INDICATIONS

BONE MARROW
UNEXPLAINED

Aspiration 1. Non-regenerative anemia

&
Interpretation 2. Neutropenia
Mary Anna Thrall
Maryanna.thrall@colostate.edu 3. Thrombocytopenia

Indications Reversible Stem cell injury

4. Suspected neoplasia
eg, patient with hypercalcemia,
or monoclonal gammopathy
Drugs and Chemicals (eg, estrogen)
Microorganisms (ie, parvovirus)
Immune mediated

5. To better classify leukemia

Possible complication: myelofibrosis

Irreversible Stem Cell Injury Irreversible stem cell injury

Etiology
Myelofibrosis

Usually not known (immune mediated?)

Aplasia

Drugs and Chemicals (eg, Benzene) Dysplasia

Microorganisms (eg, FeLV) Neoplasia (myeloproliferative disorder)

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 Commercially available bone marrow biopsy needles
Technique
• Trochanteric fossa or humerus
• Local or general anesthesia
• Bone marrow biopsy needle (16-22 gauge)
• Avoid diluting with blood
• Use EDTA or make slides quickly (pull films)
• Air dry, Wright’s stain
• If can’t obtain aspirate, take core biopsy for
histopathology

Bone marrow aspirate from wing of ilium

Bone marrow aspirate from humerus

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 Diluting a bone marrow aspirate with blood Bone marrow

Stop
STOP when you have two or three drops.

Bone marrow slide preparation Bone marrow pull film

Preparing
pull film: Bone marrow films
Place one
drop of marrow
on slide.
Place another
slide on top
of the drop,
pull apart
gently

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 Appropriate appearance of bone marrow film Bone marrow interpretation
• Cellularity
• Presence of megakaryocytes
• Presence of iron stores
• Myeloid:Erythroid ratio
• Orderliness & completeness of maturation
• Presence of other cells, ie plasma cells
• Presence of abnormal cells
• Presence of microorganisms (rare)

Adequate
cellularity
Megakaryocyte

Megakaryocytes
present

1 2
1. 2. Inadequate
Megakaryocytes Cellularity
ranging from
small immature
(panels 1 and 2)
to mature
(panel 3)

3.
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3

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Iron stores M:E RATIO
present
Number of granulocytes:
nucleated erythrocytes

1:1 up to 3:1 is normal

Erythroid maturation sequence

Myeloid
precursors
ranging from
progranulocyte
(P)
/ progranulocyte/ myelocyte /metamyelocyte / band /seg to myelocytes &
metamyelocytes
(arrows)
to
Myeloblast progranulocyte myelocyte metamyelocyte band seg bands and segs
myeloblast (arrowheads)

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 Eosinophils Basophils
Myeloid
To
Erythroid
Ratio

INCREASED M:E RATIO

ALWAYS INTERPRET Could mean either
ERYTHROID HYPOPLASIA or
BONE MARROW APLASIA
FINDINGS and/or
GRANULOCYTIC HYPERPLASIA
GRANULOCYTIC LEUKEMIA
AS THEY CORRELATE
TO CBC RESULTS.

Granulocytic hyperplasia, mild erythroid hypoplasia

ERYTHROID HYPOPLASIA, APLASIA

HYPOPLASIA CBC
PCV - 35%
Anemia of inflammatory disease
Anemia of renal disease, etc NCC - 96,000

PURE RED CELL APLASIA Neuts - 83,000

Bands - 8,000
FeLV in cats
Monos - 5,000
Immune mediated in dogs

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 CBC
DECREASED M:E RATIO Erythroid hyperplasia
PCV - 20%
REGENERATIVE ANEMIA
ERYTHROID LEUKEMIA NCC - 15,000

NEUTROPHIL CONSUMPTION Neuts - 12,000

Lymphs - 3,000
or DESTRUCTION

Orderliness
of Presence of other cells
maturation

• Plasma cells
• Lymphocytes
• Macrophages
• Mast cells
• Osteoblasts and Osteoclasts

Plasma cells Osteoblasts

Plasma cells

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 Osteoclast Mast Cells

Osteoclast

Mott cells

Immune mediated destruction? Hemophagocytic syndrome? Macrophages:

1. 2.
1. Phagocyted
nucleated RBCs

2. Phagocytized RBCs

3. Phagocytized
leukocyte (green arrow)
3.

Histoplasmosis
Microorganisms in bone marrow

• Histoplasma capsulatum
• Toxoplasma gondii
• Leishmania donovani
• Red cell parasites

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 Toxoplasmosis

Leishmania

Leishmania

Babesia
gibsoni

Abnormal cells Myelodysplasia

Dysplasia Often pre-leukemic

Neoplasia (Leukemia)
Morphologic abnormalities
Lymphoproliferative Myeloproliferative
•Lymphoblastic leukemia •Granulocytic leukemia Usually FeLV induced
•Lymphocytic leukemia •Monocytic leukemia
•Plasma cell leukemia •Myelomonocytic leukemia
(multiple myeloma) •Erythroid leukemia
(erythremic myelosis)

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Myelodysplasia
1.1. 2 Dysplastic
2.
neutrophils
1. Dysplastic
erythroid cells

2. Dysplastic
giant platelets

Myelodyplasia LEUKEMIA
Dysplastic immature
megakaryoctes
PRESENCE OF NEOPLASTIC CELLS
IN THE BLOOD OR
BONE MARROW
NUCLEOLUS

Lymphoproliferative LEUKEMIA
Lymphoblastic leukemia LYMPHOID LEUKEMIA

Lymphocytic leukemia

Plasma cell leukemia

(multiple myeloma)

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 Lymphoblasts (arrow) Bone marrow

Dog with
Chronic
Lymphocytic
Leukemia.

Lymphocytes
indicated by
arrow heads.

Lymphoblasts
indicated by
arrows.

Bone marrow
Polymerase chain reaction (PCR)
Dog with
lymphoma
involving • detects antigen receptor rearrangements
bone marrow
used to identify a clonal, neoplastic
Lymphoblasts population of cells
(arrows)

• differentiates non-neoplastic
lymphoproliferative disorders from those
that are neoplastic.

9 yr old dog: Anorexia, lethargy, nose bleeds

ref range Interpretation and plan
PCV (%) 22 37-55
• Non regenerative anemia.
Retics (x 103/µl) 18 0-60
T Prot (g/dl) 11.0 6.0-8.0 • Increased total protein - likely due to increase in globulin, rather
than dehydration. Protein electrophoresis indicated.
NCC (103/µl) 10.0 6.0-17.0
Segs 4.4 3.0-11.5 • Inflammatory and stress leukogram
Bands 1.2 0-0.3
• Thrombocytopenia. DIC? Immune mediated? Lack of production?
Monos 2.5 0.2-1.4
Lymphs 0.7 1.0-4.8 • Combination of non-regenerative anemia and thrombocytopenia
NRBCs 1.2 0 should trigger bone marrow aspirate.
Platelets (x103/µl) 60 150-900

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 Bone
Marrow
Monoclonal gammopathy Aspirate:

Many
Plasma
Cells

Multiple
Myeloma

Bone marrow aspirate, dog with multiple myeloma Bone marrow aspirate, dog with multiple myeloma

Lytic lesions in dog

with multiple myeloma.

Spontaneous fracture in
dog with multiple myeloma
Indications of multiple myeloma:
Monoclonal gammopathy
Increased plasma cells in bone marrow
Lytic lesions in bones (+/-)
Bence-Jones protein (light chains of immunoglobulin)
in urine

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 Myeloproliferative LEUKEMIA
• Granulocytic • GRANULOCYTIC (NEUTROPHILIC)
• Myelomonocytic
• Monocytic
• Erythremic myelosis
• Erythroleukemia
• Megakaryocytic

Very poorly differentiated myelogenous or myelomonocytic leukemia

M1 cannot be differentiated, based on morphology, from lymphoid leukemia

Granulocytic
(myeloblastic)
Leukemia

Type 1
Myeloblasts
(arrows)

Type II
Myeloblasts
(Arrowhead)

M2

Granulocytic
Leukemia
Granulocytic
leukemia
Cell in mitosis
(arrowhead)

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 Lymph node aspirate Chronic Myelogenous Leukemia

Myeloblasts (arrows)
Dog with granulocytic Note that cells are
leukemia involving maturing to neutrophils.
lymph nodes
Presence of blasts in
P - progranulocyte blood and severe anemia
arrow - small lymphocyte
aided diagnosis

Myelomonocytic leukemia. Monoblasts (arrows) & progranulocyte (arrowhead)

Blood film, dog with chronic myelogenous leukemia

CBC

PCV - 16%
NCC - 150,000/µl

Note blast (Type

II myeloblast or
progranulocyte
(arrow) and cell in
mitosis (red arrow)

Bone marrow aspirate, cat with non-regenerative anemia

Leukemia Rubriblasts (arrows)

Red Cell Leukemia (Erythremic myelosis)

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 Undifferentiated leukemia in a cat.
Has features
of both erythroid Cytoplasm fragment
and myeloid
Leukemia

Undifferentiated cell
Myeloid cell
(arrows)
rubricyte

Erythroid cells
(arrowheads

Undifferentiated leukemia

Megakaryoblastic leukemia
Additional tools to characterize leukemia

• Cytochemistry

• Immunophenotyping

chloracetate esterase (CAE), a granulocyte marker

Cytochemistry
Granulocyte markers

Peroxidase
Sudan Black B
Chloracetate esterase
M4
Myelomonocytic leukemia

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 alpha-naphthyl butyrate esterase (ANBE), a monocyte marker
Cytochemistry
Monocyte markers

Alpha-naphthyl acetate esterase

Alpha naphthyl butyrate esterase, inhibitable
by sodium fluoride

Problem: Sudan Black B may stain M4

Myelomonocytic leukemia

Cytochemistry
Alkaline Phosphatase
Positive staining rare in normal immature
granulocytes
Immature neoplastic granulocytic &
monocytic cells commonly stain
Lymphoid cells may stain
Immunophenotyping
Monoclonal antibodies directed against
cell surface antigens
Conjugated to fluorescent molecules, mixed
with the cells, analyzed by flow cytometry
Cell size
Protein expression and concentration

Other types of bone marrow neoplasia Mast Cell Leukemia

• Mast Cell leukemia (mastocytosis)

• Malignant histiocytosis

• Metastatic Carcinomas

• Metastatic Sarcomas

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 Malignant Histiocytosis (Histiocytic Sarcoma) Mammary carcinoma

Hemangiosarcoma

Caution: Immune mediated

neutropenia
can appear similar to
granulocytic leukemia

166288 3 yr old Great Dane, lame Immune mediated neutropenia and thrombocytopenia
CBC

WBC - 43%

NCC - 3,000

Neuts: 1,000
Lymphs: 2,000

Platelets: 53,000
Interpretation
Granulocytic hyperplasia
Disorderly maturation

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