1. Hematology 425 Hematopoiesis
Russ Morrison
September 18, 2006
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2. Peripheral Smear Examination – Preparation of Smear
Review Chapter 1 of Atlas
Properly prepared blood smear is essential to accurate assessment of cellular morphology
Wedge smear preparation – well-prepared
2/3 to ¾ of slide length covered by smear
Slightly rounded at featheredge
Lateral edges should be visible
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3. Peripheral Smear Preparation
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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4. Peripheral Smear Staining
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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5. Peripheral Smear Staining
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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6. Peripheral Smear Staining
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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7. Peripheral Smear Staining
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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8. Peripheral Smear Examination
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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9. Peripheral Smear Examination
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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10. Peripheral Smear Examination
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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11. Peripheral Smear Examination
WBC abnormalities (toxic changes, Dohle bodies, reactive lymphs, Auer rods, etc) also reported, if observed
If present, NRBCs are counted and reported as #/100WBCs
RBC,WBC and PLT morphology performed
Platelet estimate performed (avg PLTs in 10 fieldsX20,000), compare to machine count
All abnormal findings verified under the 100x objective
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12. Morphology & Function of Cellular Components
Cells are the structural units that constitute living organisms
Most cells have three basic parts
Membrane
Cytoplasm
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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13. M & F, Cell Membrane
A semipermeable outer boundary separating the cellular components from the environment
Three basic functions
restricts/facilitates interchange of substances with the environment
Detects hormonal signals facilitating cell-to-cell recognition
Supports the blood groups, histocompatibility loci and receptors of cellular identity
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14. 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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15. M & F, Cell Membrane Proteins
Two types of proteins in cell membranes
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.
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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16. 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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17. M & F, Nucleus The nucleus is made up of 3 components 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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18. M & F, Nucleus Chromatin 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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19. 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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20. 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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21. 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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22. M & F, Cytoplasm Golgi Complex
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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23. M & F, Cytoplasm Endoplasmic Reticulum
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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24. M & F, Cytoplasm Ribosomes
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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25. M & F, Cytoplasm Mitochondria
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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26. M & F, Cytoplasm Lysosomes
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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27. M & F, Cytoplasm Microfilaments
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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28. M & F, Cytoplasm Microtubules
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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29. M & F, Cytoplasm Centrioles
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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30. 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 2
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31. M & F, Review Questions
What cellular component of the erythrocyte is its control center?
1. Heterochromatin
2. Golgi complex
3. Nucleus
4. Cytoplasm 3
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32. 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 4
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33. M & F, Review Questions
What cellular component "directs traffic" for smooth functioning?
1. Mitochondria
2. Ribosomes
3. Golgi complex
4. Nucleoli 3
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34. 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 2
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35. M & F, Review Questions
Protein synthesis occurs in what part of a cell?
1. Ribosomes
2. Nucleus
3. Mitochondria
4. Lysosomes 1
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36. M & F, Review Questions
What cellular component is responsible for energy production and metabolic processes?
1. Ribosomes
2. Nucleus
3. Mitochondria
4. Lysosomes 3
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37. M & F, Review Questions
The granules of phagocytic white cells are composed of:
1. Microfilaments
2. Lysosomes
3. Microtubules
4. Centrioles 2
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38. 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 4
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