clinical chem fall exam 1

Explain the principles of dilutions calculations.

Dilutions are usually expressed as: 1:2, 1:5 etc  = the ratio of V1:V2

If C2 is measured then C1 = C2 x dilution factor

If C1 is measured then C2 = C1 ÷ dilution factor

Calculate dilutions.→ formula

(C1)(V1) = (C2)(V2)

Define molarity,

number of moles per liter of solution

One mole = gram atomic weight or gram MW of the atom/molecule

Note that mole/L = M; mmole/L = mM

Define molality

1 mole/Kg solvent

Define normality.

the concentration expressed in equivalent weights per liter.

1 normal solution = one equivalent weight of solute/liter solution.

Recall the three basic forms of concentration.

mass percent molarity and molality

Define pH.

measures how acidic or basic a liquid is on a scale from 0 to 14

Recall the Henderson-Hasselbalch equation for pH.

pH = pKa + log ([A-]/[HA]) or pH = pKa + log ([salt]/[acid]) pK for the bicarbonate buffer system in serum or plasma is 6.1 at 37° C

Define absorbance and state equation.

Absorbance is a value directly proportional to concentration, defined as follows: A = - log T = -log (%T/100) = 2 - log %T

Define Beer’s Law and state equation.

relates absorbance (A) to concentration (c) and light path (b) in cm A = a*b*c

Explain the utility of a calibrator in laboratory assays.

used to prime instruments for percision and accuracy → a known concentration or value used to show the baseline between instrument and analyte

Samples of known concentration (standard or calibrator) are used to define the linear relationship between analyte concentration and final measured absorbance.

Explain the principle of spectrometric analysis, including bandwidth, bichromatic measurements and the components of a spectrophotometer.

Light source determines the range of available wavelengths; Tungsten or tungsten halogen lamp for visible spectrum; Hydrogen or deuterium lamps

Spectral bandwidth (a measure of spectral purity) = (λ2−λ1) where λ2 and λ1 = 1/2 peak transmittance

Correction of background absorbance by other compound- bichromatoc 2 measures

components: light source monochrometer sample and light detector

Describe the physical and chemical principles of flame photometry and atomic absorption

Uses a hollow cathode lamp to produce light by electric current freeing ions from lamp allowing to collide with inert gas where the loss of energy produces specific wave length for the metal can have flame or no flame

flame photometry is similar except doesnt have lamp relying on only the flame

The specimen is vaporized by heat (flame or flameless). Absorption is measured at the detector

Explain the utility of the hollow cathode lamp in atomic absorption.

emits metal specific light to excite the specific target analyte

Explain the need for an internal standard in atomic absorption.

correct for variation in extraction, injection volumes, etc.... These internal standards are chemically similar, and are added in constant amount to samples and calibrators Calibrator and sample peaks are first normalized by ratio to the internal standard peaks before calculating the value

Explain the principle of Fluorometry, including chemiluminescence and fluorescence polarization.

an analytical technique that measures the intensity of fluorescent light emitted by a substance after it absorbs light of a specific wavelength

Chemiluminescence: chemical reaction

With fluorescence polarization the excitation light is polarized,and the fluorophor’s rotational time determines % polarized light emitted

Fluorescence polarization is directly proportional to fluorophor binding Thus, fluorescence polarization is indirectly proportional to specimen [analyte]

Relate excitation and emission wavelengths of light in fluorometry.

Excitation light polarization is easily measured by passing the light through a polarizer that is rotated to allow a vertical and horizontal oriented measurement. With depolarized emission of light, roughly equal measurements in both planes are found.

Explain the principles of nephelometry and turbidimetry.

Both measurements relate to light scattering of molecules in solution Placement of the detector defines the type of measurement Turbidity detector is in direct line with the light source (0°) May be performed on a standard absorption spectrophotometer Nepholometry detector is not in direct line with the light source (>0°) Nephelometers measure light scattered usually at 90°

Compare the limitations of nephelometry and turbidimetry.

particle dynamics, concentration ranges, and interference

Define electrophoresis.

Migration of charged solutes under the influence of an electrical field. Zone electrophoresis uses a porous supporting medium such as agarose gel. Electrophoresis is typically used for separation of proteins in serum, urine, and CSF

Explain the principles of agar gel electrophoresis, including the factors which affect the rate of migration of molecules during electrophoresis.

Charged molecules migrate to the pole of opposite charge Negatively charged proteins (anions) migrate to the positive pole (anode) Positively charged molecules (cations) migrate to the negative pole (cathode). Net molecular charge may be varied by altering the ph of the buffer solution ↑ph will increase the net negative charge ↓ph will have the opposite effect A molecule's isoelectric point is the pH where it has a net charge of zero

Rate of migration of a molecule is dependent on many factors, such as: net molecular electrical charge - directly proportional ,strength of the electrical field - directly proportional ,size and shape of the molecule - inversely proportional ,supporting medium viscosity - inversely proportiona

Explain the phenomenon of endosmosis and give an example.

Gammaglobulins migrate towards the cathode despite negative charge

Electroendosmosis – due to flow of buffer and weak electromotive force

Explain the principle of isoelectric focusing.

Uses a medium which provides a ph gradient Proteins migrate to the point where gel pH = protein’s isoelectric point

Define potentiometry

measures the potential difference between 2 electrodes of electrochemical cell which develops across a selective membrane (the membrane selectively interacts with the ion in solution)

Correlate potentiometry, amperometry, and coulometry to clinical applications.

potentiometry- elecrolytes

amperometry- arterial blood gas

coulometry - chloride lvls

Define amperometry

Amperometry is defined as a technique that measures the current intensity resulting from electrochemical reactions of electroactive species when a constant potential is applied, allowing for the determination of the concentration of target substances through current variations over time

Define coulometry.

an electroanalytical method that measures the total electrical charge (in coulombs) consumed during the complete electrolysis of an analyte

Describe membrane selectivity in ISE measurement of electrolytes.

determined by the membrane composition Hydrogen membrane (ph electrode) is glass of very specific composition Sodium membrane is also a glass electrode, of differing composition Potassium membrane uses the antibiotic valinomycin, which selectively binds K+

Explain the principles of osmometry, including the physical parameters of its measurement

Measure of concentration of solute particles in dilution

Concentration is expressed as osmolality = the total number of solute particles per mass of solvent

Size of the particle does not matter

Osmolality is usually measured by the level of freezing point depression

Define and list colligative properties.

Osmotic pressure - minmum pressure to stop net flow of pure solvent across membrane, boiling point- point liquid become gas , vapor pressure- the pressure of the vapor resulting from evaporation of a liquid (or solid) above a sample of the liquid (or solid) in a closed container, and freezing point- point liquid become solid

Define freezing point depression and recall the formula, which states the relationship bbetween freezing point depression and osmolality.

Sample is supercooled and the temperature monitored by thermistor probe Heat is rapidly removed from the sample T° of the sample goes below its freezing point, and crystalization occurs Crystalization releases heat, causing a temporary plateau in sample T° The plateau reading is the true freezing point

mOsm/kg =( freezing point/ -1.858 )(1000 mosms/kg h2o) )

Explain the principles of separation of molecular species by chromatography.

Chromatography is the separation of molecules in a mobile phase which is flowing through a stationary phase This separation depends on differential physical interaction with the stationary phase Chromatography classification may be made by several of the parameters of the separation, including by types of: Mobile phase (liguid or gas) Containment system (thin layer, flat, column..) Mechanism of action (absorption, ion exchange....)

Classify chromatography based on the parameters of separation and give examples.

Chromatography classification may be made by several of the parameters of the separation, including by types of:

Mobile phase (liguid or gas)

Containment system (thin layer, flat, column..)

Mechanism of action (absorption, ion exchange....)

Compare normal and reverse phase chromatography.

"reverse phase" means the stationary phase is less polar than mobile phase

both show movement of molecules for solid phase to meaure seperation of molecules

Describe the utility of the Rf value in thin layer chromatography (TLC).

Rf value of compound = distance of spot ÷ distance of mobile phase migration Rf values generate a list of potential compounds with the same Rf value Final identification depends on method of detection, type of extraction, etc

Compare and contrast competitive and non-competitive immunoassays, including the relationship between analyte and signal measurement.

non-competitive immunoassay is a sandwich assay, which is composed of immobilized antibody-analyte-labeled antibody Labeled antibody signal is proportional to amount of analyte Signal may be fluorescence, enzyme generated product, isotope counts...

competitiive binding assay uses labeled analyte sample wash add labeled antibody then wash excess and measure signal generated by the label analyte concentration signal antibody coated well = label = antibody = analyte Sandwich type immunoassay S-S- bonds hold heavy and light chains together COOH end variable region 26 Labeled analyte is first mixed with sample Limited immobile antibody then binds both labeled and specimen analyte Unbound analyte, labeled and unlabeled, is removed by washing Residual bound labeled analyte produces measured signal Signal is inversely proportional to specimen analyte concentration

Define mean

the average of n data values (xi / n).

Define Gaussian distribution

aka normal distibution

fully described by the mean and SD

◦ +1 SD = central 68.3% of data

◦ +2 SD = central 95.5% of data

◦ +3 SD = central 99.7% of data

Define standard deviation

sqrt(variance) = sqrt( Σ(xi - mean)2/(n-1))

measures how far the values in a data set are spread out from the average (mean

Define Coefficient of variation (CV).

(standard deviation x 100%)/mean

statistical measure of how spread out data points are around the average value, relative to the average itself

Compare and contrast mean, median, and mode in Non-Gaussian distribution.

the mean, median, and mode pull apart from one another, reflecting the shape and tail behavior of the data rather than sitting at a single central point

Define reference values.

Establishment of reference ranges requires statistical analysis of test results from "healthy" population, defined as individuals who are free of disease which may affect the test result Consists of a cross-section of people of mixed sex, race and age

Explain the process of determining reference values from a set of test results performed on a mixed population.

Subpopulations should be evaluated for statistically significant differences, with the most commonly significantly different values found when the data is stratified by sex, pregnancy, and age

Define sensitivity

true positives TP +FN

Define positive predictive value.

positive predictive value = true positives / all positives

Define specificity

true negatives FP + TN

Calculate sensitivity, specificity and predictive value if given the results of a test on a mixed population of people.

Sensitivity, specificity, & positive predictive value are parameters of each test Two groups (+disease) and two results (pos or neg) produce a 2 x 2 grid

Explain Baye’s theorem.

positive predictive value depends on sensitivity, specificity, and very importantly, the population disease prevalence

Using a formula, evaluate the effect of prevalence on predictive value.

Positive predictive value =(prevalence x sensitivity)/ (( prevalence x sensitivity) + (1-prevalence)(1-specificity))

Describe the method of calculating quality control limits based on repeated analysis of a control material.

from daily analyses the mean and standard deviation are calculated The established control limits for each analyte evaluated will be = mean + 2SD Since mean + 2SD represents 95% of all expected results from the analyte test performed on the control material, it is expected that 5% of results will be beyond control limit

Define 2 SD and 3 SD confidence limits.

+2 SD = central 95.5% of data +3 SD = central 99.7% of data

define the boundaries within which a certain percentage of data points are expected to fall in a normal distribution

State the percentage of data points within 1, 2, and 3 SD ranges in Gaussian Distribution

Normal distribution fully described by the mean and

SD

◦ +1 SD = central 68.3% of data

◦ +2 SD = central 95.5% of data

◦ +3 SD = central 99.7% of data

Compare and contrast precision and accuracy with attention to mean and CV of data results

precision term used to describe reproducibilty of the test or any other procedure. This term and measurement are reflected by the data's measured CV.

Accuracy is the term used to describe closeness of the test's measurements to the true value (or any other procedure) A test or procedure may be very precise, without necessarily being accurate. Similarly, the procedure may be accurate, but not precise.

Describe the function of Levy-Jennings Charts.

key to graph out qc

control charts were introduced as a graphical means of evaluating data, which is displayed as date/time on the x axis vs test control result on the y axis Control charts are a graphical illustration of performance of the analysis system Highlighted are the mean + 2SD range, which indicates acceptable performance

Recall the Westgard Quality Control rules.

developed a series of rules for acceptance or rejection of test results based on a series of control rules for interpreting control results. The Westgard control rules are designed to decrease the false rejection rate, while improving error detection. These rules involve evaluation of more than one control substance in most instances (three controls would consist of low, middle, and high range levels). Two control values beyond the +2SD limits or one value beyond +3SD limits indicates probable analytic error. Ten consecutive control values all above or all below the mean, or four consecutive control values all above or all below 1SD, would indicate a shift in the analysis mean and a need for corrective action. Potential problems which should be evaluated include calibration, dated or bad reagents, or instrument problems.

an automated CK assay gives a reading that is above the linits of linearity. a dilution of the serum sample is made by adding 1mL of serum to 9mL water. the instrument now reads 350U/L. the correct report on the undiluted serum should be

3500U/L

a standard solution is a chemical solution that has a percisly known concentration. standard solution conc is commonly expressed with which units of measurement

mol/L

a glucose determination was read on a spectrometer, the absorbance readinf standard was 0/3 the absorbance readinf of the unknown was .2 the value of the unknown is

2/3 of the standard

a technichian is asked by the supervisor to prepare a standard solution from the stock standard what is the glassware of choice for thos solution

volumetric flask

how many mL of rbc are used to make 25mL of a 4% rbc suspension

1mL

the volume of 25% stock sulfosalicylic acid needed yo prepare 100mL of 5% working solution

20mL

to prepare 25mL of 3% acetic acid how much glacial acetic is needed

0.75mL

how many gram nacl are needed to prepare 1L 0.9% normal saline

9

to prepare 40mL of 3% working solution a tech would need to use what volume of stock

1.2mL

a technician is preparing a 75% solution what volume of stock solution would be used to make 8mL

6mL

a new method is being evaluated. a recovery experiment is performed w/ the results in image

the percent recovery of the added anayte standard is

90%

when .25mL is diluted to 20mL resulting dilution is

1:80

a serum glucose is too high to reas so a 1;5 is dilution using aline. dilution a was stil too high so a 1:2 was made using saline to calculate the result the dilution b must be mult by

10

in performing spinal fluid protein determination the specimen is diluted 1 part spinal to 3 saline to obtain a result low wnough to measure to calculaye the [protein] must be

divided by 3

how many mL anti D reagent are needed to prepare 5mL of 1:25

1:450

if .5mL of 1:300 contains 1 antigenic unit 2 antigenic units would be contained in .5mL solution of

1:150

a 2% slaine erythtocyte suspension contains how many mL packe erythrocyte per 5mL of isotonic saline solution

.1

600mg/dL glucose solution diluted to 1;20 the concentration of the final solution in mg/dL is

20

how many mL 30% bovine albumin are needed to make 6mL 10% bovine albumin solution

2

what is the formula for calculating the dilution of a solution

v1c1=v2c2

a colorimetric method calls for the use of .1mL of serum 5mL reagent and 4.9mL h2o what is the dilution of the serum in the final solution

1:100

dilution is the process by which the conc or activity of a given solution is decreased by the addition of a solvent. what is the dilution factor of 1mL serum in 4mL water

1:5

what is the formula for calculating a percetn (w/v)

grams solute/ volume solvent x 100

a solution containing 20g solute disolved in .5L h2o what is % of solution

4

how many g of sulfosalicyclic acid (MW 254) are requires to prepare 1 L of a 3% (w/v) solution

30

how many mL of a 3% solution can be made if 6g solute is available

200mL

the nanometer is a measurement of

wavelength of radiant energy

to make 1: of 1 N NaOH from a 1.025N NaOH slution how many mL of naoh should be used

975.6

the sodium content in g in 100g of NaCl (atomic wieght Na= 23 cl =35.5)is approx

40

calculate molar absorbativity

absorbance = .5

lp = 1cm

conc = .2mol/L

2.5

what is the formula for calculatong the gram equiv of a chemical

MW/ oxidation num

80 g NaOH (mw 40) how many mol

2

a serum potassium (mw 39) is 19.5mg/100mL this value is equal ti how many mEq/L

5

what is the formula for calculating number mol of a chemical

g/MW

1 molal solution is equiv to

num mol of solute per 1kg solvent

formula for calculating molarity of a solution

mol solute/ L solution

what is the molarity od a solution contaning 18.7g KCl (mw 74..5) in 500mL h20

.5

25g NaOH (mw 40) added to .5L h2o what is molarity of solution is addition .25L h2o added

.83M

what is the N of a solu contian 280g NaOH (mw 40) in 2000mL solu

3.5

how many g h2so4 (mw 98) in 750mL 3N h2so4

110g

how many mL .5N NaOH needed make 100 mL .05Nsolution NaOH

20mL

a pH of 7 represents a [H+] of

100nmol/L

whwn the exact conc of the solute of a sol is known and is used to evaluate the conc unknown solutio the known sol is

standard

the mean value of a series of hgb controls was found to be 15.2g/dL and SD was calculated .2. acceptable control range is +- 2 SD which is allowable limit of conrol

14.8-15.6g/dL

measure of dispersion arounf mean = SD

an index of percision is statostically known as the

coefficient of variaition