Structure and Function - Lecture 1 Flashcards
What are the steps to looking at blood cells with a microscope?
Get some blood, don’t let it clot (use EDTA - lavendar), put drop on a slide, use another slide to spread the blood out, let it dry, stain it with “wright giemsa” stain
Describe the eosin stain
negatively charged and aromatic (acidic), soluble in ethanol but not water - why you have to let the slide dry before adding
what does eosin stain
hydrophobic basic macromolecules like hemoglobin
what is the charge of the heme group
positive (aromatic)
what do fluroescin isothiocyanate FITCs bind
amines
what percent of leukocytes are eosionphils
less than 5 % (rare in blood)
what do eosinophils do
mediators of innate immunity
when do you see a lot of eosinophils
parasite infection or allergic reaction or as a part of neoplastic malignancy
what is the starting point for diagnosing any increase in any type of blood cell
reactive vs neoplastic
what does methylene blue stain well
nucleic acids and some protein species
what are the properties of methylene blue
flat and positively charged, aromatic and basic
what is methylene blue soluble in
water or methanol
what does methylene blue stain
hydrophobic acidic macromolcules
what percent of leukocytes are basophils
less than 1 percent (rare)
what are the functions of basophils
degranulation in allergic reactions (related to tissue mast cells); increase in important neoplastic events; only rarely increase in non neoplastic events
What cell types have few or no cytoplasmic granules
monocytes and lymphocytes
what percent of leukocytes are monocytes
3 to 8 percent
what is the role of monocytes
phagocytes which present foreign antigen via MHC class 2
where do macrophages derive from
most (but not all) derive from monocytes
what does monocytosis signify
nothing - not specific at all
what cells do monocytes share info with and where
lymphocytes in the lymphatic tissues
what percentage of leukocytes are lymphocytes
20-30%
when do you see an increase in lympochytes in the blood
neoplastic events (leukemia) or viral syndromes
how long do lymphocytes live
long, days to years
what are the predominant lymphocytes
t cells then b cells then nk cells
what is a reactive lymphocyte
has a nucleus that kind of looks like a monocyte - if you cant tell then call it a mononuclear cell - increase in nmber in viral syndromes
what are large granular lymphocytes
NK cells and cytotoxic t cells sometimes show basophilic cyotplasmic granules
what is the first differential diagnosis in patient with reactive lymphocytes
viral syndrome
how od neutrophils stain
very lightly in cytoplasm with both eosin and methylene blue
what do neutrophils nuclei look like
segmented
what is lifespan of a neutrophil
less than 1 day
what percent of leukocytes are neutrophils
40-70%
when do neutrophils numbers increase
10 fold in bacterial infections
what are neutrophils weapons
phagocytosis, degranulation, formation of NETS
what is a downside to neutrophils NETS
sepsis associated conditions like pre-eclampsia
what is essential in evaluating any infectious disease
knowing the neutrophil count
what are the neutrophil requirements
migrate in response to chemokines, recognize and swarm the enemy, degrade and immobilize the enemy, and minimize collateral damage
what is leukocyte adhesion defect
deficiency in CD18
what does a def in cd18 cause
leukocyte adhesion defect
what is CXCR2
the il-8 receptor on neutrophils that allows the neutrophil to migrate in response to il-8
what can failure of cytoskeleton to response during adhesion result in
immunodeficiency
what is the wiskott aldrich syndrome
most of problems in t cells but some in neutrophils; have trouble initiating cytoskeletal reorganization via actin polymerization in response to signals from the cell surface
what are the mechanisms that enable neutrophils to migrate in response to chemokines
il-8 receptor, grab and hold devices: integrins (cd11a/cd18 complex), very active cytoskeletons
what is excess chemokine secretion associated with
excess inflammation
how do neutrophils recognize and swarm the enemy
toll like receptors (original equipment), complement receptors (old), Fc receptors (latest technology); secrete more chemokines: CXCL2, IL-8, TNF
How do neutrophils degrade and immobilize the enemy
phagocytose it via same receptors that recognized it; chew up polysaccharide cell wall with lysozyme; chew up proteins with various proteases; kill it with bleach (hypochlorite)
how do neutrophils bleach
with myeloperoxidase (mpo)
what do defects in the hypochlorite generating system result in
neutrophils can recognize but not kill; granuloma formation - chronic granulomatous disease
what do neutrophils do when they have accumulated and done their job
apoptose to minimize excess damage
what happens in neutrophils do not apoptose
chronic inflammatory conditions
how much space in bone marrow does neutrophils production take up
2/3
what are the percentages of what left shift could mean
19/20 - reactive (bacterial)
1/20 - neoplastic
what are immature neutrophils
non segmented nuclei (collectively called left shift) horshoeshaped nuclei = bands bean shaped (less mature) = metamyelocytes round nuclei = myelocytes
what are primary granules in neutrophils
seen in myeloid precursors in bone marrow - blue
what are secondary granules in neutrophils
pink ones
what does toxic granulation mean
increased cytoplasmic granules in the neutrophils - suggests bacterial infectio
what are platelets
tiny anucleate fragments
compare platelets in blood to white cells
high concentration of platelets (100 times more than white cells)
when does platelet count tend to increase
iron deficient patients
what is the lifespan of platelets
9-10 days
what are the functions of platelets
primary hemostatic plug, stimulate coagulation cascade, stimulate wound healing, immune function
what is the primary hemostatic plug function of platelets
adherence, activation, aggregation
what is involved in platelets stimulating coag cascade
fibrin formation, clot retraction
how do platelets stimulate wound healing
fibroblast growth and migration
how do platelets provide immune function
antigen presentation and pathogen inactivation
what is the mean of the broad normal range of platelet concentration
300,000 per microliter of blood
what is the average normal blood volumer
4.5 L
what is the normal number of platelets in a person with 4.5 L of blood
over a trillion
what cytokine is secreted by platelets
platelet factor 4 PF4 that actually kills the pathogen that causes malaria
what are the platelet requirements
must be present in large numbers, need to recognize damaged blood vessels, need to swarm onto the damage fast, patch the damage, rebuild the vessel wall
how many platelets do you need in order to not bleed spontaneoulsy
10,000 per microliter
how many platelts do you need to stop small bleeds
50,000 per microliter
where do you store platelets and when are they released
spleen, when you are scared
how much can epinephrine increase platelet count by
50%
what percent of platelets die without doing anything
90
What else do platelets express that is weird
FcR
what is the adaptor
von willebrand factor
how do platelets bind subendothelial collagen
surface protein called Gp1b
describe how vWF works
sticks to exposed collagen and changes shape slightly; flaps in the breeze - platelets bind to it
what happens if vWF is in short supply
patient bleeds a lot
what happens if vWF is too short
patients bleed alot
what happens if vWF is too long
clot excessively
where are reserve supplies of vWF
plasma, endothelial cells and platelets
describe VWF
homo polymer
what is VWF disease
excessive bleeding
what causes platelet activation
binding collagen or VWF
what do activated platelets do
bind VWF and fibrinogen more actively and cause aggregation via crosslinking of platelets
what is fibrinogen the precursor to
fibrin
what does autocatalytic mean
activation/aggregation is autocatalytic; activated platelets secrete mediators that augment activation/aggregation
what does activation and aggregation also promote
coagulation cascade
what is the coagulation casacde
series of plasma proteases that when activated deposits a fibrous mesh made up of mostly fibrin
if the coag cascade gets activated first what happens
it causes platelet aggregation
what is the first differential diagnosis for a patient with broad spectrum reduced production of coagulation factors
liver disease since many of them are made in the liver
what do activated platelets secrete that stimulate fibroblasts and smooth muscle cells
PDGF, TGFbeta, and VEGF
when are giant platelets seen
peripheral blood when platelet production is ramped up or when production occurs abnormally with a disease affecting bone marrow
what are the requirements for red blood cells
flexibile shape, durable membrane, maximum Hgb/O2 carrying capacity, offset osmotic pressure, anti-oxidant system, energy supply, ability to tune down complement
describe the flexibile shape of red blood cells
biconcave disk; 360 deg fold capacity
what happens if main cargo of RBC (hemoglobin) precipitates
impaired RBC, obstructed blood vessel, ruptured red cell
how long do RBC last
100 days
mutations in rbc shape to give ellipsoid or spherical shape do what to the durability
reduce it
why is it so easy for hemoglobin to precipitate
b/c hemoglobin is packed at the max highest concentration it can be at before it precipitates
what can cause hemoglobin to precipitate
minor changes in globin amino acid sequence or oxidative stress
what is a good bacterial growth medium
hemoglobin
what is the most important microorganisms that grows on hemoglobin
plamodum family of protozoa (4 species cause malaria)
what are some mutations that select against infection with bacteria that grow on hemoglobin
disabling red blood cells in stress
what is the consequence of getting rid of things like nuclei in rbc
limits response to changing conditions and pathogens
describe the rbc pump system
use atp to pump out sodium and in potassium
what happens if the RBC pump fails
cell swells and bursts
what can cause the rbc pump to fail
mutations that impair production of RBC
what are concentrations of sodium and potassium in the plasma
sodium 140 - potassium 4
what are the concentrations of sodium and potassium in the rbc
sodium 11 - potassium 100
why is an anti oxidant system needed
o2 is powerful oxidant (via generation of h2o2) - oxidized-sh groups on hemoglobin become crosslinks (R1-SS-R2) which dentaures Hgb or causes it to precipitate and oxidized iron (Fe3+) cant carry O2 which results in hemoglobin containing Fe 3+ (methemoglobin) and patient is hypoxic
how does the antioxidant system work
GSH eliminates peroxide before it can do damage by using NADPH; cytochrome b5 reductase reduces methemoglobin back to normal hemoglobin using NADH
what happens when you have mutations in the anti oxidant system (energy supply) and you are in an oxidatively stressful state
clumps of oxidized hemoglobin; can lyse (hemolytic anemia)
what is hemolytic anemia
when excess red cell lysis occurs
how do macrophages help in hemoglobin mutation situations
take big bites out of rbc and remove hemoglobin - bite cells
describe the energy supply system of the RBC
simple, don’t take up cargo space; chuck mitochondria, use glycolysis to make atp and nadh; use pentose shunt to make nadph
what is the first potential failure point in the energy supply making of RBC
Glucose 6 phosphate dehydrogenase
what resistance can happen with G6PD mutations
malaria resistance
why do we need to tune down complement fixation
alternative complement fixation pathway is active at low rate all the time; normal blood cells have to slow it down to survive
what does RBC use to slow down complement fixation
DAF - CD55; which binds the c3 convertase c4bc2a and does not allow alternative pathway to continue
what can not enough hemoglobin mean
hypochromic and/or microcytic
what does hypochromic mean
lack of color
what does anisocytosis mean
refers to abnormal distribution of red cell sizes
what does poikilocytosis mean
abnormal red cell shapes
what does bluish rbc mean
making a lot of residual mrna which means making a lot of cells (good) bc losing a lot (bad)
what are reticulocytes
bigger than normal just out of bone marrow RBC
when are rbc polychromatic
usually with accelerated production
what can cause precipitated hemoglobin
genetic defect in hemoglobin, altered shape (sickle cell); oxidized hemoglobin
what are Heinz bodies
small clumps of oxidized hemoglobin
what causes sickle cell
substitution in 6th amino acid in beta globin
what 2 diseases give heterozygotes the malaria advantage
g6pd mutation and sickle cell
what are shistocytes
red cell fragments
what causes shistocytes
mechanical lysis or microangioplastic processes
how is hemoglobin measured
spectrophotometry; lyse cells and add cyanide (blue color) and quantify non blue light
what can mess up the hemoglobin measurement
excess lipoproteins after a fatty meal which gives a false high level of hemoglobin measurement
how are RBC measured
manual methods; centrifuge anticoag blood in tube and spin down cell; sometimes capillary tube; also by conductivity in coulter chamber
what is hematocrit
A= red cells B = blood volume hematocrit = A/B
describe the coulter chamber
red blood cells pumped through large container through aperture to smaller container with same fluid as large container; electrode 1 in large electrode 2 in smaller container; voltage gradient applied and resistance across aperture measured
what happens to the resistance of the aperture as cells move from large to small container
increases; increase directly proportional to cell size
how is the red cell count calculated by conductivity
flow rate into outer container and resistance spikes per second
what is the size of each cell proportional to in the coulter chamber
peak height
how can we get mean cell volume
by peak height
how do you get hematocrit
multiply number of cells times MCV
what could fragmented rbc be counted as
platelets
what can clumped red cells (maybe by antibodies?) do to hematocrit?
lower it
what can clumped red cells (maybe by antibodies?) do to red cell count?
lower it
what can clumped red cells (maybe by antibodies?) do to mcv?
increase
how can leukocytes be counted
manually or automated (by coulter chamber)
what other info can be found using coulyter chamber for leukocytes
what’s inside cells by measuring resistance to radio freq alt current
what is formula for peak height for rbc
peak height = k * volume
what is formula for peak number for rbc
peak number = k * cell count
what is formula for peak height for leukocytes
peak height = k *cell complexity
what cells are hard to differentiate between in coulter chamber
basophils and eosinophils
what is the flow cytometer used for
hit cells with laser and get side scatter measurement
how do you get reticulocyte count
red cells + nucelic acid binding fluorescent dyes -> flow cytometer
what are benefits of reticulocyte count with flow cytometer
assess whether anemia is due to impaired red cell production
what does the hematology analyzer not reliably count
band and other immature granulocytes are counted as neutrophils; blasts counted as lymphocytes of monocytes; red cell fragments may not be detected or counted as platelets; platelet clumps not counted and may show false thrombocytopenia
what do platelets clump when sampling sometimes; how can this be fixed
presence of EDTA may expose antigens; sample with citrate
what is an important differential diagnosis for thrombocytopenia
lab artifact
what are blasts
early blood cell precursors; can correlate with bone marrow diseases such as acute leukemia
