Aplastic Anemia and PNH

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Aplastic Anemia and PNH

• Kristen Desranleau
• Anika Bystrom
• Daphne Kwan
• Alaa Rahme

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Patient Information

• 17 year old male
• Fatigue
• Sore throat
• Bleeding gums
• Petechial rash
• Fever

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Labs

• Hb 89 g/L
• RBC 3.0 x 1012/L
• MCV 103 fL
• Plt 18 x 109/L
• WBC 2.2 x 109/L
• Neuts 0.2 x 109/L
• Lymphs 2.3 x 109/L

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Aplastic Anemia

• Characterized by
• Replacement of bone marrow with fat
• Peripheral pancytopenia
• Delayed plasma iron clearance
• Classified as
• Primary (congenital)
• Secondary
• Idiopathic

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Pathophysiology

• Immune mediated
• Oligoclonal expanded Tc destroy autologous stem
  cells by inducing apoptosis
• Upregulated T-bet
• Decreased levels of T regulatory cells

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Shortened Telomeres

• Small proportion of acquired cases share
  pathophysiology with inherited disease
  shortened telomeres

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What is a Telomere?

• TTAGGG tandem repeats located at the ends of
  chromosomes
• Serve to solve the end-replication problem and to
  protect the functional sequences of the
  chromosome

• http//docinthemachine.com/2006/11/28/live-to-be-1
  00-ditm-tells-you-how/

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Telomerase

• Cells with high turnover rates
• RT enzyme that catalytically adds TTAGGG repeats
  to the 3
• Comprised of
• telomerase reverse transcriptase (TERT)
• RNA template (TERC)
• Dyskerin (DKC1)

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Dyskeratosis Congenita

• Mucocutaneous abnormalities and aplastic anemia
• X-linked
• Mutations in DKC1
• Autosomal dominant
• Mutations in TERC
• Autosomal recessive
• Mutations in TERT and NOP10

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Aplastic Anemia and Telomeres

• One third associated with short telomeres
• Mutations in TERT or TERC
• Environmental factors play a role
• Other factors
• Aging, smoking, psychological stress and
  stressed hematopoiesis

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Treatment

• Allogenetic BM transplant from matched sibling
• Most effective and can be curative
• Matched unrelated donor BM transplant
• Not as successful

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Treatment

• Immunosuppressive therapy
• Antithymocyte globulin with Cycolosporin A
• Androgens
• In patients with shortened telomeres
• Supportive care
• RBC, platelet transfusions, cytokines (EPO and
  G-CSF)

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Fanconis Anemia
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Signs Symptoms

• Sx of anemia fatigue, weakness, pallor etc
• BM failure Pancytopenia
• May have congenital malformations microcephaly,
  skin pigmentations, short height, malformed
  thumbs, crossed eyes, mental retardation and
  various others characteristics.
• Developmental problems low birth weight, poor
  appetite etc

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Diagnosis

• Often found in childhood, a complete medical
  history is important, because it is an inherited
  disorder

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Lab Findings

• Peripheral Blood
• Similar to AA, the pancytopenia is seen at 5-10
  years of age.
• HbF is elevated, which results in a decrease in
  HbA
• Osmotic fragility is increased
• Chromosomal instability is common

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• Bone Marrow Aspirate
• Hypocellularity, with a decrease in myeloid and
  erythroid precursors as well as decreased megs.
• Dyserythropoiesis (multinuclearity, ringed
  sideroblasts), dysmyelopoiesis (hyposegmentation),
  and abnormal megakaryocytes.

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Genetic Testing

• Chromosome breakage test diepoxybutane (DEB) or
  mitomycin C (MMC) is added to the sample, this
  increases the number of breaks, gaps and
  rearrangement of the chromosomes in FA
• Imaging
• View the skeletal system to identify
  abnormalities
• Cardiac ultrasonography

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Myelodysplastic Syndrome
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• MDS consists of hyper or hypo cellular BM,
  impaired morphology and maturation, and
  peripheral blood cytopenia.
• Primary MDS Idiopathic
• Secondary caused by chemotherapeutic agents such
  as radiation, alkylating agents or topoisomerase
  2 inhibitors
• The stem cells are damaged then the mutated clone
  fills the BM, suppressing the healthy stem cells.
  
• This condition often develops into AML (like FA)

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Classifications of MDS

• MDS can be classified into different categories
  depending on the cells present in the peripheral
  blood and BM.
• Refractory Anemia
• RA with ringed sideroblasts
• RA with excess blasts
• Chronic myelomonocytic leukemia
• RA with excess blasts in transformation

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Hypoplastic Myelodysplastic syndrome (h-MDS)

• This is a type of MDS seen in adults, it has a
  very hypocellular bone marrow. h-MDS can be seen
  in any of the categories.
• The tendency to progress to Acute leukemia is
  somewhat reduced

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Signs Symptoms

• Sx of anemia fatigue, weakness, pallor etc
• Fever, cough, increased infections
• Petechiae, nose and gum bleeding, decreased
  number of functional platelets.

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Diagnosis

• This is a disease seen more often in adulthood
• Lab Findings
• Peripheral Blood
• h-MDS patients often have a lower Hb level, and a
  more severe leucopenia. Than normo or
  hypercellular MDS
• Chromosomal analysis is often normal

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Lab Findings

• Bone Marrow Aspirate
• The marrow is significantly hypocellular, and
  often has signs of dysplasia. It looks very
  similar to aplastic anemia

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What is PNH??Paroxysmal Nocturnal Hemoglobinuria

• Clinically manifests as RBC breakdown with
  release of Hb into the urine.
• Once believed that hemolysis is triggered by
  acidosis during sleep, activating complement.
• Now been shown that hemolysis occurs throughout
  the day.
• Diminished blood cell production with a
  hypoplastic bone marrow ? Deficient
  hematopoiesis.
• PNH patients have a 10-20 chance of developing
  Aplastic Anemia, and 5 of AA patients eventually
  develop PNH.

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Symptoms of PNH

• PNH syndrome clinically presents 3 types of
  symptoms
• Acquired Intravascular Hemolytic Anemia due to
  abnormal susceptability of RBC membrane to
  complement activity.
• Thrombosis in large vessals such as the hepatic,
  abdominal, cerebral, and/or subdermal veins.
• A deficiency in hematopoeisis which can be mild
  or severe, such as pancytopenia in Aplastic
  Anemia.
• Physical symptoms of PNH include
• Pallor - Anemia
• Fever - Infection
• Bleedingskin ecchymoses in thrombocytopenia.
• Specific symptoms involved with vein thrombosis.

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Laboratory Studies

• Flow cytometry to detect CD59, and CD55 in
  regulation of complement action ? Gold Standard.
  
• Bacterial toxin Aerolysin binds to RBCs via GPI
  anchor and initiates hemolysis. important to
  scan both RBCs and granulocytes.
• Historical tests
• Acidified serum lysis Ham test Demonstrates
  that RBCs in PNH are lysed by complement when
  serum is acidified or activated by
  alloantibodies.
• Complement lysis sensivity test RBCs are
  sensitized with a lytic ant-i and hemolyzed.
• Sucrose lysis test Uses the ionic strength of
  serum, activating the classic pathway, and
  complement-sensitive cells are lysed.
• Other tests run for assessement of disease
  affects
• LDH Serum Haptoglobin CBC count
• Retic count Hemoglobinuria BM examination

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What actually causes PNH??

• Clonal expansion of a mutated early stage,
  multipotent hematopoeitic stem cell
• Mutation in the X-linked gene known as PIG-A ?
  GPI Anchor Protein (GPI-AP) deficiency

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What is PIG-A??

• Single copied gene on the short arm of the X
  chromosome
• No mutual mutational hot spots.
• Most are small insertions or deletions resulting
  in a frame shift in the coding region ?
  shortened, nonfunctional product.
• Involvement from all lineages myeloid, eryroid,
  and lymphoid.
• Both B and T cells derive from the mutated stem
  cell.
• Not exclusive to PNH and can be found in the
  blood of any healthy individual.
• - Mutations in healthy individuals are not
  significant enough to cause PNH.
• -PIG-A mutations do not occur in a multipotent
  hematopoietic stem cell ? occur during
  differentiation.

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PNH Association with Aplastic Anemia

• PNH patients have a 10-20 chance of developing
  Aplastic Anemia, and 5 of AA patients eventually
  develop PNH.
• In upto 70 of patients with acquired Aplastic
  Anemia, mutated PNH clones are found in their
  blood ? Pathophysiological link between the two
  disorders.
• Mechanisms of Clonal Expansion
• There are three proposed models to explain
  clonal expansion of the PNH stem cell
• Model 1 PNH cells evade immune attack, possibly
  because a missing cell surface GPI-AP which can
  be the target of the attack or the absence of a
  stress-inducing cell surface protein, allowing
  PNH cells to preferentially survive and immune
  attack
• Model 2 PIG-A mutation is intrinsically
  resistant to Apoptosis.
• Model 3 A second anti-apoptotic mutation occurs
  in PNH clones.

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Pure Red Cell Aplasia (PRCA)

• type of anemia affecting red cell precursors
• decreased red cell count

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PRCA versus AA

• Complete blood cell count and differential
• Low red cell and reticulocyte counts
• Normal platelet and WBC counts
• Bone marrow
• Lack of mature erythroid precursors
• Normal numbers of megakaryocyte and WBC
  precursors
• May see giant pronormoblasts

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Etiological Agents of PRCA

• Congenital
• Diamond-Blackfan anemia
• Several genetic defects affecting erythropoietic
  lineage
• May be more than one pathogenic mechanism for
  erythroid failure

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Etiology Cont

• Acquired
• Most acquired PRCA cases are caused by
  autoimmune-mediated suppression of erythropoiesis
• IgG Abs against erythroblasts
• Inhibit Hb production or RBC destruction
• Abs against erythropoietin
• Inactivation and removal from circulation
• T-cells and NK cells

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Etiology Cont

• PRCA has been associated with other disorders
• Neoplastic disorders ? lymphomas, CLL, thmoma
• Autoimmune disorders ? SLE, rheumatoid arthritis
• Drugs and toxins
• Pregnancy
• Allogeneic bone marrow or stem cell transplants
  between ABO-incompatible individuals

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Parvovirus B19

• Acquired PRCA can also be induced by chronic
  parvovirus B19 infection
• Targets erythroid progenitor cells through P
  blood group antigens
• Replicates in and lyses erythroid progenitor
  cells
• Immunocompetent individuals
• Transient aplastic crisis or fifth disease
• Self-limiting, neutralizing Abs
• Immunocompromised individuals
• Recurrent or chronic infection
• Weak Ab response, non-neutralizing Abs

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Treatment - IVIG

• IVIG is often used to treat parvovirus
  B19-induced PRCA
• Contains significant amount of anti-B19
• Used to neutralize the virus
• May require repeat administration
• Side effects
• Allergic reactions
• Fever, chills, headache, myalgia, hypertension,
  chest pain
• Acute renal failure

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Back to the case Labs

• Hb 89 g/L
• RBC 3.0 x 1012/L
• MCV 103 fL
• Plt 18 x 109/L
• WBC 2.2 x 109/L
• Neuts 0.2 x 109/L
• Lymphs 2.3 x 109/L

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Patient Information

• 17 year old male
• Fatigue
• Sore throat
• Bleeding gums
• Petechial rash
• Fever

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Differential Diagnosis Aplastic Anemia h-MDS

• Age of the patient
• Family History
• Immunostaining may be used to differentiate h-MDS
  from AA.
• CD34 cells are normal h-MDS and decreased or
  absent in AA.

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Aplastic Anemia Fanconis Anemia

• Family History
• Chromosome analysis

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PNH and Aplastic Anemia

• Although there are some simlarites between PNH
  and AA, the treatment for these diseases are
  differenet.
• In order to rule out PNH in this patients
  diagnosis, specialty tests such as flow cytometry
  should be run.

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Pure red cell aplasia

• PRCA is not indicated because of the low
  platelets and WBC counts.
• A bone marrow aspirate is needed for
  confirmation.

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References

• Rodak BF. Hematology Clinical principles and
  applications. 2nd ed. Philadelphia (PA)
  Saunders 1995.
• Young NS, Scheinberg P, Calado RT. Aplastic
  anemia. Curr Opin Hematol. 2008 May15(3)162-8.
• Calado RT. Young NS. Telomere maintenance and
  human bone marrow failure. Blood. 2008 May
  1111(9)4446-55.

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References

• Fisch P, Handgretinger R, Schaefer HE. Pure red
  cell aplasia. Br J Haematol 2000
  Dec111(4)1010-22.
• Mouthon L, Guillevin L, Tellier Z. Intravenous
  immunoglobulins in autoimmune- or parvovirus
  B19-mediated pure red cell aplasia. Autoimmun Rev
  2005 Jun4(5)264-9.
• Geetha D, Zachary JB, Baldado HM, Kronz JD, Kraus
  ES. Pure red cell aplasia caused by Parvovirus
  B19 infection in solid organ transplant
  recipients a case report and review of
  literature. Clin Transplant 2000
  Dec14(6)586-91.
• http//emedicine.medscope.com/article/207468-overv
  iew.