Clinical Chemistry Lab Calculations: Spectrophotometry, Beer's Law, and Acid-Base Balance

this lecture will cover chapter 6 which covers the clinical chemistry laboratory calculations

depth of what spectrophotometry is because you do go over that in more detail in your other courses however in

general spectrophotometry is used to quantify the concentrations of various analytes based on the amount of light

that the analyte absorbs and so this is based on the theory of Beer's Law which states that the amount of absorbance of

a solution is directly proportional to that solution's concentration so in Practical times the darker the solution

means the higher the absorbance and the more concentrated that that particular solution is

so Bear's law states that a represented by absorbance or absorbance equals a times B times C which equals

absorptivity coefficient the path length as well as the concentration the absorbativity coefficient is the

amount of light absorbed by an analyte at a specific wavelength and it is constant for a particular analyte at a

particular wavelength if certain conditions such as temperature solvent pH remain constant now the path length

is the distance that the light actually travels through the solution and if the analysis is performed correctly it is

also a constant thus if we remove the two constants in an equation then we will come up with a with an additional

equation so as you can see here absorbance is directly proportional to the actual concentration Beer's Law is

actually possible because of the concept of transmittance so when a cuvette containing a solution

that we're analyzing is actually placed inside of a spectrophotometer the light can actually show be shown through it

the solution will absorb some of that light so this is the absorbance but the remainder of the light will actually be

transmitted through that cuvette and then be um

be transmitted onto a photodetector so when that photodetector actually gets that light on it it actually

measures that particular light or the transmittance of that light that light on that photo detector is

known as the incident light so that particular light we can use that to plug into an equation

where the ratio of the amount of transmitted light is divided by the amount of that incident light which is

also known as transmittance so this is demonstrated mathematically where transmittance equals the light

that is transmitted over the light of the incident light so that that is detected on that photodetected photo

detector so transmittent ratios range from 0 up to 1.0

by multiplying by a hundred percent transmittent values can be obtained ranging from 0 to 100 on an actual

linear scale so if all the light is transmitted through the solution that is that no light is absorbed by the actual

solution then the transmits transmittance ratio is 1.0 and the percentage percent transmission

is actually 100 because a hundred percent of it transmits through the solution

if no light is transmitted through the solution then the transmittent ratio is zero or

zero transmittance so all that light has been absorbed 100 percent absorbed in the solution which means no light is

coming through it mathematically transmittance and absorbance are related by the formula of

a equals negative log times transmittance so we can actually take that formula of absorbance equals

negative log times transmittance and manipulate it a little bit to get an equivalent formula and so you see that

listed here and then that can further be converted from a transmittance to a percent transmitting transmittance and

then from there you will end up with the equation that absorbance equals 2.0 minus log percent transmittance

now in contrast to transmittance ratio absorbance values range from 0.000 to Infinity on a logarithmetic

scale so you can see here the pictures that indicate an absorbance in percent transmittance meteor meter so you can

notice how much easier it would be to actually interpret something on a linear scale for percent transmittent versus a

logarithmic absorbance scale and so by a convention absorbance values are reported to the third decimal place

so that will be like 0.000 whereas percent transmittent values are actually reported to the

nearest tenth with um that being a percentage student was doing a manual creatinine

essay using the jaffeine method and so 45 transmittance reading was obtained from the cuvette

that had the level one creatinine control and reagent that were mixed in this to get this reaction so we want to

know what is the absorbance value well to solve this we would use the formula that we that we just discussed where

absorbance equals 2.0 minus log percent transmittance so that would be the 45. so we'd use a calculator or you could

actually use a logarithmic table and there is there is a copy of this in your textbook but of course using a

calculator is going to make this a whole lot easier and so you would take the log of 45

and plug that into the equation which is going to give you 1.65653 and subtract that from the 2.0

which is going to give you your absorbance of 0.347 this means that a percent transmittance

value of 45 is going to have an absorbance value of 0.347 so you may be trying to figure out well

why is all of this important well this is what's going to be the basis of establishing your standard curve that

you will use on your instrumentation to actually determine what your unknown values are for your patient sample

so to do that we first must actually make a standard curve now in The Clinical Laboratory most

essays do not actually use a single standard to to quantify your unknown so instead you use four to six standards of

different concentrations that are spread out evenly across your linear range of that particular method so by doing this

more the linear range of that assay is actually covered by your standards when compared with a single standard method

so many automated instruments you'll have a series of Standards or calibrators

that are used to actually set the standard skirt standard curve that is stored in that particular instrument's

computer data and then what will happen you'll have an electronic signal that's generated by the instrument

based on that transmittance value of that particular sample and then that's compared with the computer stick

computer's standard curve for that analyte that you're actually measuring from that sample and then the computer

is going to generate a quantitative result for that particular sample of that particular analyte in that

sample and then of course it's going to send that message to your the analyzer's computer or to your Lis your laboratory

information system or to your printer or whatnot now as a student you're going to learn

the concepts of actually constructing a standard curve and being able to graph it on some linear graph paper to

determine the concentrations of standards are using the concentrations of those standards and graphing those on

the x-axis and then taking the absorbance of whatever that standard is on your spectrophotometer and graphing

that on the y-axis and of course this is going to give you a standard curve so what we're going to do is kind of

talk a little bit more about how to construct a standard curve now anytime you do a manual essay using

the spectrophotometer you're going to follow Beer's Law you're actually going to use a piece of linear grad paper and

then the standard curve of the absorbance obtained from the standards is going to actually give you a straight

line so the concentrations of the standards should range between the highest and the

lowest range the linear range of what you're trying to measure for that particular analyte and two to three

concentrations evenly spaced between the two outermost limits and let's face it we don't exactly live

in a perfect world so although our standard line should be a straight line occasionally you have to do a line

that's best fit so it's not exactly point to point connect the dots because this can be skewed just a little bit but

you basically try to attempt to draw a line that best fits and best hits all of those data points

additionally this line should never extend beyond that highest point that's actually on that standard patient

samples that have absorbances higher than that highest concentration of that standard need a dilution in order to

determine that absorbance if it falls outside of that linear range and of course once you make that dilution you

need to reanalyze it and then factor in for that actual dilution that's being made so standard curves are not limited

to just the chemistry Department you also use these in hematology Department as well

um so do keep that in mind so just a side note a standard curve can actually be plotted using percent

transmit values however if you plot it on a linear graph paper then the percent transmittance standard curve is actually

it has a curve to it it's a curvy linear and you can see an example of that here so in order to form a straight line the

percent values must be plotted on a the semilogrammatic graph paper and so that

that we'll see here next and here you see that same plot just on a different graph paper you can see the

difference in how those lines are drawn on that graph paper so it gives you that straight line so obviously you'd have to

have special graph papers to do this so we don't really use this a lot click an example of how to actually

construct that standard curve and then what you do with it after you've constructed it so in this particular

example we are doing a manual glucose assay in which we have standards that measure out at 50 milligrams per

deciliter 100 to 200 and 300 and of course once we mix that solution and get that chemical reaction perform that test

we know that this is the expected concentration of these we know that the 50 milligram per deciliter should read

50 milligrams per deciliter on this particular analyzer on the spectrometer so once we get that absorbance reading

we know that that is 0.150 and so then what we'll do is we'll actually plot that value for the 50 milligrams so

again at the bottom you've got your glucose concentration in milligrams per deciliter and then of

course over here on the side we have our absorbance so we have our different

absorbance regions that are measured out here and then we plot the 50 milligrams you can see where that first one here

has been plotted and then we do that for each of them we plot them whatever their absorbance reading is once we get that

plot then we do our straight line which is our best fit line and for this particular one it actually looks like it

hits on each of the um each of our data points pretty pretty

evenly but if it didn't we would start at the zero and we would try to draw a line that best fits hitting all of those

data points to give us that straight line so now that we've got our standard curve

established we know that this particular concentrate and we'll read at this particular absorbance and

so with that we can do the same for our patients so with our patient we we don't know how much of that glucose

is in there so we got to figure that out so if we run the test the same way what we did with those standards put it in

the spectrophotometer and read it then we are going to get the absorbance of that particular patient sample so that

we can know what's the concentration of the glucose in that patient sample so what we would do is actually use that

standard curve on that graph paper that we just made and we will figure out where does that absorbance of

0.0400 hit on that scale so with that absorbance reading of 0.400 we would actually find it on our graph

paper and we would use a straight edge and we would go all the way across until we hit our points of interception so we

can see here where it intercepts on that line and then if you go all the way down then you would figure out what the

actual concentration is so in this particular patient our concentration of where it intercepts that is 135

milligrams per deciliter so this would be what would be reported out for this patient's glucose concentration

so chemical reactions in chemistry can have either an endpoint or kinetic reaction so with endpoint assays the

measure it actually measures the absorbance of the reaction at the completion of the reaction so when it

ends and this this means that many of your manual chemistry tests that are wet chemistry meaning that it's done on a

liquid Matrix versus using dry reagents when they're performed by a student in like a Clinical Laboratory these are all

endpoint type essays where you actually have some sort of incubation period could be a few minutes to 15-20 minutes

that allows that reaction to actually take place now with endpoint essays you can

actually use a single standard a standard curve or you can use another method known as a molar absorbs

absorption absorptivity method in order to calculate the concentration of that patient's sample now with endpoint

essays they may use an enzymes within the reaction as part of that reagent but

analysis of the enzyme itself is not performed as an endpoint reaction so in many Laboratories analytes are actually

measured using a very large multi-channel Random Access analyzer and so and this kind of helps with the speed

process of actually doing those reactions because multiple samples can be be done at one time and so in order

to do that you actually need kinetic reactions so a kinetic reaction differs from the endpoint reactions and this is

because that the reaction does not go all the way to completion but rather the absorbents are taken at various

intervals for short periods throughout that testing process so some analyzers will actually continuously monitor the

absorbance readings instead of actually monitoring the various fixed intervals in order to improve accuracy so in any

reaction whether it's kinetic or endpoint there are various phases that also have to be that we have to pay

attention to phases are going to include the Lac phase in which the reactant reagents or

first starting to react together and then you have the reacting phase in which the product is actually formed so

whatever you're looking for and then the reagent depletion phase which you can see here on this graph so in the Lac

phase the absorbance of the product is not constant so in the reactant phase the absorbance actually starts to change

if there is more reactant than reagent can react with then the third phase which a reagent is actually start

starting to be depleted this is when this happens so in this phase the absorbance values will remain the same

in an endpoint assay the reagents are in excess so you have too much of the actual reagent that the reagent

depletion phase doesn't actually happen so rather the reaction is allowed to proceed past that phase until all of all

of that particular reactant has been used up so at this point the absorbance is stable but in a kinetic reaction the

absorbance regions are taken during the second phase and so um

that change in absorbance or that Delta absorbance is what is actually measured let's talk about

enzymatic type reactions in the enzyme kinetics of those with first order reactions these are

reactions in which the enzyme is in excess and the substrate concentration or the concentration of the analyte that

we're looking to measure is the actual limiting factor so in first order reactions the substrate

concentration is low relative to the enzyme concentration so a low substrate concentration at a low substrate

concentration these rates of reaction is dependent on the substrate concentration so first order reactions tend to be used

when non-enzyme analytes are being measured in an enzyme an enzyme or other enzymes are used in

that particular reaction sequence sequence as part of your actual reagent now when the activity of an enzyme needs

to be measured conditions of the essay are maintained in order to allow for zero order of order kinetics to actually

occur so with zero order kinetics the rate of the reaction is directly proportional to

the enzyme the enzyme concentration and it's independent of the substrate concentration

so with this the substrate is actually kept in excess so you have too much of it this means that the rate limiting

factor is the concentration of these of the enzyme that you're actually looking for in measuring

so zero order kinetics gives us the following reaction that will actually take place so when you have the enzyme

and the substrate mixed together that's going to yield an enzyme substrate complex which then will additionally

yield the enzyme plus the product now keep in mind that the temperature of the ph and any other variables are kept

at a constant during these reactions whereas the um

the any kind of secondary enzymes that you use in this reaction are also kept in excess along with

any of the substrates that are also used and so there is a special equation that's actually used to do this

calculation now this is not something that I'm going to make you know it's just so that you can understand how the

analyzers actually use this process to to actually get the calculations for these results when you're you when you

have a sample put on the machine and it actually runs through an enzymatic type type reaction so if your analyzer uses a

zero order kinetics it's actually using this to do those calculations so again I'm not going to go a lot into

how these cons this calculation is done but you can see here that the curve of the Velocity versus the substrate

concentration for the zero order kinetics kind of helps to illustrate that particular equation

um and here it kind of lists some of the variables depending on that Now While most of your Chemistry tests

do have standards that are made for to be able to establish those limits just know that standards may not be available

for many of your enzymes so it may be possible that you have to actually do a determination of the activity so this is

something that could be done within the analyzer but there is there is an equation to do that of course

an example of this type of reaction would be a kinetic creatinine kinase assay in which you know it was manually

performed using a coenzyme nadh as part of that as part of that reaction indicator

and so you know in this particular example we'd want to figure out what is the concentration of the level 1 QC so

you would have your volumes of your various reagents your absorbance readings and whatnot you'll have your

your set values that you will know and you would plug get those plugged into the equation that ultimately would give

you your result for that particular QC reaction buffers are solutions of weak acids or

bases and their salts that resist changes in PH so for example during serum protein electrophoresis the pH of

the solution that conducts the current is maintained by a buffer so buffers are actually used in other areas of Clinical

Laboratory as well as including your molecular Diagnostics Department so the pH of any solution is determined

by its acidity so a simple definition of an acid is that that of a substance that will

donate a hydrogen ion or a proton and a base is a substance that will accept that particular hydrogen ion or donate a

hydroxyl ion so this definition is known as the bronsted theory and with the bronsted

acids that has donated a hydrogen ion it will will then become a bronsted base the acid is termed a conjugate acid in

the newly formed base is termed the conjugate base so some examples of conjugate acids and conjugate base pairs

carbonic acid forming bicarbonate now our acid or a base can be termed stronger wheat depending on its tendency

to disassociate so strong acids or bases will completely disassociate when they're actually in a solution so

example this would be hydrochloric acid it's a strong acid so when placed in water it will it will completely

disassociate the hydrogen and the chlorine ions and now this disassociation is a constant Ka

which determines the relative strength of an acid or a base and so here we have the equation for

calculating KA for weak acid or base so again if you kind of do some manipulations and move some things

around and substitute some things in then you can actually um

fix this equation so that something that's a little bit more understandable which comes gives you the

henderson-hasselbolic equation where pH equals PKA plus log of the salts of the acid over the UN un disassociated acid

so this helps us to understand that if the ratio of salt to acid is one then the pH is going to equal the pka

or the disassociation constant this means that the buffer is at a maximal buffering capacity now in an

acid solution raising the pH above the pka it's going to cause an increase it's an

increase in the disassociation of the acid it's going to increase the amount of hydrogen ions and it's also going to

lower the pH now in a basic solution if the pH is below the pka the more of the hydroxyl

ions will actually be released and this is going to actually lower the pH so here we have an example where

technologist is to prepare a phosphate buffer by adding 5.874 grams of monopotassium phosphate

and 1.191 grams of its salt which is the dipotassium phosphate and you would add

this to water to make a 1.0 liters of water now the pka of the buffer is known to be

7.2 so we want to know what is the pH of the phosphate buffer so we would use our equation

we'd figure out what the conjugate acid is and what the conjugate base is now this is this all goes back to chemistry

organic chemistry for this so when all that's plugged in we get our equation that's noted here to the side

so then our next step is to actually determine the molarity of each of those compounds based on the quantity of grams

of each of them so we would get those actual molecular weights for them and then we would use our molarity formula

to do that calculation for for each of those from there we plug those values in

continue to work our problem out using the rules of logarithm and then ultimately come to the pH of our

solution so why is all of this important well this kind of helps us with understanding

acid-base balance and how to get those acid-base calculations because the pH of the body is strictly

controlled by the carbonic acid bicarbonic buffer system and so when you kind of plug all this stuff into these

equations then it gives us that a PKA for Carbonic acid bicarb bicarbonic buffer system is going

to be 6.10 at 37 degrees Celsius which is body temperature and so we know that the waste product of

cell metabolism is CO2 and that is carried within the plasma from tissue to our lungs in different forms but the

three most important are a gas dissolved in plasma so um

that's going to be your your carbonic acid so um and then it can also be further

broken down into carbunum carbamino hemoglobin which is going to exist within the red blood

cells so this whole system is just a balance of um

a balance of status that's going to be dependent upon the particular actions of the lungs and the kidneys and being able

to actually do the adjustments to that pH as things kind of shift throughout the body now the ratio of bicarbonic to

carbonic acid is about a 20 to 1. but with within the blood the pH of the blood is directly affected by that

particular concentration of the bicarbonate and inversely affected by the concentration of that carbonic acid

so with your blood gas instruments the partial pressure of CO2 can be measured as pco2 now there is a relationship

between the bicarbonate and the pco2 um and so you can see that listed here with the solubility coefficient of P CO2

under equilibrium conditions carbonic acid forms that dco2 now we plug everything in and substitute

it it gives us it lets us know that dco2 is um

is actually equal to the solubility coefficient of P CO2 and so we can plug that in to get our

equation for pH based off of this so how this is actually used within the lab is that you know we can measure to

figure out how much bicarbonate concentration there is so we would do this by for example here we actually

have our pco2 values and our pH if we plug that into the equation then once that equation is worked out

then it's actually going to give us our bicarbonate level for that particular patient in that sample

now the PH of blood is usually maintained between 7.35 and 7.45 now I'm not going to go in

the pathophysiology of how this all happens but it is a balance between the kidneys and the lungs working together

to make sure it maintains that balance so if one or the other or both is off then obviously you're going to have some

changes now if the pH drops too low it's considered an acidosis and if it goes too high it's considered an alkalosis so

these changes you can actually you know again kind of plug those into into these equation and then it kind of you can

kind of start getting a visualization of how this whole process is is taking place within the body so a way to

visualize that is the relationship between bicarbonate and carbonic acid which is actually your relationship

between your kidneys and your lungs so again you're going to learn a little bit more about this in your actual

chemistry course but just be aware that there are some disorders that can actually happen when there's an

imbalance that takes place so that's why it's important for us to do these blood gas measurements in order to figure out

what exactly is going on so with respiratory acidosis your pH of your blood is actually acidic the respiratory

part of it means that the major calls of it is the actual acidosis now if the lungs cannot adequately

adequately remove that CO2 that CO2 is going to start to build up which is going to mean that that pco2 level is

going to increase which is actually going to result in a lowering of that pH a reciprocal relationship between the

CO2 concentration the pH means that if that pco2 is going up that means that pH is actually coming down now with

respiratory alkalosis just as it suggests you have an increase in PH that's caused by a drop in that those

CO2 levels and so then some compensation can take place where your body is just kind of decreasing the respiration rates

or the kidneys actually starting to retain some of those hydrogen ions so that it can try to actually start to

balance some stuff out with metabolic acidosis this is caused by a metabolic increase in acid so this

is seen a lot with your your diabetics when they're in diabetic ketoacidosis so the body's actually compensating by

trying to increase your respiration rate to get rid of that CO2 and it's actually raising the ph and then the kidneys are

going to respond by increasing those acid secretions and the and have that bicarbonate retention so this is a lot

of times why diabetics will test for those ketones and stuff within doing those urine strips now with metabolic

metabolic alkalosis this is primarily caused by an excess of your bicarbonate so this may result from an insufficient

excretion of bicarbonate by the kidneys so there could be something going on there in the kidneys that's causing it

not to filter that out an increase ingestion of bicarbonate such as like with your anti-acids or if a patient has

been vomiting a lot which can lead to a loss of hydrogen ions and a buildup of bicarbonate as well now the body will

actually compensate for this by what the lungs are actually starting to decrease respiration to kind of try to

retain that CO2 and then the kidneys will excrete more that bicarbonate so this whole thing is really kind of a

seesaw mechanism of balance there but in order to determine the acid-base status we've got to look at the pH so again

patients that are above 7.45 those are considered to be an an alkalosis type State whereas if they're

below 7.34 or 5 they're in acidic state so we look at those bicarbonate values does it fit the pH in other words the

bicarbonate values move in the same direction as the pH so if pH is is alkaline then is the bicarbonate

elevated above the normal if the pH is acidic is that bicarbonate below the normal range so we've got to also look

at the pco2 is it what is expected giving the pH and this is where we kind of look at

compensation so when a metabolic problem affects the pH those lungs are going to immediately react to try to bring that

pH back into a normal range and your kidneys can take a little bit longer to get that pH back into a normal range but

they do they do try to work on that by through that excretion now the body never over encompensates so

in other words it's not going to take you too far in One Direction after you've been too far in another Direction

the body has a comp the body's able to compensate when that pH it's once it's back in normal range then

it's already done its job so it's going to kind of shut off those mechanisms it's not going to keep going so the

bicarbonate and P CO2 levels are very um they could be very abnormal but as long as the pH in the body is within

range then the body has actually done its job so you could still see those values kind of off but everything's kind

of brought back into line so what all this does it helps us to determine what actually caused the

initial problem and what had to happen in order for that compensation to take place so whereas the analyte that has

moved in the anticipated direction of the pH is actually the culprit the analyte that has moved opposite of what

you would expect to see of the pH is actually the one that's doing the compensation

so let's look at an example so a patient comes to the ER and they have an an asthma attack trouble and they're having

trouble with with exhaling so blood gas is performed we get those values here so we want to determine what is this

patient's acid-base status and we want to know is this patient compensating for it now it's not necessarily something

that you're going to do in the lab you're basically just going to send out the results but this is what the

physician is looking for so for this particular patient pH 7.5 which means the patient is in an

alkaline state so at this point they have not brought that pH into normal range so the pco2 is

abnormally low and the bicar bicarbonate is normal So based on this with the patient's asthma we can determine that

they've not compensated they're in respiratory alkalosis and they have not yet compensated for this

so just like the body straps to maintain that 20 to 1 balance between the bicarbonate and the carbonic acid the

body also tries to maintain the electrolyte neutrality balance so the concentration of anions should be equal

to the concentration of the cation cations within the body now the measurement of this balance is

done by actually getting the anion gap so what the anion gap does is it doesn't actually measure all of the anions and

cations within the body but just those are the highest concentration that could actually significantly alter any sort of

balance that's taken place in the body so A cation is found in the highest concentration in Blood and that that

particular one that is in the highest is actually sodium now chloride is the most abundant anion within the blood

bicarbonate is also an anion and it is included within that anion gap now some Laboratories will use potassium

in this calculation however the concentration of potassium is relatively low compared to the others but here you

can see the equation listed for both so here we have an example where a 58 year old woman who's insulin dependent

she's known diabetic she comes to the ER and she's in a comatose type state so they run her electrolytes most likely

they're just going to do this in a basic chemistry panel or cement panel but they run it and get those values

um and then once they get those values they'll plug those into the anion gap now your analyzers will actually do this

for you and which is also a good check um when you're reviewing results just to make sure that this this anion gap is

not a negative number if you've got a negative number or something that could be a good indicator that something's

wrong with your machine or your sample and you might need to rerun it but getting back to what we're discussing

here with your anion gap when you plug those values in you can get the calculation and we see here we've got

the calculation using with the potassium without they're pretty close so whatever your lab uses it will establish the

reference line just for this of what's acceptable and not acceptable now how are we going to use these values

what are we going to do with them once we get them well what this says it actually helps

you to be able to detect any sort of changes and concentrations of anything that's been unmeasured any anions or

cations that haven't been measured so for example if it's elevated it could be due to the presence of other anions such

as various proteins most commonly would be your acids such as like your keto acids like when we're diabetic patients

go into that diabetic ketoacidosis state and a decreased anion gap could be seen when there is an increase in unmeasured

cations such as magnesium or calcium now in addition the anion gap could be like a set of quality assurance measurement

to make sure that you you are you have reliability in those electrodes and determining that electrolyte

concentration so this is one of those numbers you're going to take a quick quick peek at before you send out those

results just as a quality check so the osmolality of a solution is based on the number of dissolved particles in

a solution not on the actual size weight or ionic activity of those particles now a one molal solution of glucose is

also a one osmolal solution because glucose does not disassociate whereas a one malial solution of sodium chloride

is equal to a two osmal solution because the sodium chloride disassociates into sodium particles and chloride particles

now osmolality measures the total concentration of all the ions and molecules present in serum or urine so

sodium glucose and urea are major compute contributors to the total osmolality of serum now but

now because major contributors to serum osmolola to your tightly regulated the osmolality of serum can actually be

calculated so the number 1.86 is used because each of the sodium ions is balanced by an

anion but there is not a perfect disassociation so 18 is used because of the molecular weight of glucose is

approximately 180 and the factor of 18 converts milligrams to deciliters to millimoles per liter so the molecular

weight of bun is approximately 28th therefore 2.8 is used this formula can't be used for

calculating urinal osmolality because the concentration of those particles vary greatly and so depending on the

structure or depending on the hydration State it's going to actually play some factors into this

so here we have an example where 70 78 year old male is emitted to the Ed with a heat stroke

We've ran our our labor work lab work there and we're trying to determine based off these values what is the serum

osmolality so to do that calculation you plug all those values in and then you come up

with your serum osmolality for this particular patient now the osmo Gap is the difference

between the calculated osmolality and the measured osmolality so the average osmogap is about 0 to 10

when the Gap is elevated it's usually due to other particles besides the sodium glucose or bun so the presence of

ketones or alcohol such as like an ethanol in the serum can cause that to be elevated so as us osmo Gap may be

very useful as a quality assurance type indicator when you're when you're looking to whether or not to release

these results so just to kind of make sure that there's nothing that could be interfering so here we have an example

54 year old female found unconscious admitted to the hospital we've got our let her lab results here

and of course we will need to plug all of that into our equation and we also have her her serum

[Music] etoh or her ethanol

so when we plug all of those in we get those calculations first we get that osmolality and then we get the odds the

osmo Gap so the difference between the calculated and the measured once we figure that out we've got that

value in this particular patient it is indicative of the presence of other dissolved particles in the serum so that

leads us to believe that that ethanol that's present in her Serum is what's causing that to be so increased

so coronary artery disease is one of the leading causes of death today so consequently patients and Physicians are

always trying to figure out you know how to determine their risk factors and using laboratory work to be able to

determine that so what we use is actually measurements of total cholesterol HDL cholesterol LDL

cholesterol until triglycerides to help us assess that risk while there are only a few methods out

there that will actually measure the LDL we're able to actually perform that calculation using um

the federal formula to actually do that calculation so that that calculations listed here which uses the total

cholesterol HDL and the triglycerides divided by five now just a note that triglycerides divided by 5 is actually

an estimate to a vldl and so the formula is not accurate if the the actual triglyceride

concentration is greater than 400 so and we have to assume that no other sources of triglycerides are present like

calamicrons so in order to ensure that you just want to make sure that your patient is

fasting when you collect these results or collect collect these labs to obtain these results

now again our analyzers are starting there are some methods that are starting to be developed that will actually

analyze it directly but you still have some Labs where the calculations will be performed so here we have an example 43

year old a man with a family history of coronary artery disease and then he's had his lipid profile performed we've

got those those values here we're trying to figure out the LDL if you remember HDL that's considered

your healthy cholesterol and then your LDL is your lousy cholesterol so

figuring out those values based off of this calculation is going to kind of help you assess that so if you plug all

of these in do the calculations then you get an LDL of 200.