Original subtitles

my name is purveyor boxia I'm one of the

back clinical faculty over here at

Russia number University Medical Center

in Chicago it's my pleasure to be

introducing our session today as with

our other Guam comm sessions this is

just a reminder that it is a Harvard

approved GME talk so please login with

their full name to be eligible at the

end so it's really my pleasure to get

our next long con session started here

and it's it's I'll start with

introducing our speaker for today dr.

jay Seltzer so dr. seltzer intended

medical school at the University of

missouri-kansas in Kansas City he

returned to st. Louis for his internship

and residency in internal medicine at

the barnes-jewish hospital at wash

Washington University School of Medicine

we also served as the chief resident he

subsequently went on to do as nephrology

fellowship training at Wash U where you

mean that as a full-time faculty and

serves as a clinical director of the

kidney stone Center and also associate

director of the fellowship training

program at Jewish Hospital in 1996 dr.

Susman moved his practice to Missouri at

the Baptist Medical Center where he was

appointed chief of Nephrology and a

medical director of renal and I Allison

services he has co-authored several

textbook chapters on natural diocese and

is also part of the real fellow Network

your analysis pediment of the month

series today he will be speaking to us

on the art and science of urine

microscopy which is a date which is a

must for all of us in our daily practice

and absolutely an important part of

training for our residents and fellows

so without further ado I will go ahead

and stop sharing my screen I have dr.

Saltzer start our session today thank

you very much for the kind introduction

over the next 45 minutes or so we're

going to review during microscopy really

focusing on its use in daily practice

we'll start out by talking about why

nephrologist should look at the urine

themselves and not solely rely on the

laboratories

interpretation then how to look at the

urine both in terms of using a

microscope properly preparing the

specimen staining techniques

illumination techniques and generally

how to use the microscope to get the

best image as you possibly can to

interpret will spend the majority of the

time talking about the different

elements of the urine sediment and then

how to interpret the findings and then

apply them to several clinical cases and

then at the end we'll have some time to

answer some questions so the question

that I often get asked is why take the

time to look at the urine and the lab

when the laboratory doesn't thorough

your analysis and they can do a

microscopic exam themselves first of all

is a nephrologist you know what you're

looking for you know the details of the

clinical case therefore you can provide

and perform a very focused exam of the

sediment the laboratories at a

disadvantage they may do several hundred

urine specimens a day and they don't

know any of the clinical information of

the case they're looking at all so you

have the expertise to interpret the

results whereas the laboratory may not

always have that expertise in terms of

its usefulness and clinical practice

there are several good articles about

this showing the utility in helping

differentiate the cause of acute renal

failure or Aki it also often helps you

determine whether a real biopsy is

necessary or not and then over time

assessing the response to therapy and in

terms of preparing the sample for

microscopy it's best to use a clean

catch specimen or catheterize urine if

you are collecting it from a Foley

catheter it's important to get it from

the tubing not from the bag you're in

sitting in the bag will result in

[Music]

decomposition of several of the formal

elements it's important to perform a

dipstick test making note of the pH and

the specific gravity as well as the

presence or absence of protein blood

white cells

you should use a conical bottom test

tube filled with urine and of course

don't forget to put a counterbalance in

the centrifuge I imagine we've all

learned that the hard way in terms of

centrifuging many labs have a protocol

with a specific rpm and time but that's

based on guidelines from the

accreditation agencies in terms of

standardization for cell counts so

there's no hard and fast rule what we as

nephrologists need to do if you have a

dilute specimen and it may benefit to

spin it for ten minutes then I'll do

that rather than what I typically would

do which would be five minutes 1,500 to

1,800 rpms or a relative centrifugal

force of 400 that's generally been shown

to give the best yield without risk of

destroying any formed elements by overly

aggressive centrifugal after that's done

you would pour off the supernatant

generally by just inverting the tube due

to the surface tension of the liquid

about a half a milliliter will remain in

the tube and you can use that to gently

resuspend the sediment at the bottom by

ever flicking the tube gently with your

finger or tapping it against the counter

use a pipette then to transfer the

sediment to a glass slide and carefully

apply a glass coverslip trying to

prevent air bubbles from forming ideally

what you want to achieve is a thin film

where all of the formed elements are

going to be viewing or in the same focal

plane air bubbles can change that and

make your images less clear staining I

think is very important not everyone

takes the time to do this step that is

very very simple the best way to point

out the utility of the stern himer

Malbin stain which is the most common is

to remind everyone that the best

resolution images are achieved using

bright field microscopy with the stains

and it can be inherently stained with

say bilirubin or hemoglobin or anything

else but using these super vital stains

like this mixture of crystal violet and

safranin really give the image a better

quality better resolving power under

bright field and it facilitates

identifying different cells which can be

difficult otherwise with the stern himer

Malbin stain you can generally readily

differentiate white blood cells from

tubular epithelial cells and it very

much highlights the cast material making

casts quite visible when they otherwise

might not be except under phase contrast

it's very simple after the usual

preparation all you have to do is add

one drop of the stain to the resuspended

sediments gently swirl it around and

allow one to two minutes for this stain

to be taken up and then prepare the

slides it's not like other stains that

require fixing and tedious steps very

simple these stains are also readily

available and have a good shelf life the

Soudan 3 stain is not necessarily used

routinely but especially if you don't

have polarization available it's very

helpful to identify lipids in the urine

again it's very simple after the usual

preparation you just add 2 to 3 drops of

the Soudan 3 staying mix I'll clear on

the slide but it takes a little longer

when the stain I want to spend a little

bit of time just reviewing microscope

Anatomy again in order to get the best

quality images it's important to have

the microscope set up and adjust it

properly

you see if I can grab a pointer here

are you able to see my pointer very

small let's see see okay the things I

want to point out is at the bottom where

the light sources is a field diaphragm

it's an iris diaphragm that controls the

amount of light that shines up into the

condenser the condenser maybe one of the

most important parts of the microscope

that's often overlooked it's job is to

focus the light on a single point on the

exact plane that you're viewing on the

slide the condenser is where all of the

different illumination modalities are

changed and we'll talk in a lot more

detail about that in the condenser there

is a separate iris diaphragm that

especially with brightfield allows you

to increase contrast the condenser focus

knob raises or lowers the condenser

allowing you to focus the light on the

specimen I'm called achieving Koller

illumination which we'll talk about in

more detail and then the more obvious

features are the mechanical stage

adjustment which moves the slide back

and forth the fine focus knob and then

the objectives and the eyepieces

in order to interpret your findings you

have to be able to tell what you're

looking at I think this might be the

most frustrating part of people doing

you're in microscopy is finding that

they can't tell what they're looking at

because the images aren't clear some of

this has to do with the quality of the

microscope and the objectives but much

of it also has to do with proper

technique so ideally you should have a

microscope of reasonable quality the

typical minimum magnifications you would

like to achieve or a hundred times 400

time in a thousand you want to make sure

the microscope objectives the eyepiece

and the condenser lenses are clean and

if you're using a camera or iPhone or an

adapter make sure those lenses are clean

and then as I refer to earlier before

viewing you want to make sure that

you've achieved proper cooler

illumination and we'll talk about that

next choose the appropriate illumination

modality for what you're looking at

review that in more detail and then very

importantly always use glass slides and

glass cover slips never use the plastic

multi chamber slides that the lab uses

these are of poor optical clarity their

thickness prohibits use of an oil

immersion lens it scatters the light

from the condenser and results in very

poor image quality again remember that

the highest resolution images are

usually obtained with bright field

illumination of a stained specimen for

instance using the stern Humber Melvin

stain and ideally if available with an

oil immersion lens so Kohler

illumination refers to the act of

focusing the light source on the

specimen that's being viewed with the

intent being to limit the light to a

pinpoint covering only the field of view

for that objective and the light should

be focused in the plane that you're

focused on if you don't do this the

light if it has VOC

is not on the plane you're looking at

some of the light is scattered through

the glass or the coverslip

or reflected and it clouds the image

overall your goal is to restrict the

light to just the specimen in the

observed area and way you achieve that

and of course this is not newer

microscopes have a fixed condenser this

is not necessary but for most scopes

including older ones with an adjustable

condenser this is applicable so the way

this is achieved as using the low power

objective you focus on the specimen we

see in the top image here and then you

close the field diaphragm which is the

iris diaphragm on the bottom light

source you close it so you can see kind

of the outline of the iris diaphragm

being octagonal or something like that

and then you focus the condenser up and

down until you see the edges of the iris

diaphragm very crisp and in focus and in

this image here you see it's kind of

off-center your goal then is to Center

the light source using these centering

screws that are present on the condenser

until the image is right in the middle

and then when it is you open up the

field diaphragm but just enough to fill

the field you're looking at no more than

that now if you scatter too much light

onto the slide it will cloud the image

that should be repeated when you change

objectives ideally what we went through

was just on the low power objective so

the illumination or deities will talk

about our bright field which all

microscopes are inherently set up to

achieve dark field which most

microscopes can achieve right out of the

box phase contrast requires specific

add-ons to the condenser and the

objective which we'll talk about and

then polarized light is also an add-on

but it's simple generally to add on

after the fact so bright field is the

simplest of all illumination techniques

in this modality the

is illuminated from below light shines

from the light source the condenser

lenses focused the light on the specimen

the light is gathered by the objective

lens and sent to the ocular lens dark

field is a little bit different in this

modality you are excluding the direct on

scattered light from the image and as a

result you see the image illuminated

against a dark background this is

achieved by a dark field disc present in

the condenser that blocks the central

portion of the light but allows the

peripheral light to hit the condenser it

is then focused but it ends up being

reflected away from the center of the

objective so the objective only gathers

light from the specimen and not the

peripheral light what you're left with

as you can see in these two images is

the difference between a bright field

and a dark field of the same exact

specimen bright field gives you much

better resolution dark field is very

helpful for bringing out items with a

low refractive index that might

otherwise be invisible or difficult to

see on bright field certain things light

up on dark field specifically lipids and

crystals so it's very helpful in

scanning on low power looking for the

presence of liquids or crystals also

many casts will light up and be quite

visible dark field I use a lot of times

just preliminary for scanning a specimen

just to get an idea of what's there

these are some different examples of

dark field images these are calcium

oxalate monohydrate crystals in the

upper left yeast in the middle upper a

granular cast with tubular epithelial

cells adherent to the exterior on the

top right the bottom right a red blood

cell cast next to that are some

dysmorphic red blood cells they can't

the sites and then on the bottom row you

can see these lipids in this case just

light up as do these calcium oxalate

monohydrate and dihydrate crystal

on the bottom-left so dark field is

useful but you don't get the resolution

that you get with bright field here's a

low-power image of a whole field and you

can see how certain things light up this

calcium oxalate monohydrate crystal

these oval fat bodies which contain

lipids and you can see other items here

gives you a good overview but again the

resolution is not as sharp as with

bright fuel phase contrast is quite a

bit different this illumination modality

actually won the Nobel Prize in Physics

in 1953 it's a means by which you alter

the course of the light and enhance the

contrast along the periphery of items in

your specimen giving kinda the halo

around each border each item that

enhances the contrast and makes them not

only more visible but also lending

somewhat of the three-dimensional

characteristic to them at times this is

achieved by using two separate discs to

alter the course of the light one of the

discs is in the condenser itself it's

called the condenser annulus and it is a

dark ring with a dark ring in the middle

and then in between the two light is

able to pass so you get a cone of light

passing between the outer disc in the

central disc this cone of light is then

focused through the condenser across the

specimen and in the objective you have a

matching phase plate that is a structure

such that light passes where there was

dark below light doesn't pass where like

did below and then the central light is

allowed to pass so it's kind of the

inverse of the condenser annulus what

this does is it alters the light so that

it's hitting this specimen

only the peripheral light that goes

through the cone and then it's altered

giving it enhanced contrast

as I mentioned you have to have a phase

disc in the condenser and a matching

disc in the objective and microscopes

fitted with this they will be labeled as

such so the phase to plate has to be

used when you're using the phase 2

objective in this case this is the 40

time objective on the condenser you

would put it to phase 2 to achieve phase

contrast using this objective so here

you see some sample images and if you

look closely especially at the top row

middle where you see some dysmorphic red

cells and normal red cells floating by

you clearly see the bright halo around

the structures that really enhances the

edge and you really see the contrast in

the top right you can see this a can't

the sight with the halo around it that

otherwise might be difficult to see you

can see the matrix of the cast if there

weren't this halo around it it would

blend in with the background so a lot of

times phase contrast allows you to see

island casts and cast matrix that's

otherwise invisible on bright view on

the video on the bottom right you see

some granular motility in a white cell

again these would be very difficult to

see without phase contrast this

cholesterol crystal in the bottom middle

you see the bright halo around the

periphery that really makes it stand out

and then in the bottom left all of these

mucous strands these would be invisible

under bright field it's this little halo

and enhanced contrast that makes it

visible so that's the real utility of

phase contrast unfortunately as is the

case usually when you increase contrast

you decrease resolution so phase

contrast cannot achieve the same

resolution as Brightview

but it is useful in many situations

polarized light is really helpful in

identifying lipids and crystals it's

fairly simple in this form this would be

called simple polarization there

Moorcock

methods of polarization with

compensators but for our purposes the

simple polarization is adequate in order

to do this you have to have a filter

below the condenser on the light source

and then another filter somewhere in the

optical path way above the specimen

usually between the objective and the

observation to to the eyepiece when you

use these you will rotate the bottom

filter until it is oriented 90 degrees

from the top at which time you are able

to then see items that are birefringence

in other words scattering light in one

direction now this is very useful for

identifying lipids crystals and also

contaminants that really light up

microscopes that are fitted for this

generally have a slot as pictured here

where the top filter goes in and then

usually an extra space over the light

source where the bottom filter that can

be rotated goes in and you can retrofit

scopes you can actually just buy two of

these put one over the light source and

one on top of your slide and of course

this limits being able to use 40 and 100

time objectives but at least it's doable

here you see some examples of polarized

light on the top left you see under

phase contrast a lipid cast but if these

lipid droplets were all of uniform size

you might not be able to be sure that

they're not red cells but right below it

on the polarized image you see these

line up with a characteristic maltese

cross pattern this one's using a red

compensator so it's in color instead of

black and white but this shows you that

these are actually lipid droplets the

same with the image next to it which is

another fatty cast you see the typical

Maltese Cross pattern under polarization

here the this is using a simple

polarizer lots of lipids here and again

the Maltese Cross pattern same with

these others

on the bottom two images on the right

are fibers artifacts especially fibers

light up and usually very vivid colors

makes it very easy to spot what's an

artifact and what's not so the question

is often which modality should you use

and the answer is you should use all of

them if they're available to you but all

for different purposes now a bright

field will give you the best resolution

especially with the stained specimen

dark field is very useful for

identifying structures with a low

refractive index it's also useful for

scanning under low power things light up

and you can easily spot crystals lipids

and casts phase contrast greatly

enhances the contrast of items and is

very useful for identifying dysmorphic

red cells it's also useful in helping

you figure out what you're looking at

getting a different view of cells if

there's any question what they are and

polarized light again very useful for

determining whether something is lipid

or crystalline or an artifact this is a

good example that illustrates the

different modalities and kind of a

real-world application on the top left

what we see here under bright field are

what looked like some by concave discs

they almost look like little doughnut

shaped things and you might not on a

less clear image be able to tell whether

these were red blood cells or whether

they were crystals under dark field on

the top right they really light up which

gives you some idea that these are

probably crystals and not red cells

which would not light up on dark field

on the bottom left on phase contrast you

see lo and behold these are not just

free-floating in the urine they're

actually within a cast which you could

not see under bright field or dark field

so base contrast is very helpful at

showing you the outline of the protein

matrix here and then on the bottom right

under polarization it shows you these

are indeed crystals in this case

calcium oxalate monohydrate so a good

example of the different illumination

modalities and their usefulness so we'll

move on now and talk about the elements

in the urine sediment first of all the

cells of importance to us then lipids

and castes crystals will very briefly

talk about that could be a topic in and

of itself but I'll point you to some

resources for more information on those

and then very briefly talk about some

common artefacts determining what you're

looking at the size is quite helpful

this gives you a kind of a proportional

scale of the difference in size between

bacteria red blood cells white blood

cells tubular epithelial cells

transitional cells and the large

squamous epithelial cells whenever

you're not sure what you're looking at

remember that the size can really help

you narrow it down especially if you

can't tell which are epithelial cells

which are white cells red cells

generally have a homogeneous appearance

there you do not have any granular

cytoplasm white blood cells have a

granular cytoplasm and the more common

one is at polymorphonuclear leukocytes

with segmented nucleus tubular

epithelial cells generally have a large

eccentric nucleus with a very distinct

border along the nucleus they can be

small they can be medium they can be

large vary quite a bit in size depending

upon their origin in the nephron they

can be cuboidal they can be trapezoidal

they can be spherical much of it depends

on where they've come from along the

nephron and whether they've been in the

urine for a while transitional

epithelial cells differ in their

appearance depending upon whether their

superficial layer or deep layer they're

often in a caudate shape as pictured

here but they can be spherical and

sometimes very difficult to

differentiate from tubular epithelial

cells squamous epithelial

cells are fairly easy to identify they

have an irregular shape a very small

central nucleus much larger than the

other cells here's an example of a few

together in the same screen what you see

here on the bottom left is a red blood

cell with kind of a homogeneous

cytoplasm this is under phase contrast

so you see kind of the halo around them

you see tubular epithelial cells in the

middle with the eccentric nucleus with

the very distinct border on the top

right you see a white blood cell with

the segmented nucleus and then the

bottom is a different type of white

blood cell with granular motility noted

in the cytoplasm we'll talk a little bit

more about that here's another good

example of the difference in size here

on the Left we see a red blood cell cast

with some embedded tubular epithelial

cells where you can clearly see the

difference in size the image on the

right you know you see a little of

everything you can see the red blood

cells compared to the appearance of the

white blood cells both dark staining and

pale staining and then a tubular

epithelial cell with the large eccentric

nucleus in this case with lipid

inclusions so called oval fat body here

with the stern heimer of Malbin stain

which you see can clearly allow you to

see the different structures different

images here phase contrast transitional

epithelial cells compared to red blood

cells tubular epithelial cells and white

blood cells and then the larger squamous

epithelial cells on the right red blood

cells generally about 78 microns in

diameter

they are generally round or slightly

oval and have a biconcave profile and

hypertonic urine they can become

cremated as seen in the upper right as

they are present for a while in the

urine they can lose some of their

characteristics and become kind of go

cells especially in hypotonic urine

dysmorphic forms can occur here's a

cartoon just showing a normal appearance

kind of the ghosted cell created and

then so-called dysmorphic cells there

are a lot of things that can mimic red

blood cells particularly air bubbles

lipid droplets as we saw earlier calcium

oxalate monohydrate crystals rarely

pollen starch isn't used much on gloves

anymore but sometimes it can be mistaken

for red cells neutrophils that are

degenerating and smaller and hypertonic

urine sometimes can look similar and

yeast can very often look like red blood

cells and in fact budding yeast can

sometimes look like a campus sites which

we'll talk about a little more sperm

that have lost their tails also can look

like red blood cells so in terms of

figuring out the origin of the red blood

cells you see in the urine it's helpful

to be able to determine whether they are

a glomerular origin or non glomerular

and one of the specific findings in

urine that will lead you towards

determining their plumeria origin is the

identification of dysmorphic red blood

cells specifically a kantha sites so

when you have damage to the glomerular

basement membrane red blood cells can

squeeze through and in the process they

have a slight deformation of their

membrane but it's not until they

actually flow through the tubular and

they're subjected to osmolar shifts that

the membrane actually develops these

cytoplasmic blasts or protrusions this

one is so called Mickey Mouse looking

cell g1 cells were acanthus sites and

these are found to be the most reliable

indicator of glomerular origin of

hematuria there are lots of different

dysmorphic red blood cell forms but the

one specifically we're going to talk

about that seemed to be the most

lemare euler origin or the acanthus

sites which can vary from kind of a ring

form but generally have these

cytoplasmic gloves here seen under

bright field in the bottom phase

contrast but you can see these

cytoplasmic Bloods this is what you're

looking for to tell you whether the red

cells you're seeing or from glomerular

origin here's an example I've circled a

some of the acanthus sites but you can

see them hopefully with these

cytoplasmic blobs here under phase

contrast which really is the best

illumination modality to look for

dysmorphic red cells the normal ones

appear kind of as the bright white disks

the dysmorphic ones will often look

darker and smaller and again you're

looking for these cytoplasmic gloves

here pictured on the right these are on

stainless Stern Hiram Alban and enlarge

but these are typically kantha sites

neutrophils in the urine can occur

either from infection or inflammation

anywhere in the urinary tract although

any white cells can appear in the urine

it's generally the segmented

polymorphonuclear neutrophil that we see

these are generally identified by the

granular cytoplasm in the segmented

multi lobe nucleus they're generally 10

to 12 microns in diameter often

spherical but if they're left on the

slide for awhile baby can develop pseudo

pods and look almost amoebic the stern

himer Malbin stain greatly facilitates

identifying these there's generally two

different staining patterns that you

observe dark staining neutrophils have a

translucent or granular cytoplasm and

very avid magenta red staining of the

segments and nucleus these cells these

dark staining ones are older and

generally no longer viable

the pale staining ones that appear kind

of with a pale and distinct blue

staining pattern of the cytoplasmic

nucleus are viable young cells as such

they often have visible cytoplasmic

granular movement

whether or not you see this cytoplasmic

granular motility depends on whether the

viscosity is low enough and this usually

occurs in hypotonic urine these pale

staining white cells with granular

motility are called glitter cells

sometimes called stern himer Malbin

cells who were the original they

originally described these or granular

motility cells and these were once

thought to be indicative of

pyelonephritis but they're now generally

agreed upon to be a nonspecific finding

and again these are younger viable pale

staining white blood cells glitter cells

you can see their appearance here on the

top under bright field in the middle

under phase contrast and hopefully you

can see under dark field now I'm East

can be visible in the urine it's very

easy to see under dark field is pictured

on the lower row on the top you can see

how these yeast these budding yeast can

look very similar to an adjacent red

blood cell and you can see how these

could be misconstrued break antha sites

in fact one of the cases we'll talk

about in the end the laboratories auto

analyzer reported three plus budding

yeast on the specimen it turned out they

had glomerular nephritis and they recant

the sites the yeast generally have a

different coloring they tend to be a

little more oval shaped and not circular

and of course if you see pseudohyphae

formation that makes it obvious that

these are yeast bacteria are obvious

they're generally modal you can see them

especially under phase contrast and you

see how easy they are to see under dark

field on the left so

you'll have an unexpected visitor in

your sediment here trichomonas these are

about the same exact size as a white

cell and if they're no longer living

they look very similar to a white cell

of course when they're alive their modal

you can usually see the flagella and

very typical movement here under

different imaging modalities lipid Juri

is a very important finding damaged in

the glomerular basement membrane can

allow the plasma lipids to escape into

the urinary space these lipid droplets

are absorbed by tubular epithelial cells

and at some point these cells become

engorged with lipids and then slough off

and pass into the urinary space becoming

what are termed oval fat bodies these

lipid droplets can also be taken up by

macrophages and again referred to as

oval fat bodies cholesterol crystals can

form not as commonly but they're

pictured here on the bottom right they

have a very unique geometric shape very

neat now these are under on the top

phase-contrast on the bottom this is

kind of achieve appearance by just

shifting the condenser daytime to give a

little more 3-dimensional appearance but

this is a cholesterol crystal with some

red cells adjacent oval fat bodies again

are indicative of lipid area they can

vary in size and appearance with the

polarizer you can identify or verify

that these are indeed lipid droplets a

lot of times these oval fat bodies will

start disintegrating and the free lipids

will be released when these oval fat

bodies are in a cast and the lipids are

released as the tubular epithelial cells

to generate you end up with a lipid cast

a lot of times you'll see oval fat

bodies within a cast here on the left

with the stern himer Malbin stain you

can see these oval fat bodies within

this cast same thing on the right this

is overexposed a little bit so you can't

see the protein matrix of the

as well but lots of oval fat bodies here

and for comparison of red to generating

red cell the Soudan 3 stain you can see

what it does on the bottom row here it

colors the lipids and orange makes them

very easy to identify again this is

useful if you don't have polarization

available castes are formed by the

solidification of Tam horsfall mew

caprile team when it solidifies it molds

into a caste and in doing so it

incorporates any cells present in the

tube you'll into the structure and as

the caste passes into the urine it

preserves evidence of what was in the

tube you'll I'm helping clarify the

nature of whatever is going on in the

kidney anything can be incorporated in

there that can be in the urinary space

crystals red cells white cells tubular

epithelial cells lipids but not

transitional epithelium cells not

squamous epithelial cells

hyaline casts are the most common thing

that you'll see they are not pathologic

they are normal in states of low urine

flow or volume depletion or whenever the

urines concentrate there is simply the

solidified tam horse-pond mukha protein

under bright field microscopy you see

here they're almost invisible now these

images are courtesy of Florian at Swiss

macro we'll talk a little more about his

resource but under bright field they're

almost invisible under phase contrast

quite obvious a very common finding

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granular casts result from the breakdown

either of cellular castes or

degenerative products from the tubular

cells and proteins depending upon the

size of the inclusions they can be

considered coarser finally granular but

that really of no significance they can

be seen in the absence of renal disease

after exercise but generally they're

indicative of some type of tubular

injury especially when seen in

conjunction with free tubular epithelial

there are tubular epithelial cells

incorporated within the cast here we see

just examples of some granular casts on

staying with stern himer Malbin stain

more coarsely on the bottom and then in

the middle what's been termed muddy

brown casts these are dense granular

casts there's not a clear consensus on

what the actual pigment is here it's

thought to be like the fuchsine but

could be other pigments associated with

cellular degeneration castes can become

pigmented i here in the bottom left this

was a case from yesterday you see a

brightly Billy Rubin stained cast with

some tubular epithelial cells within it

I'm on the bottom middle these muddy

brown casts again on the right staining

from rifampin on the top right staining

from hemoglobin or myoglobin would look

about the same in this case it's

hemoglobin now you can see some I can't

the sites adjacent to this

and then this I think this was Vinay's

of purity not a staining white blood

cell castes are indicative of

inflammation or infection they can

contain just a few white cells or be

densely packed although there were kind

of associated usually with interstitial

nephritis or pyelonephritis they're

actually not an uncommon finding and

glomerular nephritis especially

proliferative genes it's very very

important with white cell castes before

you call them a caste to make sure

you're differentiating a caste from a

clump of white cells white cells in the

urine tend to clump together and when

they clump in kind of a linear fashion

they can look very much like a caste but

before you call it a caste you want to

see this protein matrix surrounding the

cells and forming a clear cylindrical

structure so just be aware of

interpreting pseudo castes mistakenly as

castes especially with white cells in

these images you see good examples of

the difference between the dark staining

and pale staining Western heimer Malbin

again the dark staining or older

non-viable white cells the light

staining ones are viable younger stain

younger cells red blood cell casts are

an elusive finding this is what we all

kind of strive to find when we're

looking at a new case suspected GN they

usually signify the presence of a

proliferative glomerular process or

vascular disease less commonly they can

be seen in acute earnest ditional

brightest renal infarction even in

diabetic nephropathy especially when you

get masanjia lysis you get red cells

passing into the urinary space and

forecasts so they can enter the tubular

lumen either via damage from regular

capillary membranes or damaged tubular

basement membranes they have a variable

appearance you can sometimes see only a

few cells other times they're so densely

packed you can't even see the protein

matrix usually the cells retain their

shape and hemoglobin content that as

they degenerate their appearance can

change considerably they can become

depigmented appear as ghosts

in that case just the outlines apparent

but the cytoplasmic pigment is no longer

visible as they degenerate the

hemoglobin forms Heinz bodies which

appear is small dense granules along the

cytoplasmic membrane and as the heme is

released during this process it pigments

the cast of reddish brown color and

eventually results in a hemoglobin caste

here we see different examples these are

bright filled unstained you can clearly

see the red blood cell outlines in this

case they are quite visible because of

the hemoglobin pigment released as you

see in the middle of the bottom row some

degeneration occurring and it's really

kind of transforming into a kima globin

cast phase-contrast it's a little harder

to see because of the high contrast but

you can see it at different cells

outline clearly within the cast from a

few sparse ones on the top left to the

very dense just to the right of that

and again as you can see on the bottom

left the more items you have next to

each other all with a halo around them

you get a little less resolution but

this is a typical appearance under phase

contrast with stern Homer Mountain

staining you get higher resolution

images you can clearly see the

morphology of the red cells here you can

see how different they can appear

depending on how densely packed they are

on the stage of degeneration as these

cells to generate you end up seeing at

times just the outline and then further

along you see these dense dark granules

these are these so-called Heinz bodies

which is denatured hemoglobin occurring

as part of the generating process on the

left here you see still clear red cells

within the cast but on the right a

little later you see really what's

becoming a hemoglobin cast with a few

red cell remnants

tubular epithelial cell casts our renal

tubular epithelial cells embedded within

the protein matrix these are generally

indicative of acute tubular injury very

helpful when looking at a case of a ki

when you see these it really leans you

more towards tubular injury as a part of

the process remembering of course that

you can have tubular injury along with

every other finding so it doesn't imply

that it's just tubular injury but at

least it gives you an idea to what

extent the tubules are involved on the

top right here you can see not only

tubular epithelial cells but some white

cells for comparison and then some red

cells in the middle bottom you see a few

lipid droplets in there along with the

tubular epithelial cells and a white

cell just for comparison

waxy casts are seen primarily in chronic

renal failure they're thought to

represent kind of the end product of

cast evolution when castes are trapped

in the tubulin spend a long time they're

in their passage flowing from smaller

into larger ducts and eventually forming

these broad casts with a very different

refractive index they have a waxy

character they are more rigid they often

have sharp appearing edges kind of

fractured ends here are different

appearances under phase contrast with

different staining and different

adjustments of the condenser lipid casts

we talked a little bit about these are

the results of lipid droplets ending up

in the cast you can have some with just

a few droplets and others that are so

densely packed they almost look like a

coarse granular cast in the top left

here you can see how if these were all

uniform size you might mistake this for

a red blood cell cast and that's where

polarization is helpful to help you

verify these are lipid droplets that

seem to the right with a typical Maltese

Cross pattern pseudo casts beware of

these again before you call something a

cast you want to make sure that there's

visible protein matrix around the

inclusions on the bottom left this looks

like a red blood cell cast but it's

actually red blood cells that are up

against a mucous thread that has kind of

created a dam blocking all of these red

blood cells from moving on the slide but

on the left side you don't see any

protein matrix surrounding them so this

is a pseudo cast on the second image

from the left on the bottom this is a

mucus strand with white blood cells

adherent to it again it's almost

tempting to call this a white blood cell

cast same thing with these others these

are cells that are adherent to or up

against a mucus strand or mucus thread

I'm in the top middle again courtesy

events with snuff row a typical image of

amorphous debris here these are

amorphous phosphates in the urine when

they a grenade like this they can look

like a granular cast but again you don't

really see a cylindrical structure these

mucus strands are just ribbon like mucus

thread they're of no pathologic

significance but sometimes they confuse

matters when they form pseudo casts so

we'll briefly just talk about different

patterns I think these are fairly

obvious and by no means specific

generally when you see he maturely in

association with red blood cell cast and

proteinuria that's indicative of clam

Arowana Friday's heavy proteinuria and

lipid urea usually a non proliferating

process granular cast tubular epithelial

cells tubular cell casts tubular injury

or a TN pyuria and white cell casts

pyelonephritis and interstitial

nephritis but don't forget white cell

casts are a common finding in

proliferative genes a normal UA with

just Highland castes can be seen with

low urine flow rates volume depletion

and when you have normal urine sediment

no casts no cells no proteinuria that's

when you would be on the lines of pre

relays of tinea vascular cause

obstructions something else crystals

just briefly you can see all kinds of

different crystals unfortunately this is

a huge topic and we're not going to

cover I do want to point you to the is n

Atlas on your in microscopy this is by

Jose Tesla Bologna

an excellent resource not only on

urinary sediment findings but really

good coverage of crystals also on

Florian buck rumors at Swiss necro site

lots of coverage on crystals here we see

a typical appearance of cysteine in the

top left calcium oxalate monohydrate and

dihydrate the dihydrate on the top right

uric acid below that in the middle into

the right calcium oxalate monohydrate on

the bottom right an unusual variant of

uric acid on the bottom that looks

almost like a sunflower

and then a case from yesterday a typical

leucine crystal here various artifacts I

think we've talked about it's important

if you see something that just doesn't

look typical it's probably an artifact

sometimes polarization can help you

differentiate here's a red blood cell

cast that's wrapped around a fiber

artifact and then various fibers that

can end up and then some crystalline

artifact probably some powder

so now we'll finish up with a few cases

I think we've had about seven cases I'll

go through quickly and then we'll have

some time for questions I picked these

cases to help illustrate situations

where I think that urine sediment helped

figure out what was going on

helped guide to a diagnosis and

treatment plan and then lastly a case

that illustrates almost every abnormal

finding in one case so this one is from

2014 this was a 76 year old woman that

came in with a history of renal failure

skin rash a few weeks after taking

antibiotics amoxicillin levofloxacin for

a sinus infection creatinine was 3.1 and

when we're going through the history I

thought this was going to be

interstitial nephritis rash a few weeks

after taking an antibiotic the lab UA

showed two plus blood there were some

white cells the lab didn't report any

casts but when I looked at the sediment

there were red blood cell casts and a

campus sites this led to a renal biopsy

which showed Crescenta necrotizing g-n

not interstitial nephritis as I thought

it was going to be with her history the

next case this was actually not a

patient this is the mother of one of my

patients who ended up in the hospital

and I went by simply to make a social

visit and when I was talking to her she

said the doctors told her she had some

blood in her urine she asked me to look

through her case and it turns out she

was admitted with recurrent pneumonia

this was the fourth time she had

pneumonia despite getting antibiotics

several times her creatinine was 1.4 and

looked like her baseline was 1.0 and

indeed the UA was reported as two-plus

blood with five to ten red cells but

nothing else I looked at her urine and

much to my surprise she had a few a

kantha sites in fact most of the red

blood cells that were there where he

can't the sites and then piecing it

together with her history it kind of

made sense and I rented up formally

consulting on her and we

committed a biopsy and indeed her NPO

came back positive and she had a focal

necrotizing GN that might otherwise have

been missed that was a good case this

was a fairly typical one it was an older

Asian woman that had a history of

microscopic hematuria but she also had a

history of bladder cancer that she

declined treatment and her hematuria for

probably five or six years had been

attributed to the bladder cancer until

her creatinine started to rise and

that's when she was referred for urine

sediment showed red cell casts and some

tubular cell casts as you can see here

on the bottom right we ended up by op

singer and as might be predicted she had

IgA nephropathy but a good example of

the can't always attribute red blood

cells to a bladder source this was an

older man that came in with heart

failure and Aki granted 2.6 where his

baseline was about 1.8 his UA had white

cells and his history when he came in

was attributed to CHF and a UTI but when

I looked at his urine he actually had

white blood cell casts and looking

through his history and talking to him a

little further it turned out he had been

on sulfasalazine for a few months and

the dose was recently increased he

wasn't a good candidate for a biopsy

there was really no indication to do so

we stopped the drug treated him with

enteric steroid and his creatinine

within about three to four weeks drop

back down close to his baseline

this case was a little puzzling at first

this was from a while ago 63 year old

guide that had a baseline credit of 1.5

came in with bacteremia and Aki granted

a 3.9 he had bacterial endocarditis was

treated with cefazolin and nafs Ilyn who

was volume depleted and you can see

starting on the right here on March 16th

his creatinine went from 3.9 and down to

1.4 with hydration but then it started

going back up and went to 1.5 2.0 and

when we saw this happening knowing that

he had been on Massillon for about a

couple weeks at this point we looked at

his urine and lo and behold he had white

cell casts we switched his antibiotics

around and his renal functions started

to drift back another example of going

back and looking at the sediment when

things are changing this was a very nice

man that came in with chronic sinus

infections had been on antibiotics three

or four times over the course of the

year and again when I was talking to him

I thought this was going to be

interstitial nephritis but then I saw

this rash on his legs with palpable

purpura and looking at the lab UA this

is the case I referenced earlier he had

reported three plus budding yeast in the

urine but when I looked at his urine

these are actually a campus sites and

indeed he had a whole bunch of red blood

cell casts his biopsy showed Crescenta

Chi GA but a good example some of the

flow integers that are used in automated

labs will detect budding yeast as a

kantha sites I'm sorry it can't the site

says budding yeast and this is the last

case now this was a 66 year old man this

is from a month ago came in with four

months history of fatigue chronic sinus

infections and antibiotics his exam was

fairly benign he just had some lower

extremity edema no history of coffered

hemoptysis bu n 88 creatinine

twelve albumen 3.5 looking at is urine

sediment the first thing I saw were

white blood cell casts here you see some

dark staining white blood cells and then

paler staining white blood cells so at

first when I saw that again I was

thinking interstitial nephritis but

right away looking around he had all

kinds of red blood cell casts the most I

think I've ever seen very good examples

of red blood cell casts here under

bright field with stern heimer Malbin

staining but he also had tubular

epithelial cell casts here with oval fat

bodies under bright field here oval fat

bodies under phase contrast here perfect

tubular epithelial cells and here

tubular epithelial cell but you can also

see in a campus site here in red blood

cells in the same cast and he had cast

that had everything all at the same time

red blood cells tubular epithelial cells

white blood cells everything his

serologies were all negative his anti

GBM antibody was negative MPO PR 3 an A

complements all normal

of course we proceed with a renal biopsy

and interestingly it showed linear IgG

he had cellular crescents in about 45%

of the glomeruli and a nice correlative

finding on the biopsy here those red

blood cell casts so visible in his

urinary sediment and even a white blood

cell cast on cross section within a

tubular

so he was treated for anti GBM disease

and just came off dialysis last week

he's actually I think going to do well

he had a I think interstitial fibrosis

and tubular atrophy of less than 10%

surprisingly so he may actually do okay

let me go over a few resources I want to

point you to as you've probably seen on

your own most text books the pictures of

urine sediment are not that clear so

it's a little hard to learn with good

resources but these are the two best

that I found this is by Giovanni Fugazi

has published numerous articles on urine

sediment fantastic reviews this is an

excellent book it's no longer an active

print but I think you can still find it

and this might be the single best

resource the color atlas of urinary

sediment by Merrill Hebert he has an

older textbook that's also quite useful

well this is really good and then of

course on Twitter my feed Florian buck

Kramer at Swiss Neff Roe excellent

resource

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at Bellas Neff hepato really excellent

case reports urinary findings at close

8's are lots of interesting urinary

findings and the hash tags that are used

commonly urinary sediment you're in

microscopy Samira Farooq has done a

great job coordinating on the renal

fellow Network the urine sediment of the

month every month someone contributes a

specific topic these are very concise

reviews and then again the is n Atlas on

urine microscopy very thorough so with

that I'll stop and see if anyone has any

questions

Thank You dr. sell so that was an

amazing talk and we had ton of comments

on this and the chat in terms of the

great images and definitely a thank you

from everyone there are a few questions

I'll start with the first one from dr.

glass ik and then it's many years ago

this is from dr. glasses many years ago

Ken Fairley showed that hundred percent

of our sites and the normal urine were

just more 'fuck if true then a subject

with low level non-blue marie-laure

mature hematuria about two to three

times above normal can exhibit a 50/50

mixture of dysmorphic and normal morphic

hematuria

is that do you agree with that

formulation I think that's probably true

I think that's why generally I only rely

on a campus sites as being indicative of

the glomerular etiology there's so many

other dysmorphic variants that can occur

in the absence of glomerular disease

cremated cells are dysmorphic but there

are normal finding and hypertonic urine

so I think really focusing on just the

acanthus sites or g1 cells gives you the

best opportunity to single out what

cases may be at kumarila origin another

question that was in the chat from dr.

Ling was in terms of the consensus for

you know cells for high-power field what

is the what is the consensus in terms of

is that 10 X 20 X 40 X what is the

general I think that's decided by the

ACP I believe when they're talking about

high-power field it's a 40 X objective

it's also contingent upon

Standardization of the centrifuge the

volume that's resuspended the volume of

the well of the plastic slide that they

use they're all standardized by ACP and

most accredited labs have to use those

protocols I generally find cell counts

not of much use it's more useful to me

to know whether their cells they're not

necessarily whether there's 3 to 5 5 to

7 7 to 10

but the labs unfortunately are held by

following these protocols that also kind

of prohibits the central labs from using

stains using glass slides instead of

their plastic well slides I think that's

why I find that in many labs they're not

able to see the things that we see with

a focused exam because of what they're

held to and the accrediting agencies

any other questions from our audience

today feel free to unmute yourself and

we have time for a couple more questions

so dr. Saltzer this is Mario Rubin from

Houston first of all I'd like to

congratulate you on an outstanding

presentation I mean I was trained by

George Reiner and who taught me quite a

bit about urinalysis until today I

thought I knew a lot and you have shown

me there is a way ways to go number two

is one thing that I have found that has

really misled me many times is the

presence of oval flat bodies in women

that use vaginal talk and that's

something else too I think keep in mind

interesting

thank you I wasn't aware of that that's

that's good to point out thank you

you're welcome

I have a quick question if you got time

yes

vancomycin has recently been suggested

to be due to the formation of

obstructive cast in the nephron can you

identify a vancomycin cast by any of the

optical techniques you have described

very good question me I have not seen

that I have looked in cases I've

suspected it

I believe Juan Carlos Velez maybe has

and I think he may have actually posted

that online or may have had a abstract

about that but for me personally I have

not seen it I'm I'm not sure what the

specific findings would be that you

could have reassurance that's what

you're looking at so if I may comment

that this was discussed at glumly con by

the group at Houston Methodist by dr.

Sookie Antoine long probably a year ago

and they have reported cases or finding

crystals in the biopsy of patients with

vancomycin nephrotoxicity the French

have also reported the same and they

look exhaustively at Union serving

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