HEMATOPOIESIS AND LABORATORY TESTS

replicate DNA

mitosis

Cell Cycle

The purpose of the cell cycle is to _ _ once and distribute identical chromosome copies equally to two daughter cells during _

10

8

4

1

Cell Cycle Phase		Length (hours)	Description
G0			Quiescence; non-dividing cell
Interphase	G1	_	Preparation for replication
	S	_	DNA replication
	G2	_	Checking for damage
M		_	Cell division

Hematopoiesis

Is the process of blood cell production, differentiation, and development

Hematopoietic Stem Cells

Are the foundation of the adult hematopoietic system

CD34

Hematopoietic Stem Cells

Classical marker of hematopoietic stem cells:

Totipotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Present in the first few hours after an ovum is fertilized

Totipotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Most versatile type of stem cell

Can be any type of cell

Pluripotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Present several days after fertilization

Pluripotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Can develop into any cell type except into fetus

Multipotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Derived from pluripotent stem cells

Multipotential Stem Cells

Hematopoietic Stem Cells

Types of Human Stem Cells

Can be found in adults but they are limited to specific types of cells to form tissues

Monophyletic Theory

Hematopoietic Stem Cells

Theories about Hematopoietic Progenitor Cells Origin

Suggests that all blood cells are derived from a single progenitor stem cell called a pluripotential stem cell

Monophyletic Theory

Hematopoietic Stem Cells

Theories about Hematopoietic Progenitor Cells Origin

Most widely accepted

Polyphyletic Theory

Hematopoietic Stem Cells

Theories about Hematopoietic Progenitor Cells Origin

Suggests that each of the blood cell lineages is derived from its own unique stem cell

19th

Yolk sac

Primitive erythroblast

intravascularly

Hematopoietic Stem Cells

Three Phases of Hematopoiesis

Mesoblastic/Megaloblastic

• _ day of gestation

• Site: _ _

• Main cell: _ _

• Occurs _

5th to 7th

Fetal liver

extravascularly

Hgb F

Hematopoietic Stem Cells

Three Phases of Hematopoiesis

Hepatic

• _ to _ week

• RBC, WBC, platelets

• Primitive hematopoiesis declines in the first stages

• Site: _ _

  • Other organs like spleen helps

• Occurs _

• Predominant Hgb: _ _

• Hgb A is also present

5th

Bone marrow

Hematopoietic Stem Cells

Three Phases of Hematopoiesis

Hepatic

• Around _ month

• Site: _ _

Bone Marrow

Hematopoietic Stem Cells

Hematopoietic Tissues

Contains developing erythroid, myeloid, lymphoid, and megakaryocytic cells

Medullary

2.5

1

2.5

Hematopoietic Stem Cells

Hematopoietic Tissues

Bone Marrow

It is estimated to be capable of generating around

_ billion RBCs

_ billion granulocytes

_ billion platelets

Liver

Hematopoietic Stem Cells

Hematopoietic Tissues

Main site during hepatic stage

Extramedullary (adult)

Spleen

Lymph Nodes

Hematopoietic Stem Cells

Hematopoietic Tissues

Secondary lymphoid organs

Thymus

Hematopoietic Stem Cells

Hematopoietic Tissues

Primary lymphoid organ

Site of T cell maturation

First organ to be developed in the fetus

Retrogression

Hematopoietic Stem Cells

It is the process of replacing red marrow by yellow marrow during development

Ribs

Sternum, skull, shoulder blades

Vertebrae

Pelvis, proximal ends of the long bones

Hematopoietic Stem Cells

Retrogression

Eventually results in restriction of the red marrow in the adult to the: (4)

5-7

Hematopoietic Stem Cells

Retrogression

_ yrs – retrogression starts

50:50

Hematopoietic Stem Cells

Retrogression

_ – red marrow:yellow marrow

Developing hematopoietic stem cells

Macrophages

Mast cells

Osteoblasts

Osteoclasts

Hematopoietic Stem Cells

Normal Marrow Cells (5)

Myeloid:Erythroid (M:E) ratio

Hematopoietic Stem Cells

Normal Marrow Cells 

proportion of myeloid cells to nucleated erythroid precursors in the bone marrow

500

1000

Hematopoietic Stem Cells

Normal Marrow Cells 

M:E Ratio

Count at least _ cells; _ cells is more ideal

3:1-4:1

10:1

Hematopoietic Stem Cells

Normal Marrow Cells 

M:E Ratio

Normal value

If with leukemia

Erythropoiesis

It occurs in the bone marrow and is a complex, regulated process for maintaining adequate numbers of erythrocytes in peripheral blood

Iron deficiency anemia

Erythropoiesis

Insufficient erythropoiesis

megaloblastic anemia; impaired DNA synthesis

Erythropoiesis

Ineffective erythropoiesis

Progenitor Cells

Erythropoiesis

Immature hematopoietic cells that are committed to a cell line but cannot be identified morphologically

-forming units

Precursor Cells

Erythropoiesis

Immature hematopoietic cells that are morphologically identifiable as belonging to a given cell line

Burst Forming Unit-Erythroid

Erythropoiesis

Committed Erythroid Progenitors

Earliest committed progenitor

1 week

Erythropoiesis

Committed Erythroid Progenitors

BFU-E to CFU-E: _

18-21 days

Erythropoiesis

Committed Erythroid Progenitors

BFU-E to mature RBC: _

1 week

Erythropoiesis

Committed Erythroid Progenitors

CFU-E to Rubriblast: _

Rubriblast

Prorubricyte

Rubricyte

Metarubricyte

Reticulocyte

Erythrocyte

Erythropoiesis

RBC Stages of Maturation

Rubriblastic Nomenclature

Pronormoblast

Basophilic normoblast

Polychromatic normoblast

Orthochromic normoblast

Polychromatic erythrocyte

Erythrocyte

Erythropoiesis

RBC Stages of Maturation

Normoblastic Nomenclature

iron uptake, protoporphyrin

Globin production

8-32

Erythropoiesis

RBC Stages of Maturation

Rubriblast/Pronormoblast

Nucleus:Cytoplasm ratio: 8:1 Nucleus: Round to oval, 1 to 2 nucleoli Cytoplasm: Dark blue Proteins and enzymes necessary for _ _ and _ synthesis are produced _ _ begins Earliest recognizable precursor under the light microscope Capable of mitosis

Nucleus:Cytoplasm ratio: 4:1 Nucleus: Large, round with 0 to 2 nucleoli with fine chromatin pattern Cytoplasm: Distinctive blue color and lacks granules Most iron destined for hemoglobin synthesis is taken into cell at this stage Gives rise to _-_ RBCs

cytoplasmic organelles

nucleolus

24

pink

Erythropoiesis

RBC Stages of Maturation

Prorubricyte/Basophilic normoblast

Nucleus:Cytoplasm ratio: 6:1 Nucleus: Chromatin begins to condense, nucleoli may be present early in this stage but disappear later Cytoplasm: Deeper, richer blue than in pronormoblast Detectable hemoglobin synthesis occurs but the minute amount of hemoglobin pigmentation is masked by _ _ Last stage with _ Lasts slightly more than _ hrs

Nucleus:Cytoplasm ratio: 4:1 Nucleus: Clumped nuclear chromatin, nucleoli no longer apparent Cytoplasm: Basophilic No evidence of _ color that indicates hemoglobin development

pink, gray-blue

Hemoglobin synthesis

mitosis

30

Hemoglobin

Erythropoiesis

RBC Stages of Maturation

Rubricyte/Polychromatic normoblast

Nucleus:Cytoplasm ratio: 4:1 at the beginning and ends with 1:1 Nucleus: Chromatin pattern varies during this stage of development but becomes more condensed by the end Cytoplasm: First stage in which the _ color can be seen, murky _-_ color _ _ increases Last stage capable of _ (3rd stage of RBC/WBC maturation) Lasts approx. _ hrs

Nucleus:Cytoplasm ratio: 1:1 Nucleus: Increased clumping of chromatin Cytoplasm: Muddy, light gray appearance _ appears for the first time

Salmon-pink

Hemoglobin production, nucleus

48

nucleus, pyknotic nucleus

Erythropoiesis

RBC Stages of Maturation

Metarubricyte/Orthochromic normoblast

Nucleus:Cytoplasm ratio: 1:2 Nucleus: Completely condensed Cytoplasm: _-_ color _ _ continues, _ is ejected from the cell in this stage Lasts approx _ hrs Last stage with _ (_ _)

Nucleus: Chromatin pattern is tightly condensed Cytoplasm: Reddish-pink cytoplasm In the later part, nucleus will be extruded from the cell

Salmon-pink

residual messenger RNA

Hgb synthesis

bone marrow

circulation, spleen

deep blue

salmon pink

Stress/shift

Erythropoiesis

RBC Stages of Maturation

Reticulocyte/Polychromatic erythrocyte

Cytoplasm: Predominant color of hemoglobin. _-_ It completes the production of hemoglobin from the _ _ _ Last stage capable of _ _ 3 days 1st 2 days – _ _ 3rd day – _ → _ (removes residual reticulum) Young reticulocytes – _ _ Mature reticulocytes – _ _; full hemoglobinization >3 days: _/_ retics

Cytoplasm: Deep-blue, meshlike network using supravital stain Diffuse reticulum, loss of ribosomes and mitochondria, along with full hemoglobinization of the cell, marks the transition from the reticulocyte to mature RBC

salmon pink

central pallor

Erythropoiesis

RBC Stages of Maturation

Erythrocyte

Cytoplasm: Biconcave, 7 to 8 μm, _ _-staining cell with a _ _

Average diameter: 6 to 8 μm

decreases

decreases, condensed

Nucleoli

Erythropoiesis

RBC Stages of Maturation

As the erythroid precursors mature:

Overall diameter of the cell _

Nuclear diameter _ and the chromatin pattern becomes coarser, clumped, and _

_ disappear

Cytoplasm changes from blue to gray-blue to salmon pink

Felix Seyler

respiratory pigment

65%

25%

1.34 mL

3.47 mg

Erythropoiesis

Hemoglobin Synthesis

It was identified by _ _ in 1862 by discovering the characteristic color spectrum of hemoglobin

Main component of the RBC

Also known as _ _

_ Hgb synthesis = before nucleus is ejected; before metarubricyte

_ = early retics

1 gram of hemoglobin can carry _ _ of oxygen

1 gram of hemoglobin can carry a constant _ _ of iron

protoporphyrin ring

pyrrole rings

iron

Erythropoiesis

Hemoglobin Synthesis

1 Hgb = 4 heme, 4 globin

1 heme = 1 _ _ (4 _ _) + _

Ferroprotoporphyrin IX

Bone marrow

mitochondria

Erythropoiesis

Heme Synthesis

• Heme also known as _ _

• _ _ (85%) & liver

• Occurs in all metabolically active cells with _

ribosomes, cytoplasm

Ribosomes

Alpha & Zeta

Epsilon, Beta, Gamma, Delta

Erythropoiesis

Globin Chains

• The globin portion of hemoglobin is produced on specific _ in the _ of the red blood cells

• The globin in each hemoglobin molecule consists of four polypeptide chains which determine the type of hemoglobin formed

• Site: _

• Chromosome 16: Dictates the production of _ & _

• Chromosome 11: Dictates the production of (4)

zeta, epsilon

alpha, epsilon

zeta, gamma

COMPARATIVE CHAIN COMPOSITION OF HEMOGLOBIN TYPES
Hemoglobin Type	Polypeptide (Globin) Chains
Embryonic (yolk sac) Gower-1 Gower-2 Portland	2 _ + 2 _ 2 _ + 2 _ 2 _ + 2 _
Hemoglobin F (hepatic/newborn)	2 alpha + 2 gamma
Hemoglobin A (adult)	2 alpha + 2 beta
Hemoglobin A2	2 alpha + 2 delta

alpha, gamma

alpha, beta

alpha, delta

COMPARATIVE CHAIN COMPOSITION OF HEMOGLOBIN TYPES
Hemoglobin Type	Polypeptide (Globin) Chains
Embryonic (yolk sac) Gower-1 Gower-2 Portland	2 zeta + 2 epsilon 2 alpha + 2 epsilon 2 zeta + 2 gamma
Hemoglobin F (hepatic/newborn)	2 _ + 2 _
Hemoglobin A (adult)	2 _ + 2 _
Hemoglobin A2	2 _ + 2 _

Oxyhemoglobin

Deoxygenated Hemoglobin

FUNCTIONAL HEMOGLOBINS
_	_
Hgb with Fe+2 bound to oxygen	Hgb with Fe+2 not bound to oxygen
Arterial blood	Venous blood
Bright red	Dark red

Carboxyhemoglobin

Erythropoiesis

Dyshemoglobins

Hemoglobin with Fe++ bound to carbon monoxide

Cherry red

Erythropoiesis

Dyshemoglobins

Carboxyhemoglobin

Color of skin/blood

Carbaminohemoglobin

Erythropoiesis

Dyshemoglobins

Carboxyhemoglobin

Hgb bound to carbon dioxide

Reversible

Erythropoiesis

Dyshemoglobins

Carboxyhemoglobin

Reversible or irreversible?

Methemoglobin

Erythropoiesis

Dyshemoglobins

Hemoglobin with Fe3+ not bound to oxygen

Ferrihemoglobin

Erythropoiesis

Dyshemoglobins

Methemoglobin

Other name

Chocolate brown blood

Bluish skin

Erythropoiesis

Dyshemoglobins

Methemoglobin

Color of skin/blood

Reversible

Erythropoiesis

Dyshemoglobins

Methemoglobin

Reversible or irreversible?

Sulfhemoglobin

Erythropoiesis

Dyshemoglobins

Mixture of oxidized, partially denatured forms of hemoglobin

Greenish tinge Hgb

Mauve lavender blood

Erythropoiesis

Dyshemoglobins

Sulfhemoglobin

Color of Hgb/blood

Irreversible

Erythropoiesis

Dyshemoglobins

Sulfhemoglobin

Reversible or irreversible?

Embden-Meyerhof Pathway

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Maintains cellular energy by generating ATP

Pyruvate kinase

burr

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Embden-Meyerhof Pathway

_ _ deficiency

Presence of _ cells

Hexose-Monophosphate Shunt

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Prevents denaturation of globin of the hemoglobin molecule by oxidation

Bite cells

Culling

Heinz bodies

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Hexose-Monophosphate Shunt

_ _ (pitting) – denatured Hgb to bite cell

_ – engulfing by splenic macrophages

_ _ – denatured Hgb due to lack of G6PD

Methemoglobin Pathway

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Prevents oxidation of heme iron

Luebering-Rapoport Pathway

Erythropoiesis

Metabolic Pathways in the Erythrocyte

Regulates oxygen affinity of hemoglobin

Produces 2,3 DPG

Oxygen Dissociation Curve

Erythropoiesis

Changes in oxygen affinity of the molecule are responsible for the ease with which hemoglobin can be loaded with oxygen in the lungs and unloaded in the tissues

Increased

Decreased 

Decreased

Decreased

Increased

OXYGEN DISSOCIATION CURVE
Shift to the Left (Lungs)	Factors	Shift to the Right (Muscles)
(left only)	pH
	pCO2
	2,3-DPG
	Temperature
	Hemoglobin affinity to oxygen

Leukopoiesis

Is the process by which white blood cells from and develop in the bone marrow and lymph nodes

Basophils

Eosinophils

Neutrophils

Leukopoiesis

Granulocytes

Lymphocytes

Monocytes

Leukopoiesis

Agranulocytes

Basophils

Eosinophils

Neutrophils

Leukopoiesis

Polymorphonuclears

Lymphocytes

Monocytes

Leukopoiesis

Mononuclears

Basophils – minor phagocyte

Eosinophils

Neutrophils

Monocytes

Leukopoiesis

Phagocytes

Lymphocytes

Leukopoiesis

Immunocytes

condensed

Nucleoli

nonspecific, scant, specific

segmented

decreases

Leukopoiesis

• In general, as granulocytes mature:

  • Nuclear chromatin becomes more _

  • _ disappear

  • Abundant basophilic cytoplasm with _ granulation progresses to more _ cytoplasm containing _ granule

  • Nucleus indents and becomes _

  • Overall cell size _ (except promyelocyte > myeloblast)

Myeloblast

Promyelocyte

Myelocyte

Metamyelocyte

Band

Mature Granulocyte

Leukopoiesis

Granulocyte Stages of Maturation

blue, non-granular

Type I

Type II

Type III

Leukopoiesis

Granulocyte Stages of Maturation

Myeloblast

Nucleus:Cytoplasm ratio: 4:1 Nucleus: Round or slightly oval, 2 to 5 nucleoli Cytoplasm: Usually a moderate _ in color and _ Earliest morphologically identifiable granulocytic precursor using light microscope

_ _ myeloblast – no visible granules when observed under light microscopy _ _ myeloblast – shows presence of dispersed primary granules in the cytoplasm (does not exceed 20 granules per cell) _ _ – contain more than 20 granules that do not obscure the nucleus

primary

Leukopoiesis

Granulocyte Stages of Maturation

Promyelocyte

• Nucleus:Cytoplasm ratio: 3:1 to 2:1

• Nucleus: Oval or round with 2 or 3 nucleoli present

• Cytoplasm: Pale blue to basophilic

• Appearance of _ or nonspecific granules/azurophilic

secondary

mitosis

Early myelocytes

Late myelocytes

Leukopoiesis

Granulocyte Stages of Maturation

Myelocyte

Nucleus:Cytoplasm ratio: 1:1 Nucleus: Oval or round with more coarser and condensed chromatin pattern, no nucleoli Cytoplasm: May contain a few patches of blue Appearance of _ or specific granules Last stage capable of _

_ _: May look very similar to promyelocytes except that patches of grainy pale pink cytoplasm representing secondary granules start to become obvious in the area of golgi apparatus _ _: Smaller than promyelocytes

kidney

indentation

Juvenile

Tertiary

½

bone marrow

Leukopoiesis

Granulocyte Stages of Maturation

Metamyelocyte

• Nucleus: Indented or _-shaped

• Cytoplasm: A few nonspecific granules

• 1st stage of nuclear _

• _ cell

• _ granules

• Less than _ indentation

• Seen in _ _

band

circulation

Leukopoiesis

Granulocyte Stages of Maturation

Band

• Nucleus: Elongated or _-shaped, deeply indented

• Cytoplasm: Same as metamyelocyte

• Youngest granulocytic precursor

• Seen in _

• Greater than ½ indentation

Segmented Neutrophil

Eosinophil

Basophil

Leukopoiesis

Granulocyte Stages of Maturation

Mature Granulocyte (3)

7-10

2.5

12

Neutrophils	Remains in the maturation-storage phase for _ days
Eosinophils	Remains in the maturation-storage phase for _ days
Basophils	Remains in the maturation-storage phase for _ hours

Primary (Azurophilic) Granules

Leukopoiesis

Neutrophils

Formed during the promyelocyte stage

Last to be released (exocytosis)

Myeloperoxidase

Leukopoiesis

Neutrophils

Example of primary granule

Secondary (Specific) Granules

Leukopoiesis

Neutrophils

Formed during myelocyte and metamyelocyte stages

Third to be released

B2-microglobulin

Leukopoiesis

Neutrophils

Example of secondary granule

Tertiary Granules

Leukopoiesis

Neutrophils

Formed during metamyelocyte and band stages

Second to be released

Gelatinase

Leukopoiesis

Neutrophils

Example of tertiary granule

Secretory Granules (Secretory Vesicles)

Leukopoiesis

Neutrophils

Formed during band and segmented neutrophil stages

First to be released (fuse to plasma membrane)

Alkaline phosphatase

Leukopoiesis

Neutrophils

Example of secretory granule

Primary Granules

Leukopoiesis

Eosinophils

Formed during promyelocyte stage

Charcot-Leyden crystal protein (degraded eosinophil granules)

Secondary Granules

Leukopoiesis

Eosinophils

Formed throughout remaining maturation

Major basic protein

Eosinophil cationic protein

Eosinophil-derived neurotoxin

Eosinophil peroxidase

Lysozyme, Catalase, B-glucoronidase, Cathepsin D, Interleukins 2, 4, 5, and 6, Granulocyte-macrophage CSF