Clin Path 3 Unit 2

Where is the site of granulocyte production in adults vs. juveniles?

1. Adults = active bone marrow

2. Juveniles = extra medullary (spleen, liver, lymph nodes, etc)

What instances would extra medullary responses be seen in adults for granulocyte production?

Inflammatory responses

• Most prominent in spleen, includes liver + lymph nodes

What do granulocytes originate from? What is the subpopulation?

Pluripotent stem cells (granulocyte/monocyte stem cells)

• Subpopulation differentiates into neutrophils, eosinophils, and basophils

What is the difference between orderly production and disorderly production from the bone marrow? What is each indicative of? What is myeloproliferative disorders means?

1. Orderly production = relatively few immature to increasing numbers of mature cells

• Important to ID demand from bone marrow

2. Disorderly production = disproportionally high # of immature cells compared to mature cells

• Important to ID disease (myeloproliferative disorders = 1 or more of the cell line is uneven (WBCs, RBCs, platelets))

What is the order of orderly production in the bone marrow?

1. Myeloblast divides into 2 promyelocytes

2. Promyelocytes divide into 2 myelocytes

3. Myelocytes divide twice = 8 metamyelocytes

4. Metamyelocytes divide into either band cells, eosinophils, segmented neutrophils, or mature basophils

What factors induce proliferation of WBCs? What specific factors from the already stated factors achieve this process?

1. Cytokines

2. Growth factors

   • Colony stimulating factor (CSF) produced by mononuclear cells + interleukins stimulate release of cells from bone marrow

What defines neutrophil kinetics? How long do they circulate before being needed elsewhere?

• Time for production (myeloblast to segmented neutrophil) = 7 days normally

• Inflammatory state alters to 2-3 days

1. Circulation life = time in circulation until migration to tissue = 6-10 hours normally

2. Inflammation may decrease due to rapid rate of consumption by tissue (tissue needs for whatever purpose)

What is the definition of hematopoiesis? How are cells removed and replaced?

Hematopoiesis = formation of blood into cellular components

• Cells removed by phagocytosis + spleen

• Cells replaced by: bone marrow production = medullary sites + extra medullary sites = spleen, liver, lymph nodes

What are the 4 general rules of maturation seen in erythrocytes + granulocytes?

1. Cell decreases in size as it matures

2. Nucleoli lost (nucleoli indicative of dividing cells)

3. Condensation of nuclear chromatin

4. Cytoplasm becomes less basophilic

What is the most immature stage in the myeloid phase of grnulopoiesis?

Myeloblasts

What are the 3 types of cells of the granulocyte lineage?

1. Neutrophils

2. Eosinophils

3. Basophils

What are identifying factors for myeloblasts (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = large

2. Nucleus = round to oval, 1 or more prominent nucleoli

3. Chromatin = finely granular (wadded up hairnet)

4. Cytoplasm = small to moderate amount, more blue than monocyte

What are identifying factors for promyelocytes (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = same or larger than myeloblast

2. Nucelus = perinuclear clear zone ± nucleoli

3. Chromatin = fine chromatin

4. Cytoplasm = primary granules (pink/purple, azurophilic)

5. Only cell that breaks the rules of maturation

What are identifying factors for myelocytes (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = smaller than promyelocyte

2. Nucelus = round to slightly oval

3. Chromatin = slightly granular

4. Cytoplasm = moderate amount, blue, secondary granules (light pink)

What are identifying factors for metamyelocytes (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = smaller than myelocyte

2. Nucelus = kidney-shaped (indented)

3. Chromatin = moderately granular + more condensed

4. Cytoplasm = blue, secondary granules

What are identifying factors for banded neutrophils (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = round + smaller

2. Nucleus = horseshoe shaped

3. Chromatin = more condensed

4. Cytoplasm = moderate amount, light blue

What are identifying factors for segmented neutrophils (cell size, nucleus, chromatin, cytoplasm)?

1. Cell size = smaller

2. Nucelus = segmented

3. Chromatin = coarsely granular + clumped

4. Cytoplasm = moderate amount, faint blue to pink

What are some identifying factors for hyperhsegmented neutrophils? What do they indicate?

1. 6 or more lobes

2. Aged neutrophil normally removed from circulation (can be caused by increased levels of endogenous corticosteroids)

3. Often indicates B12/folate deficiency

How does development of eosinophils/basophils differ from the myelocytic stage?

1. Secondary granules in myelocytic eosinophil = red to reddish orange

2. Secondary granules in myelocytic basophil = purple

3. Both slightly larger than segmented neutrophil

What characterizes an acute vs chronic leukemia? What is leukemia and what are 2 tests to ID?

Acute leukemia 

• ^{20% blasts}

Chronic leukemia

• <20% blasts

Leukemia = neoplastic proliferation of cells

• Immunochemistry (certain stains to ID cells)

• Cell cytology/morphology

What is a left shift and what are some outcomes of one? What indicates orderly and disorderly left shifts?

Left shift = increased concentration of immature neutrophils

Can be seen with neutrophilia/neutropenia

• Orderly = Inflammation

• Disorderly = Possible bone marrow disease

What is the difference between lymphoma and leukemia?

1. Lymphoma = in lymphoid tissue

2. Leukemia = in bone marrow

What is the difference between lymphoma/lymphosarcoma and lymphocytic leukemia?

1. Lymphoma/lymphosarcoma = confined to solid mass

2. Lymphocytic leukemia = involved in blood/bone marrow

What is a plasma cell?

Mature B cell that makes antibodies

What is a myeloproliferative disorder?

Group of diseases of the bone marrow in which the stem cells that normally produce blood cells start proliferating too much (myeloid cells in BM + stem cells)

What are the most common types of myeloproliferative disorders?

1. Granulocytic

2. Erythrocytic

3. Monocytic

4. Lymphoproliferative mentioned in slideshows

What are the 3 most common toxic changes that happen to a cell?

1. Toxic granulation (seen first)

2. Dohle bodies (seen second, more common in cats)

3. Vacuoles (seen third, active phagocytosis)

What is neutrophil hypersegmentation associated with?

• Seg been around too long

• Associated w/ endogenous steroid effect

• Can be sign of B12/folate deficiency 

What is the hematologic response to inflammation? What is the inflammatory cycle?

• Vasodilation

• Cytokines

Inflammatory cycle

• Consumption

• Production

• Release from bone marrow

What animals commonly respond with neutrophilia to inflammation? What can the counts get to? Is neutropenia a medical emergency for small animals? What animals is it common in?

1. Dogs = 120,000

2. Cattle = 25,000

3. Ruminants (small marrow storage, initial is probably neutropenia, then neutrophilia)

• Dogs have high stores of extra neutrophils (medical emergency when neutropenia observed)

• Somewhat in cats/horses

• Common in cattle

What is a circulating pool vs marginal pool?

1. Circulating pool = neutrophils that are freely flowing in bloodstream

2. Marginal pool = neutrophils that are loosely attached to the walls of small blood vessels (can detach quickly when stimulated by epinephrine, corticosteroids, or inflammation)

What are 2 main causes of lymphocytosis?

Excitement

• Morph = small + normal

• Small increases in #

Lymphocytic leukemia

• Larger increases

• Abnormal morph = large, fine chromatin, nucleolus, increased cytoplasm

What are 3 other responses to inflammation that can occur?

1. Access to site (walled off lesions vs accessible

2. Excitement response (epinephrine release can double leukocytes + increase blood flow)

3. Stress response = most common (corticosteroid release + lymphopenia (redistribution) + doubling of neutrophils (lack of stress response can indicate Addison’s disease))

What is bovine persistent lymphocytosis?

• Present w/ bovine leukemia virus (BLV)

• May progress to lymphocytic leukemia or lymphosarcoma

What are some other accessory causes of lymphocytosis?

1. Chronic inflammation

2. Chronic canine ehrlichiosis (lymphocytosis w/ monoclonal gammapthy; large granular lymphs)

What are 3 main causes of neutropenia? What is another major affect of neutropenia from injury and what cells are harmed in order?

1. Acute inflammatory consumption

2. Immune-mediated consumption

3. Neutropenia resulting stem cell injuries (acute, transient, long term/permanent)

• All cell lines affected when neutropenia occurs (seen first in neutrophils due to high turn over, thrombocytes next, non-regenerative anemia last)

What are 2 types of reversible neutropenia?

1. Viral component

• Fe panleukopenia, Ca parvovirus

2. Chemicals

• Chemotherapy

• Estrogen overdose (ferrets)

• High doses of estrodiol (dogs, not cats)

What is one cause of irreversible neutropenia?

Proliferative bone marrow disorder

• FeLV

• Idiopathic hypoproliferative disorders

• Myelodysplasia

• Myeloproliferative disorders

What are 3 main causes of lymphopenia?

1. Usually steroid response

2. Lymphocytic acute virus

3. Combined immunodeficiency in Arabian foals

What are 2 main causes of monocytosis?

1. Acute + chronic inflammation

2. Steroid response in dogs

• Unimportant change

What are 2 causes discussed in lecture for leukocyte agglutination?

1. Cold agglutinin (occurs after blood collection)

2. Immunoglobulin mediated agglutination of neutrophils/lymphs

• White cell clumps in body fluid can be indicative of cancer

• Must be resolved, can lead to falsely low WBC counts

What are 6 inherited abnormalities of leukocyte morphology?

1. Pelger-huet anomaly

• Mature hyposegmented neutrophils, function normally

• Must be differentiated from left shift

2. Birman cat neutrophil granulation anomaly

• Neutrophils contain fine eosinophilic granules, function normally

• Must be differentiated from toxic granulation + mucopolysaccharidoses (MPS)

3. Mucopolysaccharidoses

• Neutrophils contain purple/magenta granules (alder reilly bodies)

• Lysosomal storage disorder

• Granules do not stain w/ Diff-Quik

4. Chediak-Higashi syndrome

• Neutrpohils have large fused lysosomes in cats, abnormal function

• Platelet dysfunction (bleeders)

• Cannot properly release ADP

5. Bovine leukocyte adhesion deficiency (BLAD)

• Lethal disorder in Holstein cattle (CD18 gene)

• Abnormal leukocyte adherence + emigration into tissue

• Recurrent infections

6. Inherited abnormalities of lymphocyte morphology

• Most common = cytoplasmic vacuolization in lymphs

What are 5 things bone marrow biopsies helpful in diagnosing?

1. Non-regenerative anemia

2. Neutropenia

3. Thrombocytopenia

4. Gammapathy

5. Neoplasias (lymphoma)

What are the 4 cell lines w/in bone marrow?

1. Erythrocytic

2. Granulocytic (myeloid series)

3. Monocyte series (few)

4. Other (macrophages, osteoblasts (build bone), osteoclasts (destroy bone), mast cells, fibroblasts, fibrocytes)

What is the official name for an nRBC seen in a peripheral blood smear?

Metarubricyte

Where does hemosiderin come from?

RBC breakdown product containing iron

What is LAP stain?

Stains abnormal granules in neutrophil which can ID inflammatory response vs. granulocytic leukemia

What is RBC morphology for B12 deficiency? Iron deficiency?

B12 deficiency

• Microcytic hyperchromic

Iron deficiency

• Macrocytic for RBC + hypersegmentation for neutrophils

What are some characteristics of the monocyte series?

• Decreased # in bone marrow

• Difficult to distinguish from myeloid series

• Precursors only recognizable w/ monocytic leukemia

• Difficult to distinguish from myeloid series

What are the 3 stages of the megakaryocytic series in the bone marrow?

1. Megakaryoblast - large, multi-lobulated (16 or more) structure in center of cell, does not divide, deeply basophilic

2. Promegakaryocyte - 2-4 nuclei connected by thin strands of nuclear material, deep blue agranular cytoplasm

3. Megakaryocyte - very large + lighter cytoplasm

What are the 3 cells covered in lecture in the lymphocytic series in the bone marrow?

1. Normal lymphs

• Round + indented nucleus occasionally

• Diffuse chromatin pattern w/out visible nucleoli

2. Plasma cells

• Produce immunoglobulin

• Eccentric nucleus*, perinuclear zone, dense chromatin

• Flame cell, Mott cell

3. Lymphoblasts rare

• Indicative of lymphoproliferative disorders

What are some characteristics of macrophages in the bone marrow?

• Will turn into monocytes later

• Decreased # in bone marrow

• Commonly phagocytize cellular debris - often contain hemosiderin + increased vacuoles

• Round to kidney bean nucleus + blue/gray cytoplasm

What are some cytochemistry stains for the myeloid series, monocytic series, and variable series?

1. Myeloid

• Peroxidase

• Sudan black B (fat)

2. Monocytic

• Alpha napthyl acetate esterase

• Nonspecific esterase

3. Variable

• Alkaline phosphatase (rarely + in immature neutrophils)

What is immunophenotyping?

Use of monoclonal antibodies directed against membrane antigens to determine phenotypic profile of cells

What is cellularity of bone marrow? What is the term for increased and decreased numbers?

Cellularity = 50% cells vs 50% fat (how full marrow is, fat increases w/ age)

1. Hyperplastic = normal in cell loss, destruction consumption; abnormal is proliferative disorders

2. Hypoplastic = infectious agents (FeLV, Ehrlichia) + common for 1 cell line to be affected

What is the myeloid:erythroid ratio?

• 0.5-3:1 is normal (can differ between species)

• Increased M:E - shifts can be seen in peripheral blood, animal will be anemic, decreased RBC production, increased WBC

• Decreased M:E - Increased RBC production, regenerative anemia, decreased neutrophil production, combo of both

• 80-90% should be mature forms (meta, band, seg)

• Disorderly = leukemia, arrested marrow maturation

• 300-500 nucleated cells

• Myeloid = neutrophils, basophils, eosinophils

Where is hemosiderin notable? When are macrophages noted in bone marrow?

1. Hemosiderin notable in macrophages (rare in cats, abundant in dogs/horses)

2. Macrophages not numerous in bone marrow, increased in autoimmune, marrow necrosis, drugs, toxins

• DAT coombs = antibody to IGg (main antibody in blood for infections)

What are 4 neoplastic disorder in the bone marrow other than lymphoproliferative and myeloproliferative?

1. Epithelial + mesenchymal tumors

• Rarely in bone marrow

• Epithelial tumors = carcinomas

• Mesenchymal tumors = metastatic sarcoma (large, discrete spindle-shaped tumor), must be differentiated from fibroblasts

2. Mast cell leukemia

• Seen in dogs and cats w/ systemic mastocytosis (marrow involvement is rare)

3. Malignant histocytosis (dogs)

• Rapidly progressive

• Proliferation of large, pleomorphic, single + multinucleated histocytes

• Phagocytosis of RBC + WBC

• Involved bone marrow, liver, spleen, lung, lymph nodes, CNS