Essentials of Clinical Lab Science Exam 2 (updated)

Accuracy

describes how close a test result is to the true value. Reference samples and standards with known values are needed to check accuracy.

Urine for pregnancy testing

first-voided morning specimen

Specimen Collection

Blood is the type of specimen most frequently analyzed in the clinical laboratory. Urine specimens, body fluids, and stool specimens are also frequently analyzed. Other specimens such as throat cultures and swabs from wound abscesses are sent to the microbiology laboratory for study. Knowledge of proper collection, preservation, and processing of specimens is essential. A properly collected blood specimen is crucial to quality performance in the laboratory. In addition to specimen procurement, related areas of specimen transportation, handling, and processing must also be fully understood by anyone who collects or handles blood specimens. Strict adherence to the rules of specimen collection is critical to the accuracy of any test. The major potential sources of error include identification errors, both of the patient and the specimen.

Box 3-6

~Examples of Errors in Laboratory Testing~ Preanalytical (Preexamination) Phase = • Test order inaccuracy • Error in order entry • Wrong patient identification • Blood culture contamination • Type of evacuated tube used for blood collection • Specimen labeling errors • Improper specimen handling • Adequacy of specimen information Analytical Phase (Examination) = • Poor quality control • Accuracy of point-of-care testing • Instrument malfunction • Analysis inference, e.g., hemolysis, lipemia • Incorrect results resulting from drug interference Postanalytical (Postexamination) Phase = • Failure in critical value reporting • Increased turnaround time • Missing laboratory test results resulting from discontinuity of care • Incorrect interpretation of results • Lack of clinician follow-up

Guaiac Slide Test sampling p. 220

-*Hemoccult II* is used and contains filter paper with hum guaiac -thin film in two boxed on front side and on a wooden applicator: in kit -two of 3 consecutive samples collected: three slides in kits -test slides dry overnight and sent to lab= label properly -if mailed, must be on a mailing pouch, NOT envelope -*test opposite side or back* of test slide controls are revealed and mist air dry before solution applied -blue color* The American Cancer Society (ACS) recommends that for colorectal screening, two samples from three consecutive specimens be collected. Therefore test kits are usually supplied to patients in groups of three slides. The patient is instructed to allow the test slides to dry overnight, then return them to the physician or laboratory. If the slides are to be mailed, they must be placed in an approved U.S. Postal Service mailing pouch (not a standard paper envelope). The reaction requires that blood cells be hemolyzed for proper release of peroxidase. This usually takes place within the GI tract. If whole, undiluted blood is applied to the test paper, the RBCs may not hemolyze, and the reaction may be weak or atypical. The test is significantly more sensitive to the presence of occult blood if the specimen is allowed to dry on the slide before the developing solution is applied. The ACS recommends that slides be tested within 6 days of preparation and that the slides not be rehydrated. Also, a single positive smear should be considered a positive test result, even in the absence of dietary restriction.

General Protocol of Blood Collection

1. Phlebotomists should pleasantly introduce themselves to the patient and clearly explain the procedure to be performed. It is always a courtesy to speak a few words in a patient's native language if English is not his or her first language. Ethnic populations vary geographically, but many patients are now Spanish speaking. If a patient doesn't understand English and the phlebotomist is not fluent in the patient's native language, an interpreter should be requested. 2. Patient identification is the critical first step in blood collection. Patient misidentification errors are potentially associated with the worst clinical outcomes because of the possibility of misdiagnosis and mishandled therapy It is necessary to have the patient state and speak his or her name. If a patient cannot provide this information, he or she must provide some form of identification or must be identified by a family member or caregiver. Check the identification band that is physically attached to the patient. Wristbands with unique bar-coded patient identifiers have great potential for reducing patient misidentification. Unfortunately, wristband errors do occur. A study conducted by the College of American Pathologists (CAP) identified six major types of wristband errors, as follows: • Absent wristband • Wrong wristband • More than one wristband with different information • Partially missing information on the wristband • Erroneous information on the wristband • Illegible information on the wristband When the patient is unable to give his or her name, or when identification is attached to the bed or is missing, nursing personnel should be asked to identify the patient physically. Any variations in protocol should be noted on the test requisition. A CAP recommendation is that phlebotomists should refuse to collect blood from a patient when a wristband error is detected. The current requirement for two patient identifiers usually includes the patient stating his or her full name and the date of birth. The name of the patient's physician should be verified to avoid any possible confusion when test results are sent to the ordering physician. 3. The patient should be asked if he or she is taking any medications, including over-the- counter medications such as aspirin. It is particularly important that patients inform personnel if they are taking an anticoagulant such as warfarin (Coumadin). When therapeutic drug levels are being determined, it is important to ask when the last dose of a medication was consumed. 4. Test requisitions should be checked and the appropriate evacuated tubes assembled. All specimens should be properly labeled immediately after the specimen is drawn. Prelabeling is unacceptable. 5. The patient's name, unique identification number, room number or clinic, and date and time of collection are usually found on the label. In some cases, labels must include the time of collection of the specimen and the type of specimen. A properly completed request form should accompany all specimens sent to the laboratory.

Checking a Reagent Before Use

After the prepared reagent is in the reagent bottle, it must be checked before it is put into actual use in any procedure. New lots or batches of reagents are generally run in parallel testing with existing reagents. Controls, standards, and calibrators are means of testing the new lot or batch with the existing reagent. In addition, a reagent log should be kept to indicate date in use and date of expiration. This log should also note the lot numbers of controls. After the reagent has been checked, this is indicated on the label, and the solution can be used for laboratory testing.

Order of Draw Table 4-4

1. Yellow | Blood cultures: SPS- aerobic and anaerobic | 8-10x 2. Light blue | Citrate Tube | 3-4x | 4x 3. Gold or red/gray | BD Vacutainer SST gel separator tube | 5x - Red | Serum tube (plastic) | 5x | 5-10x - Red | Serum tube (glass) | None - Orange | BD Vacutainer RST | 5-6x 4. Light green or green/gray | PST gel separator tube with heparin | 8-10x | 5-10x - Green | Heparin | 8-10x | 5-10 5. Lavender | EDTA | 8-10x | 5-10 6. White | PPT Separator tube (K2EDTA with gel | 8-10x | 8-10x) 7. Gray | Fluoride (glucose) tube | 8-10x Closure color | Type of Tubes | Mixing by Inverting BD Tubes | Mixing by inverting Greiner Tubes

Control Specimens

A QC program for the laboratory makes use of control specimens, which are or resemble a serum sample with a known concentration of the analyte being measured in the testing procedure. Most clinical laboratories use multiconstituent controls because these require less storage space, offer ease of inventory, and increase manufacturer services through peer laboratory comparisons. Lyophilized (freeze-dried) and liquid control materials offer good stability and reasonable expiration dating. Liquid controls may offer greater reproducibility between bottles because, unlike lyophilized controls, no pipetting error is added on reconstitution. Suppliers often offer to sequester a specific quantity (estimated usage) of control material to be sent to the laboratory at the customer's request. This ensures that the customer can continue to receive the same lot over time. A control specimen must be carried through the entire test procedure and treated in exactly the same way as any unknown specimen; it must be affected by all the variables that affect the unknown specimen. Control specimens are used because repeated determinations on the same or different portions (or aliquots) of the same sample will not, as a rule, give identical values for any particular constituent. Many factors can produce variations in laboratory analyses. With a properly designed control system, it is possible to monitor testing variables. According to CLIA regulations, a minimum of two control specimens (negative or normal and positive or increased) must be run in every 24-hour period when patient specimens are being run. Alternately, when automated analyzers are in use, the bi-level controls are run once every 8 hours of operation (or once per shift) Assaying control specimens and standards along with patient specimens serves the major functions of: 1. Detecting errors in equipment, reagents, or individual technique. 2. Confirming the stability and accuracy of testing compared with reference values. 3. Detecting an increase in the frequency of both high and low minimally acceptable values (dispersion). 4. Detecting any progressive drift of values to one side of the average value for at least 3 days (trends). Slow deterioration of reagents, controls, or light source can produce this type of systematic error. 5. Demonstrating an abrupt shift or change from the established average value for 3 days in a row (shift).

Environmental Factors

A variety of environmental factors can affect the quality of evacuated tubes used to collect blood. These factors can then influence the published expiration dates of the evacuated tubes. Environmental factors affecting evacuated tubes include the following: • Ambient temperature • Altitude • Humidity • Sunlight 1. If an evacuated tube is stored at low temperature, the pressure of the gas inside the tube decreases, leading to an increase in draw volume. Conversely, higher temperatures can cause reductions in draw volume. Increased temperatures in evacuated tubes also can have a negative effect on the stability of certain tube additives, such as biochemicals or gel. Gel is a compound that potentially can degrade when exposed to high temperatures. 2. In situations where blood is drawn at high altitudes (> 5000 feet), the draw volume may be affected. Because the ambient pressure at high altitude is lower than at sea level, the pressure of the residual gas inside the tube will reach this reduced ambient pressure during filling earlier than if the tube were drawn at sea level. The resulting draw volume will be lower. 3. The effect of storage at various levels of humidity can affect only plastic evacuated tubes because of the greater permeability of these materials to water vapor relative to glass. Conditions of extremely high humidity could lead to the migration of water vapor inside a tube that contains a moisture- sensitive material, such as a lyophilized additive. Conditions of extremely low humidity could hasten the escape of water vapor from a tube containing a wet additive. Such storage conditions may compromise the accuracy of clinical results. 4. A special additive mixture for coagulation testing that is sensitive to light and found only in glass evacuated tubes is called CTAD (citric acid, theophylline, adenosine, and dipyridamole). The CTAD mixture minimizes platelet activation after blood collection. Normally, this additive has a slightly yellow appearance that becomes clear when no longer viable. These tubes are generally packaged in small quantities to minimize exposure to light.

Volumetric Pipets

A volumetric, or transfer, pipette has been calibrated to deliver a fixed volume of liquid by drainage. These pipettes consist of a cylindrical bulb joined at both ends to narrow glass tubing. A calibration mark is etched around the upper suction tube, and the lower delivery tube is drawn out to a fine tip. Some important considerations concerning volumetric pipettes are that the calibration mark should not be too close to the top of the suction tube, the bulb should merge gradually into the lower delivery tube, and the delivery tip should have a gradual taper. To reduce drainage errors, the orifice should be of a size such that the outflow of the pipette is not too rapid. These pipettes should be made from a good-quality Kimax or Pyrex glass. Volumetric pipettes are suitable for all accurate measurements of volumes of 1 mL or more and are calibrated to deliver the amount inscribed on them. This volume is measured from the calibration mark to the tip. A 5-mL volumetric pipette will deliver a single measured volume of5 mL, and a 2-mL volumetric pipette will deliver 2 mL. The tolerance of volumetric pipettes increases with the capacity of the pipette. A 10-mL volumetric pipette will have a greater tolerance than a 2-mL pipette. The tolerance of a 5-mL volumetric pipette is 0.01 mL. When volumes of liquids are to be delivered with great accuracy, a volumetric pipette is used. Volumetric pipettes are used to measure standard solutions, unknown blood and plasma filtrates, serum, plasma, urine, cerebrospinal fluid, and some reagents. Measurements with volumetric pipettes are done individually, and the volumes can be only whole milliliters, as determined by the pipette selected (such as 1, 2, 5, and 10 mL). To transfer 1 mL of a standard solution into a test tube volumetrically, a 1-mL volumetric pipette is used. To transfer 5 mL of the same solution, a 5-mL volumetric pipette is used. After a volumetric pipette drains, a drop remains inside the delivery tip. The specific volume the pipette is calibrated to deliver is dependent on the drop left in the pipette tip. Information inscribed on the pipette includes the temperature of calibration (usually 20° C), capacity, manufacturer, and use (TD). The technique involved in using volumetric pipettes correctly is very important, and a certain amount of skill is required.

Percent

Another expression of concentration is the percent solution (%), although in the International System of Units (SI system) the preferred units are kilograms (or fractions thereof) per liter (w/v) or milliliters per liter (v/v). A description of the percent solution follows, because this expression of concentration is still used in some instances. Percent is defined as parts per hundred parts (the part can be any particular unit). Unless otherwise stated, a percent solution usually means grams or milliliters of solute per 100 mL of solution (g/100 mL or mL/100 mL). Recall that 100 mL is equal to 1 deciliter (dL). Percent solutions can be prepared using either liquid or solid chemicals. Percent solutions can be expressed either as weight per unit volume percent (w/v%) or as volume per unit volume percent (v/v%), depending on the state of the solute (chemical) used; that is, whether it is a solid or a liquid. When a solid chemical is dissolved in a liquid, percent means grams of solid in100 mL of solution. If 10 g of sodium chloride (NaCl) is diluted to 100 mL with deionized water, the concentration is expressed as 10% (10 g/dL). If 2.5 g is diluted to 100 mL, the concentration is 2.5% (2.5 g/dL). The following is an example of concentration expressed in percent: 10 grams of sodium hydroxide (NaOH) is diluted to 200 mL with water. What is the concentration in percent? A proportion can be set up to solve this problem, as follows: 10 g / 200 ml = ?g / 100 ml ? = 5% solution (preferably expressed as 5 g/dL) If a liquid chemical is used to prepare a percent solution, the concentration is expressed as volume per unit volume percent, or milliliters of solute per 100 mL of solution. If 10 mL of HCl is diluted to 100 mL with water, the concentration is 10% (preferably expressed as 10 mL/dL). If 10 mL of the same acid is diluted to 1 L (1000 mL), the concentration is 1% (preferably expressed as1 mL/dL).

Processing Blood Specimens

Blood specimens must be properly handled after collection. Blood samples should be analyzed as promptly as possible. Institutional protocol should be followed for conditions of storage (such as room temperature, refrigerated, or frozen), depending on the analyte to be measured. In general, specimens should be analyzed within 24 hours of collection. It is important that the proper evacuated tube be used, especially if a specimen is being analyzed for glucose, which requires a preservative. If no anticoagulant is used, the blood will clot, and serum is obtained. The serum is then removed from the clot by centrifugation. To prevent excessive handling of biological fluids, many laboratory instrumentation systems can now use the serum directly from the centrifuged tube, without another separation step and without removing the stopper. It is important to remove the plasma or serum from the remaining blood cells, or clot, as soon as possible. Because biological specimens are being handled, the need for certain safety precautions is stressed. The Standard Precautions policy should be used because all blood specimens should be considered infectious and must be handled with gloves. The outside of the tubes may be bloody, and initial laboratory handling of all specimens necessitates direct contact with the tubes. To separate the serum and plasma from blood cells or a blood clot, tubes must have stoppers for centrifugation. When a stopper must be removed from the tube to obtain plasma or serum, it must be removed carefully and not popped off, because this could cause infection by inhalation or by contact of the infectious aerosol with mucous membranes. Stoppers should be twisted gently while being covered with protective gauze to minimize the risk from aerosolization. This processing step can be done using a protective plastic shield to prevent direct splashes. It is generally best to test specimens as quickly as possible. Specimens should be processed to the point where they can be properly stored so that the constituents to be measured will not be altered. It must be guaranteed that specimens collected at stations away from the testing laboratory are delivered in less than 2 hours from collection, and that they have been stored properly, with refrigeration or freezing, if necessary. Testing laboratories provide specific instructions on the collection, processing, and delivery requirements for all assays that they perform.

Table of Contents

Chapter 3 Regulation of Laboratories CLIA p.58 ISO 15189 Box 3-1 p. 59 Lean Principles p. 59 Preanaytical errors Fig 3 p. 61 Box 3-6 p. 61 Box 3-7 p. 62 Control Specimens p. 63 Quality Control p. 64 (two bulleted items) Accuracy p. 64 Sensitivity p. 65 Determination of Control Range p. 68 Sources of Variance or Error p. 68 Monitoring QC - Levy Jennings and Westgard p. 68 Westgard Rules p. 69-71 Nonanalytical Factors in Quality Assessment p. 72 Chapter 4 Quality Assessment p.83 Patient Care Partnership p. 84 Standard Precautions p. 85 Specimen Collection p.86 Blood Collection Variables p.86 Layers of Anticoagulated Blood p. 86 Environmental Factors p.87 Expiration Dates of Evacuated Tubes p. 87 Altitude p. 87 EDTA p.89 Order of Draw Table 4-4 p. 91 General Protocol of Blood Collection p.92 Processing Blood Specimens p.95 Hemolyzed Specimens p.96 Blood Spot Collection for Neonatal Screening Programs p.97-98 Chapter 6 Metric Volume p. 140 Metric length p. 140 Temperature Conversion - Fahrenheit and Celsius p. 142 Volumetric Pipets p.146 Analytical Balance p. 150 Type I Reagent Water p. 153-154 Distilled Water p. 154 Deionized Water p. 155 Analytic Reagent Grade p. 155 Reagent defined p. 155 MSDS sheets p. 156 Standard Solutions p. 156 Storage of Chemicals -Flammables p. 156 Checking a Reagent Before Use p. 157 Chapter 7 Rounding Off Numbers p. 170 Significant Figures p. 170 Percent p. 171-172 Molarity p. 172 Dilutions and Diluting Specimens p. 174-175 Chapter 9 Waived Testing p. 219 Beta-Human Chorionic Gonadotropin p. 222 Specimen Collection for Beta hCG p. 222 Urine for pregnancy testing p. 222 Clinical Significance of FOBT p. 224 False negative FOBT p. 224-225 False Positive p. 224-225 Guaiac Slide Test sampling p. 225 Handheld Automated Equipment p. 225 Characteristics of POCT devices p. 225 (bulleted items) Overview of LIMS p. 228 Components of a Computer System p. 228-230 2D Bar Codes p.229 Computer Interfaces p. 230 Benefits of Automation p. 235 Steps in Automated Analysis p. 235 Most automated areas of the lab p. 236

Waived Testing

Diagnostic testing not performed within a traditional laboratory is called waived testing by TJC. CLIA '88 subjects all clinical laboratory testing to federal regulation and inspection. According to CLIA, test procedures are grouped into one of the following four categories: waived tests, moderately complex tests, highly complex tests, or provider-performed microscopy (PPM) tests A site performing only waived tests must have a Certificate of Waiver license from CLIA but will not be routinely inspected. The site must, however, adhere to manufacturers' instructions for performing the test. Good Laboratory Practice according to CLIA dictates appropriate-quality testing practices as outlined under the moderate- and high-complexity test requirements. These include the training of testing personnel, competency evaluation, and performance of QC. The Veterans Administration, College of American Pathologists (CAP), and The Joint Commission (TJC) do not recognize the waived category. These accreditation organizations have guidelines for waived testing and other POCT that must be met Test complexity is determined by criteria that assess knowledge, training, reagent and material preparation, operational technique, QA/QC characteristics, maintenance and troubleshooting, and interpretation and judgment. Any over-the-counter test approved by the U.S. Food and Drug Administration (FDA) is automatically placed into the waived category. POCT falls within either the waived or the moderately complex category. However, the examination and interpretation associated with it, such as collecting and Gram-staining a specimen, can be classified as highly complex testing.

Expiration Dates of Evacuated Tubes

Expiration dates are determined by testing shelf life under known environmental conditions. Shelf life of an evacuated tube is defined by the stability of the additive and vacuum retention. Most evacuated tubes on the market have at least a 12-month shelf life. It is important that tubes be stored under recommended conditions. The expiration dates of glass tubes are generally limited by the shelf life of the additives, because vacuum and water vapor losses are minimal over time. Exposure to irradiation during sterilization of tubes and to moisture or light during the shelf life of the product can limit the stability of biochemical additives. The expiration dates of evacuated plastic tubes are also limited by the same factors that affect glass tubes. Evacuated plastic tubes do sustain a measurable loss of vacuum over time, and some evacuated plastic blood collection tubes may have their expiration dates determined by their ability to ensure a known draw volume. It is important to understand that evacuated blood collection tubes are not completely evacuated. There is a small amount of gas (air) still residing in the tube, at low pressure. The higher the pressure of the gas inside the tube on the date of manufacture, the lower the intended draw volume will be for a tube of a given size. The draw volume specified for a given tube is achieved by manufacturing the tube at a designated evacuation pressure. The dynamics of blood collection inside the tube are based on the ideal gas law: PV = nRT where P is the pressure inside the tube, V is the volume that the gas occupies, n is the number of moles of gas inside the tube, R is the universal gas constant, and T is the temperature inside the tube. According to the equation, if the moles of gas and the temperature do not change, the product of pressure and volume is a constant. When blood starts filling the tube, the residual gas inside is confined into a decreasing volume, causing the pressure of the gas to increase. When the pressure of this gas reaches ambient pressure, the collection process is completed for that tube. The inability of an evacuated tube to fill correctly, referred to as a "short draw" or quantity-not- sufficient specimen, is a cause of rejection of a blood specimen. In fact, underfilling has been cited as the second most frequent cause of specimen rejection, with a rate of underfilling of 16% to 21%. To reduce the number of rejected specimens as a result of underfilling because of an expired shelf life, a laboratory can initiate a kaizen ("good change" in Japanese) to improve the inventory. A strategy that can be implemented is to survey the inventory in all phlebotomy supply centers.

Clinical Significance of FOBT

Fecal Occult Blood Test (FOBT) Tests for hemoglobin in fecal specimens are often referred to as tests for occult blood. This is because hemoglobin may be present in the feces, as evidenced by positive chemical tests for blood, but may not be detected by the naked eye. In other words, occult blood is hidden blood and requires a chemical test for its detection. Occasionally, enough blood will be present in the feces to produce a tarry black or even bloody specimen. However, even bloody specimens should be tested chemically for occult blood. In such cases the outer portion is avoided, and the central portion of the formed stool is sampled. The detection of occult blood in feces is important in determining the cause of hypochromic anemias resulting from chronic loss of blood and in detecting ulcerative or neoplastic diseases of the gastrointestinal (GI) system. Blood in the feces may result from bleeding anywhere along the GI tract, from the mouth to the anus. Tests for occult blood are especially useful for early detection and treatment of colorectal cancer. Such tests are useful because more than half of all cancers (excluding skin) are from the GI tract. Early detection results in good survival. Persons over age 50 should be screened annually for occult blood. They sample their own stool specimens for three consecutive collections, apply a thin film to the test slides, and mail or bring them to the laboratory for testing. Dietary considerations are important to avoid false-positive results, and special instructions are generally included with the test slides. It is now less common practice for the laboratory to receive the actual fecal specimen to be tested for occult blood. Bleeding at any point in the GI system and representing as little as 2 mL of blood lost daily may be detected by the tests for occult blood. However, false-negative results occur for unknown reasons, possibly because of inhibitors in the feces. Implications of both false-positive and false-negative tests are important clinically. Early diagnosis and treatment of serious disease might be missed with false-negative results, resulting in poor prognosis and death. Positive results are serious and require extensive further testing to determine the cause of bleeding or to rule out false-positive reactions. Further testing is both unpleasant for the patient and expensive.

Storage of Chemicals -Flammables

Flammable solvents (such as alcohol and chloroform) should be stored in specially constructed, well-ventilated storage units with appropriate labeling in accordance with OSHA regulations. Flammable solvents such as acetone and ether should always be stored in special safety cans or other appropriate storage devices and in approved storage units.

Beta-Human Chorionic Gonadotropin

For the first 6 to 8 weeks after conception, β-hCG helps maintain the corpus luteum and stimulate the production of progesterone. In a normal pregnancy, detectable amounts of about 25 mIU/mL β- hCG are secreted 2 to 3 days (48-72 hours) after implantation, or approximately 8 to 10 days after conception or fertilization. Peak levels are reached approximately 2 to 3 months after the last menstrual period. Levels rise rapidly after conception. If a test is negative at this stage, the test should be repeated within 1 week. Most specimens will contain enough β-hCG for detection by the twelfth day after a missed period. Some test methods (such as enzyme-linked immunosorbent assay [ELISA]) use serum and detect increases in β-hCG much earlier, often within days of conception.

Temperature Conversion - Fahrenheit and Celsius

From Fahrenheit to Celsius To convert from degrees Fahrenheit to degrees Celsius, subtract 32° from the temperature and multiply by 5/9 From Celsius to Fahrenheit To convert from degrees Celsius to degrees Fahrenheit, multiply the temperature by 1.8 (9/5) and add 32°:

Hemolyzed Specimens

Hemolysis in specimens is the most common cause of an abnormal appearance. Hemolyzed serum or plasma is unfit as a specimen for various assays, including coagulation testing and chemistry assays (such as potassium and hemoglobin measurements), blood banking, and immunology testing. A specimen that is hemolyzed appears red (usually clear red) because the RBCs have been lysed and the hemoglobin has been released into the liquid portion of the blood. Often the cause of hemolysis in specimens is the technique used for venipuncture. A poor venipuncture with excessive trauma to the blood vessel can result in a hemolyzed specimen. Other causes include inappropriate needle bore size and contact with alcohol on the skin. Hemolysis of blood can also result from freezing, prolonged exposure to warmth, or the serum or plasma remaining on the cells too long before testing or removal to another tube. A determination of whether the hemolysis is in vitro or in vivo is also useful. Although relatively rare, in vivo hemolysis is a clinically significant finding. Hemolyzed serum or plasma is unsuitable for several chemistry determinations because substances that are usually present within cells (such as potassium) can be released into the serum or plasma if the serum is left on the cells for a prolonged period. In addition, several other constituents, including the enzymes acid phosphatase, lactate dehydrogenase, and aspartate transaminase (or aminotransferase [AST]; formerly glutamic oxaloacetic transaminase), are present in large amounts in RBCs, so hemolysis of red cells will significantly elevate the value obtained for these substances in serum. Hemoglobin is released during hemolysis and may directly interfere with a reaction, or its color may interfere with photometric analysis of the specimen. The procedure to be performed should always be identified to determine whether abnormal-looking specimens can be used.

Layers of Anticoagulated Blood

If left undisturbed in the tube, this mass will begin to shrink, or retract, in about 1 hour. Complete retraction normally takes place within 24 hours. Whole blood that is allowed to clot normally produces a pale-yellow fluid called serum that separates from the clot and appears in the upper portion of the tube. During the process of coagulation, certain factors present in the original blood sample are depleted, or used up. Fibrinogen is one important substance found in circulating blood (in the plasma portion) that is necessary for coagulation to occur. Fibrinogen is converted to fibrin when clotting occurs, and the fibrin lends structure to the clot in the form of fine threads in which the red blood cells (RBCs, erythrocytes) and the white blood cells (WBCs, leukocytes) are embedded. To assist in obtaining serum, collection tubes with a separator gel additive are used. Serum is used extensively for chemical, serologic, and other laboratory testing and can be obtained from the tube of clotted blood by centrifuging. When fresh whole blood is mixed with a substance that prevents blood clotting, called an anticoagulant, the blood can be separated into plasma, a straw-colored fluid, and the cellular components: erythrocytes, leukocytes, and platelets (thrombocytes). When an anticoagulated blood specimen is allowed to stand for a time, the components will settle into three distinct layers, as follows: 1. Plasma, the top layer, a liquid that normally represents about 55% of the total blood volume 2. Buffy coat, a grayish-white cellular middle layer composed of WBCs and platelets, normally about 1% of the total blood volume 3. Erythrocytes, the bottom layer, consisting of packed RBCs and normally about 45% of the total blood volume

Characteristics of POCT devices

Important characteristics of POCT devices are: • Small blood sample • Rapid turnaround time • Easy portability with single-use disposable reagent cartridges or test strips • Easy-to-perform protocol with one or two steps • Accuracy and precision of results comparable to those with central laboratory analyzers • Minimal QC tracking • Storage at ambient temperature for reagents • Bar-code technology for test packs, controls, and specimens • Economical equipment cost and maintenance free • Software for automatic calibration, system lockouts, and data management • Hard copy or electronic data output that interfaces with a laboratory information system or other tracking software

Regulation of Laboratories CLIA

In 1988 the U.S. Congress enacted the Clinical Laboratory Improvement Amendments of 1988 (CLIA'88) in response to concerns about laboratory testing errors. The final CLIA rule, Laboratory Requirements Relating to Quality Systems and Certain Personnel Qualifications, was published in 2003. Enactment of CLIA established a minimum threshold for all aspects of clinical laboratory testing. The introduction of routine QC in the clinical laboratory was a major advance in improving the accuracy and reliability of clinical laboratory testing. Various agencies monitor laboratory quality and have commonality in many of the noted top 10 deficiencies. Effective in 2003, all laboratories must meet and follow the final QC requirements. These regulations established minimum requirements with general QC systems for all nonwaived testing. In addition, a controversial 2004 regulation allows the Centers for Medicare and Medicaid Services (CMS) to consider acceptable alternative approaches to QC practices, called equivalent quality control, for laboratory testing.

Sources of Variance or Error

In general, it is impossible to obtain exactly the same result each time a determination is performed on a particular specimen. This may be described as the variance, or error, of a procedure. These factors include limitations of the procedure itself and limitations related to the sampling mechanism used. Sampling Factors = One of the major difficulties in guaranteeing reliable results involves the sampling procedure. Sources of variance that involve the sample include the time of day when the sample is obtained, the patient's position (lying down or seated), the patient's state of physical activity (in bed, ambulatory, or physically active), the interval since last eating (fasting or not), and the time interval and storage conditions between the collection of the specimen and its processing by the laboratory. The aging of the sample is another source of error. Procedural Factors = Other sources of variance involve aging of chemicals or reagents, personal bias or limited experience of the person performing the determination, and laboratory bias because of variations in standards, reagents, environment, methods, or apparatus. There may also be experimental error resulting from changes in the method used for a particular determination, changes in instruments, or changes in personnel.

Metric Volume

In the clinical laboratory the standard unit of volume is the liter (L). It was not included in the list of base units of the SI system, because the liter is a derived unit. The standard unit of volume in the SI system is the cubic meter (m3). However, this unit is quite large, and the cubic decimeter (dm3) is a more convenient size for use in the clinical laboratory. In 1964 the Conférence Générale des Poids et Mesures (CGPM) accepted the litre (liter) as a special name for the cubic decimeter. Previously, the standard liter was the volume occupied by 1 kg of pure water at 4° C (the temperature at which a volume of water weighs the most) and at normal atmospheric pressure. On this basis, 1 L equals 1000.027 cubic centimeters (cm3), and the units, milliliters and cubic centimeters, were used interchangeably, although there is a slight difference between them. One liter is slightly more than 1 quart (qt) in the English system (1 L = 1.06 qt). The liter is divided into thousandths, called milliliters (mL); millionths, called microliters (μL); and billionths, called nanoliters (nL). The following examples show volume equivalents: 500 mL = 0.5 L 0.25 L = 250 mL 2 L = 2000 mL Because the liter is derived from the meter (1 L = 1 dm3), it follows that 1 cm3 is equal to 1 mL and that 1 millimeter cubed (mm3) is equal to 1 mL. The former abbreviation for cubic centimeter (cc) 3has been replaced by cm . Although this is a common means of expressing volume in the clinical laboratory, milliliter (mL) is preferred.

Deionized Water

In the process of deionization, water is passed through a resin column containing positively (+) and negatively (−) charged particles. These particles combine with ions present in the water to remove them; this water is known as deionized water. The only substances that will be removed in the process of deionization are those that can ionize. Organic substances and other substances that do not ionize are not removed. Further treatment with membrane filtration and activated charcoal is necessary to remove organic impurities, particulate matter, and microorganisms to produce type I water from deionized water.

Distilled Water

In the process of distillation, water is boiled, and the resulting steam is cooled; condensed steam is distilled water. Many minerals are found in natural water, most often iron, magnesium, and calcium. Water from which these and other minerals have been removed by distillation is known as distilled water. The process of distillation also removes microbiological organisms, but volatile impurities such as CO2, chlorine, and ammonia are not removed. Water that has been distilled meets the specifications for water types II and III.

Overview of LIMS

Informatics software was traditionally divided into the sample-centric laboratory information management system (LIMS) A LIMS represents transmission of sample-centric information with the ultimate goal of providing accurate information in a timely manner to clinicians. LIMS is used because it can routinely integrate automation and data handling, provide uniform methodology with complete visibility, and lead to increased productivity and process integrity. The essential requirements of an LIMS include secure login, flexibility to add-ons and software upgrades and, most importantly, data management. The number of laboratory tests has increased as a result of the development of new diagnostic assays and the increased use of automated, high- volume instruments and handheld devices. Because of the dramatic increase in the number of assays performed in the clinical laboratory and by POCT, and because these assays have produced so much analytical information, the ability to process this information efficiently and accurately has become essential. LabWare of Wilmington, Delaware, is an example of an LIMS product with features to support clinical laboratory workflow, instrument interfacing, sampling, and aliquoting. This product allows for the addition of patient management features that enable the system to be patient-centric, while maintaining all sample management and tracking capabilities. It is not possible to discuss LIMS without discussing automation. Automation describes an instrumental system that involves the mechanization of discrete processes and is "noninterventional" of self-regulating and self-timing. Many automated techniques make use of robotized units, as is typically seen in a QC laboratory for procedures that are susceptible to gross method errors. Robotics describes the use of instrument management systems and the way in which information is handled. More recent revisions have produced automated robotized systems capable of handling multichannel information sources while running selected instruments.

MSDS sheets

Information and training regarding hazardous chemicals must be provided to all persons working with them in the clinical laboratory. Occupational Safety and Health Administration (OSHA) regulations ensure that all sites where hazardous chemicals are used comply with the necessary safety precautions. Any information about signs and symptoms associated with exposures to hazardous chemicals used in the laboratory must be communicated to all persons. Reference materials about the individual chemicals are provided by all chemical manufacturers and suppliers by means of the safety data sheet (SDS). This information accompanies the shipment of all hazardous chemicals and should be available in the laboratory for anyone to review. The SDS contains information about possible hazards, safe handling, storage, and disposal of the particular chemical it accompanies

Dilutions and Diluting Specimens

It is often necessary to make dilutions of specimens being analyzed or to make weaker solutions from stronger solutions in various laboratory procedures. A laboratory professional must be capable of working with various dilution problems and dilution factors. In these problems, one must often be able to determine the concentration of material in each solution, the actual amount of material in each solution, and the total volume of each solution. All dilutions are a type of ratio. Dilution is an indication of relative concentration. In performing a laboratory assay, it may be necessary to dilute a specimen because of the high concentration of a constituent. The needed dilution will vary according to the procedure

Type I Reagent Water

Laboratory reagent-grade water is water that is suitable for use in a specified procedure and does not interfere with the specificity, accuracy, and precision of an assay procedure. Process definitions alone, e.g., distilled or deionized water, do not adequately define required water quality. Laboratory water needs to have inorganic or organic impurities in the water removed before analysis. Type I Reagent Water = Type I reagent water is the purest and should be used for procedures that require maximum water purity. Type I must be used for preparation of standard solutions, buffers, and controls, and in quantitative analytical procedures (especially when nanogram or subnanogram measurements are required), electrophoresis, toxicology screening tests, and high-performance liquid chromatography. Type I water should be used immediately after it is produced; it cannot be stored.

Handheld Automated Equipment

Microprocessors in small and often handheld instruments provide automated, easy-to-perform testing with calibration and on-board QC. Both handheld and small instruments may be used for testing

Blood Collection Variables

Most clinical laboratory determinations are done on whole blood, plasma, or serum. Blood specimens may be drawn from fasting or nonfasting patients. The fasting state is defined as having no food or liquid other than water for 8 to 12 hours before blood collection. Fasting specimens are not necessary for most laboratory determinations. Blood from fasting patients is usually drawn in the morning before breakfast. Blood collected directly after a meal is described as a postprandial specimen. In the case of blood glucose, a sample may be collected 2 hours postprandially. After 2 hours, blood glucose levels should return to almost fasting levels in patients who are not diabetic. Blood should not be collected while intravenous (IV) solutions are being administered, if possible. Food intake, medication, activity, and time of day can all influence the laboratory results for blood specimens. It is critically important to control preanalytical variables such as timed drawing of a specimen, peak and trough drug levels, and postmedication conditions. Other controllable biological variations in blood include the following: • Posture (whether the patient is lying in bed or standing up) • Immobilization (such as resulting from prolonged bed rest) • Exercise • Circadian/diurnal variations (cyclical variations throughout the day) • Recent food ingestion (such as caffeine effect) • Smoking (nicotine effect) • Alcohol ingestion

Computer Interfaces

Most current systems use the Health Level 7 (HL7) standard for their interfaces. The goal in using this standard is to prevent misunderstandings between the computers by defining messages and their content. The HL7 standard is used primarily for financial and medical record information. It does not address many types of clinical information or other data, such as raw data from instrument interfaces. For laboratory use, the interface specification should include what data will be transferred, where data will be transferred, when data will be transferred, and security and encryption considerations. Interfaces are important to the laboratory because they contribute to the overall effectiveness of the computer support of laboratory operations. It is critical to remember that interfaces pass patient information between computers without direct human intervention. Interfacing of the workstation with the analytical testing instrument allows the test result to be entered directly into the computer information system or LIS and saves laboratory time. The test results data are transferred directly over a single wired or wireless interface. A unidirectional interface transmits or uploads results; a bidirectional interface allows for simultaneous transmission or downloading of information and for the reception of uploaded information from an instrument. An example is the LifeScan Accu-Chek, a bidirectional interface that patients and laboratories can use for the management of diabetes testing. Patients can both upload their test results from a point- of-care device and download information related to their care. An LIS is often interfaced with other information systems, most often the HIS; interfacing allows electronic communication between two workstations. The HIS manages patient census information and demographics and systems for billing, and the more complex systems process and store patient medical information. The interfacing of the HIS and the laboratory workstation facilitates the exchange of test request orders, return of analytical results (the laboratory report), and charges for the tests ordered and reported. When the data are verified, nurses or physicians in the patient care areas can retrieve results through workstations, monitors, tablets, and printers.

Components of a Computer System

One of the most common peripheral input/output (I/O devices is the video display monitor. Monitors are usually cathode ray tube or liquid crystal display screens; important features include the diagonal dimension, the number of pixels (picture elements), resolution, and inclusion of a touch-sensitive surface. Touch screens allow interaction with the software application and CPU through a menu. Menus are lists of programs, functions, or other options offered by the system. A cursor is moved to the point on the list (such as a list of tests) that is the option of choice and placed on the test desired. The position of touch on the screen determines the choice. A stylus, the operator's finger, or a mouse can be used to interact with a touch-sensitive screen to indicate a menu choice.

Monitoring QC - Levy Jennings and Westgard

Most laboratories plot the daily control specimen values on a QC chart. Currently, many instruments automatically generate QC charts on each day of testing. Out-of-control specimen results are automatically flagged. If an instrument does not generate QC charts, the laboratory professional must perform this task Levey-Jennings (Shewhart) QC charts have traditionally been used to identify unacceptable runs and then to evaluate the source and magnitude of the deviation to decide whether results are to be released to patient charts. Software designed for laboratory information systems and personal computers is available to automate the plotting of control values. The software's complexity and capabilities (for multiple QC options) will vary among suppliers, but all types typically provide a graphical presentation of data using the traditional Levey-Jennings chart. The main purpose for control charting in the clinical laboratory is to aid in maintaining stability of the analytical measuring system

Specimen Collection for Beta hCG

Most of the kits currently can be used for both serum and urine β-hCG but show better sensitivity with serum, because the concentration of β-hCG in serum is not subject to the wide variation found in urine β-hCG as a result of changes in urine concentration. Urine for the hCG assay (pregnancy test) must be collected at a suitable time after fertilization to allow the concentration of the hormone to rise to a significant detectable level. The first morning urine specimen is required because it contains the highest concentration of hormone. It should have a specific gravity of at least 1.015. The urine specimen is collected in a clean glass or plastic container. It may be refrigerated for up to 2 days or frozen at − 20°C for at least 1 year. Thaw frozen samples by placing the frozen specimen in a water bath at 37°C, and then mix thoroughly before use. If turbidity or precipitation is present after thawing, filtering or centrifuging is recommended. Specimens containing blood, large amounts of protein, or excessive bacterial contamination should not be used. Do not refreeze.

Blood Spot Collection for Neonatal Screening Programs

Most states have passed laws requiring that newborns be screened for certain diseases that can result in serious abnormalities, including mental retardation, if they are not diagnosed and treated early. These diseases include phenylketonuria (PKU), galactosemia, hypothyroidism, and hemoglobinopathies. CLSI12 has set standards for filter paper collection, or blood spot collection, of blood for these screening programs. Blood should be collected 1 to 3 days after birth, before the infant is discharged from the hospital, and at least 24 hours after birth and after ingestion of food for a valid PKU test. There is an increased chance of missing a positive test result when an infant is tested for PKU before 24 hours of age. When infants are discharged early, however, many physicians prefer to take a sample early rather than risk having no sample at all. In most neonatal screening programs, the specimen is collected on filter paper and sent to the approved testing laboratory for analysis. Special collection cards with a filter paper portion are supplied by the testing laboratory; these are kept in the hospital nursery or central laboratory. These cards have a section where, as for any other request form, all information requested must be provided. The filter paper section of the card contains circles designed to identify the portion of the paper onto which the specimen should be placed, where the filter paper will properly absorb the amount of blood necessary for the test. Collection is usually done by heel puncture following the accepted procedure for the institution. When a drop of blood is present, the circle on the filter paper is touched against the drop until the circle is completely filled. A sufficiently large drop should be formed so that the process of filling the circle can be done in only one step. The filter paper is allowed to air-dry and then is transported to the testing laboratory in a plastic transport bag or other acceptable container. The procedure established by the testing laboratory should be followed for the collection step.

Determination of Control Range

Once a control solution has been purchased unassayed, it is necessary for the laboratory to determine the acceptable control range for a particular analysis, and there are various ways of establishing it. One method to establish a control range is to assay an aliquot of the control serum with the regular batch of assays for 15 to 25 days. In testing the control sample, it is important to treat it exactly like an unknown specimen; it must not be treated any more or less carefully than the unknown specimen. Repeated determinations on different aliquots of the same sample often will not give identical values for any particular constituent. It has been shown that if a sufficient number of repeated determinations are made, the values obtained will fall into a normal bell-shaped curve. When a statistically sufficient number of determinations have been run (the number is different for averaged duplicate determinations and single tests), the mathematical mean (x) or average value can be calculated. The acceptable limits or variation from the mean for the control solution are then calculated on the basis of the SD from the mean, using statistical formulas Most laboratories use 2 SD above and below the mean as the allowable range of the control specimen, whereas others use this range as a warning limit. According to the normal bell-shaped curve, setting 2 SD as the allowable range for the control sample means that 95% of all determinations on that sample will fall within the allowable range and that 5% will be out of control. It may not be desirable to disallow this many batches, however, and the third SD may be chosen as the limit of control. Once the range of acceptable results has been established, one of the control specimens is included in each batch of determinations. If the control value is not within the limits established, the procedure must be repeated, and no patient results may be reported until the control value is acceptable. Control procedures may include the following: 1. Review the procedures used. 2. Search for recent events that could cause change, such as a new reagent kit or lot, component replacement, or environmental condition (such as temperature and humidity). 3. Prepare new control materials. 4. Follow the manufacturer's troubleshooting guide. 5. Contact manufacturers of instruments, reagent materials, and controls.

False negative and False Positive FOBT

Positive = Several interfering substances may give false-positive results for occult blood, including dietary substances with peroxidase activity, especially myoglobin and hemoglobin in red meat. Vegetable peroxidase, as found in horseradish, can also cause false-positive results. Several foods have been identified as causing erroneous reactions, including turnips, broccoli, bananas, black grapes, pears, plums, and melons. Cooking generally destroys these peroxidases, and therefore patients are generally instructed to eat only cooked foods. White blood cells (WBCs) and bacteria also have peroxidase activity that might result in false-positive reactions. Various drugs, including aspirin and aspirin-containing preparations and iron compounds, are known to increase GI bleeding, causing positive results. Patients are generally instructed to eat no beef or lamb (including processed meats and liver) for 3 days before collecting the first specimen and to remain on this diet through the collection of three successive samples. They may eat well-cooked pork, poultry, and fish. They are also instructed to avoid raw fruits and vegetables, especially melons, radishes, turnips, and horseradish. Cooked fruits and vegetables are acceptable. Ingestion of high-fiber foods, such as whole-wheat bread, bran cereal, and popcorn, is encouraged. Negative = Vitamin C and other oxidants may give false-negative results. The ingestion of more than 250 mg/day of vitamin C is to be avoided because it may cause false-negative results. The mechanism of this interference is the same as for reagent strips used in urinalysis (UA). Aspirin and other nonsteroidal antiinflammatory drugs (NSAIDs) should be avoided for 7 days before and during the test period.

Quality Control

Two types of Quality control: • Internal QC, or statistical QC, which evaluates the daily precision of assay measurements • External QC, which evaluates the accuracy of assay measurements. This type of QC uses data generated from voluntary submission of internal QC control assay results (peer-groups) and data generated from required submission of specific control specimens (PT) For tests of moderate complexity, CLIA states that laboratories must comply with the more stringent of the following requirements: • Perform and document control procedures using at least two levels of control material each day of testing. • Follow the manufacturer's instructions for QC.

Rounding Off Numbers

Test results sometimes produce insignificant digits. It is then necessary to round off the numbers to a chosen number of significant value so as not to imply an accuracy of precision greater than the test is capable of delivering. Some general rules may be used in rounding off decimal values to the proper place. When the digit next to the last one to be retained is less than 5, the last digit should be left unchanged. When the digit next to the last one to be retained is greater than 5, the last digit is increased by 1. If the additional digit is 5, the last digit reported is changed to the nearest even number. Examples are as follows: 2.31463 g is rounded off to 2.3146 g. 5.34659 g is rounded off to 5.3466 g. 23.5 mg is rounded off to 24 mg. 24.5 mg is rounded off to 24 mg.

Analytic Reagent Grade

The AR-grade chemicals are of a high degree of purity and are used often in the preparation of reagents in the clinical laboratory. The American Chemical Society (ACS) has developed specifications for many reagent-grade or AR-grade chemicals, and those that meet its standards are designated by the letters ACS.

EDTA

The International Council for Standardization in Haematology (ICSH) and CLSI recommend the salts of the chelating (calcium-binding) agent EDTA as the anticoagulant of choice for blood cell counting and sizing, because EDTA produces less shrinkage of RBCs and less of an increase in cell volume on standing. EDTA is spray-dried on the interior surface of evacuated plastic tubes. The proper ratio of EDTA to whole blood is important because some test results will be altered if the ratio is incorrect. Excessive EDTA produces shrinkage of erythrocytes, thus affecting tests such as the manually performed packed cell volume (microhematocrit). K2EDTA and K3EDTA are found in evacuated tubes. The mode of action of this anticoagulant is that it removes ionized calcium (Ca2 +) through the process of chelation. This process forms an insoluble calcium salt that prevents blood coagulation. EDTA is the most frequently used anticoagulant in hematology for the complete blood cell count (CBC) or any of its component tests: hemoglobin, packed cell volume, total leukocyte count and leukocyte differential count, and platelet count. The modified Westergren erythrocyte sedimentation rate (ESR) method uses EDTA as the anticoagulant of choice. In addition, EDTA is used routinely for testing in blood banking such as blood grouping, Rh typing, and antibody screening. K2EDTA with gel is used for testing plasma in molecular diagnosis. K3EDTA may be used for viral marker testing.

Reagent defined

The accuracy of laboratory analyses depends to a great extent on the accuracy of the reagent, a solution used for performing a chemical test. A reagent is defined as any substance used to produce a chemical reaction. In highly automated clinical laboratories, very few reagents are prepared by laboratory staff. In many cases, only water or buffer needs to be added to a prepackaged reagent. However, in some cases, clinical and research laboratories may need to prepare a reagent or solution for method validation or specialized analyses. In-house reagent preparation may be required because of reagent deterioration, supply and demand, or verified cost containment.

Analytical Balance

The basic principle in the quantitative measurement of mass is to balance an unknown mass (the substance being weighed) with a known mass. The electronic analytical balance is a single- pan balance that uses an electromagnetic force to counterbalance the load placed on the pan. Electronic balances permit fast, accurate weighing with a high degree of resolution. These are easy to use and have replaced the traditional mechanically operated analytical balance in most clinical laboratories. The analytical balance should be cleaned and adjusted at least once a year to ensure its continued accuracy and sensitivity. Its accuracy is what makes this instrument so essential in the clinical laboratory. The accuracy to which most analytical balances used in the clinical laboratory should weigh chemicals is usually 0.1 mg, or 0.0001 g. With the electronic balance, the weights are added by manipulating a series of dials. Weighing errors will occur if the analytical balance is not properly positioned. The balance must be level; this is usually accomplished by adjusting the movable screws on the legs of the balance. The firmness of support is also important. The bench or table on which the balance rests must be rigid and free from vibrations. Ideally, the analytical balance should be in an air-conditioned room. The temperature factor is most important. The balance should not be placed near hot objects (such as radiators, flames, stills, and electric ovens) or near cold objects and especially not near an open window. Sunlight or illumination from high-power lamps should be avoided in choosing a good location for the analytical balance. The analytical balance is a delicate precision instrument that will not function properly if abused. The following general rules apply: 1. Set up the balance where it will be free from vibration. 2. Close the balance case before observing the reading; any air currents present will affect the weighing process. 3. Never weigh any chemical directly on the pan; a container of some type must be used for the chemical. Weigh an empty container first to establish the "tare" weight. This weight must be added to the desired weight of dry chemical. 4. On completion of weighing, clean up any chemical spilled on the pan or within the balance area. 5. Weighed materials should be transferred to labeled containers or made into solutions immediately.

Patient Care Partnership

The delivery of health care involves a partnership between patients and physicians and other health care professionals. When collecting blood specimens, it is important that the phlebotomist consider the rights of the patient at all times. The American Hospital Association has developed the Patient Care Partnership document, which replaces the former Patient's Bill of Rights. This document stresses the following: • High-quality hospital care • A clean and safe environmen t• Involvement by patients in their care • Protection of patients' privacy • Help for patients when leaving the hospital • Help for patients with billing claims Patients themselves or another person chosen by the patient can exercise these patient rights. A proxy decision maker can act on the patient's behalf if the patient lacks decision-making ability, is legally incompetent, or is a minor. The partnership nature of health care requires that patients—or their families or surrogates—take part in their care. As such, patients are responsible for providing an accurate medical history and any written advance directives, following hospital rules and regulations, and complying with activities that contribute to a healthy lifestyle.

Standard Precautions

The first tier of infection control is the practice of Standard Precautions. The Standard Precautions theory recognizes the need to reduce the risk for microbial transmission, including human immunodeficiency virus (HIV), from both identified and unidentified sources of infection. These precautions require that protective protocols be followed whenever contact is made with blood and body fluids.

Benefits of Automation

The major benefits of laboratory automation follow: • Reduction of medical errors • Reduced specimen sample volume • Increased accuracy and precision (reduced coefficient of variation) • Improved safety for laboratory staff (such as stopper removal or piercing) • Faster turnaround time of results • Partially alleviating the impending shortage of skilled laboratory staff According to a U.S. Institute of Medicine report, medical errors in the United States may contribute to up to 98,000 deaths and more than 1 million injuries each year. New TJC guidelines highlight the importance of proper identification of patient samples; a mistake in labeling or misidentification can lead to critical medical errors such as transfusion of blood products or medication to the wrong patient. The report identified several critical errors concerning laboratory processes, including delay in diagnosis. Patient safety benefits from automation include workflow standardization for more precise, consistently reliable test results and improved test turnaround time for faster diagnosis and better patient care. Automated specimen processing and testing also improve safety for laboratory technologists. Because technologists are now handling blood samples less frequently, their exposure to pathogens and sharps injuries is reduced dramatically. In addition to alleviating the impending shortage of laboratory staff, automation can produce a more dynamic and robust laboratory. Clinical laboratory professionals can spend more time on difficult cases while automated instruments handle routine work.

Steps in Automated Analysis

The major steps designed by manufacturers to mimic manual techniques are as follows: • Specimen collection and processing • Specimen and reagent measurement and delivery • Chemical reaction phase • Measurement phase • Signal processing and data handling

Molarity

The molarity of a solution is defined as the gram-molecular mass (or weight) of a compound per liter of solution. This is a weight-per-unit-volume method of expressing concentration. A basic formula follows: Molecular weight x Molarity = grams/liter Another way to define molarity is number of moles per liter (mol/L) of solution. A mole is the molecular weight of a compound in grams (1 mole = 1 gram-molecular weight). The number of moles of a compound equals the number of grams divided by the gram-molecular weight of that compound. One gram-molecular weight equals the sum of all atomic weights in a molecule of the compound, expressed in grams. To determine the gram-molecular weight of a compound, the correct chemical formula must be known; then the sum of all the atomic weights in the compound can be found by consulting a periodic table of the elements or a chart with atomic masses of the elements.

2D Bar Codes

The newest development in bar codes is the growing popularity of two-dimensional (2D) bar codes in the United States and Japan. A 2D bar code is nonlinear and consists of black-and-white "cells" or "modules" arranged in a matrix pattern—typically a square—which in turn encapsulates a "finder" pattern and a "reader" pattern. These 2D symbols are omnidirectionally scannable— upside down, backward or forward, and even diagonally. A 2D bar code can store every bit of patient information in a tiny symbol that is 2 to 3 mm square, because 2D bar codes can store thousands of characters. The symbology of 2D bar codes is not as susceptible to printing defects or errors as traditional 1D bar codes. The coding pattern has a high level of redundancy, with the data dispersed in several locations throughout the symbology. This enables the bar code to be scanned correctly even if a portion of it has printed lightly or is missing altogether. Specialized equipment is required to print 2D bar codes. The best printer for 2D labels is a direct thermal or thermal transfer printer. In addition, 2D bar codes cannot be read by a laser scanner but must be scanned by an image-based scanner employing a charge-coupled device or some other digital-camera sensor technology

Preanaytical errors

The preanalytical phase of testing is particularly error prone. A major reason that the preanalytical phase is so error prone is that it is especially susceptible to human error. Frequently, specimen collection is performed outside of the laboratory and involves nonlaboratory personnel, who may not be properly trained. One study reported that the majority of laboratory errors now occur in the preanalytical phase. To reduce and potentially eliminate laboratory errors, a QA program is mandated. A QA program can be divided into two major components: nonanalytical factors and the analysis of quantitative data (QC). CAP6 includes a variety of considerations in QA management. The Institute for Quality in Laboratory Medicine (IQLM) has developed 12 measures to evaluate quality in the laboratory, based on the phase of testing

Sensitivity

The sensitivity of a test is defined as the proportion of cases with a specific disease or condition that give a positive test result (i.e., the assay correctly predicts with a positive result), as follows: Sensitivity (%) = ( True positives / True positives + False negatives ) x 100 Practically, sensitivity represents how much of a given substance is measured; the more sensitive the test, the smaller the amount of assayed substance that is measured.

Metric length

The standard unit for the measurement of length or distance is the meter (m). The meter is standardized as 1,650,763.73 wavelengths of a certain orange light in the spectrum of krypton-86. One meter equals 39.37 inches (in), slightly more than a yard in the English system. There are 2.54 centimeters in 1 inch. Using the system of prefixes previously discussed, further common divisions and multiples of the meter follow. One-tenth of a meter is a decimeter (dm), one-hundredth of a meter is a centimeter (cm), and one-thousandth of a meter is a millimeter (mm). One thousand meters equals 1 kilometer (km). The following examples show equivalent measurements of length: 25 mm = 0.025 m 10 cm = 100 mm 1 m = 100 cm 1 m = 1000 mm Other units of length that were in common usage in the metric system but that no longer are recommended in the SI system are the angstrom and the micron. The micron (μ), which is equal to 10^−6 m, has been replaced by the micrometer (μm).

Quality Assessment

The term quality assessment, or the alternate term quality assurance, encompasses policies that maintain and control processes involving the patient and laboratory analysis of specimens. Quality assessment includes monitoring the following specimen collection measures: • Preparation of a patient for any specimens to be collected • Collection of valid samples • Proper specimen transport • Performance of the requested laboratory analyses • Validation of test results • Recording and reporting the assay results • Transmitting test results to the patient's medical record • Documentation, maintenance, and availability of records describing quality assessment practices and quality control measures The accuracy of laboratory testing begins with the quality of the specimen received by the laboratory. This quality depends on how a specimen was collected, transported, and processed. A laboratory assay will be no better than the specimen on which it is performed. If a preanalytical error occurs, the most perfect analysis is invalid and cannot be used by the physician in diagnosis or treatment. Venous or arterial blood collection, phlebotomy, and capillary blood collection remain an error- prone phase of the testing cycle. In the United States it is estimated that more than 1 billion venipunctures are performed annually, and errors occurring within this process may cause serious harm to patients, either directly or indirectly. Leading causes of preanalytical errors include the following1: 1. Specimen collection tube not filled properly 2. Patient identification error 3. Inappropriate specimen collection tube or container 4. Test request error

Nonanalytical Factors in Quality Assessment

To guarantee the highest-quality laboratory results and to comply with CLIA regulations, a variety of preanalytical and postanalytical factors must be considered. Nonanalytical factors that support quality testing include the following: 1. Qualified personnel 2. Established laboratory policies 3. Laboratory procedure manual 4. Test requisitioning 5. Patient identification, specimen procurement, and labeling 6. Proper procedures for specimen collection and storage 7. Specimen transportation and processing 8. Preventive maintenance of equipment 9. Appropriate methodology 1) The competence of personnel is an important determinant of the quality of the laboratory result. Only properly certified personnel can perform nonwaived assays. CLIA '88 requirements for laboratory personnel in regard to levels of education and experience or training must be followed for laboratories doing moderately complex or highly complex testing 2) Laboratory policies should be included in a laboratory reference manual available to all hospital personnel. Each laboratory must have an up-to-date safety manual. This manual contains a comprehensive listing of approved policies, acceptable practices, and precautions, including standard blood and body fluid precautions. Specific regulations that conform to current state and general requirements, such as Occupational Safety and Health Administration regulations, must be included in the manual. Other sources of mandatory and voluntary standards include TJC, CAP, and the CDC. 3) A complete laboratory procedure manual for all analytical procedures performed within the laboratory must be provided. The manual must be reviewed regularly, in some cases annually, by the supervisory staff and updated as needed. The minimal components are as follows: • Title of the assay • Principle of the procedure and statement of clinical applications • Protocol for specimen collection and storage • QC information • Reagents, supplies, and equipment • Procedural protocol • Reference ranges • Technical sources of error • Limitations of the procedure • Proper procedures for specimen collection and storage 4) A laboratory test can be requested by a primary care provider or, in some states, by the patient. The request, either hard copy or electronic, must include the patient identification data, time and date of specimen collection, source of the specimen, and analyses to be performed. The information on the accompanying specimen container must match exactly the patient identification on the test request. The information needed by the physician to assist in ordering tests must be included in a database or handbook. 5) Patients must be carefully identified. For outpatients, identification may be validated with two forms of identification. Once obtained from the patient, the clinical specimens must be properly labeled or identified using established information requirements. Computer-generated labels help ensure that proper patient identification is noted on each specimen container sent to the laboratory. An important rule to remember is that the analytical result can only be as good as the specimen received 6) Maintaining an electronic database or handbook of specimen requirement information is one of the first steps in establishing a QA program for the clinical laboratory. Current information about obtaining appropriate specimens, special collection requirements for various types of tests, ordering tests correctly, and transporting and processing specimens appropriately should be included in the database 7) Specimens must be efficiently transported to the laboratory. Some assays require special handling conditions, such as placing the specimen on ice immediately after collection. Some analyses require that specimens be refrigerated or frozen immediately or kept out of direct light. Specimens should be tested promptly, preferably within 2 hours of collection, to produce accurate results. Documenting specimen arrival times in the laboratory, along with other specific test request data, is an important aspect of the QA process. It is important that the specimen status can be determined at any time—that is, where in the laboratory processing system a given specimen can be found. 8) Monitoring the temperatures of heat blocks and refrigerators is important to the quality of test performance. Microscopes, centrifuges, and other pieces of equipment must be cleaned regularly and checked for accuracy. A preventive maintenance schedule should be followed for all automated equipment. • Reagent lot change • Major component replacement • Instrument maintenance • New software installation 9) When a new method is introduced, it is important to check the procedure for accuracy and variability. Replicate analyses using control specimens are recommended to check for accuracy and to eliminate factors such as day-to-day variability, reagent variability, and differences between technologists.

Significant Figures

Using more digits than necessary to calculate and report the results of a laboratory determination has several disadvantages. It is important that the number used contain only the digits necessary for precision of the determination. Using more is misleading in that it ascribes more accuracy to the determination than is actually the case. There is also the danger of overlooking a decimal point and making an error in judging the magnitude of the answer. Digits in a number that are needed to express the precision of the measurement from which the number is derived are known as significant figures. A significant figure is known to be reasonably reliable. Judgment must be exercised in determining how many figures should be used. The following rules can assist in making such decisions: 1. Use the known accuracy of the method to determine the number of digits that are significant in the answer, and as a general rule, retain one more figure than this. For example, a urea nitrogen result was reported as 11.2 mg/dL. This would indicate that the result is accurate to the nearest tenth and that the exact value lies between 11.15 and 11.25. In reality, the accuracy of most urea nitrogen methods is ± 10%, so the result reported as 11.2 mg/dL could actually vary from 10 to 12 mg/dL and should be reported as 11 mg/dL. In addition, if the decimal point were omitted or overlooked, the result could be taken as "112" mg/dL. 2. Take the accuracy of the least accurate measurement, or the measurement with the least number of significant figures, as the accuracy of the final result. In doing so, certain adjustments must be made in the addition and subtraction or multiplication and division of numerals. With addition or subtraction, for example, to add the following numerals: 206.1 7.56 0.8764 rewrite them as: 206.1 7.6 0.9 [In this example, the least accurate figure is accurate to one decimal place; this is therefore the determining factor. To determine the least accurate figure, use this rule: In a column of addition or subtraction in which the decimal points are placed one above the other, the number of significant figures in the final answer is determined by the first digit encountered going from left to right that terminates any one numeral.] In multiplication or division, using this example: 32.973 / 4.3 = 7.668 the result should be reported as 7.7, following this rule: The number of significant figures in the final product or quotient should not exceed the smallest number of significant figures in any one factor.

Standard Solutions

Various organizations (such as CAP) supply certified clinical laboratory standards. The highest- grade or purest chemicals are available from NIST. Very few such compounds, standards, clinical type, are available to the clinical laboratory. Chemicals used to prepare standard solutions are the most highly purified types of chemicals available. This group includes primary, reference, and certified standards. Primary standards meet specifications set by the ACS Committee on ARs. Each lot of these chemicals is assayed, and the chemicals must be stable substances of definite composition.

Most automated areas of the lab

Versatility and flexibility are often just as important as high volume and speed of testing, but automation is also desirable for less frequently ordered tests. In the case of large-volume hospital and reference laboratories, a completely automated laboratory system may be used. Each automated instrument can operate separately or integrated with other laboratory instruments. Instruments can be linked into a single continuous operation that can include robotic specimen processing. Initially, highly automated systems were introduced in larger-volume clinical chemistry and hematology laboratories. Today, automation and semiautomation exist in other clinical laboratory sections including UA, blood bank, and microbiology.

Westgard Rules

Westgard rules are often formulated to analyze data in control charts based on statistical methods. These rules define specific performance limits for a particular assay and can be used to detect both random and systematic errors. If QC is out of control, testing must stop until the problem is identified and remediated. Patient results cannot be reported until control specimens meet performance requirements. A Levey-Jennings chart can be interpreted using the Westgard rules, Two single rules are usually applied: 12s or 13s The 12s refers to the control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean ± 2 SD. This rule is used as a warning to trigger careful inspection of the control data by the following the rejection rules. There is a false-alarm problem with a 12s rule, as is shown in the Levey-Jennings chart with 2s control limits; when N = 2, it is expected that 9% of good test runs will be falsely rejected.

Lean Principles

focuses on reducing waste When either of these systems was used to redesign workflow in high-volume core hematology and chemistry laboratories, a 50% reduction in average test turnaround time, a 40% to 50% improvement in labor productivity, and a comparable improvement in the quality of results were observed Lean principles of reduction of unnecessary and non-value-added activities to decrease total production time and effort can be appropriately applied in all sections of the laboratory (such as urinalysis). Lean tools focus on identifying steps in a procedure that are error prone. If these steps cannot be eliminated, they must be controlled. Lean principles support the concept of performing tasks correctly the first time, with minimal wasted time and effort. One highly effective tool in applying Lean principles is a process map of external and internal activities related to a specific laboratory assay. This mapping allows for a step-by-step analysis. Knowledge of a detailed process allows for improvement in outcomes ~Key LEAN Lessons~ • It is not possible to overcommunicate .• Continuously focus on improvement. • Engage all facets of an organization, not just a core team. • Actions speak louder than words. • Ideas flow from the bottom up. • Be respectful to every individual. Listen to and seriously consider everyone's ideas. • A feedback loop is critical to overcoming challenges. • To achieve success, staff must be accountable.

Box 3-7

~Examples of Potential Preanalytical, Analytical, and Postanalytical Errors~ Preanalytical (Preexamination) = Incorrectly ordered assay Specimen obtained from wrong patient Specimen procured at the wrong time Specimen collected in the wrong tube or container Blood specimens collected in the wrong order Incorrect labeling of specimen Improper processing of specimen Analytical (Examination) = Oversight of instrument flags Out-of-control quality control results Wrong assay performed Postanalytical (Postexamination) = Reporting the wrong result Verbal reporting of results Instrument: laboratory information system incompatibility error Confusion about reference ranges

ISO 15189 Box 3-1

~Management requirements~ Organization and management Quality management system Document control Review of contracts Examination by referral laboratories External services and supplies Advisory services Resolution of complaints Identification and control of nonconformities Corrective active Preventative action Continual improvement Quality and technical record Internal audits Management review ~Technical Requirements~ Personnel Accommodation and environmental conditions Laboratory equipment Preexamination procedures Examination procedures Assuring quality of examination procedures Postexamination procedures Reporting of results