Transcript Document 7422971

Hematology 425
Hematopoiesis
Russ Morrison
September 18, 2006
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Peripheral Smear Examination
– Preparation of Smear
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Review Chapter 1 of Atlas
Properly prepared blood smear is
essential to accurate assessment of
cellular morphology
Wedge smear preparation – wellprepared
2/3 to ¾ of slide length covered by smear
Slightly rounded at featheredge
Lateral edges should be visible
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Peripheral Smear Preparation
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Smooth without irregularities, holes,
streaks
Featheredge should have a “rainbow”
appearance
The whole drop is picked up and spread
Figure 1-2 is the desired smear, examples
of unacceptable smears are provided in
figure 1-3
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Peripheral Smear Staining
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Purpose of staining is to identify cells
and recognize morphology easily through
the microscope
Most commonly used stains are Wright
stain or Wright-Giemsa stain which both
contain eosin and methylene blue
Stains which contain more than one dye
are called polychrome stains as are the
stains above
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Peripheral Smear Staining
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Cells are fixed to the glass slide via the
methanol that is present in the stain
pH-dependent staining, optimal pH 6.4
Buffered stain of appropriate pH
Free methylene blue is basic, stains
acidic cellular components on the slide
(RNA) blue
Free eosin is acidic and stains basic
components (Hgb, Eo granules) red
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Peripheral Smear Staining
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Optimally stained smear characteristics:
RBCs pink to salmon in color
Nuclei dark blue to purple
Cytoplasmic granules of neutrophils are
lilac
Cytoplasmic granules of eosinophils are
red to orange
Cytoplasmic granules of basophils are dk
blue to black
Area between cells should be clean (no
ppt)
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Peripheral Smear Staining
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Best staining results are obtained from
freshly made slides prepared within 2-3
hours of blood collection
Slides must be allowed to dry thoroughly
before staining
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Peripheral Smear Examination
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Now that you have the perfect smear,
stained well:
Begin scanning on low power (10x) to
assess quality of the smear and stain
Look for abnormal red cell distribution
(rouleaux, auto-agg), disproportionate
number of large nucleated cells at the
edges of the smear (make another smear
for the later case)
Look for abnormal cells (blasts, reactive
lymphocytes, parasites)
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Peripheral Smear Examination
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Using the 40x (high dry) objective, find
an area of the smear where the RBCs are
evenly distributed
Scan 8-10 fields in this area and
determine the avg WBCs per field
Check the WBC count by multiplying
this avg by 2000 (approx.WBC
count/mm3)
Discrepancies between instrument count
and estimate should be resolved (QC!)
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Peripheral Smear Examination
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Using the 100x (oil) objective, in same
area of slide as above count 100
consecutive WBCs
Classify the WBCs
Report as a %
Use “battlement” track (Figure 1-6 of
atlas) to minimize WBC distribution
errors
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Peripheral Smear Examination
4. WBC abnormalities (toxic changes, Dohle
bodies, reactive lymphs, Auer rods, etc) also
reported, if observed
5. If present, NRBCs are counted and reported
as #/100WBCs
6. RBC,WBC and PLT morphology performed
7. Platelet estimate performed (avg PLTs in 10
fieldsX20,000), compare to machine count
8. All abnormal findings verified under the
100x objective
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Morphology & Function of
Cellular Components
 Cells are the structural units that constitute
living organisms
 Most cells have three basic parts
1. Membrane
2. Cytoplasm
3. Nucleus
 Each of these parts has components that
assist in varied functions
 Table 5-1 in the text is an excellent summary
of cell components and functions
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M & F, Cell Membrane
 A semipermeable outer boundary separating
the cellular components from the
environment
 Three basic functions
1. restricts/facilitates interchange of substances
with the environment
2. Detects hormonal signals facilitating cell-tocell recognition
3. Supports the blood groups,
histocompatibility loci and receptors of
cellular identity
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M & F, Cell Membrane
 Cellular membranes must be resilient and
elastic
 Achieves the above properties by being a
fluid structure of globular proteins floating
in lipids
 Membrane proteins are mostly glycoproteins
found floating in lipid bilayers
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M & F, Cell Membrane Proteins
 Two types of proteins in cell membranes
1. Integral proteins – may traverse the lipid
bilayer and penetrate to the outside of the
membrane. “Transmembrane” proteins
provide communication and transportation
between the cell’s interior and the external
environment.
2. Peripheral proteins – found only on the
cytoplasmic side, form the cell’s
cytoskeleton. Attach to the cytoplasmic ends
of integral proteins to form a reticular
network that maintains structural integrity
and holds integral proteins in place.
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M & F, Cell Membrane Carbos
 Occur in combination with proteins and
lipids (glycoproteins and glycolipids)
 The carbohydrate portion almost always
extends beyond the outer cell surface
 Gives the cell a carbo coat referred to as the
glycocalyx
 Carbos function in cell-to-cell recognition,
provide a negative surface charge, surface
receptor sites and cell adhesion capabilities.
 Many of the RBC antigens are carbos, genes
code for proteins (transferase enzymes).
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M & F, Nucleus
 The nucleus is made up of 3 components
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Chromatin
Nuclear envelope
Nucleoli
the control center of the cell
The largest organelle in the cell
Made largely of DNA
Site of DNA replication and transcription
Responsible for cellular chemical reactions
Responsible for cellular reproduction
Stains blue because of its acidic nature
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M & F, Nucleus
 Chromatin
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Consists of nucleic acids and proteins
Two types of protein
Histones, negatively charged
Nonhistones, positively charged
Two types of chromatin
Heterochromatin – genetically inactively, dense
and darkly staining
Euchromatin – genetically active, diffuse,,
loosely coiled and stains pale blue, RNA
transcription is able to occur here
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M & F, Nucleus
 Nuclear Envelope
1. surrounds the nucleus
2. consists of an inner and outer membrane
 Outer membrane is continuous with an
extension of the endoplasmic reticulum,
between the 2 membranes is a diaphragm 50
nm in thickness continuous with the lumen
of endoplasmic reticulum. Nuclear pores
penetrate the nuclear envelope allowing
communication between the nucleus and
cytoplasm. (#pores decrease as cell matures)
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M & F, Nucleus
 Nucleoli
- the nucleus contains one to four nucleoli
-these organelles contain a large amount of
RNA and other proteins in a loose fibrillar
form
- the site for the synthesis of various forms
of RNA, which is then transported through
the nuclear pores for ribosomal assembly
and protein synthesis
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M & F, Cytoplasm
 The cytoplasmic matrix is a homogenous
continuous aqueous solution called cytosol
 It is the environment in which the organelles
join and function
 The organelles of the cytoplasm will be
discussed individually
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M & F, Cytoplasm
 Golgi Complex
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A series of stacked, membrane-bound, flattened
sacs referred to as cysternae
Involved in modifying, sorting and packaging
macromolecules for secretion or delivery to other
organelles
The “traffic cop” of the cell
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M & F, Cytoplasm
 Endoplasmic Reticulum
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Lace-like network found throughout the
cytoplasm
Specializes in making and transporting lipid and
membrane proteins
Rough endoplasmic reticulum has ribosomes
engaged in the synthesis of proteins on its outer
surface
Smooth ER does not include ribosomes and may
serve as a storage site for new proteins
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M & F, Cytoplasm
 Ribosomes
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Small particles composed of near-equal amounts
of protein and RNA
Found free in the cytoplasm, on the surface of
RER, and in the nucleus and nucleoli of cells
May exist singly (monoribosome) or form chains
(polyribosome)
The more ribosomes present within the cell, the
darker blue the staining characteristics
Serve as the site of protein synthesis
accomplished with the assistance of transfer
RNA, for amino acid transport to the ribosome,
and messenger RNA, which provides sequencing
information for the amino acids.
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M & F, Cytoplasm
 Mitochondria
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Responsible for the metabolic processes of
energy-producing reactions and electron transfer
– oxidative reactions.
The oxidative systems described within the
mitochondria are the Krebs cycle, fatty acid
cycle and the respiratory chain.
Also present in the mitochondria are proteins,
phosphorylase, ribosomes and DNA.
Mitochondria are capable of self-replication and
their number is directly related to the amount of
energy required by the cell.
Do not accept color with routine stains
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M & F, Cytoplasm
 Lysosomes
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Contain hydrolytic enzymes bound within a
membrane and are involved in the cell’s
intracellular digestive process
The membrane prevents the enzymes from
attacking the protein, nucleic acids,
mucopolysaccharides, lipids and glycogen within
the cell itself
The hydrolitic enzymes become active when
lysosomes bind to the phagocytic vacuole and
the membrane ruptures, allowing the escape of
the enzymes into the phagosome
May require special staining techniques
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M & F, Cytoplasm
 Microfilaments
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Are solid structures approximately 5 nm in
diameter consisting of actin and myosin proteins
These fibrils or groups of fibrils are located near
the nuclear envelope or close to the nucleus and
assist in cell division
Are present near the membrane to assist in
cytoskeletal support and motility
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M & F, Cytoplasm
 Microtubules
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Are approximately 25 nm in diameter and of
variable length
Organized from tubulin through self-assembly
Have several functions
1. help maintain the cell’s shape and assists
movement of some intracellular organelles
2. makes up the mitotic spindle fibers and the
centrioles during mitosis
Not seen in blood smears
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M & F, Cytoplasm
 Centrioles
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Paired structures consisting of nine bundles of
three microtubules within each bundle
Serve as insertion points for the mitotic spindle
fibers during metaphase and anaphase of mitosis
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M & F, Review Questions
What component of the erythrocyte is most
responsible for its cytoskeleton and cellular
integrity?
1. Integral protein
2. Peripheral protein
3. Nucleus
4. Golgi complex
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M & F, Review Questions
What cellular component of the erythrocyte is its
control center?
1. Heterochromatin
2. Golgi complex
3. Nucleus
4. Cytoplasm
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M & F, Review Questions
How does the genetically active portion of the
nuclear chromatin look when stained with
Wright's stain?
1. Condensed, darkly stained
2. Clumped, dark purple
3. Ropey, pink or red
4. Diffuse, uncondensed and pale blue
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M & F, Review Questions
What cellular component "directs traffic" for
smooth functioning?
1. Mitochondria
2. Ribosomes
3. Golgi complex
4. Nucleoli
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M & F, Review Questions
If the cytoplasm of a cell is very basophilic or
blue it contains:
1. No ribosomes
2. Large number of ribosomes
3. Small number of ribosomes
4. Only polyribosomes
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M & F, Review Questions
Protein synthesis occurs in what part of a cell?
1. Ribosomes
2. Nucleus
3. Mitochondria
4. Lysosomes
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M & F, Review Questions
What cellular component is responsible for
energy production and metabolic processes?
1. Ribosomes
2. Nucleus
3. Mitochondria
4. Lysosomes
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M & F, Review Questions
The granules of phagocytic white cells are
composed of:
1. Microfilaments
2. Lysosomes
3. Microtubules
4. Centrioles
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M & F, Review Questions
What is the purpose of the granules in
phagocytic cells?
1. Maintain cell shape
2. Manufacture enzymes
3. Provide surface receptor sites
4. Digest foreign material such as microorganisms
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