w/v – solute in grams and solvent in ml to total 100 ml.  

v/v - ml of solute in 100 ml of total solution. Both solute and solvent must be liquids.

Molality – (mol/1000g)  
Normality – (eqwt/L or eq/L)  

 Serial Dilutions 

­Multiple progressive dilutions ranging from more concentrated to less concentrated solutions

 Mean -  Average 

 Mode – Most frequent value in a data set 

 Range – Difference between the largest and smallest values in  a data set 

Coefficient of Variation –  Best statistic to use when finding the most precise samples with different means

             CV (%) = (100 x SD)/Mean

­A symmetrical statistical distribution

­How close the value obtained lies to the “true” value.

 Precision 

­How close one value is to another 

 F-test

Parameters:

 Predictive value of a negative result – Probability an individual with a negative test result is free of disease

Diagnostic specificity - ­% of disease-free individuals who test negative

To pass

­Types of standards:

Two Types:

Always document out of control QC and take corrective actions including: 

• Lean =  “does this step need to exist?”

• Six Sigma = “how can this process be improved?”

Glycogenolysis – the breakdown of glycogen to form glucose

Gluconeogenesis – formation of glucose from non- carbohydrate sources like amino acids, lactate, or the glycerol portion of lipids

Glucagon -Primary hormone for increasing glucose levels

Epinephrine - Increases blood glucose 

Cortisol - Increases blood glucose 

CSF: 40-70 mg/dL

Lab findings show increased:

Characteristics:

Symptoms

Complications

~95% of diabetes cases and related to obesity

Treatment: 

Impact on infant:

Neuroglycopenic Symptoms - brain is dependent on glucose in the CSF, shortage leads to

Serum or plasma

CSF and urine specimens analyzed ASAP

In the presence of glucose oxidase:

Glucose concentration is proportional to the oxygen consumed or the hydrogen peroxide produced

-->Oxygen measured using an oxygen electrode

Trinder Method is another method through glucose oxidase
-->H2O2 measured by reacting with peroxidase
Coupled reaction: in the presence of glucose oxidase: 

and in the presence of peroxidase: 

Limitations:

In the presence of hexokinase:

and in the presence of  glucose-6-p dehydrogenase:

-->Measures the rate of appearance of NADPH, proportional to glucose concentration

Advantage: Specific for glucose

Limitations: Hemolysis causes low results due to RBCs use of NADP

Reference range: 

If 2 hour glucose is > 200 mg/dL and the pt has an elevated random or FPG, diabetes is diagnosed

Diagnostic = 3H OGTT with a 100g dose of glucose

Positive diagnosis with any 2 of the  following results:

Shows average blood glucose 2-3 months prior to blood collection (RBC lifespan)

Glycohemoglobin attaches to the boronate group of the resin, selectively eluted with a buffer.

HPLC 

In a pt with hyperinsulinemic hypoglycemia: C-peptide low or undetectable = Exogenous insulin is likely (lacks C-peptide)

Most common autoantibody tests:

Alternatives to HbA1c with decreased RBC lifespan 

3 ketone bodies 

Increase with carbohydrate deprivation or decreased carbohydrate use:

Ketonuria - increase in ketones in the urine

Check levels in type 1 diabetes with:

Sodium nitroprusside – used with urine reagent test strips and Acetest tablets

Enzymatic method for B-hydroxybutyric acid

Specimens: 

D-xylose is absorbed in the intestine and excreted unmetablozied in the urine

Test is used to distinguish between malabsorption due to pancreatic insufficiency and malabsorption by the intestines

Test procedure:

Procedure: 

Normal response = an increase of at least 25mg/dL above baseline

Patient Prep: 

Procedure:

Lab findings:

At least 13 types (I-XIII)

GSD I--AKA: von Gierke disease

Accumulation of glycogen in the liver, kidneys, small intestines impairs the organs ability to function

Principle:  As the concentration of a photon absorbing compound in solution increases, more light is absorbed by the solution and less is transmitted

Wavelength - distance between the 2 crests of the wave(nm)

Entrance slit – focuses the light on the grating

Wavelength selector/ monochromator

Exit slit- makes the light parallel, reduces stray light
Cuvette

Detector- converts light into an electrical signal proportional to number of photons striking its surface

Nephelometry - Measures the light scattered 

Five Components:

Use: 

Used in micro too

cytochromes, and other enzymes

overproduction and build-ups of heme precursors

Amount of antibody-bound radio-labeled antigen is measured

Uses - Hormones, enzymes, drugs

Concentration of Ag is directly proportional to the bound-labeled AB

1.) Specimen Preparation and Identification

2.) Specimen Measurement and Delivery

3.) Reagent Systems and Delivery

4.) Chemical Reaction Phase

5.) Measurement Phase

6.) Signal Processing and Data Handling

Lab’s responsibility - support, assist, provide oversight for testing

Characterized by bond between carbons:
Saturated

Monounsaturated

Polyunsaturated 

Cis fatty acids

Trans fatty acids 

Triglycerides containing cis unsaturated fatty acids have bends in their structure

Amphipathic

Found on the surface of lipid layers

Apolipoprotein B

Apolipoprotein C

Apolipoprotein D

Apolipoprotein E

Apolipoprotein (a)

Atherosclerosis - deposition of lipids in veins and arteries

Xanthomas - lipid deposits in the skin as nodules

Peripheral vascular disease (PVD) - Plaque develops in arteries of the arm or legs
Coronary artery disease (CAD) - Plaque develops in arteries in the heart

Cerebrovascular disease (CVD) - Plaque develops in the vessels of the brain

Based on: 

Due to increased VLDL production in the liver, decreased VLDL clearance, or a combination of both

Hypoalphalipoproteinemia 

Hypobetalipoproteinemia

Presents in infancy with:

Most common for measuring cholesterol, 3 coupled reactions

In the presence of... 
cholesteryl esterase:

cholesterol oxidase:

peroxidase:

In the presence of... 
bacterial lipase:

glycerokinase:

glycerophosphate oxidase:

peroxidase:

Homogeneous enzymatic methods

Reference method: ultracentrifugation

Direct method exist but are usually only used when specifically order for patients with triglycerides >400 mg/dL

Quantitative Analysis - 72-hour stool collection

Methods:

Acute - an excess of the early precursors (ALA- aminolevulinic acid, PBG-porphobilinogen, or both)

Secondary- Result of another disorder, toxin, or drug interference. Often due to anemias, liver diseases, and toxins 

Examples: 
PCT : Porphyria cutanea tarda 

CEP: Congenital erythropoietic porphyria

Others: EPP, XLPP 
Treatment:

Examples:
HCP: Hereditary coproporphyria & VP: Variegate porphyria:

Others: ADP: ALA dehydratase deficiency porphyria,

AIP: Acute intermittent porphyria 

Examples

Watson-Schwartz - qualitative screening test for porphyria

Biliverdin is reduced to bilirubin and transported to the liver bound primarily to albumin as unconjugated or indirect bilirubin

Infants: 

Treatment for neonatal hyperbilirubinemia - UV light therapy to photo-oxidize the bilirubin, exchange transfusions, and drug therapy

Lab findings: 

Conjugated hyperbilirubinemias:

Lab findings:

Jendrassik – Grof 

Methods used today are all just modifications of the classic diazo reaction

Total bili and conjugated (direct) bili are measured, unconjugated (indirect) bili is calculated:
Total Bilirubin – Direct Bilirubin = Indirect Bilirubin

 

Principle: Urobilinogen reacts with p-dimethyl amino-benzaldehyde (Ehrlich’s reagent) to form a red color measured spectrophotometrically

Concentrations are held within very narrow ranges

Extracellular fluid (ECF) 

Variations in the concentration of these solutes results in variation in the water distribution between compartments

Useful for ssessing electrolyte and acid-base disorders

Increased water retention due to:

Water imbalance due to:

Symptoms: 

Decreased water intake:

Increased intake or retention:

Symptoms:

Treatment: 

Atomic absorption

ISE

Uses 2 electrodes - Reference and sodium electrode with a semi permeable glass membrane 

2 types of ISE measurements

Increased cellular uptake:

Renal loss:

Decreased intake:

Symptoms:

Treatment:

Cellular shift: 

Increased intake:

Artifactual:

Symptoms: 

Treatment: 

Biologic:

Pre-analytic:

Analytic:

ISE

Amperometric – coulometric titration

Mercurimetric titration

Mercuric thiocyanate

ISE

Sweat Chloride

Decreased levels 

Increased levels 

ISE

Enzymatic

Reduced intake: 

Decreased absorption: 

Increased renal excretion: 

Increased endocrine excretion: 

Drug-induced increased excretion: 

Symptoms:

Treatment:

Increased intake:

Symptoms:

Treatment:

Atomic absorption spectrophotometry 

Colorimetric

Regulated by:

Symptoms: 

Treatment:

Symptoms:

Treatment;

Total calcium:

Ionzied calcium: 

Regulated by:

Symptoms:

Treatment:

Increased intake:

Increased breakdown of cells:

Lymphoblastic leukemia 
Symptoms:

Formation of an ammonium phosphomolybdate complex

Type B(metabolic) - associated with disease, drugs/toxins, or inborn errors of metabolism

Coupled Enzymatic Method:

Formula

Body must control and excrete H+ produced through normal metabolism to maintain pH 

Arterial pH controlled by:

Bicarbonate-carbonic acid system:

pH = pK + log (conc. A- /conc. HA)

Normal ratio of bicarbonate to PCO2 is 20:1.  

Metabolic acidosis or alkalosis caused by 

Acid-base balance is considered to be fully compensated when the blood pH is returned to normal

Causes increased loss of bicarb directly or through the buffering of excess acid:

Compensation

Anion Gap

Causes:

Compensation

Causes: 

Compensation:  

Causes:

Compensation

pCO2 = 35 mm Hg

HCO3 = 15 mmol/L

2.) pH = 7.30

pCO2 = 50 mm Hg

HCO3 = 25 mmol/L

3.) pH = 7.32

pCO2 = 30 mm Hg

HCO3 = 18 mmol/L

4.) pH = 7.30

pCO2 = 49 mm Hg

HCO3 = 30 mmol/L

5.) pH = 7.48

pCO2 = 38 mm Hg

HCO3 = 29 mmol/L

6.) pH = 7.50

pCO2 = 18 mm Hg

HCO3 = 24 mmol/L

7.) pH = 7.49

pCO2 = 52 mm Hg

HCO3 = 32 mmol/L

8.) pH = 7.48

pCO2 = 33 mm Hg

HCO3 = 20 mmol/L

Carbon dioxide is also transported by hgb

O2Hb (oxygenated hgb)

Total O2 content

Analyzed immediately(15-20min) or on ice to reduce cellular metabolism, or will lead to:

Calculates SO2 (oxygen saturation) better than pulse oximetry with abnormal hemoglobins(methb and cohb)

Clarke electrode - amperometric

Potentiometric system

Severinghaus electrode - potentiometric

Lab findings:

Methods:

Homocystinuria

Characteristics:

Decrease:

Increase:

Functions:

Decrease:

Analbuminemia – a genetic disorder where no albumin is produced
Bisalbuminemia - albumin that has unusual molecular characteristics
Increase:

Methods of measurement

Methods:

Increase:

Decrease:

Methods

Decrease

Increase

Increase

Decrease

Decrease

Increase

Decrease

Increase

Methods

Decrease

Increase

Methods

Increase

Levels correlate with cardiovascular disease risk  

hsCRP – high sensitivity CRP

Methods

Decrease

Increase

Methods:

Decrease

Increase

Methods

Increase 

Methods

Troponin Methods

Clinical Significance

Functions

Increased protein loss

Decreased synthesis

Decreased protein intake

More common than hyperproteinemia

Electroendosmosis - The force of water being drawn to the cathode, opposing protein migration to the anode and causing some g-globulins to be displaced
5 bands: 

Strip immersed in an acid solution--denatures and immobilize proteins on support medium
Stain proteins with: 

Quantitation: Scanning densitometers compute area under the curve for each band and the percentage of total dye that appears in each fraction

Increase

Method

Increase

Method

High protein concentration allows for standard serum protein methods

Exudate

Principle pathway used to excrete excess nitrogen

Concentration of urea in the blood is determined by:

Decreased renal blood flow  

Increased protein diet or increased protein catabolism 

Decreased renal function, compromised urea excretion

Obstruction of renal flow in urinary tract

Normally 10:1 – 20:1 

Step 1) Conversion of urea to ammonia by urease(image above)

Step 2) Ammonia measured by: 

OR

Urease-UV method

Step 1) same as above

Step 2) Coupled reaction with L- glutamate dehydrogenase (GLDH)

Elevated serum creatine 

Increased serum creatinine

CrCl=  [(Ucr(mg/dL)x24 hr u ml)/ (Scr(mg/dL)x1440 min/24hr)] x [(1.73 m2/ pt SA)]

Or:

(Ucr/Scr) x (vol urine per minute) x (1.73/A)

Female 88-128 ml/minute (per 1.73 m2)

Two most common equations:

In pediatric patients:

24-hour urine 250 – 750 mg/day

Increased dietary intake

Increased urate production

Decreased excretion

Catabolic pathway enzyme defects

uric acid + phosphotungstic acid + O2 --> allantion + tungsten blue + CO2

In the presence of uricase:
uric acid + 02 + 2 H20 --> allantion + CO2 + H2O2

Increase

Reye’s syndrome

Testing uses unstable serum based control materials**

In the presence of glutamate dehydrogenase (GLDH):

Example:

enzyme-substrate reaction:

E = enzyme

S = substrate
ES = enzyme-substrate complex

P = product 

Zero-order kinetics:  Reaction rate depends only on the concentration of enzyme

 v = velocity of the reaction, Vmax = maximum velocity, S = concentration of substrate, Km = Michaelis-Menton constant of enzyme for specific substrate

Inhibitors- 

Calculation of enzyme activity

Combine to form three different CK isoenzymes.

Atypical CK isoenzymes exist but don't seem important 

pyruvate kinase:

lactate dehydrogenase:

Decrease in absorbance = enzyme activity

Reverse reaction (Oliver-Rosalki method)
in the presence of... 
creatine kinase:

hexokinase:

glucose-6-phosphate dehydrogenase:

Electrophoresis

Immunoinhibition

Sources of Error

CK-MB: < 6% of total CK  

In the presence of  LD and NAD:

Tissue source = Heart, liver, skeletal tissue, and erythrocytes

Five major fractions

Relative amounts of LD isoenzymes in normal serum:

For total LD activity
In the presence of LD:

Sources of error

In the presence of AST:

Pyridoxal phosphate = coenzyme
Tissue source = Cardiac tissue, liver, skeletal muscle

malate dehydrogenase: 

Monitors decrease in absorbance as NADH is consumed

Sources of error

lactate dehydrogenase:

Monitors decrease in absorbance as NADH is consumed

Transfers a phosphate group to an acceptor molecule in an alkaline pH

Non-specific

In the presence of ALP:

Tissue source=Intestine, liver, bone, spleen, placenta, and kidney

Heat stability

Inhibitors

For total ALP
Bowers-McComb method

In the presence of ALP at a pH of 10.2:

Increase in absorbance is directly proportional to ALP activity

In the presence of ACP:

Tissue Source=Prostate, bone, liver, spleen, kidney, erythrocytes, platelets, seminal fluid 

Chemical inhibition 

Immunochemical methods

For total ACP activity 
In the presence of ACP at a pH of 5.0:

Same as ALP but at an acidic pH

In the presence of GGT:

Tissue source=Kidney, brain, prostate, pancreas and liver

Help to evaluate the source of an elevated ALP value  

Biliary tract obstruction or inflammation

Monitoring of alcohol consumption

p-nitroaniline chromogenic product

Digestion of starch and glycogen

In the presence of amylase:

Activators = calcium and chloride
Tissue source= Salivary glands (S isoamylase), Pancreas (P isoamylase)

Saccharogenic

Chromogenic

Continuous monitoring

In the presence of lipase:

Tissue source= pancreas, stomach, small intestine.

Acute pancreatitis

in the presence of lipase

Substrates:

As the triglycerides break down, turbidity decreases

Turbidimetric methods

Colorimetric