Chem Exam I Labware + Intro

Serological (TD with blow-out) frosted ring indicates blow-out

Blow-out pipette?

Capacitive deionization (CDI) is a technology to deionize water by applying an electrical potential difference over two porous carbon electrodes. Anions, ions with a negative charge, are removed from the water and are stored in the positively polarized electrode. Likewise, cations (positive charge) are stored in the cathode, which is the negatively polarized electrode

what is Deionized water?

relatively little amount of solutes in solution.

what is Dilute solution?

Distilled water is water that has many of its impurities removed through distillation. Distillation involves boiling the water and then condensing the steam into a clean container.

what is Distilled water?

Reverse osmosis can remove many types of molecules and ions from solutions, and is used in the production of potable water. The result is that the solute is retained on the pressurized side of the membrane and the pure solvent is allowed to pass to the other side. To be "selective", this membrane should not allow large molecules or ions through the pores.

what is Prefiltered water?

Generally "screening" procedures are qualitative 2. Usually answers the question: is it present or not? 3. Often are quick methods: urinalysis dipstick, pregnancy screening

what is Qualitative in relation to chemistry?

A solution that has excess amount of solute particles such that the solution contains some undissolved solute particles.

what is Saturated solution?

usually concentrated solutions, or of high purity; working standards and working reagents are often prepared from stock standards/reagents so that the stock solutions do not become contaminated from every day use.

what is Stock standard?

High or Highest Purity High purity needed for procedures such as chromatography, atomic bsorption, immunoassays, molecular diagnostics,standardization procedures

Ultra-Pure, Spectroscopic Grade, Chromatographic Grade?

Unknown Used to manufacture drugs; not for laboratory use.

United States Pharmacopeia (USP), National Formulary (NF)?

Ultrafiltration (UF) is a variety of membrane filtration in which forces like pressure or concentration gradients lead to a separation through a semipermeable membrane.Suspended solids and solutes of high molecular weight are retained in the so-called retentate, while water and low molecular weight solutes pass through the membrane in thepermeate.

what is Ultrafiltrated water?

working reagent: usually enough is prepared from stock solutions for the assay to be completed.

what is Working standard?

1. Name. 2.Concentration. 3. Intended use. 4. Date made. 5. Expiration date. 6. Initials of technologist who prepared. 7. Storage instructions. 8. Hazardous precautions (ex: not hazardous to critical organs).

what is the 8 pieces of information that is required on all reagent, chemical, and solution labels for use in the clinical laboratory?

Classified according to design, number of pans, mechanical or electronic, operating range b. Requires accuracy: standardized (calibrated) using analytical weights traceable to the NIST.

what is the general use and care of Balances?

Highest Exceptionally pure for standardizing solutions and preparing reference standards.

ACS Primary Standard, NIST-certified Standard Reference Material (SRM)?

1.Analytic Reagent Grade (AR). High standards set by American Chemical. Society (ACS) For general lab use.

Analytic Reagent Grade (AR).

Terminology • Micropipet - measures volumes less than 1 mL • Macropipet - measures volume greater than 1 mL Types Air displacement Positive displacement Dispenser/Diluters

Automatic pipette?

Impurity limitations are not stated, non-uniform preparation Generally better than USP or NF but of lower quality than AR Grade; not for laboratory use.

Chemically Pure (CP)?

Most laboratories will use only class A macro-pipettes, so that in house calibration checks are not needed. • manual pipettes • Do not require calibration checks • Only manual pipet that can be used for quantitative work without calibration checks

Class A?

• If an 'A' is not present on the pipet it is NOT a Class A pipet! • Class B or unclassified pipets • Used for non-quantitative work such as bulk reagent prep (buffer, mobile phases, substrates) • CANNOT be used for quantitative work such as: • preparing standards, calibrators, and controls • dispensing of samples in assays • example • Beakers • Ehrlenmeyer flasks • Transfer pipettes • Test tubes • Reagent bottles • Photometry cuvettes

Class B?

Hemolyzed: pink to red, depends on extent of RBC lysis Icteric: Orange color, indicates presence of bilirubin is likely Lipemic: Sample appears milky or cloudy due to presence of fats (lipids). Specimens are often collected 'fasting' to eliminate lipemia which may interfere with procedures based on spectrophotometric principles

Differentiate the appearance of normal, hemolyzed, lipemic and icteric serum/plasma samples?

1. All supplies and equipment including the specific instrument needed to complete the assay are listed 2. Laboratory supplies such as pipettes and test tubes need not be included since these supplies are considered to be standard in all laboratories. However, if a pipette size or type is required that is not routinely supplied, then that should be listed. 3. Example: 37oC water bath; Turner model 340 visible wavelength spectrophotometer

Equipment and supplies?

a. Contains a 'clot activator' b. Contains an 'inert gel' to physically separate the cells from the liquid layer c. Does not contain an anticoagulant e. Centrifugation must be done to separate the clot from the liquid portion called serum f. After centrifugation, the 'inert gel' is positioned such that it provides a physical barrier between the clotted cells and the liquid portion, eliminating the need to aliquot the specimen. Called a SST = serum separator tube

GOLD TOP TUBE: what type of anticoagulant it contains or does not contain, the action of the tube contents,name of the liquid component (serum or plasma) and identify if whole blood or clotted blood is obtained?

a. Contains the anticoagulant Heparin b. Heparin acts to inhibit/neutralize thrombin; no coagulation occurs, no clot is formed, blood remains fluid c. Most common sample type submitted to chemistry lab for analysis because this sample type decreases turnaround time, thus increasing efficiency in the lab. Turnaround time is decreased by at least 40 minutes because we do not have to wait for the blood to clot before analyzing the plasma e. Centrifugation must be done to separate the cells from the liquid portion called plasma f. After centrifugation, the plasma must be aliquotted into a separate tube. g. Error: failure to separate plasma from the cells will result in falsely decreased analytes such as glucose; and falsely increased analytes such as potassium.

GREEN TOP TUBE: what type of anticoagulant it contains or does not contain, the action of the tube contents,name of the liquid component (serum or plasma) and identify if whole blood or clotted blood is obtained?

Contains anticoagulant and glycolytic agent that preserves glucose levels in the specimen b. Very limited use in chemistry lab c. Analyze whole blood: glucose d. Analyze plasma: glucose, lactic acid

GREY TOP TUBE: what type of anticoagulant it contains or does not contain, the action of the tube contents,name of the liquid component (serum or plasma) and identify if whole blood or clotted blood is obtained?

1. Any interfering substances will be listed here 2. The limits of linearity will be listed here 3. The lower limit of sensitivity (analytic sensitivity) will be listed 4. The procedure to follow in the event a patient sample exceeds linearity or sensitivity is listed here and how to report test results. 5. Example: Hemolyzed test samples will falsely increase test results.

Limitations of the procedure?

• Used for titrations • Capable of delivering multiple volumes • Are identified by frosted rings ( a pair of rings near the upper end)- this indicate that the pipette is a blow out pipette • Has a uniform diameter with volume graduations • Measurement from upper fill mark to lower mark • Partial measurement using the last few millimeters of the pippet in or near the delivery tip are in accurate • When delivering o Drain by gravity • Blow out the fluid remaining while bottom tip is touched inside surface of the receiving container • Tip of pipette SHOULD NEVER COME IN CONTACT WITH ACCUMULATING FLUID

Measuring pipette; serologic?

The curved upper surface of a liquid in a tube

Meniscus?

1. Explains how the method is standardized (calibrated) and how the results of the unknowns are determined 2. Stability and frequency of calibration is indicated. 3. How the calibration is verified to be accurate 4. Example: A standard curve is prepared for each run using the absorbance values obtained from the following standards: 0.0 mg/dl, 10.0 mg/dl, 50.0 mg/dl, 100.0 mg/dl. Values of all controls and patient samples are determined using this standard curve.

Method of calibration?

This is a TD (to deliver) pipette, and has 2 plunger stop positions: primary and secondary 1. As the plunger knob is depressed, the first stop is encountered. This first depression of the plunger knob is the primary or calibrated movement that 'aspirates' the specified volume 2. The plunger knob may then be depressed to its lowest position. This is the secondary or blow out movement (similar to the 'blow-out' step when using a serologic pipette)

Micropipette? (automatic)

used to measure reagents

Mohr?

Contains anticoagulant called EDTA = ethylenediamine tetraacetic acid b. EDTA binds/precipitates calcium; no coagulation occurs, no clot is formed, blood remains fluid c. Limited use in chemistry lab d. May analyze whole blood: cyclosporine, FK506, glycosylated hemoglobin, complete blood count e. EDTA plasma does not contain calcium

PURPLE TOP TUBE: what type of anticoagulant it contains or does not contain, the action of the tube contents,name of the liquid component (serum or plasma) and identify if whole blood or clotted blood is obtained?

Explains what type of chemical reaction is taking place, what is being measured, how it is measured, how the response being measured relates to the test result 2. Example: Glucose in the test sample reacts with glucose oxidase causing an increasedabsorbance. The increase in absorbance is directly related to the concentration of glucose in the test sample

Principle?

1. Explain in detail how to do the assay.

Procedure?

1. List what controls and what levels are required 2. Indicate the frequency of running controls 3. Include how to interpret control values and what should be done in the event of a control failure 4. List stability of quality control material (expiration) 5. Example: Level 1 and Level 2 Lyphocheck controls should be included on each run. Both controls should be within the 95% confidence interval. If a control falls outside of 3 SD from the mean, the run should be rejected and repeated.

Quality Control requirements?

a. Contains a 'clot activator' b. Does not contain an anticoagulant d. Centrifugation must be done to separate the clot from the liquid portion called serum e. After centrifugation,The serum must be aliquotted into a separate tube. f. Error: failure to separate serum from the cells will result in falsely decreased analytes such as glucose; and falsely increased analytes such as potassium. Often used for drug testing

RED TOP TUBE: what type of anticoagulant it contains or does not contain, the action of the tube contents,name of the liquid component (serum or plasma) and identify if whole blood or clotted blood is obtained?

All reagents needed to complete the assay should be listed. 2. If any of the reagents need to be prepared, the complete instructions (including recipes) must be listed in its entirety 3. Expiration dates of all reagents should be clearly listed. 4. Example: Reagent #1 = 0.9 % sodium chloride solution: take 0.9 grams sodium chloride and add enough d-H2O to equal 100 ml total volume. Mix well. Store in brown glass bottle, appropriately labeled. Stable for one year after preparation date.

Reagents?

Normal values (also called reference values) and critical limits are reported here, as well as the procedure to follow in the event a critical result is obtained.

Reference values?

1.Package insert: this contains all the information needed to write a procedure following NCCLS guidelines. 2. If any textbooks were used, it should be documented here as well.

References (or bibliography)?

1. This explains the laboratory protocol used to report test results. It should also explain how to report using a computerized format and provide detailed directions to help you navigate through the computer 2. Example: After verifying all controls are within limits, check all test values for linearity and sensitivity flags. To report, use worksheet: UAM, test name: GLU, and enter the test result at the appropriate prompt.

Reporting results?

Not as high as primary standardConcentration is determined by comparison with primary standard

Secondary Standard?

To drain by gravity.

Self-draining pipette?

Used for titrations

Serologic?

Explains the medical use of the analyte being tested for 2. Example: quantitative plasma glucose levels are increased in hyperglycemia caused by diabetes mellitus.

Significance of measurement (Clinical Significance) ?

This explains what types of specimens can be used in the test procedure, how to process the sample, what type of sample is unacceptable and any special handling requirements needed, stability, etc 2. Example: Serum or heparinized plasma is acceptable. 8-10 hour fasting or random specimens are acceptable. Draw the specimen by routine collection techniques and process following established laboratory protocol. Specimens containing fibrin strands or particulates (RBC, etc) are not acceptable and should be centrifuged prior to testing. Stable at room temperature up to 8 hrs, stable at refrigerated temperatures up to 2 weeks.

Specimen collection and handling requirements?

TC - to contain or "rinse out". Calibrated to contain a volume of liquid. Must be rinsed out to ensure complete delivery of sample. A small amount of liquid will remain in tip after rinsing. Does not meet Class A specifications.

TC; To contain?

Calibrated to deliver a volume of liquid by gravity with the tip against the side of the receiving vessel (i.e. self-draining)

TD; to deliver?

Undetermined; non uniform preparation Suitable for general industrial use; not for laboratory use.

Technical Grade, Commercial Grade?

• Volumetric flasks (TC) - used to prepare accurate volumes of reagents, dilutions • Delivers a single specified volume of fluid • IS MOST ACCURATE PIPETTE AND SHOULD BE USED WHEN DILUTING STDS AND CONROLS • Has a mark indicating proper filling • When delivering o Content from the pipette to drain by gravity DO NOT TOUCH INSIDE WALLS OF THE VESSEL o After fluid stops flowing The bottom tip of the pipette is touched inside the surface of the receiving vessel to allow CAPILLARY DRAW. Fluid remained after the capillary is allowed to remained in the pipette. DO NOT BLOW OUT o NEVER TOUCH THE ACCUMULATING FLUID IN THE VESSEL o ERROR BLOWING RESULTS - FALSE DECREASE OF RESULTS TOUCHING THE FLUID RESULTS - INACCURATE FLUID VOLUME DELIVERY

Transfer pipette; volumetric?

• Volumetric o Error: blowing all the liquid out of this pipette into the receiving vessel resulting in too much reagent in reaction tube = falsely decreased test results. o Error: when delivering sample to receiving vessel, the pipette tip is allowed to touch the liquid already in the receiving vessel, resulting in an inaccurate fluid volume delivery. • Serologic o Error: failure to blow out the pipette resulting is less reagent in reaction tube = falsely increased test results. • Mohr o Error: fluid remaining in the tip after drainage is 'blown' into the receiving vessel, resulting in extra volume of liquid being delivered: delivering too much reagent = falsely decreased test results o

What are some errors due to improper pipetting technique?

a. Sample variables include physiologic variation, patient preparation, and problems with collecting, transporting, processing and storage of the sample a. Physiologic variation refers to changes that occur within the body, such as cyclic changes (diurnal or circadian variation); may also refer to changes resulting from exercise, diet, stress, gender, age, medications, posture, underlying medical conditions (fever, asthma, obesity) b. Patient preparation errors include non-fasting specimen when the specimen should be fasting, fasting time not sufficient (8-10 hour fast, 12-14 hour fast), drugs (prescribed, over the counter, ect), smoking, diet (not inclusive list) c. Patient lifestyle choices (nicotine (cigarette smoking), caffeine (coffee, tea, cola), alcohol intake, long distance running, excessive exercise, lack of exercise. d. Patient treatment protocols: diuretics, intramuscular injections e. Specimen procurement techniques: prolonged tourniquet application, repeated clenching of fist during venipuncture f. It is VERY GOOD PRACTICE to write down specific diet or other patient preparation instructions for patients. Directions/instructions can be overwhelming to patients and can be easily misunderstood; include lab contact information on written directions

What are the 3 sample variables that may affect test results?

Most laboratories discourage use of the Mohr pipette because it is often mistaken as a serologic pipette and is used improperly. Error: fluid remaining in the tip after drainage is 'blown' into the receiving vessel, resulting in extra volume of liquid being delivered: delivering too much reagent = falsely decreased test results

What are the reasons why the mohr pipette is not preferred for use in the clinical lab?

Calibration of micro-volume pipettes is required by the laboratory at periodic intervals (usually quarterly) to verify accurate delivery of a specified volume of liquid.

What are the two methods commonly used in the laboratory to check calibration of micro-pipettes?

Class A.

What class of labware is preferred for use in the clinical laboratory?

a. Used to establish the accuracy or correctness of a screening test by ruling out false positive or false negative results; often are/may be quantitative. b. Characteristics of ideal confirmatory test i. Different methodology or chemical reaction for analysis ii. Better diagnositc specificity (rules out false positive test results) iii. Better diagnositc sensitivity (rules out false negative test results

What is Confirmatory test in relation to chemistry?

Allows sample to be stored in initial collection tube after centrifugation; helps minimize specimen 'mixup' errors that may occur when transferring processed samples into an aliquot tube.

What is PST and what is it used for?

a. Demands accurate and precise technique in all phases of the analysis b. Measures the amount of substance present, e.g. weight per volume. Most chemistry assays are quantitative c. Methods more technically demanding as compared to qualitative testing d. Several 'systems' of reporting test results; both are based on the metric system i. Conventional: used most often in the U.S. ii. SI unit (Systèmes Internationale) : used internationally, preferred in scientific literature.

What is Quantitative in relation to chemistry?

Allows sample to be stored in initial collection tube after centrifugation; helps minimize specimen 'mixup' errors that may occur when transferring processed samples into an aliquot tube.

What is SST and what is it used for?

a. Determines presence or absence of disease or condition c. Methodology may be generally non-specific and may require follow-up testing to rule out false positive/negative test results ('confirm' screening results), but this is dependent upon clinical situation.

What is Screening test in relation to chemistry?

a. Not as accurate as quantitative testing, but can provide an indication of the approximate concentration of substance b. Titers in serologic testing obtained from serial dilutions; urinalysis reagent strip.

What is Semi-quantitative in relation to chemistry?

1. Blood: Most common fluid submitted for analysis. 2. Urine: Second most common fluid submitted.

What is the 2 most common types of specimens submitted to the laboratory for testing?

Plasma contains the protein fibrinogen because the coagulation cascade has been prevented from occurring.

What is the Difference between serum and plasma?

Using a safety bulb, exert gentle and continuous suction drawing the liquid into the pipette until slightly above the fill mark (sometimes called the calibration mark).

What is the proper use of the safety bulb when pipetting liquids in the lab?

Volumetric • Used to prepare accurate volumes of reagents, dilutions • capable of delivering multiple volumes of fluid Serologic • Used in chemistry, frosted ring indicates blow-out • delivers single specific volume

What is the relative volume delivery accuraccy for the volumetric and serologic pipette?

Air displacement automatic

What pipette do you use for delivering micro-volumes of patient specimen?

Mohr. Volumetric. Serologic.

What pipette do you use for delivering or measuring a macro-volume of reagent?

volumetric + Air displacement

What pipette do you use for preparing dilutions?

Serologic

What pipette do you use for reconstituting standard?

To verify accurate delivery of specified volume of liquid.

Why is it necessary to check calibration of the micropippete and not volumetric, or serologic pipette?

large amounts of solute are in the solution.

what is Concentrated solution?

Volumetric flasks (TC) - used to prepare accurate volumes of reagents, dilutions

volumetric?

a. General information 1) i. One means of separating solids from liquids. Ultracentrifugation is used when separation of different densities required ii. 2) Centrifugal force is dependent on iii. 3 variables: mass, speed, radius 3) Speed of centrifuge is expressed as RPM = revolutions per minute iv. 4) Centrifugal force that is generated is expressed as RCF = relative centrifugal force = gravities (g) v. 5) RPM is related to RCF by the equation: RCF = (1.118 x 10-5 ) x (r) x (rpm)2 vi. 6) Speed is checked with tachometer or strobe light (required by accreditation agencies) b. b. Proper use and care: i. 1) Must be clean ii. 2) Proper balance is CRITICAL (review this in the book) iii. 3) Lid/cover remains closed when in use iv. 4) Centrifuge speed must be checked

what is the general use and care of Centrifuges?

Drying agents (desiccators) are usually hygroscopic b. Hygroscopic = takes up water when exposed to air c. Purpose of desiccant: to keep chemicals from taking on water (becoming hydrated) when exposed to air)

what is the general use and care of Desiccants?

Glassware: Type A most accurate, most precise .

what is the general use and care of Glassware/Plasticware?

Used to maintain test samples, test reactions, reagents at optimal temp b. Temperature must be monitored (most often at time of use and once per day if not used) and documented as within stated limits c. Must be kept clean

what is the general use and care of Incubators/waterbaths?

Used to store reagents and test samples (pre and post analysis) b. Temperature must be monitored regularly (once/shift, once/day) and documented as within stated limits c. Must be kept clean; freezers must be kept free of ice buildup

what is the general use and care of Refrigerators/freezers?

Handling specimens inside a safety hood (or behind a splash shield) during specimen processing decreases chance of exposure to contaminated aerosols. b. Safety hoods must be periodically (yearly) checked by authorized personnel to test and verify safety-related features are properly operating c. When using a safety hood, the sash must not be higher than the intended mark d. Must be kept clean and free of clutter

what is the general use and care of Safety hoods/splash shields?

a. Used for checking the pH of body fluids, adjusting the pH of test specimens and/or reagents for analysis b. Standardized (calibrated) using buffers of specific pH in both the acid and base range. c. Must maintain the electrode's internal fill solution at a proper level d. Slope and drift of the electrode must be checked prior to use e. Temperature must be monitored because temperature changes may affect pH measurements

what is the general use and care of pH meters?

a. Critical Need 2 patient identifiers: i. patient name AND medical record number or date of birth b. Date specimen was collected c. Time of day the specimen was collected (use military time) d. Initials of person who collected the specimen e. Name of test to be performed

what is the proper sample handling techniques and their importance in regard of ID?

a. After collection, allow blood to completely clot, ~30 minutes b. Centrifuge the blood specimen for 10 minutes at 1000-2000 g (RCF). c. Do not use the "brake" to slow centrifuge: can disrupt cells into supernate d. Draw off specimen using a disposable transfer pipette (dispopette) and place into clean properly labeled container called an aliquot tube a. Patient name b. Hospital identification number (called the medical record numbered = MRN) c. Date sample was drawn (format = month, day, year) d. Time sample was drawn (format = use military time) e. Tests requested

what is the proper sample handling techniques and their importance in regard of Processing?

a. Samples must be brought to the lab shortly after collection; prompt separation of serum/plasma required to preserve components/analytes b. Perform routine tests within 1-4 hours of collection; otherwise process the specimen to a point where it can be stored without alterations to chemical constituents c. To minimize effects of evaporation and possible environmental contamination, specimen should be capped/stoppered and kept away from rapid airflow, light and heat

what is the proper sample handling techniques and their importance in regard of Timing?

a. How the sample is stored is dependent upon what test is ordered b. Room temperature: many constituents will be stable for up to 4 hours until analyzed, then must be stored refrigerated or frozen (long term) c. Refrigeration sufficient for most analytes d. Freezing: may be necessary for unstable components such as hormones, complement and samples containing multiple antibiotics. Not all freezer temps are the same (-20o C or -70o C); Avoid repeated freeze-thaw cycles (causes analyte instability) e. Tightly stopper sample to avoid evaporation, possible bacterial contamination

what is the proper sample handling techniques and their importance in regard of storage?

a. Hemolyzed specimens: ruptured RBC's interfere with some procedures especially LD, potassium, folic acid b. Sample drawn in incorrect collection tube: incorrect anticoagulant, etc c. Specimen collection and/or handling requirements not followed d. Patient's/sample ID questioned or incomplete information on label e. Insufficient quantity (QNS) obtained 6. Sample drawn from a site above an IV (error: sample will be contaminated with the IV fluid which will cause erroneous test results)

what is the proper sample handling techniques and their importance of Special handling requirements?

Contains all reagents used in the assay, except the 'reactant' is omitted 2. Used to set the baseline of the instrument to zero so that any response seen from the instrument is due to the chemical reaction we are trying to measure. 3. Matrix of blank is assay dependent

what is the purpose and proper sequence of Blank?

The assay may have only one standard if the assay is known to follow Beer's Law up to a specific concentration.1. This is a solution with an exact, known amount of the specific compound. 2. Standards are of the same as the patient sample. 3. Each run has several standards of different concentrations.

what is the purpose and proper sequence of Standard(s)/calibrator(s)?