Hematopoiesis 2

Mr. Normel Adarve) ǀǀ 08/18/20

Transcribers: Ley Andrea Arranz, Joeperl C. Vedadero

RETICULOCYTE Question. What happens to the reticulocyte when there is a

severe hypoxia/thalassemia?
• doesn’t have nucleus
- EPO is released in a VERY HIGH concentration in the blood
• not yet fully mature cell
-- reticulocytes are forced to be released early
• Ave size: 8-12µm
-- they aren’t as mature
• Cytoplasmic color: Purple, no
--metarubricyte can also be released earlier, which is
central palor
- stains usually used: abnormal
Romanowsky, Wright/Giemsa,Dipquick Method
Blood Cell Reporting
- the blueness of the cytoplasm is because of the remnants
of RNA contained in the ribosomes (inclusion bodies) • Presence of metarubricyte in blood smear and other
- reticulocyte remnants can also be called as RNA immature cells is collectively reported as presence of
remnants or Fine basophilic stippling Nucleated Red Blood cells (NRBC) in the blood smear
- mitochondria is still within the cytoplasm, collectively  ex. 10 NRBC per 100 WBC
they’re called as Reticulocyte remnants - NRBC is not part of the 100 WBC observed
* Organelles left in the cytoplasm: ribosome & mitochondria Consequences of NRBC presence
- these remnants should be removed gradually  machine sometimes cannot differentiate NRBC like
- as Reticulocyte enters the blood stream or circulatory metarubricyte to lymphocyte because of their almost
system, it should mature by going to spleen similar cytoplasmic appearance
• Spleen will remove all the inclusions in it by 24 - results to ↑ in lymphocyte count
hours - False lymphocytosis or Relative lymphocytosis
- through its Pitting function of the spleen’s (“cytosis” is the increase number of cell)
macrophage  if lymphocyte count is increased, WBC count will also
- removes the inclusions, whether fine or coarse, as be false increased
RBC should NOT have any inclusions in its cytoplasm • Do corrected WBC count.
Stipplings & Their Removal
(a) Fine basophilic stippling Stress cells – shift cells or shift reticulocytes that are still large
- physiologic or normally seen in reticulocytes and is gradually (immature) and released immediately
removed by the spleen - they mix with normal sized RBC ➟ Macrocytosis
- Macrocytes is associated with immature reticulocytes
(b) Coarse basophilic stippling -- Macrocytosis may also be seen in Reticulocytosis
- abnormal large inclusions in reticulocytes because of certain -- Reticulocytosis may also be seen in Hemolytic anemia
disorders such as
 Thalassemia MEAN CELL VOLUME (MCV)
 Megaloblastic Anemia - parameter that gives us the idea on the RBC size
 Sideroblastic anemia - measures the volume and also somehow the size of the cell
- Example of large inclusion: Howell Jolly Bodies - Reference value: 80-100 femtoliter (fL)
- unfortunately because they are large, the membrane of red -- average volume of a single RBC
cells are also bitten along with the inclusions and will lyse, this  if there is more than 100fL, it means that the red
event is called/may lead to Hemolytic anemia cells are HIGH in size – Macrocytic red cells
- RBC dies earlier than expected in the spleen.  if there is less than 80fL, it means that the red cells
- early destruction of RBC may be due to several factors are SMALL – Microcytic red cells
 if the size ranges from between 80 to 100, it means
Hemolysis – happens normally for old red cells (normal life the size is NORMAL– Normocytic red cells
span is 120 days, Red Cell Turnover)  if we see varying size of red cells, it refers to
Hemolytic Patterns Anisocytosis
 if it happens OUTSIDE the blood vessels, usually at
the spleen it refers to Extravascular hemolysis Volume Size Description
 if it happens INSIDE the blood vessels, it refers to > 100 fL Large Macrocytic
Intravascular hemolysis
< 80 fL Small Microcytic
• Reticulocytes normally stays in the bone marrow for 2 days,
80 - 100 fL Normal Normocytic
and eventually will go to the blood circulation and will stay for 1
Varying Anisocytosis
day to be mature RBC = 3 days

EPO: Cell Development EPO: Prevention of Apoptosis

Question. How is reticulocyte released from the bone marrow • It takes 18-21 days for BFUE to become a mature RBC.
to the blood circulation? - too long
- EPO continuously stimulates,helps in the release of • An abundant (excess) CFUE in our bone marrow must be
reticulocyte stored in cases of hypoxia.
(a) Blood Loss: CFUE will be used instead of BFUE(long
 In the Bone Marrow, reticulocyte has a receptor in their
development)
membrane and it consist of sticky protein called
(b) If the maturation of cells are normal, some of the CFUE
Fibronectin which helps the cells to be confined in the
will eventually die by apoptosis and will be eaten by
bone marrow
macrophages – chromatin degenaration or karyorrhexis
 but because of the stimulation of EPO, the fibronectin will
Necrosis – pathologic death of cells
gradually diminish,if receptors of fibronectin is totally gone,
reticulocyte becomes loose and goes out.
 Hematology Lec 02: Hematopoiesis 2
• More mature cells (Prorubricyte, Rubricyte) has a molecule in - staining a stain while it’s alive
their membrane FASL Ligand - the stain innervates the membrane of red cells
• The more immature cells (CFUE, BFUE, Rubriblast) what - Stain is directly added to the whole blood, fixative is not
they have in their cell membrane is death receptor known as needed.
FAS receptor where FASL ligand will bind. Two Supravital stains
(a) During normal development of Red cells, the time in 1. Brilliant cresyl blue (BCB) / Bromocresol blue - better stain
contact with CFUE is longer. FASL Ligand (Prorubricyte) 2. Methylene blue
usually bind with the FAS Ligand of immature red cell
- constant contact, cross reaction of FASL & FAS • Unlike, blood smear stain where fixation is done after
- when in contact, immature cells die smearing.
- one way Bone marrow controls RBC production - Wherein, fixation kills the cells to preserve morphology.
(b)In Hypoxia, Prorubricyte and Rubricyte are forced to
mature for early release. • Inclusion with DNA remnants is the Howell Jolly Bodies.
- shorter time for the mature - immature cell interaction - Special stain/reagent - Fuel gen Reaction - is used to
- activation of FAS Ligand & FAS to produce cell death is confirm if presence of DNA remnants.
gone.
- Immature cells will have more time to mature. Hemoglobin Synthesis
• This prevention is an External factor / Extrinsic • Starts at rubriblast - prorubricyte.
(cell-cell interaction) • Completes at rubricyte.
• EPO cells in contact with CFUE • Small hemoglobin synthesis still happens in Reticulocyte
- internally, cell death is prevented - Reticulocytes have RNA remnants (ribosome & mitchondria)
- This prevention is an Internal factor / Intrinsic
- activation of JAT II which activates the, • Heme is formed in the mitochondria of maturing RBC.
Stat pathway Heme composition: Heme ring + iron
– “Signal Transduction Activator of Transcription” - Protoporphyrin IX and ferrous iron
– activating the genes of that cell to prevent cell death (anti- (a) Heme ring is is where the Iron atom is inserted (middle)
apoptotic genes) - other name: Protoporphyrin IX (P9)
 activating the mitochondria to prevent the release of (b) Iron should be ferrous state (+2 charge), has a free
Cytochrome C, which is one of the chemical that binding site for oxygen
allows cell death - Ferric iron (+3 charge), oxygen can’t bind
 molecule that is stimulated by stat is BCL-XL gene or
BCL2 Protein Light 1 gene

Anemia
(a) Hemolytic Anemia
- early destruction of red cells,↑ Reticulocytes
- ↑ Reticulocytes = ↑ Polychromasia
* Polychromasia – associated with high level of
reticulocyte in the blood
(b) Non-hemolytic type
- growth maturation of red cell in bone marrow (production &
development)
- ↓ Reticulocytes = ↓ Polychromasia
• Ribosomes - protein synthesis, formation site of globin
- no reticulocytosis or decrease polychromasia
 Bone Marrow Failure - fails to produce enough remnants
erythrocyte;
 Kidney Failure - there will be no EPO that will Question: What’s the last stage of maturing red cells where
stimulate stem cells to be red blood cells hemoglobin is still being synthesize?
Reticulocyte
 Iron deficiency Anemia and Megaloblastic anemia
- red cells fail to mature normally, develops poorly
- ↓ production production, ↓ reticulocytes release ERYTHROCYTE
Reticulocyte cnt Type • Cytoplasm stains completely
Anemia Low Non-Hemolytic red.
High Hemolytic • no hemoglobin synthesis,
•Eg. Thalasemmia is a Hemolytic anemia - because RNA remnants are
•Significance of Reticulocyte cnt. is to classify anemia gone as it travels to the spleenic
environment
Relative Reticulocyte cnt. • main content of cytoplasm is
Adult hemoglobin
- In every 1000 red cells, only 0.5-1.5% (2%) of reticulocytes. • no organelles
Newborn or reaching 3 months - if they’re designed to have a mitochondria, they’ll compete
- In every 1000 red cells, 2-6%of reticulocytes. in consuming oxygen for ATP synthesis instead of delivering it.
• designed to produce energy without using oxygen
Severe anemia - presence of NRBC (metarubricyte) is • Life span: 120 days
possible • Normally and Pathologic death in the spleen.
• MCV is 80-100 fL. , Ave Volume: 90 fL
Internal Structure Stains
• To visualize internal structures - Fine Basophilic Stipplings,
- use supravital stain(can’t be visualized using routine stains)
 Hematology Lec 02: Hematopoiesis 2
Why is the nucleus removed?
1. Absence of nucleus allows blood transfusion easily. Question: Which of the following is not related to the effects of
• Membrane of nucleated cells have MHC molecules erythropoietin?
- Major Histocompatibility molecule A. The number of divisions of a normoblast.
- MHC is diverse in individuals. B. The formation of pores in sinusoidal endothelial cells for
-- reason why organs transplants are hard. marrow egress - ANSWER (egress or exit)
- Immune System knows the body’s own MHC. - * Bone marrow and blood vessels has tight boundaries. Any cell
-- Rejection of organs: Immune system identifies the that pass through enters the pores of sinusoid. EPO loosens these
transplanted organ as foreign. pores.
Transplant pt ➟ immunosuppressant ➟ prone to infection ➟ C. The time between mitosis of normoblasts
antibiotic ➟ kidney/liver damage D. The production of antiapoptotic molecules by erythroid
- For the body to not reject the organ, immune system is lowered. progenitor.
Individual is more prone to infection. Antibiotics is administered. LEUKOCYTOPOIESIS
Medications can damage both the kidney and immune system.
• Without the nucleus, MHC is also gone. • White cell production
• Compatibility testing in blood is also easier. • CSF factors - growth factors necessary for the formation of
monocytes and granulocytes. (CSF-G, CSF-M)
2. To not disturb - SCF, IL 3, IL 6, IL 5
• RBCs squeeze in blood vessels. Basophil IL 3, IL 6, CSF-G
- if with nucleus, they won’t be as flexible Neutrophil IL 3, IL 6, CSF-G
- may block blood vessels, won’t reach deeper tissues Eosinophil IL 3, IL 5, CSF-G
• Flexibility and prevent blockage Monocyte IL 3, IL 6, CSF-M

Cell Palor & Cell Size GRANULOCYTOPOIESIS

• 6 stages from Myeoloblast to Mature Granulocytes.
Red Cell Palor - site where nucleus was - 3 to 5 Mitosis
once located • CFU Granulocyte-Monocyte - Stem cells for granulocytes
(a) Normal Size: 1/3 of Total
General Events during Maturation
diameter
of RBC or less than 3µm- Normochromic • Decreasing size as it matures.
• Cytoplasmic color: from basophilia decreases to lilac
(b) Larger/Increased: indicates there’s not much • Granulation in cytoplasm increases.
hemoglobin in the RBC - Hypocromic • Chromatin material: fine to compact
(c) Absent/Decreasesd: Hypercromic • Nucleoli: visible to not visible
• N:C ratio decreases.
• Size of mature RBC: 6-8 µm (diameter) - Size is not a good distinguishing factor(overlaps).
- Cytoplasmic & nuclear appearance
 Large – Macrocytic red cells
 Small – Microcytic red cells
Pools of Granulocyte in the Bone Marrow
 Within the range - Normocytic red cells
• Sized can be estimated by comparing it to normal sized (a) Mitotic Pool - can divide
small lymphocytes (min. 8µm) - Myeloblast, Promyelocyte, Myelocyte
- relative cell: Small lymphocyte (b) Maturation Pool
- Metamyelocyte, Band/Stab Cell, Mature Granulocytes
Anemia MYELOBLAST
• Some anemia is characterized with normal sized red cell. • Cytoplasmic color: Blue (basophilic)
• While,some is associate with decreased / increased red Type 1 w/o granules
cell size. Type 2 w/ granules < 20 Granules
- Microcytic Anemia Type 3 w/ granules > 20 Granules
1. Iron deficiency anemia
2. Thalassemia • Nuclear Characteristic: Nucleus has
3. Anemia of Chronic disease 2-4 visible nucleoli with fine
4. sideroblastic anemia Chromatin material.
- Macrocytic Anemia • N:C Ratio: 8:1 or 4:1
1. Megaloblastic anemia • Distribution in bone marrow tissue: 1-3%
• Size: 14-20 µm
Question: Which of the following is the most mature PROMYELOCYTE
normoblast? • Distinction from Myeloblast: primary granules in cytoplasm
* Normoblast - immature cell • Cytoplasm:Primary granule synthesis (start-end)
Answer: Metarubricyte (orthochromatophilic) - Azurophilic granules = Blue
- Non-specific granules, some of its
Question: Which among the red cell precursors has almost contents can also be found in monocytes.
1:1 ratio, with a murky gray-blue cytoplasic hue and with no (Myeloperoxidase enzyme)
distinct nucleoli? - Primary granules continuously present
A. Metarubricyte - grey, blue,red cytoplasic color (1:2) • Nuclear Characteristic: Nucleus has
B. Rubricyte - ANSWER (late rubricyte) 1-3 visible nucleoli, slight compactness of Chromatin
C. Reticulocyte • N:C Ratio: 7:1 or 4:1
D. Prorubricyte • Distribution in bone marrow tissue: 1-5%
• Size: 16-25 µm
 Hematology Lec 02: Hematopoiesis 2
* Myeloblast & Promyelocyte - No indentation and the nuclear • When WBC marginal pool goes to circulating pool, ↑ WBC
orientation is eccentric or sometimes adjacent to the cell cnt.
membrane. 1. Physiologic factors:
MYELOCYTE • Anxiety, stress, exercise
• Nuclear shape & orientation: ovoid, centrally or eccentrically 2. Medications
located, not adjacent to the cell membrane • makes marginal pool detached and go to the circular pool
• Nuclear Characteristic: Nucleus has no • cortisol, epinephrine
visible nucleoli, moderate compactness • Lithium-containing drugs,phenacetin,digitalis-containing.drug
of Chromatin material. - ↑ neutrophil cnt
• Cytoplasm: Lilac
• N:C Ratio: 6:1 or 5:1 • Lobes in the nucleus of mature granulocytes are connected
• Granules: Primary + Secondary by a thin filament
- Secondary / Specific Granules NEUTROPHIL
- Reddish granule • Half-life of in Blood - 7 days
- content is specific to a cell type • Lobes: 3-5, each lobe is interconnected by a filament
- its color indicates the future fate of the cell - indicates maturity of cell
Cell Type Color Distribution in < 3 lobes - Hyposegmented neutrophil
BM > 5 lobes - Hypersegmented neutrophil
Myelocyte- neutrophil purple 6-17% * Struggle in identifying between a band cell
Myelocyte- eosinophil red 3-5% and a granulocyte , automatically report it
Myelocyte- basophil blue <1% as Mature Granulocytes.
•“Dawn of Neutrophilia” - when staining (eg. Neutrophil), lobes may
• No identation. stack making it hard to see the thin
METAMYELOCYTE filaments
• May migrate in the tissues in cases of
• Presence of identation ➟ Juvenile Cell
infection.
• Identation size: 1/3 of total diameter of
- Duration depends on the stimulation of infection
nucleus
EOSINOPHIL
• Nuclear shape & characteristics:
horse/kidney shape, no visible nucleoli, • Lobes: 2-3
highly compressed chromatin materials • Differentiate neutrophil & eosinophil
• Cytoplasm: Lilac by their granules
• N:C Ratio: 4:1 - EOSINOPHIL: Large bright red-
• Granules: Primary + Secondary + Tertiary orange granules
- Tertiary Granules (specially neutrophil) • Half-life of in Blood - 18 hours
- Contents (Acid phosphatase, alkaline phosphatase) • May migrate in the tissues for 2-5
- used by the WBC to kill ingested organisms days.
Cell Type Color Distribution in BASOPHIL
BM • Lobes: 2-3, hard to visualize as it’s
Myelocyte- neutrophil purple 3-5% covered by its blue granules
Myelocyte- eosinophil red 1-3% - Granules are water-soluble, may be
Myelocyte- basophil blue <1% wash out
• Life span -60 hours
BAND/STAB CELL • May migrate in the tissues, doesn’t
• Deeper indentation stay long in the blood
• Indentation size: 1/2 to 2/3 of total diameter of nucleus MONOCYTOPOIESIS
• Nuclear width: almost the same in all area • 3 Stages from Monoblast ➟ Promonocyte ➟ (immature)
• Cells become Band-Neutrophil, monocyte
Band-Eosinophil, Band-basophil. • Size: 12-20µm (fairly similar for all)
• Secretory granules may form here. - Monocyte: 12-18µm
• N:C Ratio: 4:1 • Cytoplasmic color: Red, Blue, Grayish
• Distribution in bone marrow tissue: 9-32% • Chromatin material: fine, lacy
• Distribution in blood (allowed): 0-5% Cell Nucleus N:C Ratio
- ↑ Band cell in Blood = Leukemoid reaction Monoblast nucleoli can still be observed 8:1
- LR: Body’s response/overreaction because of severe Promonocyte presence of identation, Nuclear 6:1 to 4:1
fissure
infection.
Monocyte evident nuclear indentation 5:1
- Immature WBC are pushed to go into the blood stream
- Band Stem or Metamyelocyte can be seen accidentally. * vacuolations in the cytoplasm may be seen in maturing
- Presence of Myeloblast, promyelocyte, or myelocyte may be monocyte
a sign of Leukemia. • Cells are not categorized in pools. Monoblast will be released
Band Cell ➟Granulocyte ➟ Blood early
• Only stays in the blood for 3 days, and goes to the tissues
GRANULOCYTES - Monocyte is NOT the end stage/cell.
- In the tissue,it becomes a macrophage (last) for several
Pools of Granulocyte in the Blood
days
(a) Marginal Pool - Macrophage assume different names
- margining the blood vessel •Kupffer cell s (Liver), Alveolar macrophage (lungs), Dendritic
- attached in the blood vessel walls cells (spleen) Liver - 21 days - 30-5) Microglial cells (CNS),
(b) Circulating Pool Langerhans cellls (skin)
- blood that’s extracted - Larger 40-50µm (diameter)
 Hematology Lec 02: Hematopoiesis 2

PROMEGAKARYOCYTE
• Size: 30-80µm
• Cytoplasmic color: blue (basophilic)
• Nucleus: 2 nucleus
• Has cytoplasmic tags
• With primary granules
- granules synthesis starts here
- slight number of granules
• N:C Ratio: 1:1 or 1:2
LYMPOPOIE
• Bone marrow & thymus are primary organs for lymphocyte MEGAKARYOCYTE
development. • Size: 30-100µm
• Lymphocyte is developed in the bone marrow • Cytoplasmic color: purple/lilac
• Some of the lymphocytes will go to the thymus for direction. • Nucleus: 2 or 4 nucleus
- nucleus MUST have even
• GF: FLT3 Ligand, IL (4,5,7)
number of maturing
• Two types of Lymphocyte: small (basis for RBC), large (eg.
• Moderate number of granules
NK cells)
- granules increases, additions
• Cytoplasmic color: Blue (basophilic)
are formed
• Chromatin Material: Highly compact
• w/o cytoplasmic tags
Cell Nucleoli Size
• N:C Ratio: 1:12
Lymphoblast 2-4 visible nucleoli 15-20µm
Prolymphocyte 1-2 visible nucleoli 12-15µm , 15-18µm
METAMEGAKARYOCYTE
Lymphocyte no visible nucleoli Small - 8-15µm
(small/large) Large - 12-15µm • Size: 30-100µm
• Lymphocyte (small) is NOT the end stage cell. • Cytoplasmic color: lilac
- they’ll transform to a more specific cell • Nucleus: 4 or more (16 -32), Highly
- eg. B-cell, T-cell, B-Memory cell, plasma cell, effector cells compact chromatin
- depending on the stimulation of the pathogenic organisms • Granulation is aggregated, high in
• Maturation of the Lymphocyte can be, (Lymphocyte numbers
differentiation) • w/o cytoplasmic tags
(a) Antigen dependent • N:C Ratio: 1:12
(b) Antigen independent • For every metamegaryocyte 2000 to 4000 platelets are
produced.

Platelets - form from the fragmentation of metamegakaryocyte

• Size: 2-4µm (diameter)
• Cytoplasmic color: (In Blood Smear) Reddish pink to purple
with purple or blue granules

MEGAKARYOPOIESIS
• Platelet production
• requires 5 -7 days to mature from megakaryoblast to
metamegakaryocyte.
• Division is only associated with the nucleus and not the Theories of Platelet release:
cytoplasm- Endomitotic Division (a) Platelet Shedding Theory
- One megakaryoblast matures to become one - Metamegakaryocyte tries to shed/extrude its cytoplasm
metamegakaryocyte. (b) Fragmentation Theory / Platelet Budding
- Number of nucleus increases, but NOT the cell number. - platelets are detached in the cytoplasm and goes to the
• Size increases.
• N:C ratio decreases. circulation
MEGAKARYOBLAST
• Size: 20-50µm
• Cytoplasmic color: blue
• Nucleus: 1 nucleus, 1-2 visible
nucleoli, with fine chromatin material
• No granules
• Has cytoplasmic tags / protrusion in
cell membrane
• N:C Ratio: 1:1