1. Hematology 425 Increased RBC Destruction Intracorpuscular Defects
Russ Morrison
November 3, 2006
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2. Intracorpuscular Defects – Hereditary Disorders of Cation Permeability and Volume
RBC hydration is determined by intracellular concentration of Na+ and K+
If the cation content is increased, water enters the cell and forms a hydrocyte, or stomatocyte
If cation content is decreased water leaves the cell and produces a dehydrated RBC or xerocyte
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3. Hereditary Stomatocytosis (Hydrocytosis)
A complex mixture of hemolytic anemias in which hemolysis may be mild to severe
Hydrocytosis is rare and usually inherited in an autosomal dominant pattern
It is characterized morphologically by stomatocytes and biochemically by the failure of the Na+ and K+ pumps and increased intracellular water
In most patients a deficiency of a membrane integral protein called stomatin occurs
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4. Stomatocytes
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5. Hereditary Stomatocytosis (Hydrocytosis)
Clinical and laboratory findings
Mild, moderate or severe hemolysis
5 to 50% stomatocytes on the PB smear
Macrocytes may be present
Reduced K+ and increased Na+ in the RBCs
Increased RBC osmotic fragility
Patients with anemia may benefit from splenectomy
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6. Acquired Stomatocytosis
Seen frequently on PB smears as a drying artifact
Acute alcoholism, some drug therapy and sometimes, marathon runners show stomatocytosis on PB smears
Rhnull patients may exhibit spherocytes and stomatocytes and osmotic fragility may be increased
It is speculated that Rh antigens associated with the RBC membrane, when lost, affects membrane skeletal stability
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7. Hereditary Xerocytosis
A rare autosomal dominant hemolytic anemia
RBCs are dehydrated, showing increased MCH and very decreased osmotic fragility
Disease mechanism is not known
RBCs demonstrate stomatocytes, target cells, spiculated RBCs and macrocytes
2,3-BPG levels is moderately decreased
Splenectomy does not reduce the hemolysis and level of anemia in these patients
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8. Acanthocytosis (Spur Cells)
A distinct form of echinocyte (burr cell) in which the cellular projections vary in width, length and surface distribution
Found in sever liver disease, abetalipoproteinemia, infantile pyknocytosis and anorexia nervosa
May be associated with blood groups and hypothyroidism
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9. Acanthocytosis (Spur Cells)
Abetalipoproteinemia is a rare autosomal recessive disorder
Associated with the disorder is retinitis pigmentosa, that often results in blindness
Disease usually progresses to death by the time a patient reaches 20s or 30s
50-100% of RBCs will be acanthocytes
Affected persons have mild hemolytic anemia and normal RBC indices
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10. Acanthocytosis (Spur Cells) – Scanning EM
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11. Acanthocytosis (Spur Cells)
RBCs have normal membrane protein composition but the membrane lipids are abnormal
Abnormal lipids cause a decrease in the lipid fluidity of the RBC membrane and result in the shape change
Shape change is not present in developing NRBCs or reticulocytes, but becomes evident as the RBCs age
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12. Acanthocytosis (Spur Cells)
Other causes of acanthocytosis
Blood group association with McLeod phenotype and In(Lu) gene presence in the Lutheran blood group system
Malnutrition as a result of anorexia nervosa and cystic fibrosis
Vitamin E deficient patients may demonstrate acanthocytes on their blood smear
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13. RBC Enzymopathies HAs may also be the result of RBC enzyme problems
Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency is one of the most common RBC enzyme related causes of HA
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14. G6PD Deficiency
The gene for G6PD is located on the X chromosome and shows characteristics of X-linked inheritance
Mutations of the associated genes, when inherited, cause G6PD deficiency either by decreasing its in vivo stability or by affecting its enzymatic functions, or maybe both
Different genetic expressions of G6PD have been divided into classes by the WHO based on clinical symptoms and amount of enzyme activity
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15. G6PD Deficiency WHO Classification of G6PD Deficiency
Class I – severe clinical symptoms of chronic, nonspherocytic HA and less than 20% G6PD activity
Class II – mild clinical expression with intermittent hemolysis and less than 10% activity
Class III – mild clinical expression with intermittent hemolysis associated with infection or drugs and 10% to 60% G6PD activity
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16. G6PD Deficiency Class IV – no clinical expression with 100% activity
Class V – no clinical expression and more than 100% activity
Within the group of syndromes, over 400 different genetic mutations and enzymes may exist which demonstrate different electrophoretic patterns
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17. G6PD Deficiency Normal G6PD has been designated G6PD-B
G6PD variants may be designated with a letter, G6PD-A or by geography, G6PD-Mediterranean
G6PD-A is the most common mutation in blacks while G6PD-Med is the most common in whites
Incidence of G6PD-Med among Kurdish Jews ranges from 3% to 50%
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18. G6PD Deficiency
Most individuals are asymptomatic except during oxidative stress resulting from drugs, foods (fava beans) or other causes
Heinz bodies typically appear along with membrane skeletal structural abnormalities
Substances that can trigger hemolysis in patients with G6PD deficiency are listed in box 21-2 on page 277 of the text
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19. G6PD Deficiency Clinical and Laboratory Findings
Most G6PD deficient persons are asymptomatic and never discover their deficiency
Clinical manifestation may only be acute hemolysis during oxidative stress resulting from ingestion of drugs or foods
Anemia during crisis may range from moderate to severe
Gives a normochromic normocytic picture
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20. G6PD Deficiency Clinical and Laboratory Findings
During crisis Heinz bodies (denatured Hgb) develop in the RBCs which may be seen using a supravital stain
Reticulocyte count may be as high as 30%
Haptoglobin level is decreased
Free Hgb may be detected in the plasma
WBC count is usually elevated
Platelet count is variable
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21. G6PD Deficiency Clinical and Laboratory Findings
Unconjugated bilirubin level is elevated
Darkly colored urine tests positive for blood
Intact RBCs do not appear in the urine sediment because the color and blood reaction result from hemoglobinuria and hematuria
G6PD levels may be assayed
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22. G6PD Deficiency
G6PD enzyme levels may be screened using UV light to detect fluorescence caused by the production of NADPH by G6PD
NADP does not flouresce while NADPH does
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23. G6PD Deficiency Therapy and Prognosis
The main treatment for G6PD deficiency is avoidance of oxidative stressors. Rarely, anemia may be severe enough to warrant a blood transfusion. Splenectomy generally is not recommended. Folic acid and iron potentially are useful in hemolysis, although G6PD deficiency usually is asymptomatic and the associated hemolysis usually is short-lived. Antioxidants such as vitamin E and selenium have no proven benefit for the treatment of G6PD deficiency.
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24. Symptoms and Laboratory Evaluation in Patients with G6PD and Acute Hemolysis
Findings in patients with G6PD deficiency and associated acute hemolysis
Back pain
CBC
Mild to severe anemia
Abdominal pain
Retic.count
Increases four to seven days after hemolysis
Jaundice
PB smear
Heinz bodies
Transient splenomegaly
Haptoglobin
Decreased
Hemoglobinuria
Liver function
Elevated indirect bilirubin
Scleral icterus
Coombs'
Negative
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25. Medications to avoid with G6PD
Incomplete Listing
Acetanilid
Doxorubicin
Furazolidone
Methylene blue
Nalidixic acid
Niridazole
Nitrofurantoin
Phenazopyridine
Primaquine
Sulfamethoxazone
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26. Enzymopathies of the Glycolytic Pathway
As a reminder, the mature RBC lacks a nucleus, mitochondria and other organelles and is unable to synthesize proteins and lipids or to perform oxidative phosphorylation
Energy requirements of the mature RBC are met by generation of ATP through glycolysis
Effective glycolysis is essential for the survival of the RBC
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27. Enzymopathies of the Glycolytic Pathway
Enzyme deficiencies or abnormalities that diminish the effectiveness of the glycolytic pathway result in hemolysis or other RBC abnormalities
The most common of these disorders is Pyruvate Kinase (PK) deficiency
Seven other defects of enzymes of the E-M pathway are have been described and are listed in Table 21-4
Our discussion will be directed toward PK Deficiency
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28. Pyruvate Kinase Deficiency
PK deficiency accounts for 90% of the enzyme defects of the glycotic pathway
Inherited as an autosomal recessive trait
HA occurs in homozygotes; heterozygotes are not anemic although their RBCs may show some enzyme alterations
Most common in people of Northern European ancestry, but reported world wide
High prevalence in the Amish of Mifflin County, Pennsylvania (traced to 1 immigrant couple)
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29. Pyruvate Kinase Deficiency
Mechanism for hemolysis in PK-deficient RBCs is not known
Alterations in PK-deficient RBCS are thought to result in a rigid cell that is removed from the circulation by the macrophages of the spleen and liver
Anemia may be severe as in neonates, requiring exchange transfusions or fully compensated in apparently healthy adults
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30. Pyruvate Kinase Deficiency
No specific therapy is available for PK deficiency except supportive treatment and RBC transfusions, as necessary
Splenectomy does not totally correct the hemolysis, but may raise the Hgb level 1 to 3 g/dL, enough to avoid transfusion
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31. Paroxysmal Nocturnal Hemoglobinuria (PNH)
Discovered in the late 1800s when a patient was described who had blood in the urine after sleeping
PNH is a hemolytic anemia, but also a myeloproliferative clonal disorder of the bone marrow
PNH results from a mutation in a hematopoietic stem cell
PNH exhibits an intravascular HA resulting from increased susceptibility of the RBCs to complement
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32. PNH
The RBC membrane defect present in PNH is also present in the platelets and granulocytes and maybe the lymphocytes
PNH is the only acquired hemolytic disorder caused by an abnormality of the RBC membrane
PNH results from a clonal somatic mutation of a gene on the X chromosome designated phsphatidylinositol glycan, class A (PIGA)
The mutation occurs at the pluripotential stem cell level
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33. PNH
In most PNH patients, this abnormal clone of cells coexists with normal hemopoiesis resulting in a dual population of blood cells
Some PNH patients have over 95% cells with the characteristic abnormality of the absence of the GPI-linked proteins on the cell surfaces
This abnormal cell line appears to originate from a damaged BM
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34. PNH
Many PNH patients have a prior history of aplastic anemia or pancytopenia that has been induced by drugs or for which the cause is unknown (idiopathic)
The main defect in PNH is an acquired membrane abnormality that causes increased susceptibility of blood cells to complement
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35. PNH
The complement connection was made when two complement regulatory proteins, CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) were found to be missing from blood cells in PNH
Because of the lack of these proteins, randomly deposited complement factors and C3 convertase complexes are not removed from the RBC membrane, leading to the formation of the membrane attack complex, pore formation, and RBC lysis
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36. PNH
CD55, CD59 and other proteins use a GPI anchor for attachment to the RBC membrane
The entire GPI-linked polypeptide is extracellular
All of the GPI-linked surface proteins that occur normally on blood cells are absent from PNH cells because of the incomplete bioassembly of the GPI anchors
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37. PNH
A mutation in the gene designated as PIGA has been identified as the gene defect in PNH
The PIGA gene resides on the X chromosome
PNH RBCs are classified into subpopulations according to their susceptibility to complement-mediated lysis
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38. PNH
Flow cytometry is used today to analyze surface expression of the GPI-linked proteins (CD16, CD48, CD55, CD59)
RBCs that are totally deficient in GPI-linked surface proteins are the PNH type III cells
Type I cells have normal amounts of the surface proteins and are normal in their response to complement
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39. PNH
The percentage of PNH-III cells determines the intensity of the clinical symptoms
Patients with less than 20% PNH-III cells have mild hemolysis
20-50% PNH-III cells will show episodic hemolysis (sleep induced)
Patients with more than 50% PNY-III cells will exhibit perpetual hemoglobinemia and hemoglobinuria
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40. PNH – Clinical Findings
Up to 30% of PNH patients may have sever aplastic anemia for several years before diagnosis
PNH has been associated with middle age or young adulthood, but can occur at any age
PNH affects both sexes equally
Passage of reddish urine on arising from sleep (hence the name) only occurs in 25% of patients
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41. PNH Some nocturnal hemoglobinuria occurs in most patients
Hemolytic episodes may be triggered by
Lower blood pH during sleep (facilitates complement binding on PNH cells)
Infections
Vaccinations
Transfusions
X-ray contrast dye exposure
Strenuous exercise
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42. PNH Other findings Hemoglobinuria Hemosiderinuria
Iron deficiency anemia due to heavy loss of iron in the urine
Infections are common due to impaired neutrophil function
Predisposition to intravascular thrombosis (brain - headaches, portal vein – abdominal pain)
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43. PNH
PNH can evolve into acute nonlymph-ocytic leukemia at an incidence of 1-3%, far greater than seen in the general population
The leukemic transformation typically occurs within the first 5 years of the disease
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44. PNH – Routine Hematologic Findings
Pancytopenia with a mild to sever anemia, leukopenia and thrombocytopenia
Hgb ranges from below 6 g/dL to normal
Reticulocyte levels are mildly to moderately elevated
Anisocytosis and poikilocytosis is absent
Osmotic fragility is normal
DAT is negative
Moderate neutropenia is present leading to infections which cause 5%-10% of the fatalities in PNH patients
Platelet count is decreased
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45. PNH – Routine Hematologic Findings
BM shows normal cellularity with RBC hyperplasia
Iron may be absent from the BM when anemia is severe
Hemosiderinuria is a constant feature of PNH and is diagnostically important
Renal problems may develop in PNH patients due to deposition of iron in the kidneys
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46. PNH – Special Tests
Suspect PNH in any patient with idiopathic pancytopenia and acquired nonspherocytic anemia accompanied by reticulocytosis
PNH screening tests include urine hemosiderin determination and the sucrose hemolysis test
If the sucrose hemolysis test is positive, the Ham test (acidified serum lysis test) was used to confirm the diagnosis
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47. PNH – Special Tests
PB RBCs and granulocytes are now analyzed by flow cytometry for surface expression of the GPI-linked proteins (CD16, CD48, CD55, CD59)
Figure in the txt shows immunophenotyping of PNH cells using flow cytometry
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48. PNH – Therapy & Prognosis
Treatment of PNH is supportive with transfusions, antibiotics and anticoagulants
Iron therapy and folate may help alleviate further development of anemias secondary to the PNH
Steroids may decrease hemolysis
Androgenic steroids may be effective in cases with prominent marrow hypoplasia
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49. PNH – Therapy & Prognosis
Anticoagulants lessen thrombotic complications
In severe cases, where suitable donors are available, bone marrow transplantation may be curative
Clinical course of PNH varies widely
Occasionally a patient dies with the first few months of diagnosis
Most patients experience a chronic course with severity changing based on the % normal cells and PNH cells present
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50. PNH – Therapy & Prognosis
PNH is a very serious disease and most patients die from its complications (thrombotic episodes, infections)
Acute leukemia may be the terminal event experienced by the PNH patient
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