Clinical Chemistry Review

Review of Clinical Chemistry

Topics & Sub-Topics

• Specimen collection is allocated 5% towards the overall grade.
• Instrumentation principles, methods, and calibration contribute 5%.
• Reagent preparation and laboratory mathematics also account for 5%.
• Quality assurance represents 10% of the grade.
• Metabolic blood tests, including principles, procedures, diseases/disorders and reference values are a substantial 50%
• Endocrinology and Toxicology, covering principles, procedures, and diseases/disorders accounts for 16%
• Laboratory safety represents 5% of the total grade.

Specimen Collection and Considerations

• Venipuncture involves obtaining a blood sample from a vein using a needle attached to a syringe or evacuated tube.
• Major veins useful for venipuncture are located in the antecubital fossa.
• The "H" pattern of veins in the antecubital fossa is observed in approximately 70% of the population.

Median Cubital Vein

• Located near the antecubital fossa center
• Typically large, close to the surface, stationary
• Generally is easiest and least painful to puncture
• Puncture in this location is least likely bruise

Cephalic Vein

• Often harder to palpate than the median cubital vein
• Is fairly well-anchored

Basilic Vein

• Last choice for venipuncture
• Isn't well anchored
• There is an increased risk of puncturing a median cutaneous nerve branch or the brachial artery

Other Veins

• Veins on the back of the hand and wrist can be used for venipuncture if necessary
• Veins on the underside of the wrist should never be used for venipuncture
• Leg, ankle, and foot veins are sometimes used with the physician's permission due to potential medical complications

Types of Blood Specimens

• Serum is the clear, pale yellow fluid separated from clotted blood after centrifugation
• Non-fasting serum can appear cloudy due to lipids
• Plasma is a clear to slightly hazy, pale yellow fluid that separates from cells when blood in an anticoagulant tube is centrifuged
• Unlike serum, plasma contains fibrinogen
• Whole the blood contains both cells and plasma, and needs to be collected in an anticoagulant tube to keep it from clotting

Methods of Venipuncture

• The evacuated tube system (ETS) is preferred because it minimizes the risk of contamination and exposure to blood.
• Use of needle and syringe is discouraged by CLSI
• Butterfly sets can be used with either ETS or syringe
• Tourniquets restrict venous but not arterial flow, and limit the time they are left on to one minute maximum

Venipuncture Equipment

• In needles, gauge and bore are inversely related
• 21 gauge is considered standard for routine venipuncture

Tube Colors and Anticoagulants

• Red-stoppered tubes (glass) have no additive and yield serum for chemistry and serology
• Red-stoppered tubes (plastic/Hemogard) contain a clot activator and also yield serum
• Lavender tubes contain K3EDTA (liquid) or K2EDTA (spray-dried) for hematology, by chelating calcium
• Light blue tubes with sodium citrate are used for plasma coagulation tests, also by chelating calcium
• Green tubes contain sodium heparin or lithium heparin for plasma chemistry, inhibiting thrombin formation

II. Reagent Preparation and Laboratory Supplies

Reagent Grades

• Analytic Reagent (AR) grade reagents meet standards set by the American Chemical Society (ACS)
• AR grade reagents are suitable for most analytic laboratory procedures.
• Ultrapure reagents have undergone additional purification steps for specific procedures like chromatography.
• Chemically pure (CP) reagents have impurity limitations not stated and are not recommended unless further purified.
• United States Pharmacopeia (USP) and National Formulary (NF) reagents are used to manufacture drugs with purity standards not based on lab needs
• Technical or Commercial Grade reagents are used primarily in manufacturing and should never be used in the clinical laboratory.

Reference Materials

• Primary standards are highly purified chemicals measured directly to produce a substance of exact known concentration and purity.
• Secondary standards are substances of lower purity, with concentration determined by comparison with a primary standard.

Water Specifications

• Type I water requires highest resistivity and is suitable for routine laboratory use.
• Type II and III water have lower resistivity and are used for less stringent applications.

Laboratory Supplies

• Three major thermometer types exist: liquid-in-glass, electronic/thermistor, and digital.

Glassware and Plasticware

• Kimax and Pyrex glassware are made of borosilicate.
• Polystyrene and polyethylene are common plasticware materials.

Laboratory Vessels and Pipets

• Lab Vessels include volumetric flasks, Erlenmeyer flasks, Griffin beakers, and graduated cylinders.
• Pipets can be designed "to contain" (TC) or "to deliver" (TD) a specific volume.
• Pipets can have blowout or self-draining drainage characteristics.

Types of Pipets

• Measuring pipets can be serologic or Mohr type.
• Transfer pipets such as volumetric or Ostwald-Folin dispense one volume only.
• Automatic macropipets or micropipets are most routinely used.

Automatic Pipets

• Air-displacement pipets use disposable tips.
• Positive-displacement pipets use reusable capillary tips.

Other Equipment

• Burets dispense a particular liquid volume during titration.
• Syringes transfer small volumes in blood gas analysis.
• Desiccants prevent moisture absorption, used in desiccators.

Balances

• Mechanical or substitution balances are required for primary standard preparation.
• Electronic balances or single-pan balances use electromagnetic force.

Centrifuges

• Horizontal centrifuges allow tubes to attain a horizontal position when spinning.
• Fixed-angle centrifuges have angled compartments.
• Ultracentrifuges separate layers of different specific gravities and are refrigerated.

Centrifuge Care

• Calibrate centrifuges every three months
• Disinfect centrifuges weekly.
• Check speed using tachometer or strobe light.

III. Laboratory Mathematics

Percent Concentration Solutions

• Weight/volume % (w/v) = (grams of solute / mL of solution) x 100
• Volume/volume % (v/v) = (mL of solute / mL of solution) x 100
• Weight/weight % (w/w) = (grams of solute / grams of solution) x 100

Molarity

• Molarity (M) = moles of solute / liters of solution

Normality

• Normality (N) = equivalents of solute / liters of solution = Molarity x valence

Molality

• Molality (m) = moles of solute / kg of solvent

Ratio and Dilution

• Ratio = amount of solute / amount of solvent
• Dilution = amount of solute / amount of solution

Conversion Factors

• Common analytes and their conversion factors to SI units are provided.

IV. Quality Assurance and Quality Control

Quality Assurance

Ensures quality results by monitoring preanalytical, analytical, and postanalytical stages.

• Preanalytical encompasses test ordering, patient preparation, and specimen handling.
• Analytical involves assay procedures, QC, reagents, and calibration.
• Postanalytical includes verification of results, reporting, and interpretation.

Quality Control

• Monitors results from control samples to verify accuracy of patient results.

Laboratory Statistics

• Measures of central tendency include mean, median, and mode.
• Measures of spread or dispersion include range, standard deviation (SD), and coefficient of variation (CV).

Gaussian Distribution

• Describes many continuous lab variables.
• The distribution is symmetric with mean, the median, same mode.
• Total area under the curve is 1.0.

Test Performance Parameters

• Accuracy is the nearness of measurement values to the true value.
• Precision (reproducibility) is closeness of assayed values to each other.
• Reliability maintains accuracy and precision over time.
• Analytical sensitivity measures the smallest analyte concentration.
• Analytical specificity measures only the analyte of interest.
• Reportable range or linearity defines the range of values over which accuracy is verified. Formerly called "normal value".

Reference Interval

• Reference interval varies for different patient populations
• Reference interval is Established by testing at least 120 healthy subjects.

Diagnostic Value of a Test

• True positive (TP): Correctly identifying diseased individuals.
• False positive (FP): Incorrectly identifying healthy individuals as diseased.
• True negative (TN): Correctly identifying healthy individuals.
• False negative (FN): Incorrectly identifying diseased individuals as healthy.

Calculations

• Diagnostic sensitivity assesses the test's ability to detect disease when present
• Calculated as: TP / (TP + FN) x 100.
• Diagnostic specificity assesses the test's ability to correctly identify absence of disease
• Calculated as: TN / (TN + FP) x 100.
• The positive predictive value (PPV) reveals the percentage of time that a positive result is correct
• Calculated as: TP/ (TP + FP) × 100.
• The negative predictive value (NPV) shows the percentage of time a negative result is correct
• Calculated as: TN/(TN+FN) × 100.

QC Samples

• Assayed regularly to verify proper lab procedure performance.
• QC samples should be like human samples
• QC samples need to be analyzed for 20 days when using a new instrument or reagent lot

Levey-Jennings Control Chart

• Called a Shewart plot
• Presents QC results, showing trends/shifts.

Errors

• Trend has increasing or decreasing values for six consecutive runs.
• Shift involves six consecutive control values on the same mean side.
• Outliers highly deviate and fall outside established limits

Westgard Rules

• Uses guidelines when there's a violation of control limits
• Initiates other rule testing for the run's control
• Type of error can be random or systematic, triggering corrective actions

Types of Errors

• Random error is present and unpredictable without means of preventing it
• Systematic error influences all observations consistently.

Proficiency Testing

• External quality assessment compares lab results with others for the same samples.

QA Program Components

Patient Identification

• Verify before specimen collection Collection of samples
• Familiarize with circumstances when the samples Testing
• Test requests should be confirmed if unclear Delta checks
• Compare patient data with old results Critical values
• Test results are a life and death situation Data reporting
• Results are checked at least twice Preventive maintenance
• Keep equipment in peak form and must be documented

Quality Management

Lean Six Sigma

• Infrastructure and improve quality The main team has
• 1: Quality improvement directors
• 2. Team people who maintain normal Jobs
• 3. People who give advice on the team

V. Concepts in Instrumentation

Spectrophotometry

• Method to determine a solution substance concetration by measuring the volume of light, after treatment
• Electromagnetic travels through photon waves

Light waves

• Wavelength is distance between 2 peaks Amplitude is the distance between peak to trough Visible light is measured Beer-Lambert’s Law
• Substance concentrations is directly proportional to volume of light or inversely proportional to logarithm of transmitted light

VI. Components to Spectrophotometer

Enrance slit

• 1 light source Existance slit
• Sampling machine Monochromator
• PM test tube
• Grating

Quality Assurance in spectrohotometry

• Wavelength measured using
• Didymium glass with peak of 600nm absorption
• Holmium oxide with multiple absorption peaks at 360nm.
• Absorbance is a performed check by using filters of glass or value solutions with a known absorbance.

Reflectometry

• Measures light by the volume in a liquid sample
• Uses a tungsten LED

Atomic absorption spectrophotometry

A comprehensive review of clinical chemistry. Key areas includie specimen collection, instrumentation, quality assurance and metabolic blood tests. Additional topics cover endocrinology, toxicology and laboratory safety.