Hematology Exam 2-- Hematopoiesis, Non-Malignant Changes

What is meant by tissue hemostasis?

The maintenance of an adequate number of cells

What are the two theories of hematopoietic origin?

Monophyletic and polyphyletic

What are the five stages of cell development during hematopoiesis?

Pluripotent, multipotent, omnipotent, precursor, matured

What is differentiation?

Generates diverse cell populations, first commitment step of an HSC

What is commitment?

The point where an HSC switches to a differentiating cell

What is maturation?

Occurs after commitment, unique cell characteristics develop

What are circulating cells?

Most mature cells, have limited lifespan, incapable of mitosis

How many RBC, WBC, and PLT are produced daily?

2 x 10^11 RBC, 1 x 10^11 WBC, and 1 x 10^11 PLT

What are the characteristics of an HSC (2, general)?

An undifferentiated pluripotent cell

How many HSCs does the average adult have?

2 x 10^4

What are the 4 potential fates of an HSC?

renew, differentiate/commit, apoptosis, quiescence

What are the uses of the osteoblastic and vascular niches in HSC hematopoiesis?

The osteoblastic niche supports and maintains quiescence and/or renewal, the vascular niche provides signals for proliferation and differentiation

What is asymmetric cell division?

One daughter cell self-renews while the other goes through differentiation

What is a progenitor cell?

The daughter cell of an HSC committed to differentiation

What is the progenitor cell phenotype?

CD34+ in early progenitor cells, CD38+ after differentiation

What are progenitor lineage restricted cells?

CFUs

What are the 5 types of CFUs?

GEMM (granulocytes, erythrocytes, monocytes, megakaryocytes)
GM (granulocytes, monocytes)
MEG (megakaryocytes)
EO (eosinophils)
BFU-E -> CFU-E (erythrocytes)

What are maturing cells?

Cells that are morphologically recognizable (blasts), measured by nuclear and cytoplasm characteristics

What are the stem cell niches?

Osteoblastic niches where differentiation-inducing signals are blocked and a hypoxic environment protects HSCs from oxidative stress

What are the lymphoid aggregates?

B cells are stored close to the endosteal surface and T cells are stored close to stromal cells in the BM which secrete IL7

What are the erythroid niches (erythroblastic islands)?

In the marrow sinusoids

Where are the megakaryocytic niches?

Near the marrow sinusoidal endothelial cells--position is key to easy platelet release

Where are the granulocytic nests?

In the endosteal compartment of the bone marrow

What are the early-acting multilineage growth factors?

KIT ligand (stem-cell-factor), FLT3 ligand, IL3, and GM-CSF

What does the KIT Ligand (SCF) do?

Promotes prolif/diff of stem cells, MPCs, CFU committed cells, and mast cell precursors

What does FLT3 Ligand do?

Inhibits apoptosis and promotes the prolif/diff of granulocytes, monocytes, B cells, and dendritic cells

What does IL3 do?

Acts on multipotential progenitor cells (MPCs) and early committed cells

What does GM-CSF do?

Promotes development in bone marrow, stimulates colony growth for all cell types except basophils, activates mature phagocytes (neutrophils, macrophages, eosinophils)

What signaling molecules are active in erythropoiesis?

IL3 and CM-CSF for blast forming units (BFU-Es) and EPO for colony forming units (CFU-Es)

What signaling molecules are active in platelet stimulation?

IL11 and TPO

What are the steps in neutrophil development?

HSC in bone marrow--> CMP/CFU-GEMM --> committed precursor cells (CFUs)

What are the 6 stages of maturation for a neutrophil?

Myeloblast, promyelocyte, myelocyte, metamyelocyte, band, seg

What are features of the maturation process for a neutrophil (general)?

Nucleus-- nucleoli disappear, chromatin condenses, nucleus indents and segments
Cytoplasm-- myeloblast is agranular, increases in size compared to nucleus

How many cells are produced by leukocytosis?

1.5 x 10^9 per hour

How can a myeloblast be identified? What is its percentage in the bone marrow?

14-20 um in diameter, high NC ratio, 0.2-1.5% in BM
Nucleus-- nucleoli visible, open and evenly stained chromatin
Cytoplasm-- blue and agranular, visible golgi, peroxidase and esterase positive, may have auer rods (fusion of primary granules)

What signaling molecules act on the myeloblast?

CD33, CD13 (both distinct to myeloid lineage)
CD38, CD45RA

How can a promyelocyte be identified? What is its percentage in the bone marrow?

15-21 um in diameter, high NC ratio 2-4% in BM
Nucleus-- nucleoli visible, open and evenly stained chromatin (slightly more condensed than in myeloblast)
Cytoplasm-- basophilic granules, primary/azurophilic granules

How can a myelocyte be identified? What is its percentage in the bone marrow?

12-18 um in diameter, 8-16% in BM, *dawn of neutrophilia*
Nucleus-- reduced size, flattened on one side, nucleoli are indistinct
Cytoplasm-- neutral pink color (less blue than pro cytoplasm), secondary granules w/o peroxidase appear first in golgi, secretory vesicles may be visible

How can a metamyelocyte be identified? What is its percentage in the bone marrow?

10-18 um in diameter, 9-25% in BM
Nucleus-- chromatin is clearly condensed, nucleoli not visible, indentation forms
Cytoplasm-- neutral pink color, secondary and secretory granules, tertiary granule synthesis begins (may be visible)

How can a band be identified?

9-15 um in diameter
Nucleus-- chromatin is very condensed, indentation breaches 50%
Cytoplasm-- neutral pink color, all granules present but primary are obscured

How can a seg be identified?

9-15 um in diameter
Nucleus-- chromatin is very condensed, has segmented lobes connected by a filament, may have a Barr body attached
Cytoplasm-- neutral pink color, all granules present but primary are obscured

What signaling molecules act on a mature neutrophil?

CD15, CD16, CD11b/18

What are the types of granules found in developing neutrophils? (What color are they, do they contain anything?)

Basophilic-- DARK BLUE/BLACK, peroxidase and antimicrobial compounds
Primary/Azurophilic-- PINK (RED/PURPLE), nonspecific
Secondary-- lysozymes, hydrolases
Tertiary--lysozymes, etc.
Secretory-- contain lipids, carbohydrates, proteins

What is agranulocytosis?

No granules present in the cell, not the same as granulocytopenia (decrease in granulocytes below 2.0 x 10^3 per uL)

What is neutrophilia? What is it caused by?

Increase in neutrophils above 7.0 x 10^3 per uL, caused by acute bacterial infections, metabolic/drug intoxication, tissue necrosis

What are the 4 steps neutrophils use to initiate an immune response?

Adherence, migration (chemotaxis via amoeboid motion), phagocytosis, and bacterial killing (degranulation)

What are the two mechanisms a neutrophil may use to kill bacteria?

Oxygen dependent (ROS, myeloperoxidase)
Oxygen independent (pH defensins, lysozymes, hydrolases, extracellular traps)

What are the stages of maturation for an eosinophil? In what stage is a baby eosinophil first recognizable?

Same 6 stages as neutrophils, first recognizable as a myelocyte

What signaling molecule is required for eosinophil maturation?

IL5

What is seen in the cytoplasm of a mature eosinophil?

Primary granules-- Charcot-Leden proteins with lysophospholipase activity
Small granules-- acid phosphatase, aryl-sulphatase
Specific granules-- source of cytotoxic and proinflammatory proteins
Proinflammatory cytokines

What are the proinflammatory cytokines in an eosinophil?

IL2, IL4, GM-CSF, IL5, TNF-alpha

What is the concentration of eospinophils in the periphreal blood?

less than 0.4 x 10^3 per uL with diurnal variation (highest conc in morning)

How long do eosinophils stay in circulation?

18 hours, then move to tissues

What is eosinophilia? What is it caused by?

More than 0.4 x 10^3 per uL in PB, caused by allergies, parasitic infection, toxic reactions, GI disease

What are the steps in basophil maturation?

Similar to neutrophils, have a gradual indentation and segmentation of the nucleus, develop large purple/black granules

What is contained in basophil granules? Mature basophil granules?

Histamine, heparin, lysophospholipase, major basic protein
Mature granules-- peroxidase, are water soluble

What signaling molecules act on basophils?

CD9, CD11a, CD13, CD40

What is basophilia?

Concentration more than 0.2 x 10^3 per uL

How long does basophil maturation in the BM take?

2.5-7 days

What is the role of basophils in immune responses?

Mediate inflammation, have receptors for IGE to activate degranulation (basoactive, bronchoconstrictive, chemotactic)

What are mast cells? Where are they found?

Have proliferative potential, live weeks-months, found in BM and tissues but not PB, nucleus is round and cytoplasm has dense granulation (contain acid phosphatase, protease, alkaline phosphatase)

What are the mononuclear cells?

Monocytes, lymphocytes, dendritic cells

What is the maturation process for monocytes (general)?

GFU-GM in BM, requires CM-CSF, IL3, and M-CSF

What is the role of monocytes in the immune response?

Stimulated by T lymphocytes and endotoxins, phagocytic

How is a monoblast identified?

12-20 um in diameter, *cannot be differentiated from myeloblast*
Nucleus-- ovoid/round, may be folded or indented
Cytoplasm-- abundant, agranular, blue-gray color, may contain vacuoles

How is a promonocyte identified?

12-20 um in diameter, *does have specific characteristics*
Nucleus-- irregular shape, indented, may have nucleoli
Cytoplasm-- blue-gray color, has azurophilic granules

How is a mature monocyte identified?

12-20 um in diameter, largest mature cell in PB, low NC ratio
Nucleus-- horseshoe/bean shaped, numerous folds, open chromatin
Cytoplasm-- blue/gray, "ground glass"

What are the two types of granules in monocytes?

1. Contain peroxidase, acid phosphatase, aryl sulfatase

2. Does not contain alkaline phosphatase

What signaling molecules act on monocytes?

CD11b, CD14

What is identifiable in the shift from monocytes to macrophages?

Nucleus and cytoplasm stay same, expression of CD68 acquired

How long do macrophages live in the tissue? Why do they go there?

Live in tissue for months, do not re-enter circulation. Shift is dictated by cytokines/chemokines (inflammation or infection), or to set up "camps" (called resident macrophages).

What are the 3 main functions of monocytes/macrophages?

Housekeeping-- direct cytotoxic effect on tumor cells, digest dead cells (removes toxins and conserves iron)

Activate innate/adaptive immunity-- inhibit growth of intracellular microorganisms (nonspecific killing activated by T cells and/or endotoxins, opsonins), act as APCs, can produce cytokines

Ingest/Kill microorganisms

What are the lymphocytes?

B cells, T cells, NK cells

What are the two stages of lymphopoiesis?

Antigen-independent and antigen-dependent

What is antigen independent lymphopoiesis?

Takes place in primary lymphoid tissue, CLP --> not yet immunocompetent T and B cells, cells migrate to secondary lymphoid tissue

What is antigen dependant lymphopoiesis?

Follows Ag-independent lymphopoiesis. Receptors on B and T cells interact with Ag to become immunocompetent (can now mediate adaptive immunity)

What are effector T cells?

*cannot be identified with microscope*
Responsible for cell-mediated cytotoxic reactions (cytotoxic T cells)
Helper activity for B cells and MO (helper T cells)
Supress other T cell immune responses (Tregs)

What is the immune function of B Cells?

Concentrate and present Ag to T cells
Precursors of Ig-secreting plasma cells
Programmed to make specific Ab for specific Ag

What is the immune function of NK Cells?

Cytotoxic lymphocytes, active in innate immunity

Responsible for cytolysis of tumor and infected cells-- kill by secreting granules containing perforin and protease

Have more cytoplasm than other lymphs, contains azurophilic granules

How are lymphocyte cells differentiated?

VDJ recombination

What are the B cell markers?

CD19, CD20, CD21, CD22

What are the T cell markers?

CD7, CD5, CD2, CD3
In lymph nodes CD4
In BM CD8
(T4 to T8 ratio is 2:1)

What are the NK cell markers?

CD56, CD16
Lack CD3, CD4, TCR

What is the concentration of NK cells in the PB/spleen?

5-15%

What is the concentration of T4 and T8 cells in the PB?

60-80% T4, 35% T8

What are the three stages in lymphocyte development?

Lymphoblast --> Prolymphocyte --> Lymphocyte

How is a lymphoblast identified?

10-18 um in diameter, high NC ratio
Nucleus-- chromatin is open, pale blue nucleoli visible, nuclear membrane is dense
Cytoplasm-- agranular, less basophilic than other WBC blasts, positive for PAS, TdT, acid phosphatase

How is a prolymphocyte identified?

lower NC ratio
Nucleus-- chromatin is open, nucleoli may not be visible
Cytoplasm-- light blue, agranular

How is a large lymphocyte identified?

11-16 um in diameter, lower NC ratio
Cytoplasm-- light blue, may have azurophilic granules, peroxidase negative
If granules are prominent cell is called a large granular lymph

How is a small lymphocyte identified?

7-10 um in diameter
May be motile, "hand-mirror" shape, cytoplasm has relatively few granules

How is a reactive lymphocyte identified?

16-30 um in diameter, low NC ratio
Nucleus-- round or elongated, chromatin more dispersed
Cytoplasm-- localized basophilia, azurophilic granules, vacuoles, may be indented or dark where touching RBCs

Also called stimulated, transformed, atypical, activated
Increase in number during viral infection

How is a plasma cell identified?

14-20 um in diameter
Nucleus-- eccentric, coarse masses of chromatin, no nucleoli
Cytoplasm-- very basophilic (RNA), visible golgi and RER, enlarged cytoplasm due to Ig production, may have azurophilic granules or rodlike crystal inclusions

What is the concentration of lymphocytes in the PB?

1-4.8 x 10^3 per uL, elevated in children
If high called lymphocytosis, if low called lymphocytopenia

How are lymphocytes distributed?

5% in PB, 95% in extravascular tissue, lymph nodes, spleen

What is the ratio of long lived to short lived lymphs?

80% long-lived (months-years), 20% live hours-days

What are examples of non-malignant cytoplasmic changes?

Dohle bodies, toxic granulation, reactivity (lymphocytes)

What are examples of non-malignant nuclear changes?

Hyper- and Hypo-segmentation (Pelger Huet)

What are examples of non-malignant changes in cell number?

Neutrophilia, neutropenia, lymphocytosis (leukocytosis, leukopenia)

What are quantitative tests done to classify non-malignant changes in WBCs?

CBC relative and absolute counts

What are qualitative tests done to classify non-malignant changes in WBCs?

Blood smear (microscopy)