H E M A T O L O G Y LECTURE

MIDTERM notes

o Colony-Forming Unit (CFU-E)

!!! REMINDERS !!!
 Another 1 week for
1. This is a complete and CFU-E to become
summarized notes from the PRONORMOBLAST, a
book and lecture (CHAPTER 8 first morphologically
ONLY) identified RBC
2. INDI PAG IPASA SA IBAN PARA precursor
LANG NI SA GROUPIES HAHAHA  Cell approximately 3-5
3. GOODLUCK, RMT SOON mitotic division before
maturing further
 Pronormoblast take
ERYTHROPOIESIS approximately another
6 to 7 days to become
TERMINOLOGY:
mature enough to enter
1. ERYTHROCYTE – red blood cell the circulation.
2. ERYTHROBLAST – nucleated precursor  TAKE NOTE: so
of erythrocyte in bone marrow (a.k.a approximately 18-21
NORMOBLAST, refers to the developing days are required to
nucleated cells with normal produce a MATURE RBC
appearance) from the BFU-E
3. MEGALOBLAST – abnormal appearance
ERYTHROID PRECURSORS
of the developing nucleated cells (large
sized cells, related to megaloblastic  NORMOBLASTIC PROLIFERATION is
anemia) similar to the proliferation of other cell
lines, is a process encompassing
THREE NOMENCLATURE FOR NAMING replication (i.e.,division) to increase cell
ERYTHROID PRECURSOR numbers and development from
 ERYTHROBLAST (used primarily in immature to mature cell stages.
Europe)  Pronormoblast - derived via the BFU-E
and CFU-E from the pluripotential stem
 NORMOBLASTIC (used primarily in
cells
United States)
 The pronormoblast is able to divide,
o Descriptive in appearance with each daughter cell maturing to the
 RUBRIBLAST next stage of development, the
o Parallel to granulocyte basophilic normoblast.
development  Each of these cells can divide, with each
of its daughter cells maturing to the
MATURATION PROCESS
next stage, the polychromatic
 Two identifiable progenitors – both are normoblast.
committed to the erythroid cell line  8 to 32 mature RBCs usually result
o Burst-Forming Unit-erythroid
(BFU-E)
 About 1 week for the
BFU-E to mature
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 H E M A T O L O G Y LECTURE
MIDTERM notes
CRITERIA USED IN IDENTIFICATION OF THE
ERYTHROID PRECURSOR
 Nuclear chromatin pattern
 Texture
 Density
 Homogeneity
 Nuclear Diameter
 N:C Ratio
 Presence or absence of nucleoli
 Cytoplasmic color
SIDE NOTES:
 In hematology, a modified
Romanowsky stain, such as
Wright or Wright-Giemsa, is
commonly used.
 The stage of maturation of any
blood cell is determined by
careful examination of the
nucleus and the cytoplasm
As RBCs mature, several general trends affect
their appearance, graphically represents these
trends.
1. The overall diameter of the cell
decreases.
2. The diameter of the nucleus decreases
more rapidly than does the size of the
cell. As a result, the N:C ratio also
decreases.
3. The nuclear chromatin pattern becomes
coarser, clumped, and condensed. The
nuclear chromatin of RBCs is inherently
coarser than that of myeloid precursors.
It becomes even coarser and more
clumped as the cell matures,
ERYTHROPOIESIS (September 9, 2019)
G. Abrera, BMLS 3
developing a raspberry-like appearance,
in which the dark staining of the
chromatin is distinct from the almost
white appearance of the
parachromatin. This
chromatin/parachromatin distinction is
more dramatic than in other cell lines.
Ultimately, the nucleus becomes quite
condensed, with no parachromatin
evident at all, and the nucleus is said to
be pyknotic.
4. Nucleoli disappear. Nucleoli represent
areas where the ribosomes are formed
and are seen early in cell development
as cells begin actively synthesizing
proteins. As RBCs mature, the nucleoli
disappear, which precedes the ultimate
cessation of protein synthesis.
5. The cytoplasm changes from blue to
gray-blue to salmon pink. Blueness or
basophilia is due to acidic components
that attract the basic stain (methylene
blue). These organelles decline over the
life of the developing RBC, and the
blueness fades. Pinkness called
eosinophilia or acidophilia is due to
accumulation of more basic
components that attract the acid stain
eosin. Eosinophilia of erythrocyte
cytoplasm correlates with the
accumulation of hemoglobin as the cell
matures. Thus the cell starts out being
active in protein production on the
ribosomes that make the cytoplasm
basophilic, transitions through a period
in which the red of hemoglobin begins
to mix with that blue, and ultimately
ends with a thoroughly salmon pink
color when the ribosomes are gone and
only hemoglobin remains.
NUCLEUS-TO-CYTOPLASM (N:C) RATIO
 Used to identify and stage red blood
cell and white blood cell precursors.
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 H E M A T O L O G Y LECTURE
MIDTERM notes

 The ratio is a visual estimate of what  CELLULAR ACTIVITY

area of the cell is occupied by the o begins to accumulate
nucleus compared with the cytoplasm. the components
 If the areas of each are approximately necessary for
equal, the N:C ratio is 1:1. hemoglobin
 If the nucleus takes up less than 50% of production. The
the area of the cell, the proportion of proteins and enzymes
nucleus is lower, and the ratio is lower necessary for iron
(e.g., 1:5 or less than 1) uptake and
 If the nucleus takes up more than 50% protoporphyrin
of the area of the cell, the ratio is higher synthesis are produced.
(e.g., 3:1 or 3). Globin production
begins.
ERYTHROPOIESIS-MATURATION
SEQUENCE  LENGTH OF TIME
1. Pronormoblast (Rubriblast) o Slightly more than 24
 First morphologically hours
identifiable RBC precursor
2. Basophilic Normoblast (Prorubricyte)
 NUCLEUS
o (N:C ratio of 8:1)  NUCLEUS
o round to oval, o chromatin begins to
containing one or two condense
nucleoli o parachromatin areas
o purple red chromatin is become larger and
open and contains few, sharper
if any, fine clumps o N:C ratio decreases to
about 6:1
 CYTOPLASM o chromatin stains deep
o dark blue because of purple-red
the concentration of o Nucleoli may be
ribosomes present early in the
o basophilic cytoplasm stage but disappear
later
 DIVISION
o undergoes mitosis and  CYTOPLASM
gives rise to two o Intense dark blue
daughter
pronormoblasts  DIVISION
o undergoes mitosis and
gives rise to two
 LOCATION daughter
o Bone marrow in healthy pronormoblasts
states  LOCATION
o Bone marrow in healthy
states

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 H E M A T O L O G Y LECTURE
MIDTERM notes

disappearance of
 CELLULAR ACTIVITY nucleoli are evidence of
o Detectable hemoglobin progressive decline in
synthesis occurs transcription DNA
o completely mask the
minute amount of  LENGTH OF TIME
hemoglobin o Approximately 30 hours
pigmentation
4. Orthochromic Normoblast
 LENGTH OF TIME (Metarubricyte)
o Slightly more than 24
hours  NUCLEUS
o completely condensed
3. Polychromatic (Polychromatophilic) (pyknotiic)
Normoblast (Rubricyte) o N:C ratio is low
orapproximately 1:2
 NUCLEUS
o N:C ratio decreases  CYTOPLASM
from 4:1 to about 1:1 o Pinkish-orange in color
o Checkerboard o reflects nearly
appearance complete hemoglobin
o no nucleoli are present production

 CYTOPLASM  DIVISION
o Murky gray-blue color o Not capable of division
or muddy light gray due to condensation of
o Combination of chromatin
multiple colors
 LOCATION
 DIVISION o Bone marrow in healthy
o Last stage of mitosis states

 LOCATION  CELLULAR ACTIVITY

o Bone marrow in healthy o the nucleus is ejected
states from the cell
o loss of vimentin, a
 CELLULAR ACTIVITY protein responsible for
o Hemoglobin synthesis holding organelles in
increases, and the proper location in the
accumulation begins to cytoplasm
be visible in the color of o The enveloped
the cytoplasm. extruded nucleus,
o Ribosome is still called a pyrenocyte,1 is
present then engulfed by bone
o condensation of the marrow macrophages
nucleus and

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 H E M A T O L O G Y LECTURE
MIDTERM notes

using supravital stain

 LENGTH OF TIME (NMB) and wright stain
o Approximately 48 hours o When so stained, the
polychromatic
5. Polychromatophilic Erythrocyte erythrocyte is called a
(Reticulocyte) reticulocyte.

 NUCLEUS  LENGTH OF TIME

o no nucleus o Bone marrow – 1 day
o Circulating blood – 1
 CYTOPLASM day
o Contains small amount
of RNA, Golgi apparatus 6. Erythrocyte
remnants, residual
mitochondria  NUCLEUS
o the cell is the same o no nucleus
color as a mature RBC
o shape of the cell is not  CYTOPLASM
the mature biconcave o Diameter: 7 to 8 mm
disc but is irregular (based on the book)
7.2-7.9 mm (based on
 DIVISION the lecture)
o Lacking a nucleus, the o Thickness: 1.5-2.5 um
polychromatic o Salmon pink in color
erythrocyte cannot o central pallor is about
divide. one third the diameter
of the cell
 LOCATION o biconcave disc
o resides in the bone o smooth and round
marrow for 1 day or edges
longer and then moves o shape facilitates oxygen
into the peripheral and carbon dioxide
blood for about 1 day transport
before reaching
maturity  DIVISION
o retained in the spleen o No division
for pitting of inclusions
and membrane  LOCATION
polishing o Mature RBCs remain
active in the circulation
for approximately 120
 CELLULAR ACTIVITY days
o Reticular network of  CELLULAR ACTIVITY
strands can be o Hemoglobin is the main
observed inside the cell cell component

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 H E M A T O L O G Y LECTURE
MIDTERM notes

o delivers oxygen to  thermostable, nondialyzable,

tissues, releases it, and glycoprotein hormone
returns to the lung to  produced by the kidney
be reoxygenated  consists of a carbohydrate unit that
reacts specifically with RBC receptors
and a terminal sialic acid unit, which is
ERYTHROKINETICS necessary for biological activity in vivo
 dynamics of RBC production  ACTION OF EPO:
and destruction o EPO is a growth factor that
 Erythron initiates an intracellular
o Name given to a message in developing RBCs
collection of all stages (signal transduction)
of erythrocytes o EPO’s effects are mediated by
throughout the body Janus-activated tyrosine kinase
 The erythron is the entirety of 2 (JAK2) signal transducers
erythroid cells in the body,  MAJOR EFFECTS:
whereas the RBC mass refers o Allowing early release of
only to the cells in circulation reticulocytes from the bone
 Unified functional tissue marrow
o Bone marrow o Preventing opoptotic cell death
o Peripheral blood o Reducing the time needed for
o Vascular spaces within cell to mature in the bone
the specific organs marrow

STIMULUS TO RED BLOOD CELL EFFECTS OF ERYTHROPOIETIN

PRODUCTION 1. PROMOTES EARLY RELEASE OF
 HYPOXIA RETICULOCYTES
 Oxygen-sensing system  Mechanism:
o Located in peritubular i. EPO induces changes in
fibroblasts of the kidney the adventitial layer of
 Detected by the the marrow/sinus
peritubular cells which barrier that increases
produces the width of the spaces
Erythropoietin (EPO) for the RBC egress into
 Erythropoietin is the the sinus.
major stimulatory ii. EPO downregulates the
cytokine for RBC expression of the
 EPO production increases by membrane receptors
transcriptional regulation if there is: for adhesive molecules
o Hemorrhage so that the cells can exit
o Increased red cell destruction the marrow earlier that
 The EPO gene has a hypoxia-sensitive the normal.
region (share ko lang)

ERYTHROPOIETIN

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 H E M A T O L O G Y LECTURE
MIDTERM notes

 Result: EFFECTS OF ERYTHROPOIETIN

o Presence of shift  INHIBITION OF APOPTOSIS
reticulocytes in the  EVASION OF APOPTOSIS
peripheral circulation o Indirect avoidance
apoptosis
2. INHIBITION OF APOPTOSIS  EPO removes
 Apoptosis – programmed cell apoptosis induction
death signal
 Increase RBC production o Death receptor Fas
 BFU-E RBC (it takes 18-  Molecule of the
21 days) external
 More CFU-Es are produced than messaging
needed system
i. 8 to 10 days headstart  Expressed in
 If Steady-state of demand Is membrane of
reached CFU-Es are allowed to earliest
die precursor cells
 PROCESS OF APOPTOSIS  FasL (Fas
o Degradation of chromatin ligand) is
into fragments of varying expressed in
sizes membrane of
o Activation of more mature
transglutamase cells
o Morphologic changes: o EPO level  dec
 Condensation of production 
nucleus apoptosis
 Nuclear o Polychromatophilic
disintegration normoblastic (FasL)
 Shrinkage of cell cross linked with
volume pronormoblast and
 Increase in cell basophilic normoblast
density (Fas-marked)
 Compaction of o Direct EPO rescue from
cytoplasmic apoptosis
organelle  Major way for
 Mitochondria EPO to increase
remains normal RBC prdxn.
 Partitioning of  EPO binds to
cytoplasm and the receptor on
nucleus into the CFU-E
membrane bound (reduce FasL
apoptotic bodies production)
(blebbing)  EPO is able to
stimulate
production of
various anti-

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 H E M A T O L O G Y LECTURE
MIDTERM notes
apoptotic
molecule (Bcl-
2like-protein)
 REDUCE MARROW TRANSIT TIME
o Mechanism:
Increase rate of cellular
processes:
-Accelerated Hb production
-Accelerated process of Bone
marrow egress (loss of adhesiiive
receptors and acquisition of egress-
promoting surface molecule)
-Reduce cell cycle time up to
20% reduction:
Pronormoblast  Reticulocyte
(6 days)
-Fewer mitotic divisions:
6 days  4 days
-Shift reticulocytes (stress)
 OTHER STIMULI
o Testosterone
o Pituitary hormone
o Thyroid hormone
ROLE OF MACROPHAGES IN
ERYTHROPOIESIS
 Provides iron directly to the
normoblasts for the synthesis of
hemoglobin
 Major cellular anchor for RBC
 Components of anchoring system:
o A stable matrix
 FIBRONECTIN
o Bridging molecule for
attachment (adhesive)
o Receptors on erythrocyte
membrane for adhesive
molecule
ERYTHROCYTE DESTRUCTION
 Deterioration of enzyme
o Decline of cellular function
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G. Abrera, BMLS 3
 Loss of glycolytic enzymes
 Senescence – process of cellular aging
which culminates in phagocytosis by
macrophages
MACROPHAGE-MEDIATED HEMOLYSIS
(EXTRAVASCULAR HEMOLYSIS)
 90%
o Spleen bone marrow
o Liver lymph nodes
 Red pulp  cell flow stagnation 
deplete glucose  slow glycolysis 
reduced ATP
 Movement through the red pulp is
sluggish
 available glucose in the surrounding
plasma is depleted quickly as the cell
flow stagnates, so glycolysis slows
 pH is low, which promotes iron
oxidation and accelerate catabolism of
enzyme
 Reduced ATP
o Oxidation of membrane lipids
and proteins
o Failure of Na-K pumps:
 Intracellular Na –
Increase
 Extracellular K –
Decrease
 Spherocytes
o Less flexible/rigid ---
phagocytosis
MECHANICAL HEMOLYSIS (Fragmentation of
Intravascular Hemolysis)
 10%
 Blood vessel
o Traumatic or mechanical
 Indicates serious condition that
must be address promptly
END OF CHAPTER 8
8