MLAB 2401: Clinical Chemistry Basic Principles and Practice of Clinical Chemistry Part One

UNITS OF MEASURE Measurement requires a numerical value and a unit Laboratory results almost always have units of measurement associated with them SI units: length ( meter ) mass ( gram ) quantity ( mole ) Volume ( liter ) Time ( second ) Basic units describe unrelated physical quantities

Unit of Measure: Prefixes Common prefixes and abbreviations that are added to units of measure: deci (d) 10-1 centi (c) 10-2 milli (m) 10-3 micro ( μ) 10-6 nano (n) 10-9 pico (p) 10-12 femto (f) 10-15 Example: A common unit of liquid measurement is a deciliter( dl ), or one – tenth of a liter Combine a prefix with a basic unit results in a statement of a specific length, weight or volume Reporting clinical chemistry results may be in units such as : mg / dL g / dL mEq / L

Scientific Notation True scientific notation format: 1.22 X 104 BUT in hemo, for example a hemoglobin result would look like = 12.2 X 103

Water Specifications Tap water is unsuitable for lab use (too many impurities) Types of water purification techniques Distillation – removes most organic matter Reverse osmosis-removes organic, ionic, microbial, and viral contaminants Ultrafiltration – removes particulate matter, bacteria, emulsified solids Deionization – ions removed Reagent Grades of water Type I Purest – Required for sensitive tests Type II Acceptable for most uses Type III OK for washing glassware CAP - QC of water : pH, electrical resistance, bacterial culture

Water filtration system for Automated chemistry analyzer.

Solutions The clinical lab almost always uses solutions. A solution means that something has been dissolved in a liquid. In the clinical laboratory the solvent we measure most of the time is human plasma. The solute is whatever the substance is we want to measure. Mixtures of substances – the substances in a solution are not in chemical combination with one another. Dispersed phase - the substance is dissolved (the solute) The substance in which the solute is dissolved is the solvent. Solute + Solvent = Solution

Concentration Amount of one substance relative to the amounts of the other substances in the solution. Concentration can be measured in many different units % Solutions: w/w, v/v , w/v (parts of solute / 100 totals parts ) Note: liquids + liquids and solids + solids alters the total parts, but solutes + solvents does not Molarity: Moles / Liter Molality: Moles / 1000 grams solvent Normality: equivalent weight/ liter

Expressing Concentration: Percent Solution (parts/100) % w/w – percentage weight per weight Most accurate method of expressing concentration, but can be cumbersome (especially with liquids), not often used in clinical labs. % w/w = gram of solute OR gram of solute per 100.0 g of solution 100.0 g of solution How many grams of NaOH are needed to make a 25.0% w/w solution using deionized water as the solvent? 25.0% w/w = X g of solute in 100 g of solution X= 25.0 g NaOH

Expressing Concentration: Percent Solution (parts/100) % w/v – percentage weight per volume Easiest & most commonly used, very accurate if temperature controlled. %w/v= g of solute OR g of solute per 100.0 mL of solution 100 mL of solution What is the %w/v of a solution that has 15.0 g of NaCl dissolved into a total volume of 100 mL deionized water? X% w/v = 15.0 g NaCl X= 15.0 %

Expressing Concentration: Percent Solution (parts/100) % v/v –percentage volume per volume Least accurate, but used when both substances are liquids Note: volumes of liquids are not necessarily additive %v/v= mL of solute OR milliliter of solute per 100 mL of solution 100 mL of solution How many milliliters of ethanol are needed to make a 75.0% v/v solution using deionized water as the solvent? 75.0% v/v EtOH = X mL EtOH in 100 mL of solution = 75.0 mL EtOH

Expressing Concentration: Molarity Three components of Molarity Gram weight of solute Solute’s gram molecular weight Solvent quantity Number of moles per one liter of solution Mole = 6.022 X 1023 number of atoms or molecules OR Mole= Molecular weight in grams

Determinig Molarity: First step Molecular Weight Sum of the atomic weights of each element in the compound What is the molecular weight of Na3PO4? Step 1: Sodium has an atomic weight of 22.99, but there are 3 molecules so 22.99*3= 68.97 Step 2: Phosphorus has an atomic weight of 30.97, and only 1 molecule, so 30.97 *1= 30.97 Step 3: Oxygen has an atomic weight of 16, but there are 4 molecules ,so 16*4= 64.00 Step 4: Add 68.97+ 30.97+ 64.00= 163.94 gram molecular weight

Determinig Molarity: Next Step How many grams are contained in one mole of Na3PO4? Use the formula for mole calculations Number grams of solute Gram molecular weight of solute 1 mole Na3PO4 = X g Na3PO4 gram molecular weight(gmw) X= 163.94 g Na3PO4 So, 163.94 grams of trisodium phosphate are contained in 1 mole of trisodium phosphate or 6.022 X 1023 trisodium phosphate molecules weigh 163.94 grams

Determinig Molarity: Final Step Molarity (M) = 1 mole of solute 1L of solution We are asked to make a 1.00 L volume of a 0.100 molar solution of trisodium phosphate. How many grams would we need? M= grams gmw 1.00 L of solution 0.100 molar= X grams of Na3PO4 163.94 gmw of Na3PO4 (0.100M)(1.ooL) = X g 163.94 gmw 0.100= X 163.94 (0.100)(163.94)= X 16.39= X

Expressing Concentration: Molality Amount of solute per one kg of solvent Expressed in terms of weight per weight or moles per 1000 grams of solvent Used to measure the physical properties of solutions Molality = 1 mole of solute 1 kg of solvent

Expressing Concentration: Normality- First Step Equivalents Weights / Liter Equivalent weight is equal to the gram molecular weight of a substance divided by its valence Valence = the electrical charge of an ion, or the number of moles that react with 1 Mole H+ Example The MW of calcium = 40 grams Calcium ions carry a +2 electrical charge ( valence = 2 ) Equivalent Weight of calcium = 40 / 2 = 20 gram equivalent weight

Normality: N= number of grams of solute Gram equivalent weight of solute 1.00 L of solution Normality (N) N = Molarity (M) x valence Molarity = N / valence M is always < N

Solution Properties Titration – Method of measuring concentration of one solution by comparing it with a measured volume of a solution whose concentration is known General formula: when you have a volume and concentration of one, and either the volume or the concentration of the other: V1 C1 = V2 C2 For Example: How many mls of 1.0 N HCl is required to prepare 25 mls of 0.5 N HCl ? ( 1.0 N ) ( ? mls ) = ( 0.5 N ) ( 25 mls) ? mls = 12.5 mls You would need to add 12.5 mls of 1.0 N HCl to 12.5 mls of deionized water ( a total volume of 25 mls) to prepare 25 mls of 0.5 N HCl

Solution Properties Density – An expression in terms (usually) of a mass per unit of volume Many examples - including specific gravity, osmolality

pH and Buffers Buffers resist change in acidity Buffers are usually weak acids ( or bases) and their salts pH is the unit used to measure acidity ( Hydrogen ion concentration ) “p” = “negative log” of the concentration of a substance in solution. Example: pH = - log [H+] The Hydrogen ion concentration of deionized H2O is 1 x 10-7 M The negative log of 10-7 = 7. The pH of H2O is 7.0 The pH scale ranges from 0 - 14 pH 7 = neutral pH > 7 = alkaline (basic) pH < 7 = acid

Temperature Measurement of temperature is an important component of the clinical lab. Instruments, refrigerators and incubators are required to operate within specific temperatures that must be maintained and monitored daily. Examples Heat blocks, water baths, and incubators shall be maintained at least +/- 1 degree C. of the desired temperature Refrigerators shall be maintained at 2 -8 degrees C. Each laboratory must have a NIST calibrated thermometer in order to ensure the accuracy of other thermometers in the laboratory Out-of-range temperatures should be addressed asap

Temperature Scientific measurement of temperature is always expressed in the Celsius ( C) scale , not Fahrenheit ( F ) Celsius scale: 0 degrees = freezing point of water 100 degrees = boiling point of water

Conversion: Temperature Conversion of Celsius to Fahrenheit and Fahrenheit to Celsius F° = ( C ° x 1.8 ) + 32 C° = ( F ° - 32 ) 1.8 For example: Your refrigerator at home is probably around 40 ° F. What is that in Celsius? Celsius= 40-32 = 4.4 Water boils at 100 ° C. What is that expressed in Fahrenheit? (1.8)(100) +32 = 212

Conversions Most conversions within the metric system occur in units of TEN where changing a unit of measure to a higher or lower designation requires moving the decimal one place either to the left or to the right. When converting measures in either the high end of the scale (example kilo to mega) or the low end of the scale (examples milli to micro, micro to nano, etc.) the decimal must be moved three places right or left as the prefix designations are assigned only to every third unit in the extreme ends.

Example of a conversion How many mls are there in 2.5 liters? The question you have to ask yourself is, what is the relationship between liters and mls? The answer : 1 liter = 1000 ml But now what? We want to get rid of the “liters’ units and end up with “mls” … Right ?

1.25 liters = _____ mls ? Remember, write a fraction that does two things: 1. Equals 1 2. Gets rid of unwanted units and / or adds needed units 100 mg = _________ ug ?

Dilutions A ratio of the concentrate to the total (final) volume. A 1:4 dilution has a 1 volume of sample and 3 volumes of diluent mixed together. Any volume can be used to create this dilution, but it must be the same unit of volume Keep in mind the sample size when making your dilution For example: a 2:3 dilution could contain: 2 mL serum: 1 mL pure water 20 µL of serum: 10 µL of pure water 0.2 mL of serum: 0.1 mL of pure water

Dilutions for the Clinical Laboratory Example: A technician performed a laboratory analysis of patient’s serum for a serum glucose determination. The patient’s serum glucose was too high to read on the glucose instrument. The technician diluted the patient’s serum 1:2 and reran the diluted specimen, obtaining a result of 210 g/dl. To correct for the dilution, it is necessary to multiply the result by the dilution factor (in this case x 2). The final result is 210 g/dl x 2 = 420 g/dl.

Examples of dilutions and dilution factors Parts Parts Total Dilution Dilution Specimen Diluent Volume Factor 1.0 1.0 2.0 1 : 2 2 1.0 2.0 3.0 1 : 3 3 1.0 3.0 4.0 1 : 4 4 1.0 9.0 10.0 1 : 10 10 0.5 4.5 5.0 1 : 10 10 0.2 1.8 2.0 1 : 10 10 0.2 9.8 10.0 1 : 50 50

Serial Dilutions In these types of questions, you are given a series of tubes. Each tube having a measured amount of a diluent. You are instructed to add a specified amount of specimen into the first tube, mix well and transfer a specified amount of the mixture to the next tube, etc.

Serial Dilutions Example: 6 tubes, each with 0.5 mL DI water Add 0.2 mL serum to first tube and serially dilute Find the dilution in tube # 6 Find the dilution factor (will be the same in each of these tubes) 1/dil factor x 1/dil factor x 1/dil factor (etc. 6 times) Result multiplying the numerator 1x1x1x1x1x1x1x = 1 Multiplying the denominators Will give the result as 1 / 1838

Resources Serial dilution http://tinyurl.com/cw7e3ok

References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. Doucette, L. (2011). Mathematics for the Clinical Laboratory (2nd ed.). Maryland Heights, MO: Saunders. Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson .