HEMA1 - Aplastic Anemia, Bone Marrow Failure and Pancytopenia

Pancytopenia

A Complete Blood Count finding in which all peripheral blood cell lineage counts (WBCs/neutrophils, RBCs and platelets) are below the reference ranges.

Bicytopenia

Decreases in only two cell lines.

Red cell cut-off

Hemoglobin <12 g/dL for non-pregnant women and <13 g/dL for men, or absolute reticulocyte count <40 × 10⁹/L.

Neutropenia

Neutrophils constitute the majority of leukocytes in blood and marrow, so nearly all cases of leukopenia manifest as neutropenia.

Absolute neutrophil count (ANC) cut-off

<1.8 × 10⁹/L.

Platelet cut-off

<150 × 10⁹/L.

Causes of pancytopenia

Peripheral conditions (destruction or sequestration); bone marrow failure (aplastic anemia and non-AA bone marrow causes).

Non-AA bone marrow causes

Bone marrow infiltration/replacement, nutritional causes, bone marrow infection.

Peripheral causes of pancytopenia

Destruction or sequestration: disseminated intravascular coagulation, thrombotic thrombocytopenia purpura, hypersplenism.

Hypersplenism

Peripheral cause of pancytopenia — e.g. from liver cirrhosis, storage diseases, lymphoma, or autoimmune disorders.

Bone marrow infiltration or replacement

Primary malignancies (MDS, AML, lymphoma, multiple myeloma, myeloproliferative neoplasms such as primary myelofibrosis); secondary malignancy (metastatic carcinoma); storage diseases (Gaucher's, Niemann-Pick); bone marrow fibrosis (secondary myelofibrosis).

Nutritional causes of marrow failure

Megaloblastic anemia (vitamin B12 and/or folate); zinc and copper.

Bone marrow infections

Disseminated tuberculosis; fungal infection (e.g. histoplasmosis); sepsis.

Bone marrow failure (BMF)

• Decreased production of one or more major hematopoietic lineages.
• Broad term: pancytopenia from marrow replacement (tumor, fibrosis), myelodysplastic syndromes, and other causes of AA.
• Leads to diminished or absent hematopoietic precursors and peripheral cytopenia(s).

Aplastic anemia (AA)

Pancytopenia with a hypocellular bone marrow in the absence of an abnormal infiltrate or marrow fibrosis.

Loss of hematopoietic stem cells

The defining feature of aplastic anemia; results in a decrease of mature blood cells.

Severe AA

Bone marrow cellularity <25%, plus at least two of:
• ANC <0.5 × 10⁹/L
• Platelets <20 × 10⁹/L
• Reticulocytes <20 × 10⁹/L

Very severe AA

Criteria for SAA and ANC <0.2 × 10⁹/L.

Non-severe AA

Hypocellular bone marrow; ANC <1.8, platelets <150, reticulocytes <40 × 10⁹/L.

Hematopoietic stem cells (HSCs)

Not morphologically identifiable (resemble lymphoid cells); recognized by immunophenotyping — a small population within the CD34+/CD38− fraction of bone marrow cells.

Etiology of aplastic anemia

Acquired (majority) or inherited.

Inherited AA causes

DNA repair defects (Fanconi anemia), telomerase defects (dyskeratosis congenita), ribosomopathies (Shwachman-Diamond syndrome), cMPL mutations (amegakaryocytic thrombocytopenia).

Inherited AA

Less common; usually presents within the first decade of life; immunosuppressive therapy is not helpful for most inherited forms.

Acquired AA

Most cases of AA; mostly immune mediated; 80% respond to immunosuppressive therapy (IST) with anti-thymocyte globulin (ATG) and cyclosporin (CsA).

Indirect HSC injury

Through autoimmune mechanisms (majority): PNH, autoimmune disease, post-transfusion GVH, viruses, drugs (idiosyncratic reactions).

Direct HSC injury

Cytotoxic chemotherapeutic drugs, irradiation, chemicals and viruses.

CD4+ve T cells in AA

Produce IFN-γ and TNF, which inhibit HSCs by inhibiting cell proliferation, inducing Fas-mediated apoptosis, and blocking hematopoietic growth factor intracellular signals.

Cytotoxic lymphocytes in AA

Activity and levels decreased with antithymocyte globulin (ATG) therapy.

Immune-mediated AA

Can be antibody mediated; associated with autoimmune conditions e.g. eosinophilic fasciitis and SLE.

Etiology of acquired AA

Drugs (direct toxicity and idiosyncratic reactions), chemicals, ionizing radiation, viruses, autoimmune conditions including PNH and post-transfusion GVHD.

Dose-dependent drugs

Predictable: cytotoxic drugs, immunosuppressive agents (azathioprine), anti-inflammatory medications (phenylbutazone, gold), certain antibiotics (chloramphenicol).

Idiosyncratic drug reactions

Less predictable (autoimmune).

Chloramphenicol

Certain antibiotics e.g. chloramphenicol — dose-dependent/predictable drug effect in acquired AA.

Alkylating agents

Busulfan, melphalan, cyclophosphamide — regularly produce marrow depression.

Antimetabolites

Antifolic compounds, nucleotide analogs — regularly produce marrow depression.

Antimitotics

Vincristine, vinblastine, colchicine.

Some antibiotics

Daunorubicin, doxorubicin (Adriamycin) — regularly produce marrow depression.

Ionizing radiation

Predictable, dose-dependent bone marrow suppressive effect (direct toxicity); BM hypoplasia with doses higher than 1.5–2 Gy to the whole body.

Toxins

Prolonged exposure to solvents/degreasing agents, industrial chemicals, insecticides (lindane), pesticides.

Benzene

Toxin; less often benzene-containing chemicals: kerosene, carbon tetrachloride, Stoddard solvent, chlorophenols.

Viral infection in AA

• Thought to alter antigens on hematopoietic cells and activate a cytotoxic T cell clone or initiate T cell release of cytokines.
• T cell mediated cytotoxicity of similar target antigens on both liver and bone marrow cells.
• Hepatitis viruses, EBV, HIV.

Posttransfusion GVHD

GVHD by the transfusion of competent lymphocytes into immunodeficient hosts.

PNH

• Acquired mutations of the PIG-A gene; absence of certain proteins (e.g. CD59) on the surface of blood cells.
• Reduced ability to resist destruction by complement.
• Also thought to initiate an immune response that damages HSCs.

Autoimmune conditions with AA

Eosinophilic fasciitis, systemic lupus erythematosus, rheumatoid arthritis, Sjögren syndrome, multiple sclerosis, immune thyroid disease, thymoma or thymic hyperplasia.

Inherited aplastic anemia

• Germline mutations, inherited from parents or arising de novo.
• Symptoms of AA (fatigue, hemorrhage, recurrent bacterial infections) plus extra-marrow features unique to each syndrome.
• Patients can present as adults.

Inherited AA syndromes

Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, congenital amegakaryocytic thrombocytopenia, Blackfan-Diamond anemia, reticular dysgenesis.

Autosomal recessive (majority)

Fanconi anemia, Shwachman-Diamond syndrome, congenital amegakaryocytic thrombocytopenia, reticular dysgenesis.

X-linked recessive (small subset)

Dyskeratosis congenita; 2% of FA.

Autosomal dominant (small subset)

Blackfan-Diamond anemia; the lecture also lists reticular dysgenesis here (reticular dysgenesis is autosomal recessive).

Most common inherited BMF syndrome

Fanconi anemia.

Fanconi anemia

• Mutations in genes coding for DNA damage repair proteins.
• 13 genes make up the FA complex; FANCA is the most common mutation.
• Unrepaired crosslinks → chromosome breaks during replication → aneuploidy.

BRCA2 in Fanconi anemia

A subset of patients has BRCA2 mutations — the lecture lists "(FANCD1, FANCG, FANCJ, FANCP)" — with increased risk of breast and ovarian cancer. (Only FANCD1 is BRCA2; FANCJ is BRIP1, FANCP is SLX4.)

Extra-marrow manifestations of FA

Low birthweight, short stature; microcephaly; renal or cardiac malformations; hypogonadism; absent radii and thumbs; café au lait spots; family history of MDS, AML, SCC at younger ages (younger than 40 years).

Dyskeratosis congenita

• Mutations in genes for proteins and RNAs involved in telomere maintenance (DKC1, TERC, TERT, NOLA2, NOLA3, WRAP53).
• Defects in telomerase → short telomeres → premature hematopoietic stem cell exhaustion and marrow aplasia.

Extra-marrow manifestations of DC

Shares FA features (short stature, low birth weight, hypogonadism, premature MDS/AML and SCC). Distinct: lacy reticulated skin pigmentation with telangiectasias (upper body); maldentition, alopecia, premature graying of hair; dystrophic nails, hyperhidrosis of glabrous skin; pulmonary fibrosis.

Shwachman-Diamond syndrome

Mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene — proteins used in ribosome biogenesis and mitotic spindle function.

Congenital amegakaryocytic thrombocytopenia

• Mutations in MPL (myeloproliferative leukemia virus) gene, coding the thrombopoietin receptor c-MPL.
• Thrombocytopenia from absent or reduced megakaryocytes in the bone marrow.

Extra-marrow manifestation of CAMT

Thrombocytopenia in infancy (bleeding of the skin, gut and mucosal surfaces); this progresses to aplastic anemia in later childhood; increased risk of MDS/AML.

Reticular dysgenesis

• One of the rarest and most severe forms of severe combined immunodeficiency (SCID).
• Mutation in mitochondrial adenylate kinase 2 (AK2).
• Profound leukopenia, especially neutropenia; early onset of infections; decreased T and NK cells with absent to low B cells.

Blackfan-Diamond anemia

• Mutations in RPL and RPS gene families (ribosomal proteins, ribosome biogenesis).
• Presents early in life with isolated erythroid failure — an inherited pure red cell aplasia.
• The lecture also lists "bone marrow failure (predominantly neutropenia)" and "exocrine pancreatic dysfunction" here — Shwachman-Diamond features.

Extra-marrow manifestations of BDA

Low birth weight, bony deformities (triphalangeal thumbs), short stature.

Acquired pure red cell aplasia

Associated with thymoma.

Clinical manifestations of inherited BMF

• Fatigue and pallor (anemia).
• Hemorrhage (thrombocytopenia).
• Recurrent fevers and bacterial infections (neutropenia).
• Extra-marrow features — their absence cannot exclude an inherited syndrome.

Laboratory evaluation of AA

CBC (pancytopenia); peripheral blood smear (including B12 and/or folate deficiency — hypersegmented neutrophils, oval macrocytes); bone marrow biopsy and aspirate (myelodysplastic morphology, clonal cytogenetic abnormalities); cytogenetic/mutational studies.

Dry tap

Common finding in aplastic anemia (DDx: myelofibrosis, metastatic Ca, multiple myeloma); confirmed by bone marrow biopsy.

Aplastic marrow

• Hypocellular with a decrease in all elements; mostly fat cells and stroma (yellow marrow).
• Erythropoiesis, myelopoiesis and megakaryopoiesis all reduced; no abnormal cells, no mitosis.
• Residual cells predominantly lymphocytes, morphologically normal; not megaloblastic.
• No malignant infiltration or fibrosis.

Flow cytometry in AA

Cell surface CD59 on peripheral blood red cells (PNH); detects coexistent myelodysplastic syndrome; rules out acute leukemia.

Conventional karyotype and FISH

In all children, and in adults with characteristic abnormalities (short stature, skeletal abnormalities, skin/nail lesions), to identify inherited genetic abnormalities.

Molecular diagnostics in BMF

PCR and next generation sequencing — screening germline DNA for genes responsible for FA, telomeropathy, and Shwachman-Bodian-Diamond syndrome.

Diepoxybutane (DEB) test

• FA chromosomes exposed to DNA cross-linkage agents (MMC and DEB) show increased breakage and abnormal patterns including radials.
• Baseline breakage recorded; then mitomycin C and DEB added and breakage recorded.
• Breakage with DEB is the most specific tool for FA and is diagnostic; MMC increases detection rate.

DEB test specimen

5–10 mL peripheral blood or bone marrow in sodium heparin (green top).

Fanconi Anemia Panel

For patients with a positive chromosome breakage study: a 22 gene panel using next generation sequencing (NGS) to identify the causative mutation(s).

FANCC IVS4+4 A>T

Mutation-specific analysis for patients of Ashkenazi Jewish descent.

Pancytopenia work-up

• BM biopsy: exclude MDS, leukemia, metastatic cancer; identify marrow hypo/aplasia.
• BM aspirate: cytogenetics/FISH; cytology confirms absence of marrow blasts.
• Peripheral blood: neutrophil count (severity), DEB test (exclude FA), GPI-negative cells, antibody-mediated cytopenias, telomere length.

Acquired AA management

HLA typing to identify HLA-matched family donors; choose treatment: ATG + CSA (androgens/growth factors) or BM transplantation.

Supportive care in AA

Infection prophylaxis/treatment; blood component transfusions; granulocyte colony-stimulating factors and/or erythropoietin.

Leukoreduced red blood cells

For Hb less than 7 mg/dL as printed — the unit should read g/dL (Hb 7 g/dL).

Platelet transfusion

Less than 10,000/microliter, or less than 50,000/microliter for active blood loss.

Growth factors in inherited severe AA

Not recommended — not enough precursor cells to generate satisfactory responses.

Hematopoietic stem cell transplant (HSCT)

The definitive treatment of inherited bone marrow failure syndromes.

Androgen therapy

Sex hormones increase telomerase gene transcription; danazol has been effective in marrow failure due to telomeropathy. Useful androgens: nandrolone decanoate, oxymetholone, danazol.

Androgen response

The hemoglobin response is frequently more impressive than improvements in granulocyte or platelet levels.

Immunosuppressive therapy in inherited AA

May not be required.

Gene therapy

May promise a cure in the future.