HE/HPP โ€” skeleton can’t recover (elliptocytes, fragments)
HE vs HPP Usually AD, no anemia, normal MCV AR, severe anemia, MCV 50โ€“65, thermal fragility
โ†‘ vs โ†“ osmotic fragility Spherocytes (HS, AIHA), HPP, OHS Target cells (thalassemia), DHS
OHS vs DHS โ†‘ MCV, โ†“ MCHC, โ†‘ OF โ†‘ MCHC, โ†“ OF, puddled Hb
Acanthocyte vs burr cell Irregular spicules, no pallor Short even projections
CD55 vs CD59 DAF: inhibits C3/C5 convertases MIRL: blocks MAC formation
PNH RBC flow vs WBC flow Underestimates clone; transfusion affects it Preferred; FLAER on granulocytes/monocytes
G6PD vs PK deficiency X-linked; episodic; Heinz/bite cells; HMP shunt AR; chronic; burr cells; glycolysis; โ†‘ 2,3-BPG
G6PD Aโˆ’ vs Mediterranean Class III; self-limited; retics protect Class II; severe; favism
Heinz bodies vs Howell-Jolly bodies Denatured Hb; supravital stain DNA remnant; visible on Wright stain
P5โ€ฒNT deficiency vs lead poisoning Hereditary; normal lead Acquired; โ†‘ lead; both inhibit the same enzyme

Timeline Table โ€” G6PD Drug-Induced Hemolysis

Marker Onset Peak Duration Best Exam Use
Hemolysis / Hb fall Hours to 1โ€“3 days after drug Days 2โ€“7* Self-limited in Aโˆ’ even if drug continues* Link to recent drug/infection
Heinz bodies Early (first days) Early Disappear as spleen removes them Supravital stain early in episode
Bite/blister cells After Heinz body pitting First week Days Film clue
Hemoglobinuria With acute intravascular component Early Short Dark urine
Reticulocytosis Day 4โ€“6 ~Day 7โ€“10* Weeks Causes false-normal G6PD
Reliable G6PD assay โ€” โ€” 2 weeks to 2 months after episode Timing of testing

*Approximate, from general references.

4 ยท Diagnostic Flowcharts

Flowchart 1 โ€” Spherocytes on the film

Spherocytes + hemolysis markers โ†’ Perform DAT
DAT positive โ†’ Immune hemolysis (warm AIHA, HDFN, transfusion reaction)
DAT negative + family history / โ†‘ MCHC / splenomegaly โ†’ Suspect HS
Confirm โ†’ EMA binding (โ†“ MFI) and/or incubated osmotic fragility (โ†‘)
Equivocal or atypical โ†’ SDS-PAGE, ektacytometry, molecular testing
โš ๏ธ Common mistake: diagnosing HS from spherocytes alone โ€” warm AIHA also causes spherocytes and โ†‘ osmotic fragility. The DAT separates them.

Flowchart 2 โ€” Reading an osmotic fragility curve

Curve normal (lysis starts 0.45%, complete 0.35โ€“0.30%) โ†’ Normal โ€” but mild HS not excluded; run incubated OFT/EMA
Shifted left (lysis starts >0.5%) โ†’ โ†‘ Fragility โ†’ HS, AIHA, HPP, OHS
Tail at high NaCl concentrations โ†’ Splenic-conditioned subpopulation (HS)
Shifted right (lysis at lower concentrations) โ†’ โ†“ Fragility โ†’ thalassemia, target cells, DHS
โš ๏ธ Common mistake: reversing the direction. Fragile cells burst at HIGHER saline concentrations (closer to isotonic).

Flowchart 3 โ€” Abnormal RBC shapes in a hereditary hemolytic anemia

Spherocytes โ†’ HS (vertical defect)
Elliptocytes โ†’ HE (horizontal); MCV very low + fragments โ†’ HPP
Rigid ovalocytes with bars, SE Asian โ†’ SAO
Stomatocytes โ†’ MCV โ†‘, MCHC โ†“ โ†’ OHS ยท MCHC โ†‘, targets, puddled Hb โ†’ DHS
Acanthocytes โ†’ Abetalipoproteinemia, McLeod, chorea-acanthocytosis, spur cell anemia
No specific shape (burr cells, stippling, bite cells) โ†’ Enzymopathy โ€” PK, P5โ€ฒNT, G6PD
โš ๏ธ Common mistake: calling artifactual stomatocytes (thick-area drying) or burr cells (old EDTA, glass) a disorder.

Flowchart 4 โ€” Suspected PNH

Intravascular hemolysis, DAT negative ยฑ cytopenias ยฑ unusual thrombosis โ†’ Order flow cytometry
Screen โ†’ Granulocytes and monocytes: FLAER + CD24/CD15/CD45 and CD14/CD64/CD45
RBCs โ†’ CD59 (+ CD235a) โ†’ type I, II, III populations
Absent GPI markers in โ‰ฅ2 lineages โ†’ PNH confirmed; size the clone (classic vs with marrow disorder vs subclinical)
Treatment โ†’ Eculizumab (after meningococcal vaccine) or HSCT
โš ๏ธ Common mistake: ordering a Ham or sucrose hemolysis test โ€” both are obsolete; flow cytometry is the standard.

Flowchart 5 โ€” Episodic hemolysis after a drug or infection

Acute Hb drop, dark urine, bite/blister cells, DAT negative โ†’ Suspect G6PD deficiency
Supravital stain early โ†’ Heinz bodies
G6PD assay during/right after crisis โ†’ May be falsely normal (retics)
Retest 2 weeksโ€“2 months later, or test family / DNA โ†’ Confirms deficiency
Management โ†’ Stop the oxidant; avoid Box 24-3 drugs, fava beans, naphthalene
โš ๏ธ Common mistake: accepting a normal G6PD level during reticulocytosis as ruling out the deficiency.

Flowchart 6 โ€” Chronic nonspherocytic hemolysis (HNSHA)

Chronic hemolysis, DAT negative, no spherocytes, normal OFT โ†’ Suspect enzymopathy
Screen G6PD โ†’ Class I variant? (males)
Screen PK (buffy coat removed) โ†’ โ†“ PK โ†’ PK deficiency (AR, burr cells, โ†‘ 2,3-BPG)
Coarse basophilic stippling, lead normal โ†’ P5โ€ฒNT-1 deficiency
Others โ†’ GPI, hexokinase, TPI, PGK โ€” specialized enzyme panel
โš ๏ธ Common mistake: forgetting leukocyte contamination โ€” WBCs are rich in PK and G6PD and give false-normal results.

5 ยท 25 High-Yield Facts

# Fact
1 RBC deformability depends on surface-to-volume ratio, membrane elasticity and cytoplasmic viscosity.
2 Hereditary spherocytosis is caused by defects in vertical membrane interactions.
3 The most common HS mutation involves ankyrin (ANK1), followed by band 3 and ฮฒ-spectrin.
4 About 75% of HS cases are autosomal dominant.
5 HS spherocytes form because microvesicles are lost, reducing the surface-to-volume ratio.
6 HS typically shows increased MCHC (35โ€“38 g/dL), increased RDW and a negative DAT.
7 More than 4% hyperdense cells (MCHC >41 g/dL) suggests HS.
8 Normal RBCs begin to lyse at 0.45% NaCl; HS cells begin to lyse above 0.5% NaCl.
9 Osmotic fragility is decreased in thalassemia (target cells).
10 EMA binding (flow cytometry) is decreased in HS, with sensitivity 93โ€“96%.
11 Aplastic crisis in HS is caused by parvovirus B19.
12 After splenectomy for HS, spherocytes persist but anemia resolves.
13 Hereditary elliptocytosis is caused by defects in horizontal membrane interactions, mostly ฮฑ-spectrin.
14 About 90% of HE patients are asymptomatic.
15 HPP has a very low MCV (50โ€“65 fL) and RBCs that fragment at 41โ€“45 ยฐC.
16 Southeast Asian ovalocytosis results from a 27-bp deletion in band 3 and needs no treatment.
17 Splenectomy is contraindicated in hereditary stomatocytosis because of thrombosis risk.
18 Abetalipoproteinemia causes acanthocytes, fat malabsorption, ataxia and retinitis pigmentosa.
19 McLeod syndrome is X-linked, with absent Kx and weak Kell antigens.
20 PNH is an acquired PIGA mutation that prevents GPI-anchor synthesis.
21 PNH cells lack CD55 (DAF) and CD59 (MIRL) and are lysed by complement.
22 Flow cytometry with FLAER on granulocytes and monocytes is the preferred PNH test.
23 G6PD deficiency is X-linked and the most common RBC enzyme defect.
24 G6PD assays may be falsely normal after a hemolytic episode because reticulocytes have more enzyme.
25 PK deficiency is autosomal recessive, lowers ATP and raises 2,3-BPG.
โญ EXAM TIP โ€” The single most testable pattern

Spherocytes + โ†‘ MCHC + DAT negative + family history = HS โ†’ confirm with โ†‘ osmotic fragility or โ†“ EMA. Bite cells + recent oxidant + male = G6PD โ†’ retest weeks later. Intravascular hemolysis + DAT negative + thrombosis/cytopenias = PNH โ†’ flow cytometry (CD55/CD59/FLAER).

Quick Interpretation Table

Lab Finding Clinical Interpretation
Hb 11.9, MCV 77, MCHC 36.7, RDW 22.9, spherocytes, polychromasia, gallstones, family history Hereditary spherocytosis (Rodak case study)
โ†“ EMA MFI, incubated OFT โ†‘ Confirms HS
OFT: lysis starts 0.40%, complete 0.20% Decreased fragility โ€” target cells (e.g., thalassemia)
FLAER-negative neutrophils 60%, CD59-negative RBCs 20% Classic PNH (RBC clone underestimated)
G6PD 6.5 IU/g Hb (normal) on day 5 of hemolysis; retics 18% Possibly false normal โ€” repeat in 2 weeks to 2 months
PK activity low, many burr cells, normal OFT PK deficiency
MCV 138, MCHC 26, stomatocytes 30% Overhydrated hereditary stomatocytosis โ€” avoid splenectomy
High serum Kโบ in a delayed sample, normal ECG, high MCHC Pseudohyperkalemia โ€” consider DHS or familial pseudohyperkalemia

๐ŸŽด Flashcards โ€” Active Recall Practice

How it works: tap the card to flip it. Then mark it โœ“ Knew It or โœ— Review Again (buttons unlock after flipping). Use ๐Ÿ”€ Shuffle to randomize and ๐Ÿ Finish anytime โ€” your summary appears at the end, and you can drill only the cards you missed.

๐Ÿ“ Practice Quiz โ€” Test Your Knowledge

How it works: tap an answer for each case. You’ll get instant feedback with the full explanation and the examiner’s trap. Your final score appears once all 10 are answered.

๐Ÿชค Trap Questions โ€” Beat the Examiner

Harder on purpose. Each question targets a real misconception. After answering you’ll see the correct reasoning, the trap explained, why the examiner wrote it, and the wrong answer most students choose.

8 ยท Rapid Revision Sheet

๐Ÿ“– READ THIS 2 HOURS BEFORE YOUR EXAM

Membrane Basics

  • Deformability: shape, elasticity, viscosity
  • Vertical: ankyrin, band 3, 4.2, 4.1 complex
  • Horizontal: spectrin dimerโ†’tetramer, junction
  • Vertical โ†“ โ†’ spherocyte; horizontal โ†“ โ†’ elliptocyte

Hereditary Spherocytosis

  • N. European 1/2000โ€“3000 ยท 75% AD
  • ANK1 > band 3 > ฮฒ-spectrin
  • โ†‘ MCHC 35โ€“38 ยท โ†‘ RDW ยท hyperdense >4%
  • DAT negative ยท splenomegaly ยท gallstones
  • Crises: hemolytic, aplastic (B19), megaloblastic

HS Tests

  • OFT: heparin, 0.85โ†’0% NaCl, 540 nm
  • Normal lysis 0.45% โ†’ complete 0.35โ€“0.30%
  • HS lysis >0.5% ยท incubated 24 h 37 ยฐC
  • EMA โ†“ MFI ยท sens 93โ€“96% ยท also โ†“ HPP, SAO
  • Thalassemia = โ†“ fragility

HE ยท HPP ยท SAO

  • HE: horizontal, SPTA1 65%, 90% asymptomatic
  • Elliptocyte % โ‰  severity
  • HPP: AR, MCV 50โ€“65, lyse 41โ€“45 ยฐC
  • SAO: 27-bp band 3 del, rigid, no Rx

Stomatocytosis

  • OHS: RHAG, MCV 110โ€“150, MCHC โ†“, OF โ†‘
  • DHS/HX: PIEZO1, MCHC โ†‘, OF โ†“, puddled Hb
  • Pseudohyperkalemia
  • NO splenectomy (thrombosis)
  • Artifact stomatocytes normal 3โ€“5%

Acanthocytosis

  • Abetalipo: AR, MTP, 50โ€“90%, RP, ataxia
  • McLeod: X-linked, no Kx, โ†“ Kell
  • Chorea-acanthocytosis: VPS13A
  • Spur cell anemia: liver disease, cholesterol

PNH

  • Acquired PIGA โ†’ no GPI anchor
  • No CD55 (DAF) / CD59 (MIRL)
  • Types I, II, III ยท I+III most common
  • Hemolysis, thrombosis (Budd-Chiari), marrow failure
  • DAT negative ยท hemosiderinuria

PNH Testing & Rx

  • Flow: granulocytes + monocytes, FLAER
  • RBC CD59 underestimates clone
  • Classic >50% ยท with AA/MDS <30% ยท subclinical <1%
  • Ham/sucrose obsolete
  • Eculizumab anti-C5 + meningococcal vaccine

G6PD Deficiency

  • X-linked ยท ~400 million ยท 10% AA males
  • Class II Mediterranean ยท III Aโˆ’
  • Triggers: infection, drugs, fava, naphthalene
  • Heinz bodies (supravital) โ†’ bite cells
  • Assay 340 nm NADPH ยท retest 2 wkโ€“2 mo

PK & Others

  • PK: AR, PKLR, โ†“ ATP, โ†‘ 2,3-BPG, burr cells
  • PK assay: โ†“ absorbance at 340 nm (NADH)
  • Remove buffy coat (WBC false normal)
  • P5โ€ฒNT-1: coarse basophilic stippling
  • PGK X-linked ยท BPGM def โ†’ erythrocytosis

9 ยท Final Memory Map

  • ๐Ÿงฌ INTRINSIC RBC DEFECTS
    • Membrane Structure
      • Deformability: geometry, elasticity, viscosity
      • Vertical (ankyrin/4.1 complexes) vs horizontal (spectrin)
    • Structural Defects
      • HS โ€” vertical, spherocytes, โ†‘ MCHC, OFT/EMA
      • HE โ€” horizontal, elliptocytes, mostly asymptomatic
      • HPP โ€” MCV 50โ€“65, thermal fragility
      • SAO โ€” rigid ovalocytes, band 3 deletion
    • Transport Defects
      • OHS โ€” โ†‘ MCV, โ†“ MCHC
      • DHS/HX โ€” โ†‘ MCHC, PIEZO1
      • No splenectomy
    • Lipid Defects
      • Abetalipoproteinemia, McLeod, chorea-acanthocytosis
      • Spur cell anemia (acquired)
    • Acquired Intrinsic โ€” PNH
      • PIGA โ†’ no GPI โ†’ no CD55/CD59
      • Hemolysis, thrombosis, marrow failure
      • Flow cytometry/FLAER ยท eculizumab ยท HSCT
    • HMP Shunt โ€” G6PD
      • X-linked, WHO classes Iโ€“V
      • Oxidants โ†’ Heinz bodies โ†’ bite cells
      • False normal after crisis
    • Glycolysis โ€” PK & Others
      • PK: AR, โ†“ ATP, โ†‘ 2,3-BPG, burr cells
      • GPI, hexokinase, TPI, PGK (X-linked)
      • BPGM deficiency โ†’ erythrocytosis
    • Nucleotide Metabolism
      • P5โ€ฒNT-1 โ†’ coarse basophilic stippling



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