---start syspath2. 9.8.97-----

Dr McManus
9.8.97
pathology 4 pm

she 
knows we are tired but will try to make this fun. Dr McManus is a 
clinical pathologist. This is the second year we've included marrow 
pathology in this class and it's early in the course based on last year's 
evaluations.

clinical vs anatomical/research pathologists - what's the 
difference? 
cytology vs biopsy?

clinical pathologist has some common 
training with anatomic pathologist, but is more into hematology and 
cytology, does more diagnostics of body fluids. anatomic pathologist will 
look more at tissues, fixing tissues, making slides. the clinical 
pathologist is more interested in dynamics of what is going on in living 
patient - looks at aspirates of masses, smears etc from living patient.


what is cytology? study of cell structure and function. but in a clinical 
way, cytology is a whole different enterprise. the cytologic evaluation 
enables us to examine structure and infer function, but has more to do 
with how cell is prepared for evaluation. 

so you have a simple cell. 
when histopath is performed, tissue is fixed in 3D state and then 
sectioned, so you see a slice through it. in cytology, you take a cell 
and flatten it on the slide. cytology requires an artifact to be created. 
you have to flatten the 3D cell. this enables you to visualize 
cytoplasmic and nuclear features. so a clinical pathologist looks at 
smushed flat cells. the anatomical pathologist looks at 2d/3d stuff.

if 
you're doing a LN aspirate and need to see if animal has LSA or whatever, 
and you spray aspirate onto slide, you look at it and can get an idea of 
what's going on within minutes. but you've lost the architecture 
completely. for a definitive diagnosis, you may have to do a biopsy. but 
cytology is fast. in some tissues you really only do cytology. you can't 
do a tissue section of pleural fluid. finally, using cytologic technique 
enables evaluation of some features you can never see on histopath. eg, 
distinction between mast cell tumor and histiocytoma.

why is this 
relevant? we're talking about cytology today, that's why.

slide: blood 
smear of animal with leukemia. we see many dark blue/purple cells. they 
look like small lymphocytes. might dx lymphoid leukemia, chronic form 
becuase they look like mature lymphs. but this smear is near the thick 
part of smear - you can't make out the features of the cells. over near 
the feathered edge you can see the purple cells are really monocytes, and 
this is a monocytic leukemia.

*** um, I thought we didn't see leukemias 
in dogs? ***

this thursday we're doing microscopy. we'll see three BM 
preps. we'll look at smear at low power, and figure out where to look, 
and THEN go to high power.

also, looking at section vs smear, things 
will look and stain differently. H&E is used for tissue sections, and 
Wright's/giemsa technique used for cytology.

slide: carcinoma - 
pleomorphism is evident, loosely adherent cell population.
slide: 
cytologic prep of same tumor. the marked pleomorphism is still clear, can 
see huge nuclei/nucleoli, etc.

now. that's an intro. now we know what 
she's talking about. 
everything from this lecture is in the handout. 
test questions are in the handout. should be able to predict them from 
this lecture.

what is meant by MYELOID and MYELOGENOUS? 
myeloid== 
derived from marrow. here, we're talking about hematopoietic elements, 
NOT lymphoid elements. that includes cells that generate granulocytes, 
monocytes, megakaryocytes, and rbcs. 
myeloproliferative diseases refer 
to these elements only.

when you do a marrow evaluation, myeloid means 
granulocytes and monocytes and their precursors, and doesn't include 
erythroid or megakaryocytic precursors. eg, you do an M:E ratio...the 
erythroid agents are separate.

this will become more clear as we go 
through slides and in lab.

the reason we stop to define this now is 
because myeloid is a term used often and loosely.

MARROW DISEASE

marrow 
is a tissue that generates very diverse cell types. that seems very 
simple and profound at the same time. here is an organ generating WBC, 
RBC and platelets, keeping us immune, oxygenated, and hemorrhage-free :). 
a multifaceted organ. it's inherently complex, and therefore some people 
make it harder than it has to be. think of those three cell types and 
their functions and you can predict the clinical problems based on those 
cell functions. if an animal is leukopenic, it will be prone to 
infection. if it is thrombocytopenic, it will bleed. that's really pretty 
simple. in lab on thursday, initial reaction will be that people get hung 
up in detail a lot of the time. do not do that! look for broad 
categories. go back to histology books. in fact, bring tht book to lab on 
thursday :)

so. in young animals, marrow is in flat and long bones. as 
animal ages, red marrow becomes yellow marrow in the distal extremities. 
this can revert to red marrow if needs increase. productive marrow 
regions usually red but can be tan or dark red. if tan, marrow is 
producing more WBCs. if dark red, producing more RBCs.

within the marrow 
elements include the hematopoietic elements, stromal cells, reticular 
cells, adventitial cells, adipocytes. stroma includes 
endothelium/microvasculature.., macrophages, plasma cells- plasma cells 
generally less than 1% of all nucleated cells. lymphocyte %age species 
specific. less than 1% in dogs, up to 10% in cats, horses/cows 3-5%. 
higher in young animals. don't memorize that.

bone cells are also 
present. 

matrix elements - fibronectin, laminin, collagen, hemonectin, 
proteoglycans.
when evaluating marrow, scan for cellularity at low power 
and look for particles - spongy cellular areas. maturation sequences of 
all cell types will also be routinely evaluated. as a given, the marrow 
we see thursday will be normal unless they have lymphosarcoma. you 
calculate the M:E ratio, percent of blasts, frequency of various 
elements, etc. so a BM exam takes a while. maybe 15-20 minutes for 
experienced clinical pathologist. 
one thing of note: cats have no 
stainable iron in the BM so you can't dx iron deficiency this way. you 
can in other species. M:E ratio in dogs/cats usually 1.4-2:1. in 
livestock, it favors erythroid elements more, so will be more like 
1.something to 1 whereas in dogs/cats more like 2:1

sampling marrow - 
can do core biopsy and do histopath, but that requires decalcification, 
or can do aspiration and make smears.

SLIDES:

* marrow section. ideal 
prep. mouse sternum. perfect fixation and plastic embedding etc. it would 
never look this good in real life :) we see a bony fragment with 
osteocytes, a megakaryocyte sitting on endothelium of sinusoid, blood 
vessels, and a whole bunch of purply cells - neutrophil, monocyte, and 
erythroid precursors. the myeloid and monocytic cells and precursors tend 
to be the light zones. the erythroid cells tend to be dark. you can 
grossly estimate the M:E ratio this way. should be 2x light zone...


erythroblasts/rubricytes==erythroid precursors. these are darker cells. 
the lighter cells are myeloid precursors.

when you are in the clinics 
here, you are not going to see those histo sections. we do not do core 
biopsies. only if we have a hypoplastic marrow, to see if that is real or 
if you got a crappy sample. mostly we do aspirates. you use a biopsy 
needle with a stylet that you remove, then you put on a syringe and suck 
out the marrow. skin is not pierced by the needle - you use a scalpel.

you apply a very abrupt suction to the syringe. if you're not forceful, 
it won't work. this is painful to the pet :(

then you squirt the marrow 
onto slides and make squash preps - drop tissue on slide, take another 
slide, put it on there, and drag them apart.
so you'll see these 
particles of marrow on the slide. if you don't see particles, and you 
have a prep that is very dispersed, it is hard to get a good smear and 
you don't see the flattening. optimum spreading is right next to the 
particles on the slide. if you move away from the particle you will not 
be able to evaluate cells correctly. so we'll scan for particles at low 
power and look next to them. you check particles for hemosiderin (storage 
form of iron)(not seen in cats) hopefully you will find nice area with 
flat cells, and you can figure out that dark cells are erythroid and 
light cells are myeloid and you can count them up and calculate an M:E 
ratio 

the RBCs lack nuclei, remember. right before they lose that 
nucleus, chromatin gets very dense. we don't count RBC in M:E ratio but 
we count the precursors with the dark, dense chromatin getting extruded. 
the other reason these cells are dark is because they have many ribosomes 
which are basophilic. the reason for the ribosomes is because they are 
making protein - the globin portion of hemoglobin. once they mature, the 
ribosomes are lost, and hemoglobin is more eosinophilic, so they become 
pink. the new RBCs are polychromatophilic - bluish/pink. 

in WBCs, 
nuclei get dense/quiescent, but are not lost. 

slide: marrow that has 
been stained with prussian blue for iron. if you can see hemosiderin in 
marrow, you don't need to do this.  sometimes you can't see it though. a 
cat will have a negative prussian blue stain normally.  if you do see 
hemosiderin in cat marrow, it's abnormal. 

other cell types: 
macrophages. these cells change morphology readily. here, we see one from 
a marrow with erythroid hyperplasia. this mphage is eating RBC nuclei to 
beat the band. they call this little collection of mphages surrounded by 
immature RBCs an erythroid island :)

plasma cells: here we see classic 
plasma cell - making immunoglobulins, so have lots of ribosomes, so have 
blue cytoplasm. round, eccentrically located nucleus with clear halo 
(golgi). 

lymphocytes - sometimes can't tell from erythroblast. but 
cytoplasm is usually not as dark a blue, and there is less cytoplasm per 
unit of nucleus, and chromatin is not as dense. 

slide showing small 
lymphocyte with an erythroblast - can see difference.

osteoblasts - oval 
nucleus, golgi off to side, eccentric nucleus, irregular cell shape


osteoclast - large, multiple round small nuclei. pinkish.

any questions?

some of that stuff is an intro to the lab. on Thursday we split into two 
halves. first hour, one half will do gross stuff w/drs haskins and wolfe 
and other half will do microscopy w/weber and mcmanus, then we'll switch. 


we're divided so that groups 1-8 go to one side, 8-16 go to another. 
we'll see one normal marrow, one hyperplasia, and one lymphosarcoma 
marrow.

profiles we may encounter are very predictable. 

erythroid 
hyperplasia - seen in response to internal or external blood loss or RBC 
destruction. you can see it in a few other situations but these are the 
main situations. if you see this, it should make you look for blood loss 
or hemolysis. what you will see is a shift in the type of marrow elements 
present. there will be more erythroid elements. overall marrow will be 
darker. calculated ME ratio will decrease as E component increases. 
anemia -->hypoxemia-->erythropoietin production--->erythropoiesis. so 
hypoxemia will stimulate this.
reasons for blood loss: trauma, 
coagulopathy, neoplasia, etc. 

one other thing - please think about 
blood destruction as category. 
don't memorize reasons.

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