Strom - Blood Cells Flashcards
What agents are added to prevent blood from clotting in a sample?
- how do these work?
Chelating agents like EDTA or Citrate
Both chelate Ca2+ which causes the blood to clot
What is used to stain blood cells?
- chemical properties?
- solubility?
- what in the cell does it stain?
- Eosin (wright-giemsa stain)
- Eosin = Aromatic and Acidic (neg. charge)
- soluble in EtOH, NOT water
- Stains HYDROPHOBIC BASIC (positivity) macromolecules (e.g. hemoglobin and some others)
What gives hemoglobin its positive charge?
Fe++ at the center of the heme group
What are the 3 key morphological features of normal red cells?
- Bi-concave Disks
- Uniform in size
- Central pallor occupies 1/3 of disk diameter
What morphological change happens to RBCs when patients are iron deficient?
Enlarged Central Pallor
What are two possible causes of eosinophilia?
- Allergies
2. Infections with parasites
Eosinophils
- presence in blood
- biological role
- what is contained in the granules?
Rare in blood (less than 5% of leukocytes)
Biological role:
- protect against parasites
Major Basic Protein (MBP) found in granules
**Increase during allergic Reaction
Methylene Blue
- Chemical Characteristics
- Solubility
- What does it stain?
- Methylene blue is Aromatic and Basic
- Soluble in Water or MeOH
Stains:
- Nucleic Acids
- Some proteins
- Basophils
Basophils
- Presence in Blood
- Biological Role
- How it works
- when are they elevated
Presence in Blood:
- Rare (1% leukocytes)
Biological Role:
- Parasite protection
How it works:
- Secretes Histamine in response to IgE-bound antigens
When are they Elevated:
- Presence in Blood increases during an allergic reaction
What is pale blue cytoplasm referred to?
Amphiphilic
Monocytes
- what do they do?
- Where do they do it?
- Distinguishing features
Job:
- Gather information and share it with lymphocytes
Where:
- Information is shared in Lymphatic Tissue
Distinguishing Features:
- Amoeboid nucleus
- lack of granules
When does monocytosis occur?
- is monocytosis usefule for a differential diagnosis?
- Low grade or cryptic infections
- autoimmune conditions
NOT useful for differential Dx
Monocytes
- Percentage of Leukocytes
- Give rise to…
- Biological Role
- How it works
3-8% of Leukocytes
Give Rise to Macrophages
Biological Role:
Macrophages bind antigens and initiate immune response
How it works:
Phagocytose invading organisms and present their antigens to helper T-cells (CD4+) via MHC-II
Lymphocytes
- Percentage of blood leukocytes
- Type of most peripheral blood lymphocytes
- when do you see an increase?
Percentage:
20-30% of blood leukocytes
- T-cells are the most common type of peripheral blood lymphocytes
Increase:
- Leukemia
- Viral Syndromes
What cell types are difficult to distinguish morphologically and are therefore referred to as mononuclear cells?
- why report mononuclear cell number?
- how could you attempt to differentiate these cell types?
- Reactive lymphocytes are often hard to tell from monocytes
- Elevated mononuclear cells helps you know granulocytes are NOT elevated
- Lymphocyte nucleus is usually round while monocyte nucleus is usually more S-shaped (amoebiod)
What is the 1st thing you should determine when beginning your differential diagnosis on any Blood Cell or Platelet?
Determine if its Reactive or Neoplastic
Compare the lifespan of lymphocytes to neutrophils.
Lymphocytes are long lived while neutrophils die in a day or so
Neutrophils
- staining characteristics
- % of leukocytes
- biological role
- how they work
Staining:
- Light staining with eosin and methylene blue
40-70% of all leukocytes
Biological Role:
- Ingest and kill invading organisms
How they work:
- Chemotaxis
- Phagocytosis
- Degranulation
- Formation of Extracellular traps (NETS)
How do dead neutrophils still help to kill bacteria?
- Forms NETS (neutrophil extracellular traps) from their chromatin
What are the benefits and downfalls of NETS formation by neutrophils?
Benefits:
- Captures Bacteria
Downfalls:
- May contribute to sepsis
What is Pre-eclampsia?
- associated cells type?
- Sepsis Associated condition in which severe threatening hypertension occurs during pregancy.
- Can be caused by NETS secreted by neutrophils
T or F: KNOWING THE NEUTROPHIL COUNT IS ESSENTIAL IN EVALUATING ANY INFECTIOUS DISEASE
TRUE
Why do oncologists follow neutrophil count so closely?
- It is the first thing to drop as a side effect of chemotherapy
- This is a result of the fast turnover (1 day) of neutrophils
where are neutrophils made?
Bone Marrow, neutrophil production accounts for over 2/3 of bone marrow space
What 3 things does chemotaxis refer to?
- Communication
- Navigation
- Propulsion
**Signal is received from environment and cells respond by moving to affected site
What causes Leukocyte Adhesion Defect?
- Congenital defects in a component of (lymphocyte function associated antigen 1) LFA-1 (CD18)
What is a CD number?
“Cluster of Differentiation”
- When 2 or more monoclonal antibodies complete for the same binding protein they are given a CD number
e. g. CD18
What are the 3 target recognition methods for neutrophils?
- are these associated with innate or acquired immunity?
- Toll-Like Receptors
- innate immune system components
- Old - Complement Fixation
- innate or adaptive immunity
- Newer - Fc Receptors that recognize antibodies bound to targets
- Newest
What weaponry to neutrophils posses?
- be specific (4 things)
**NEED TO KNOW
***COME BACK TO THIS AND FIX IT
They degraulate and release their contents
- Myeloperoxidase (MPO) - enzyme that makes hypochlorite (bleach)
- Lysozymes - break down bacterial cell wall (NAM-NAG bond break)
- Defensins
- NETS
Chronic Granulmatous Disease
- What causes it?
- Characteristic features?
Defect in Hypochlorite-generating system of neutrophils
Characteristic Features:
- Granulomas - clusters of ineffectual dying neutrophils
What is immunostaining?
- Use an antibody to find a protein of interest in the cell and attach an enzyme to the antibody that converts the soluble precursor into an insoluble pigment
What kind of antibody would you use to do an immunostain for neutrophils?
MPO (Myeloperoxidase) antibody
What is a left shift of granulocytes and why does it happen?
Left Shift:
- BACTERIAL infection causes ramped up production of nuetrophils and immature band cells get dumped into blood earlier than normal
What are two possible causes of a left shift?
- Bacterial infection (19/20)
2. Neoplastic (1/20)
Two granule types seen in neutrophils.
- when are they seen?
- where are they seen?
Primary Granules
- Dark Blue
- Usually only seen in early myeloid precursors of bone marrow but seen in blood during infection
Secondary Granules:
- Salmon-pink
What is toxic granulation?
- Presence of Primary Granules in blood
Platelets
- Concentration
- lifespan
- biological roles (4)
Concentration:
- High (100x that of white cells)
Lifespan:
- 9-10 days
Biological Roles:
- Primary Hemostatic Plug
- Stimulate coagulation cascade (fibrin)
- Stimulate would healing (fibroblasts)
- Immune Function (pathogen inactivation)
How does platelet adherence work?
- Damage to BV causes exposure of subendothelial Collagen
- Platelets adhere either directly or via vWF (von Willebrand factor - a circulating adaptor protein)
- Adherence induces “platelet activation’
vWF
- where is it located?
- structure?
- interacts with?
- what receptor binds it?
Location:
- Blood Plasma
Structure:
- Coiled Cable, multimer made of identical monomers
Interactions:
- Uncoils to bind Collagen and Platelets
Receptor:
GP1b receptor of platelets bind it
What diseases result from a lack of GP1b or vWF?
- symptoms
Deficient GP1b:
Bernard-Soullier syndrome
Deficient vWF:
Von Willebrand’s disease
Both result in mild bleeding tendencies in mucocutaneous locations
What would you expect to see in a patient with excess vWF?
- Thromboses
How does increased affinity from activated platelets come about?
- what is there increased affinity for?
Increased binding affinity for:
- Collagen
- vWF
- Fibrinogen
- Caused by:
- GPIIb/IIIa conformational change (platelet surface protein complex) - we have DRUGS that can block this
What are the two components to platelet activation?
- Increased affinity for collagen, vWF, and Fibrinogen
2. Autocatalytic activation via secretion of a number of mediators which results in activation
What happens in autocatalytic activation?
- ADP is released, it rebinds to ADP-receptor then causes more ADP and MEDIATORS to be released.
- Thromboxane A2 (TXA2) gets released then comes back to bind on TXA2-receptor, causes TXA2 production to increase and Granule secretion
What drug inhibits ADP receptor binding and TXA2 receptor binding in autocatalytic activation?
ADP receptor blocked by Clopidogrel
TXA2 receptor blocked by asprin
What 3 things are stored in alpha granules?
- vWF
- Factor V
- Fibrinogen
What are the 2 parts to platelet aggregation?
- Augmentation of the coagulation cascade
2. Crosslinking of platelets into the “platelet plug”
How do platelets augment the coagulation cascade?
Release of alpha-granuals containing vWF, Fibrinogen, Factor V
Platelet Crosslinking
- how does it occur?
- Platelet bound by Fibrinogen
2. Fibrigogen used to make fibrin
How do platelets aid in wound healing?
Secretion of Fibroblast Growth and Chemotactic Factors (e.g. Platelet Derived Growth Factor PDGF)
Why do patients with severe bacterial infections involving the bloodstream (bacteremia) and associated organ disfunction (sepsis) often show Thrombocytopenia (low platelet count)?
- TLR’s on the platelet surface cause them to bind bacteria
2. Antibodies bound to the bacteria cause uptake by macrophages
When might you see a giant platelet in the peripheral blood?
- Platelet Production is Ramped up
2. Diseases affecting the bone marrow
What are the immunological functions of platelets?
- Store and secrete defensins
- They recognize and bind bacteria via TLRs
- Activated cells stimulate expression of ICAM-1 on endothelial cells
- Bind malaria-infected red cells and kill parasite by secreting a factor
RBC
- % of cells in blood
- Lifespan
- % wt. hbg in RBCs
- 99% of Cells in blood
- 120 day lifespan
- RBCs - 90% Hb by wt.
What allows hemoglobin to bind and dump so much O2 compared to myoglobin?
Hb
- Sigmoidal curve produced by cooperative binding allows for sharp release of O2 at higher pO2 (like that in muscle)
Mb
- Not sigmoidal so only way to increase release is to reduce affinity which reduces maximal binding (100% saturation)
Bohr Effect
- cause
Low pH:
- Hb has lower Affinity for O2 at Low pH
- This causes more O2 to be dumped in O2 deficient tissues where lactate is high driving pH down.
High pH:
- CO2 blown off in lungs Raises pH
- Hb binds more tightly at high pH
What tissue type would you expect to have the highest levels of 2,3 DPG and why?
2,3 DPG would be highest in HYPOXIC tissues
why?
- Lactate builds up and glycolysis intermediates back up
- 1,3 DPG is shunted to 2,3 DPG via the action of DPG mutase
How do RBCs offset osmotic pressure due to high Hb levels?
Pumps that use ATP (Na+/K+ pump)
**If these pumps fails H2O will rush in and the cell will burst
What is the significance of the biconcave disk of RBCs?
- How is this shape maintained?
Allows for 360º Folding
Spectrin
- Dimeric Polymer is linked to…
Ankyrin
- transmembrane protein
What shape are RBCs that lack Spectrin or Ankryin?
Elliptical or Spherical
What is the most important group of microorganisms that thrive on Hb?
Plasmosdum
- 4 species in this family cause malaria
T or F: Hb concentration in RBCs is so high that even small changes from oxidative stress etc. can cause Hb precipitation
True
What are the plasma and intracellular concentrations of Na+ and K+?
- why are ion concentrations so important to RBCs?
Plasma:
[Na+] 140 mM
[K+] 4 mM
-144 mM outside
RBC:
[Na+] 11 mM
[K+] 100 mM
- 111 mM inside
**Lower Ion Concentrations in the cell prevent H2O from trying to rush in
Why is an antioxidant system important to RBCs?
- O2 is a powerful oxidant
- -SH groups on cysteine oxidize to form -S-S- crosslinks
- Oxidized Fe+++ cannot carry O2
What results from Oxidized Fe+++?
- why?
- Hypoxia, because Fe+++ cannot bind O2
* patient can be hypoxic in spite of not being anemic or having lung disease
Why do defects in glycolytic enzymes result in hemolytic anemia in children?
- RBCs only use glycolysis for ATP and ATP is needed to run the Na+/K+ pump to keep H2O from bursting the cell
What is the role of G6P dehydrogenase in RBCs?
- Involved in the Pentose Phosphate Pathway that makes NADPH
- NADPH is needed to reduce Glutathione so that cell can perform antioxidant function
What is the complement system?
- A group of proteins in the bloodstream that lyse invading organisms or host cells containing them.
Define hypochromia
lack of color
Define anisocytosis
abnormal distribution of RBC sizes
Define Poikilocytosis
Abnormal RBC shape
What does it mean if RBCs pick of methylene blue dye?
- what is the called
- when does it happen?
RBCs have lots of Nucleic Acids (RNA)
POLYCHROMIA = Called Reticulocytes
These are seen when patients are loosing RBCs somehow
What does the presence of bite cells indicate?
- how do they arise?
- G6PD deficiency
- Come about because phagocytes bite out the oxidize Hb from the cell
What are Heinz bodies?
- Oxidized Hb forms Clumps
What are scistocytes?
RBCs that look like they’ve been cut in half
- Happens via mechanical lysis (aka Microangiopathic Processes)
What are 3 forms of neutrophil that may be expelled during a bacterial infection?
- Band Forms
- Horseshoe Shaped Nucleus - Metamyelocytes
- Bean shaped nucleus - Myelocytes
- Round Nucleus
**Remember neutrophiles should have a lobular nucleus
What if you have an anemic patient but you don’t see a single reticulocyte in a blood smear?
- they may have a bone marrow defect because immature RBCs (reticulocytes) should be kicked out in response to blood loss
What would happen if oxidatitive phosphorylation occurred in RBCs?
They would consume their own product
What is DAF (CD55)?
- where is it found?
DAF - decay accelerating factor
- presented on RBCs to prevent complement from acting on RBCs, this prolongs the life of the cell
What happens if you have a mutation in your cytochrome b5 reductase gene?
Methomeglobin - from Fe+++ not being able to be reduced
NOTE: this requires NADPH
