Patozone

Bone marrow basics

 

"Liquid" organ without evaluable gross anatomy [1]

- dynamic relationship with lymphoid organs and PB

- different methods of morphologic evaluation

-- trephine biopsy and aspirate smears

- diagnosis often requires ancillary studies

 

BM evaluation requires cytology, histology, IHC, Flow cytometry, genetics (cytogenetics, FISH,, molecular) clinical information

 

BM function: site of production of hematopoietic cells

-- Myeloid cells: RBCs, platelets, neuts, eos, basos, monos, dendritic cells, mast cells

--- Lymphoid cells: T-, B- and NK

 

BM structure

- hematopoietic marrow

-- semi-solid organ in intertrabecular spaces of axial skeletal bones

-- non-hematopoietic marrow: fat,

- potential hematopoietic organs: spleen > liver ? LNs > soft tissue and other sites

 

Components of hematopoietic BM

- hematopoietic elements: stem cells, maturing myeloid, erythroid, megakaryocytic, and lymphoid elements

- stromal cells: fat cells, fibroblast-like, dendritic, macrophage

- vascular sinuses

 

Components of complete marrow evaluation:

- BMBx

- BM aspirate (clot prepared from BM aspirate)

- PBS

- Clinical info

- Ancillary studies

 

Clinical info is essential

- age and gender

- CBC and differential

- medication hx

- previous diagnoses

- lymphadenopathy and splenomegaly

- serology

- infections, esp HIV and hepatitis

- anemia-related studies (iron status, B12, folate)

 

BM aspirate

- aspirated liquid containing tiny fragments of fractured trabecular bone and fat particles (spicules)

- used to make air-dried smears

- Wright-Giemsa, iron stain, FISH and NGS

 

Dry tap scenario

- no or poor aspirate obtained either due to BM fibrosis, packed marrow, or poor technique

- touch imprint preps of fresh marrow core (WG-stain and FISH)

- BM core can be pureed or teased apart with needle to release single cells (cytogenetics or flow cytometry)

 

How to approach aspirate smears

- review and count in excellent areas of smears

- obtain count from several different areas/slides

- routine cases: 200 cell counts

- 500 cell counts in Any MDS, borderline AML, borderline Myeloma

 

Bone marrow biopsy technical aspects

- should be taken perpendicular from cortical bone surface

- formalin-based fixation: heavy metals improve nuclear features (B5, B+, Bouins AZF)

- decalcification:

- strong acids

- buffered acids

- EDTA

- paraffin embedding

- thin sections (2 um)

 

Marrow composition

- cellular elements (~50% of space)

-- hematopoietic cells at carious stages of maturation admixed with storomal cells

- adipocytes (~50% of space)

 

Cellularity (%) = (Area with cellular elements) / (Areas of cellular elements + adipocytes)

- avoid subcortical fatty marrow, crushed or hemorrhagic areas, vascular spaces, empty spaces (air bubbles)

 

Thought experiment [1]: Marrow cellularity and differential diagnosis

- Hypercellular + high blood counts

--- leukemic hematologic neoplasm

--- appropriate marrow reaction (eg infection)

 

Hypocellular + low blood counts

--- BM failure (eg aplastic anemia)

--- exogenous marrow suppression

 

Hypercellular + low blood counts

--- ineffective hematopoiesis

--- PB cell destruction

 

Hypocellular + normal counts

--- sampling artifact

 

 

 

Medium-power (20x) architecture: marrow microarchitecture

- erythroid and myeloid form clusters

- M:E ratio = 2:1 - 4:1

- immature myeloid elements adjacent to bone trabeculae, granulocytes away from bone

- megakaryocytes do not cluster

 

40x evaluation: detailed morphology

- meg number and morphology (normally 2-4 per 40x field)

- myeloid maturation (nuclear segmentation, abundant pink cytoplasm)

- erythroid maturation

 

Bone marrow stroma

Fibroblasts/reticular cells

Mesenchymal stromal cells (MSCs) - a part of the hematopoietic stem cell niche

-Support self-renewing proliferation of CD34 positive hematopoetic progenitors - hematopoietic stem cell "niche" component

 

Bone marrow collagens Reticulin fibrosis – type 3 (branching) collagen, reticulin stain,

- Internal control: perivascular Collagen fibrosis

- type 1 (nonbranching) collagen, trichrome stain

- internal positive control: osteoid seam

Aplastic Anemia

 

1-2 new cases / year / 1 million people (rare)

No gender or race differences in the USA

Marked geographic variability

>1 case in a family rare

Presentation related to pancytopenia:

- bleeding (thrombocytopenia)

- fatigue, other nonspecific symptoms (anemia)

- infection uncommon at presentation

- bimodal age distribution, one peak in the 20s, and one in the 60s (usually due to MDS)

 

Divided into:

1. Idiopathic aplastic anemia

- accounts for 70-80% of AA

- decreased numbers of stem cells

- impaired responsiveness to growth factors

- in most cases, damage is immune-mediated

 

2. Secondary aplastic anemia

- Radiation, Drugs and chemicals (cytotoxic agents, benzene), idiosyncratic reactions (chloramphenicol, NSAIDS, antiepileptics, gold, others), Viruses (EBV, hepatitis [non A through G], Parvovirus [rare], HIV [rare]), immuno diseases (eosinophilic fasciitis, hypogammaglobulinemia, thymic neoplasia), pregnancy

 

Micro:

- PB findings: pancytopenia with normal RBC morphology, macrocytosis may be present, reticulocyte index is low

- BM findings: hypocellular bone marrow, dyspoiesis, if any, is only erythroid (dysplastic neutrophils or megakaryocytes strongly suggests hypocellular MDS rather than AA)

 

Clonal abnormalities in AA

- clonal cytogenetic abnormalities in 12% of cases

- usually small, may be transient

- monosomy 7 has higher risk of morphologic MDS or AML

 

Using NGS panels, mutations indicating clonal hematopoiesis reported in 70-85% of AA

- in adults, approaches 100% with WES (whole exome sequencing) or WGS (whole genome seq)

 

Grading [3]:

Severe AA

- marrow cellularity < 30%

- At least two of:

-- ANC <500/mm3

-- Platelets <20,000/mm3

-- Reticulocytes <1% (<40,000/mm3)

-- No other hematologic disease

 

Moderate AA

- Pancytopenia

- Do not fulfill severe criteria

 

DDx:

- inherited pancytopenia syndromes (FA, DKC, esp in younger pts)

- hypoplastic MDS / AML (beware of dysplastic changes, carefully correlated with cytogenetics and FISH) - blast count is critical!!!

- lymphoma (consider doing IHC or flow (esp HCL)

 

Tx:

Bone marrow Transplantation

- immunosuppression - ATG, CsA, steroids, other

- growth factors

 

Px: natural history: moderate disease may recover spontaneously, severe disease fatal in days to months

- recent 5-year survival: 75% with immnuosuppresion or immnuotherapy

 

Proportion of successfully treated pts will relapse

 

Long-term AA survivors at risk for PNH, MDS, and AML

Paroxysmal Nocturnal Hemoglobinuria (PNH)

 

First described in 1866, William Gull, in a patient with dark urine noted only in the morning

 

Acquired clonal hematopoietic disease

- characterized by a defect in GPI glycolipid anchor

- required for the localization of many proteins to the plasma membrane

 

Median age: 40 years, M:F 1:1, 1 in 100,000

 

Patients present with intravascular hemolytic anemia (anemia with normal RBC morphology, reticulocytes may be increased, decreased haptoglobin, hemoglobinuria)

- thrombosis (20% of cases in the US)

- Hepatic vein (Budd-Chiari, cerebral vein, abdominal vein)

- Bone marrow failure

 

PNH and AA

1/3 of PNH pts will present previously with diagnosis of AA

1/3 of PNH patients will eventually develop AA

1/3 remain hemolytic

 

Pathogenesis

Somatic mutations in PIG-A gene at Xp22.1

- enzyme required for synthesis of GPI moiety

- >100 mutations identified

- leads to absence of GPI anchored proteins

 

Normal: very small % of PIG-A mutated cells

PNH: selective expansion of PIG-A mutated clone

 

- Lack of CD55 and CD59 leads to lysis of RBCs and activation of platelets

- PNH type cells also show decreased colony formation in vitro

 

GPI- Anchored Proteins

Complement: DAF (CD55), MIRL (CD59), C8-binding protein

Immune-related: CD16 (FcgR IIIa), CD58 (LFA-3), CD14, CDw52 (Campth)

 

Enzymes: acetylcholinesterase, Leukocyte Alk Phos.

 

Receptors: Urokinase receptor, Folate receptor

 

Other: CD48, CD66

 

Labs: (was initially diagnosed only on clinical grounds)

- 1930s: Hemolysis, thrombosis or pancytopenia with positive Ham's test (enhanced lysis of RBCs in acidified serum)

- 1990's: Hemolysis,thrombosis, or pancytopenia with "PNH-type" cells detected by flow cytometry

 

Flow analysis: Looks for loss of GPI anchored proteins

- Guidelines: use >1 antibody

- examine both RBC and WBC (WBCs may be less prone to spontaneous lysis, which is helpful to monitor the proportion of abnormal cells)

- can use to monitor patient therapy

 

Micro:

BM: cellularity variable from markedly decreased (AA-PNH) to hypercellular

- Dyspoietic changes may be present

-- be careful!! Hypocellular MDS should be considered

 

Cytogenetics:

Typically normal, but abnormal cases described (possibly MDS?)

 

Tx:

Complex - varies c clinical presentation

Immunosuppression - May improve cytopenias – thrombosis management (much of the mortality is due to fatal clots)

-- New! Eculizumab (Solaris) is effective anti-C5 complement inhibitor (very expensive) - WORLDS MOST EXPENSIVE DRUG!!!

- Bone marrow transplant may be considered, usually only in severe cases

 

Px:

Median survival 10 to 15 years - With flow cytometry now diagnosed much earlier - Low risk of transformation to acute leukemia (less than 3%)

Fanconi anemia 1927 - Falconi reports three brothers with fatal aplastic anemia and congenital abnormalities

- pancytopenia, hyperpigmentation, skeletal defects, small stature, hypogonadism

 

Hematologic findings Essentially all patients will develop hematologic abnormalities - At birth, but may be normal, or show only macrocytosis - Pancytopenia develops usually between 5 to 10 years (median age 7) - May be present in adults -as late as 56 years!

 

Lab tests: Defined by sensitivity of chromosomes to certain damaging agents Diagnosis established by: diepoxybutane (DEB), mitomycin C (MMC)

 

Micro: Hypocellular marrow, resembling AA - May show display dyspoietic changes - Small, often transient clonal side of genetic abnormalities recorded - May present with MDS or AML

 

Genetics: Very heterogeneous - 21 genes identified to date - Almost always autosomal dominant - rare XLR cases (2%)

- BRCA2 is one of the genes implicated in Fanconi anemia (aka FANCD1)

 

PX: Increased cancer risk, at risk for development of MDS, AML (52% by 40 years) - Risk greater than 5000 times over general population - Increased risk for a solid malignancies (squamous cell carcinoma, hepatic tumors)

 

Median cancer free survival: 29 years - Bone marrow transplant is curative for hematologic abnormalities, but increased cancer risk remains - Growth factor, androgen therapy

Hematogone hyperplasia

 

Seen post chemotherapy, post stem cell transplant, immune thrombocytopenic Purpera, Copper deficiency, viral infections, metastatic malignancy

 

Flow immunophenotype: Dim variable city 45 Very low to low side scatter CD10+, CD19 +

- Partial dim CD20 - Partial CD34 and TdT

Promyelocyte hyperplasia Response to exogenous and indigenous GCSF Danger of misdiagnosis of AML

Promyelocyte Bulge - Sepsis or posty G-CSF - Versus APL: usually some maturation,lack of folded / bilobed nuclei

--- peripheral blood: toxic granulation,Dohle bodies

Megakaryocyte Hyperplasia

 

In setting of increased megakaryocyte destruction or turnover (ITP or post chemotherapy) - May show mild clustering

Plasma cell hyperplasia

 

Reactive plasmacytosis can significantly exceed 10% Morphology can be mildly atypical (binucleation,enlargement) - Etiologies: infection,autoimmune,other

Causes of Non-malignant dyspoiesis

 

Avoid MDS diagnosis if potential secondary cause

 

-- Beware of making the diagnosis in young patients!!!!!!!! Vitamin/micro nutrient deficiencies: copper (ring sideroblasts, vacuoles), Zinc excess (ring sideroblasts), vit B12/folate dificiency Infections: HIV (multilineage dyspoiesis)

Toxins/drugs: EtOH (acanthocytosis, ring sideroblasts), MMF (pelgeroid neutrophils), recent (<6 mo) chemotherapy or radiation therapy (INH--> ring sideroblasts), arsenic/heavy metal toxin

Autoimmune/rheumatologic Congenital: dyserythropoiesis due to increased RBC fragility, baseline dyspoiesis and bone marrow failure syndromes, X-linked sideroblastic anemia, Pearson marrow-pancreas syndrome)

Neoplasms: involving marrow (esp myeloma, LGL, HCL)

Post chemotherapy bone marrow Week one: complete application - Mixed hematopoiesis absent

- proteinaceous fluid, not fat

- fibrinoid necrosis

- prominence of residual stromal cells, histiocytes, lymphocytes, plasma cells, mast cells

Week2+:early regeneration -  islands of erythroids, mild dyserythropoiesis

- left-shifted myeloids

- megakaryocytes clustered, hypolobated - increased hematogones

- mild reticulin fibrosis

Coagulative marrow necrosis

-fever, bone pain, cytopenias, leukoerythroblastic reaction -marrow plus/minus trabecular bone -May fibrose with resolution Aspirate: granular basophilic background, loss of cell detail Core: eosinophilic granular material, loss of cellular detail

 

Differential diagnosis of coagulative marrow necrosis Neoplastic: ALL most common, other leukemia's, lymphomas, carcinoma, neuroblastoma Non--neoplastic: sickle cell anemia (acute chest syndrome), infection, vascular obstruction (DIC, infarct), anti-phospholipid syndrome, acute GVHD following BMT

Serous Atrophy

 

"Gelatinous transformation"

- response to severe malnutrition/cachexia

- hypocellular marrow with loss of hematopoietic cells

- homogenous eosinophilic material surrounds, replaces fat cells

Amyloidosis

 

Esprit: pink to purple masses of homogenous material Core biopsy: waxy homogenous eosinophilic material

Hemophagocytosis

 

May be seen an infection Nucleated-cell hemophagocytosis is more specific for HLH than red cell hemophagocytosis

Osteitis fibrosa cystica due to ESRD/dialysis-related secondary hyperparathyroidism

 

Micro: complex trabecular architecture of variable width -replacement of hematopoietic space by fibrosis -deep invaginations of trabeculae lined with osteoclasts -visible unmineralized osteoid seam

 

 

DDX:

- radiologic – metastatic disease, plasma cell myeloma – clinical: primary hyperparathyroidism, renal failure/secondary hyper PTH Histologic: Paget's disease of bone, primary myelofibrosis with osteosclerosis

Cystinosis leading to ESRF and renal osteodystrophy