MTAP II: HEMATOLOGY 1


GLOBIN CHAIN COMPOSITIONS
CBC (COMPLETE BLOOD COUNT)
Quantitative determination that yields qualitative interpretations
Dependent on Sex, Age, Population
 Variables that dictate the reference ranges in a CBC
PARAMETERS OF A CBC REPORT

RESULTS SEEN ON AUTOMATED CBC:

RESULTS SEEN ON MANUAL CBC:
o Hematocrit
o RBC count  derive the value for Hemoglobin (rule of 3)
o WBC count
o Platelet count

Qualitative reporting

No numerical equivalent value
*Red cell indices NOT included on manual CBC results
🔔 Rule of Three

Applicable only to normocytic / normochromic RBCs
RBC INDICES
INDEX
MCH
(Mean Cell Hemoglobin)
MCV (Mean Cell Volume)
RDW
(Red cell Distribution Width)
CORRELATION
Color
 Color is attributed to Hgb content or RBC
Size
Uniformity / heterogeneity in size
 Indicates possible anisocytosis
🔔 Thalassemia: Disorder of globin chain synthesis

Absent globin chain is substituted by delta chain instead
CELLULARITY OF BONE MARROW
SOME CLINICAL CORRELATIONS OF RBC INDICES/ CBC RESULTS
Disease
Aplastic Crisis (aplasia)
Blastic crisis (i.e. leukemia)
Myelofibrosis
(deposition of fibrous connective tissue
= replaces bone marrow tissue)
Effect
 RBC indices
All values DECREASED
 WBC count
 RBC & Plt
Values DECREASED
REVIEW OF HEMATOPOIESIS
Child: Red bone marrow
(red-shaded areas) is
located throughout the
skeletal system
Adult: Yellow marrow
replaces red marrow in
the adult skeletal system
(Retrogression @ 4-7
y/o). Red marrow activity
occurs in the central
portion of the skeleton.
(sternum,
vertebrae,
scapulae, pelvis, ribs, skull, and proximal portion of the long bones)
and CD8+ T
cells
Suppress Treg responses,
mediator of
immune
tolerance
Indications for bone marrow studies:

Blastic crisis suspicion (High
WBC & diff Count)

Anemia (only if secondary to
leukemic disorders)

Observation of M:E ratio
🔔 Where is the most ideal site of
collection for bone marrow studies?

Superior ileac crest
It is the Safest area to
puncture
BONE MARROW STUDIES
Core
biopsy
Aspiration
Trephine
biopsy needle
Aspiration
needle
COLLECTION
Jamshidi needle
University of Illinois
sternal needle
IL-3
Taking out a
fraction of a bone
Just aspirating
bone marrow
M:E RATIO OBSERVATION
(Myeloid-to-Erythroid precursor ratio)
Normal
Infection
Leukemia
2:1 – 4:1
6:1
25:1
(ave 3:1)





NORMAL CELLS FOUND IN THE MARROW
All developing hematopoietic
cells
Macrophages
Mast cells
-Make use of blood as transport
Confused w/ basophils
Osteoblasts
-Young bone cells Confused w/
megakaryocyte
Osteoclasts
-Mistaken for Plasma cells
IL-6
Activated Tcells, NK cells
T cells
Macrophages
Fibroblasts
Hematopoieti
c stem cell
and
progenitors
Proliferation of
hematopoietic
progenitors
T cells
B cells
Liver
Co-stimulation
with other
cytokines,
cells growth/
activation of T
cells and B
cells,
megakaryocyt
e maturation,
neural
differentiation,
acute phase
reactant
IL-10
CD4+
Th2 T cells
Monocytes
Macrophages
T cells
Macrophage
Inhibits
cytokine
production,
inhibits
macrophages
IL-12
Macrophages
T cells
T cell, Th1
differentiation
IL-15
Activated
CD4+ T cells
CD4+ T cells
CD8+ T cells
NK cells
CD4+ / CD8+
T cell
proliferation
CD4+ / NK cell
cytotoxicity
IFN-a
Dendritic cells
NK cells
T and B cells
Macrophages
Fibroblasts
Endothelial
cells
Osteoblasts
Macrophage
NK cells
Antiviral,
Enhances
MHC
expression
CYTOKINES INVOLVED IN HEMATOPOIESIS
Cytokine
EPO
Source
Peritubular
interstitial cells
G-CSF
Endothelial
cells
Placenta
Monocytes
macrophages
GM-CSF
T-cells
Macrophages
Endothelial
cells
Fibroblasts
Mast cells
IL-2
CD4+ T-cells
NK cells
B cells
Target cell
BFU-E
CFU-E
Neutrophil
precursors
Fibroblasts
Leukemic
myeloblasts
Bone
marrow
Progenitor
cells
Dendritic
cells
Macrophage
NKT cells
T-cells
NK cells
B cells
Monocytes
Effect
Stimulates
proliferation of
erythroid
progenitors
and prevents
apoptosis of
CFU-E
Stimulates
granulocyte
colonies,
differentiation
of progenitors
towards
neutrophil
lineage and
maturation
Promotes
antigen
presentation,
T-cell
homeostasis,
hematopoietic
cell growth
factor
Cell
growth/activati
on of CD4+
Therapeutic
Applications
- Anemia of
chronic disease
-chronic anemia
patients
-Treatment of
anemia in cancer
patients on
chemotherapy
-Autologous
predonation blood
collection
-Anemia in HIV
infection to permit
use of zidovudine
(AZT)
-Post autologous
hematopoietic
stem cell
transplant
Chemotherapyinduced
neutropenia Stem
cell mobilization
Peripheral
blood/bone marrow
transplantation
Congenital
neutropenia
Idiopathic
neutropenia Cyclic
neutropenia
Chemotherapyinduced
neutropenia Stem
cell mobilization
Peripheral
blood/bone marrow
transplantation
Leukemia treatmen
Metastatic
melanoma
Renal cell
carcinoma
Non-Hodgkin
lymphoma
Asthma
-Stem cell
mobilization
-Postchemotherapy/
transplantation
-Bone marrow
failure state
-Stimulation of Plt
production
(but not at
tolerable doses)
-Melanoma
-Renal cell
carcinoma
-IL-6 inhibitors may
be promising
-target
lymphokines in
prevention of Bcell
lymphoma and
EBV
-lymphomagenesis
-HIV infection
- Allergy treatment
- Adjuvant for
infectious disease
therapy
-Asthma
- Possible role for
use in vaccines
-melanoma
-RA
-Adoptive cell
therapy
-Generation of Agspecific T cells
- Adjuvant
treatment for stage
II/III melanoma
- Kaposi sarcoma,
hairy cell leukemia,
and chronic
myelogenous
leukemia
PROCESS OF HEMATOPOIESIS
🔔 Progenitor cell: NON-COMMITTED immature cells (i.e. CFU-GEMM)
Precursor cell: Committed cell lineages (i.e. megakaryoblast)
ERYTHROPOIESIS
BFU-E
3.
PRONORMOBLAST
CFU-E
MATURE RED
CELL
(8-32 cells)
*Total of 18-21 days for RBC maturation
Pro-erythroblast
Basophilic
erythroblast
Polychomatic
erythroblast
Pro-rubricyte
Rubricyte
Meta-rubricyte
reticulocyte
Erythro
blastic
Orthro
Chromatic
Erythroblast
Rubri
cytic
reticulocyte
Normoblastic
nomenclature
Rubriblast
Morph.
Comments
-largest stage
-highly basophilic
-stays in BM for 24 hrs
-capable of mitotis
-accumulates
materials for globin
chain synthesis
-w. intense staining
due to high activity of
cell in synthesizing
Hemoglobin
-color unaltered
-stays in BM (24hrs)
-capable of mitosis
-Transition in color
Blue – grey – pink due
to increasing
synthesis of Hgb
-last stage capable of
cell division
-has a single color
-stage where the cell
extrudes its nucleus
-stays 48 hours before
it gives out its nucleus
-NRBC
“Polychromatophilic
reticulocyte”
-stains cytoplasm pink
-stays in BM for 1 day,
then peripheral circ for
1 day
-DNA, RNA,
Ribosomes stains
Mature Erythrocyte
4.
5.
The nuclear chromatin pattern becomes coarser, clumped, and
condensed.
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
The cytoplasm changes from blue to gray-blue to salmon pink.
Blueness or basophilia is due to acidic components that attract the basic
stain, such as methylene blue. The degree of cytoplasmic basophilia
correlates with the amount of ribosomal RNA.
Early release of
reticulocytes
Prevent
apoptotic cell
death
Reduces
maturation time
inside BM

HYPOXIA & EPO
EPO induces changes in the adventitial cell layer
of the marrow/sinus barrier that increase the
width of the spaces for RBC egress into the sinus
“Shift Reticulocytes”/ macrocytic retics
o
Larger retics that didn’t stay for 1
day in the BM

EPO is able to stimulate production of various
anti-apoptotic molecules, which allows the cell to
survive and mature
 JAK2 protein: activates the signal
transduction and activator of
transcription (STAT) pathway, leading
to the production of the anti-apoptotic
molecule Bcl-XL

EPO can remove an apoptosis induction signal
Fas (CD95)

With the decreased cell cycle time and fewer
mitotic divisions, the time it takes from
pronormoblast to reticulocyte can be shortened
by about 2 days total.
🔔 Hypoxia: lack of O2 in tissues
🔔 EPO: major stimulatory cytokine for RBCs
EPO’s effect is mediated by the transcription factor GATA-1, which is
essential to red cell survival
HOW THE BODY RESPONDS TO HYPOXIA
Chemical Reponse
Physical response
Hematologic
response
Skin appears pale since there is
EPO stimulation
 2,3 DPG (right
Redistribution of blood from the
shift)
skin to more important parts of the
body
EMP
TRENDS IN RBC MATURATION
90% ATP used
by RBCs
-anaerobic
METABOLIC PATHWAYS
HMP
LeuberingRapaport Shunt
Source of
10% ATP
-aerobic
Source of 2,3
DPG
Methemoglobin
Reductase
pathway
Converts ferric iron
to its ferrous state
RBC HEMOLYSIS
EXTRAVASCULAR HEMOLYSIS
1.
2.
The overall diameter of the cell decreases.
The diameter of the nucleus decreases more rapidly than does the size
of the cell. As a result, the N:C ratio also decreases.
INTRAVASCULAR HEMOLYSIS
RULE OF THREE
Hct
3
MCV
LABORATORY METHODS
HEMATOCRIT:
MCH
MCHC
RBC
LEUKOPOIESIS
RDW
more significant index
NEUTROPHILS
MCV
MCH
MCHC
RDW
RBC INDICES SUMMARY
Lower than RV
Reverence value
<80 fL
80-100 fL
Microcytic RBC
Normocytic RBC
<26 pg
26-32 pg
Hypochromic
Normochromic
<11.6
anisocytosis
Higher than RV
>100 fL
Macrocytic RBC
>32 pg
Hyperchromic
(Spherocytes &
Macrocytes)
PRODUCTION
32-36%
11.6-14.6
CI (COLOR INDEX)
-same as MCH
-reflects Hgb
SI (SATURATION INDEX)
-same with MCHC
VI (VOLUME INDEX)
-same with MCV
Myeloblast
MCD (MEAN CELL DIAMETER)
-size of red cell
Pro
myelocyte
Myelocyte
MCAT (MEAN CELL AVERAGE THICKNESS)
-Red cell thickness is not
uniform due to its central
pallor
Meta
myelocyte
Type II myeloblast have 15-20 azurophilic primary
granules near golgi complex & mitochondria
Larger than myeloblast (14 to 20 um)
(+) azurophilic granules
Sudden change in color
-due to secondary/ specific granule presence
Last stage capable of mitosis
D-shaped nucleus
“Dawn of Neutrophilia”
First stage to show a change in nuclear
presentation (indented nucleus)
“juvenile cell”
Increased indentation (more than half of nucleus)
Band
Segment
ed
Neutrophil
Mature neutrophil with 3-5 segments in nucleus
*Hypersegmentation: Vit. B12 / folate deficiency;
macrocytic anemias
*Hyposegmentation: Pelger-huet anomaly
FUNCTION
Pools involved in Neutrophil Maturation:
a. Mitotic Pool

Consists of cells that are actively dividing

CFU-GEMM, CFU-G myeloblast, promyelocyte, myelocyte

Location: BM
b. Storage Pool

bound for maturation ; prepares for maturation

non-dividing


Consists of metamyelocyte, band, neutrophil
Location: BM
c. Circulating Pool

Neutrophils that are
present in peipheral
circulation



FUNCTIONS
Degranulation
Regulation of immune response
Indicator of parasitic infections
 Hallmark of allergic
reactions
BASOPHILS
d. Marginating Pool

Ready to undergo
diapedesis

Exit of neutrophil
from the blood
vessel due to
chemotaxis

Facilitated by
integrin & selectin
PRODUCTION
e. Tissue Pool

Fulfills/
performs its
function:
phagocytosis

Death occurs
Granules: metachromatic, water soluble; obscures nucleus
Phagocytosis:
1. Recognition of pathogen
2. Attachment to receptors in target
bacterium
3. Engulfment of bacterium & killing
– formation of phagosome
Release
of
reactive
substances (superoxide, H2O2)
– alters pH to damage outer
membrane of bacteria
1.
2.
3.
4.
5.
6.
7.
FUNCTIONS
Responds to adrenal corticosteroids
Immediate hypersensitivity reactions
Delayed hypersensitivity reactions

Heparin *absorbs deep blue staining

Histamine

Peroxidase
Release of cytokines
Induction of Ige Synthesis
Angiogenesis
Control of helminth infections
Basophil vs. MAST CELL:
🔔 Mast cells contain proteolytic enzymes (digestion) and serotonin (induces
vasoconstriction)
NEUTROPHIL GRANULES




MONOCYTES & MACROPHAGES
Biggest cell in peripheral circulation
Show vacuolations, grey ground-glass / lace-like cytoplasm;
(+) esterase = α-napthyl acetate, or α-napthyl butyrate esterase
Scavengers of the body
PRODUCTION
Secretory granules 1st to be secreted
Primary granules last to be secreted
EOSINOPHILS
PRODUCTION
In electron microscopy: show heavy granulation & granules appear crystalloid
MBP: takes up acidic stain due to its granules being alkaline
1.
2.
3.
4.
FUNCTIONS
Innate immunity
Adaptive immunity
Housekeeping functions- ingests cellular debris
Induce cytokines that influence hematopoietic functions


LYMPHOCYTES
Innate immunity : NK cells
Adaptive immunity: B cells


Humoral: B cells
Cellular immunity: NK cells & T cells
T Cell Blastogenesis
PRODUCTION
A. B-Lymphocyte Development


TdT
HALA
earliest
recognizable
precursor
 CALLA
Naïve B cells/
hematogotes
-migrates to
2ndary lymphoid
organs
 SIG
(surface Ig)
C. NK Cells
B Cell Blasogenesis: acquisition of new function due to Ag exposure

Forms plasma cell to produce Ab’s Or produce a memory cell
FUNCTIONS
A. B-Lymphocyte
B. T Lymphocyte Development
B. T Lymphocyte
C. CD4+ T lymphocytes
CELL COUNTS
GENERAL PRINCIPLES
(Hemocytometry & Other principles)
FUCHS-ROSENTHAL HEMOCYTOMETER
USE
WBC
COUNT
NAME
2% Acetic
acid
Turk’s
solution
DILUTING FLUIDS
COMPOSITION
Glacial acetic acid
Distilled water
1% aqueous gentian
violet (1mL)
Glacial acetic acid (2mL)
Distilled water (100 mL)
COMMENTS
Glacial acetic acid
lyses RBCs
Gentian violet
stains nuclei of
WBCs (methylene
blue can also be
used)
Randolf’s
solution
EOSINOPHIL COUNT
Speirs-Levy Hemocytometer
Propylene glycol (50 mL)
Pilot’s
solution
10% acqueous solution
Na2CO3 (1 mL)
Heparin (100 units)
Phloxine
Improved Neubauer Counting Chamber
Gower’s
solution
RBC
COUNT
Hayem’s
solution
Toisson’s
solution
Dacie’s
solution
Isotonic
saline
Distilled water (40 mL)
Anhydrous sodium
sulfate (12.5 g)
Glacial acetic acid (33.3
mL)
Distilled water (100 mL)
Sodium sulfate (2.5g)
NaCl (0.5g)
HgCl2 (0.25 g)
Distilled water (100 mL)
Sodium sulfate (8 g)
NaCl (1 g)
Methyl violet (0.25 g)
40% formalin (10 mL)
3% w/v trisodium citrate
to 990 mL
Lyses RBC (acts as
stain transporter
due to its viscosity
and does not
evaporate quickly)
Lyses all WBCs,
other than the
base-resistant
eosinophils
Enhances staining
Prevents clumping
Stains eosinophilic
granules red
POISSON’S LAW OF DISTRIBUTION
Diluting Pipettes used:
SIGNIFICANCE OF MANUAL CELL COUNTS (Henry, 23rd ed)
ROUTINE COMPUTATIONS
ROUTINE DILUTION
WBC COUNT
RBC COUNT
Blood up to 0.5
mark
Blood up to 0.5
mark
DF up to 11
mark
DF up to 101
mark
PLT COUNT
Blood up to 0.5
mark;
DF to 0.5 mark
DF to 101 mark
EOSINOPHIL C
Blood up to 1
mark
DF to 11 mark
Charge
hemocytometer
Charge
hemocytometer
Charge
hemocytometer
Focus under
LPO
Focus under
HPO
Count cells in 5
intermediate
squares
Focus under
HPO
Charge
hemocytometer;
Stand for 15
mins to
allow lysis of
cells
Focus under
LPO
Count cells in all
corner squares
Count cells in all
9 squares
Count cells in all
corner squares
INVERTED L RULE:
LEUKOCYTE COUNT NORMAL REFERENCE VALUES
DIFFERENTIAL COUNT
CORRECTED WBC COUNT
Reference ranges:
ABSOLUTE LEUKOCYTE FRACTION COUNT
The Schilling Hemogram
EOSINOPHIL COUNT: THORN TEST
SHIFT TO THE LEFT:
BASOPHIL COUNT: COOPER AND CRUICKSHANK
METHOD
Fuchs-Rosenthal
or Speirs-Levy
Normal Value for
adults:
Hemocytometer
0-200/cumm
ARNETH COUNT
MEGAKARYOPOIESIS AND
THROMBOPOIESIS
Method of viewing PBS:
PLATELET QUANTITATION
Laboratory Methods for Platelet Count:
Direct
Rees and Ecker
Method
PBS
Platelet Estimation
THE PERIPHERAL
BLOOD SMEAR
Automation
MPV (Coulter)






PERIPHERAL BLOOD SMEAR EVALUATION:
RBC morphology
WBC Estimate
Differential count
Platelet estimate
Presence of immature cells
Presence of malarial parasites
PREPARATION
REPORTING