Technologies for Coagulation Instruments

Transcription

1 C E U P D A T E I N S T R U M E N T A T I O N I V John A. Koepke, MD Technologies for Coagulation Instruments Both coagulation and fibrinolysis have traditionally been measured by functional assays. For example, the overall coagulation reaction or any component involved in the reaction may be studied in an anticoagulated plasma sample by observing the time required for a visible fibrin strand, or coagulum, to form after adding calcium chloride to the sample. The smaller the concentration of procoagulant(s), the longer it takes for the clot to form. Although functional techniques provide accurate results in the hands of a skilled operator, increased demand for testing led to the development of electromechanical, turbidimetric (nephelometric), and immunologic methods for use on instruments. Methods Manual In 1935 Dr Armand Quick developed the original method for measuring prothrombin time (PT). 1 Taking advantage of the extrinsic pathway in clot formation, he mixed thromboplastin (extracted from human brain) with blood plasma, added calcium ions, and observed the time it took for clots to form at 37 C. Under these conditions, Quick found that fibrin clots usually developed within 12 seconds. (He used specimens taken from unsuspecting first-year medical students enrolled in the biochemistry course.) Later methods used a metal hook to catch the first observable fibrin formed in the reaction, and tests were routinely done in duplicate. ABSTRACT Although different technologies are available for coagulation testing, the most frequently used are based on traditional functional methods. Many semiautomated and automated instruments can process and analyze both test and quality control data. This article reviews the principles of various methods for measuring coagulation (prothrombin time [PT] and activated partial thromboplastin time [aptt]) and issues associated with quality control and quality assurance. This is the final article in a 4-part continuing education series on instrumentation. Upon completion of this article the reader will be able to identify the important aspects of PT and aptt testing and the principles of the various instruments used for these tests. Functional Quick s functional approach was the basis for automated methods developed by instrument manufacturers. The Fibrometer, the electromechanical prototype for measuring PT, offered temperature control and included a pair of electrodes (one moving, one stationary) and a timing device to detect clots at 0.5-second intervals. The Fibrometer provided a good transition from manual methods that required a water bath and, to search for clots, a magnifying mirror. Mechanical clot detectors, however, have disadvantages, particularly when analyzing blood specimens with a low concentration of fibrinogen. In these samples, an appropriately sized fibrin strand could bridge the gap between the electrodes and stop the timer; the 0.5-second cycle of the electrodes produces artifactual 0.5-second intervals in the reported clotting times. Such results are minimally imprecise, but not of clinical significance. From the Pathology Department, Duke University Medical Center, Durham, NC. Reprint requests to Dr Koepke, 3924 Saint Mark s Rd, Durham, NC ; or nckoepke@ mindspring.com Section 4 Scientific Communications APRIL 2000 VOLUME 31, NUMBER 4 LABORATORY MEDICINE 2 1 1

2 Preanalytic Factors Associated With Spurious Coagulation Test Results Improper anticoagulation of specimen due to inadequate mixing, filling tube to less than 90% capacity, or overfilling tube Unexpected heparinization of specimen (eg, heparin locks or heparin therapy) Testing delays, inappropriate storage, prolonged warming, unstoppered specimen tubes (especially if specimens are not buffered) Test Your Knowledge! Look for the CE Update exam on Instrumentation (002) in this issue of Laboratory Medicine. Participants will earn 4 CMLE credit hours. In addition, if the electrodes are not properly cleaned between tests, clotting times for subsequent samples could be erroneously shortened owing to residual activated procoagulants on the electrodes. 2 If a specimen contained significant amounts of fibrinolysin, any clot formed might be fragile and unstable, thus compromising the ability of the Fibrometer to accurately detect the end point. Some second-generation instruments have overcome these problems. Other manufacturers chose to measure the increase in turbidity when soluble fibrinogen is converted to insoluble fibrin. Their photo-optical systems, which account for most of the coagulation instruments being used in the United States today, automatically deliver reagents to cups containing measured volumes of plasma. After a predetermined mixing phase, the system monitors the changing optical density and automatically reports the end point of the reaction. 2 Although less susceptible to error in samples with low levels of fibrinogen than the electromechanical systems, these instruments cannot produce accurate clotting times in specimens with marked turbidity due to hyperlipidemia or icterus. The effects of hyperlipidemia and icterus have been minimized, however, in electromechanical clot detection instruments that measure fibrin formation around an iron ball suspended in the plasma. In the absence of fibrin, the ball oscillates in an alternating electromagnetic field, and the instrument monitors its movement. When a clot forms, the metal ball stops moving. 3 This innovative approach accounts for a recent resurgence of electromechanical clot detectors. Immunologic Proteins associated with coagulation can be measured with high specificity by immunologic methods. Some procedures (for slide testing) use latex beads coated with specific antibodies. Others rely on radial immunodiffusion, immunoelectrophoresis, and nephelometry. The highly sensitive nephelometric procedures, though seldom used routinely, detect low levels of analyte, provided that the appropriate antibodies are available for the assay. These methods are not used for PT or activated partial thromboplastin time (aptt) testing. Chemical Chemical methods use synthetic substrates chromogenic and fluorogenic to measure levels of specific coagulation factors. Although seldom used in PT or aptt testing, these methods find use in complex coagulation cases. Current Coagulation test systems include both an instrument and a reagent (thromboplastin or partial thromboplastin). Although evaluations of various systems have been published, 4-6 few studies of systems in current use have been reported. The College of American Pathologists (CAP) coagulation proficiency-testing program, which enrolls almost 3,700 laboratories, shows that participants use many different systems. A recent survey shows that approximately 25 different instruments, 25 different thromboplastin reagents, and 23 different partial thromboplastin reagents are being used. Laboratories reported results from 78 different instrument PT reagent combinations and a similar number of instrument aptt reagent combinations. Some systems LABORATORY MEDICINE VOLUME 31, NUMBER 4 APRIL 2000

3 are more popular than others. For example, 520 participants used the same PT system, and 799 used the same aptt system. Other systems were reported by fewer than 10 laboratories. The popularity of some systems was due to the convenience associated with using an instrument and reagent from the same manufacturer, and some systems were more precise than others. 7 The information in these surveys may help in selecting an instrument or reagent for coagulation testing. In addition to precision, sensitivity is important because it defines the ability of a system to discriminate between a normal and an abnormal clotting time. In coagulation testing, sensitivity refers to the slope of clotting time vs the level of anticoagulation with either warfarin or heparin therapy. Instruments can also determine the relative level of abnormality. The relationship between sensitivity and precision is shown in the Figure. A recent survey of more than 34 coagulation instruments marketed by 9 manufacturers has been published. 8 The study describes the availability of routine and stat tests, throughput, information system interfacing, prices, and other important features. Practically all instruments detect end points (clots) by turbidimetric or optical methods. An increasing number provide international normalized ratios, as well as PT, and can be interfaced with the laboratory information system with ease. An editorial 9 offers comments on vendor responses to the 77 bits of information presented about each instrument. A recent conference on laboratory monitoring of anticoagulant therapy discusses coagulation reagents. 10 Preanalytic Factors In the routine clinical laboratory, vacuum tube collection methods are quite adequate for PT, aptt, fibrinogen level, and other common coagulation tests. To avoid overanticoagulation artifacts, especially in patients with elevated hematocrits, collection tubes containing 1 volume of buffered sodium citrate (109 mmol/l) are recommended. Tubes must be filled to at least 90% of capacity to avoid falsely elevated PT or aptt results. 11 PT or PTT (s) Section 4 Scientific Communications Decreasing Procoagulant or Increasing Heparin Concentration Coagulation test sensitivity and precision. The prothrombin time (PT) and activated partial thromboplastin time (aptt) increase as the concentration of procoagulant decreases or the concentration of heparin increases. The slope of the line reflects the sensitivity of the test system, and the shaded area shows the variation, or precision. APRIL 2000 VOLUME 31, NUMBER 4 LABORATORY MEDICINE 2 1 3

4 COMING UP IN MAY Feature 21st-Century Detectives: The Laboratory Professional in the Prevention of Infectious Disease The laboratory has always served as an early warning system for the recognition and prevention of infectious disease. Acknowledging the importance of the laboratory professional and the laboratory, the Centers for Disease Control and Prevention, Atlanta, focuses particular attention on 3 areas: foodborne disease, bioterrorism, and nosocomial infections. CE Updates Point-of-Care Testing Blood Glucose The first article in the point-of-care testing series examines blood glucose monitoring: what it does and what it cannot do, what biochemical methods are used to arrive at a blood glucose number, the factors that can alter the result, and technological breakthroughs likely to alter or revolutionize how blood glucose will be tested in the future. Waste Composting: The Art and Science of Converting Organic Waste to a Valuable Soil Resource Composting is controlled decomposition and transformation of raw organic materials into biologically stable, humic substances. It is an ancient technology used in homes and industry. The final product, finished compost, is a valuable soil resource for a variety of agricultural, horticultural, and silvicultural purposes. Science Rhodococcal Bacteremia in an Immunocompetent Patient Rhodococcus species are diverse in their morphology, biochemistry, and ability to cause disease. Not particularly fastidious, they grow on most routine bacteriologic and fungal media. While most infections cause lung disease in immunocompromised patients, extrapulmonary disease and infections have also occurred in patients who are immunocompetent, as this study indicates. Profile In Concert With Progress Irina Lutinger, MPH, H(ASCP)DLM, is assistant administrative director of clinical laboratories at New York Presbyterian Hospital, New York. Her story is one of drive and determination, coupled with a love of learning. It begins in the Soviet-dominated Ukraine, where she trained to be a concert pianist.l For routine coagulation tests, it is not necessary to use the 2-syringe technique or to chill specimens to be transported. In fact, chilling may be detrimental to some components associated with coagulation. Specific factor assays and esoteric tests may have special collection requirements. Testing should be done within a few hours after the blood is collected, although longer delays are not as detrimental to accuracy as previously thought. The coagulation testing standard of the Natinal Committee for Clinical Laboratory Standards, 12 includes useful advice for collecting, storing, and transporting specimens. For example, refrigerated citrated specimens (unopened) are stable for up to 18 hours, and PT results are reliable for up to 24 hours under these conditions. Preanalytic factors that may adversely affect coagulation test results are shown in the Table. Classified as moderately complex, coagulation testing must be done by a trained technologist or technician. Quality control specimens, usually a normal and an abnormal level control, must be run at least once during each shift. Quality Control and Quality Assurance To fulfill accreditation requirements, several of the large coagulation systems generate Levy-Jennings plots and offer cumulative reporting and patient tracking features, multirule checking, and workload recording features LABORATORY MEDICINE VOLUME 31, NUMBER 4 APRIL 2000

5 As for evaluating the performance of instruments and reagents, one group 13 cautions that data from interlaboratory surveys may not be reliable for this purpose, at least for factor VIII assays. The survey information, however, may give a clue to the precision and bias of commonly used systems as well as predict how a laboratory will fare in the program (which may be used to measure the quality of its work). Most modern coagulation instruments have coefficients of variation of approximately 3% in the proficiency testing programs. 7 Laboratory professionals may also wish to evaluate in house the convenience and safety, which are locally important features, before purchasing an instrument. It is important to ensure that point-of-care test testing done by patients receiving anticoagulants or technologists provides results comparable with those of the central coagulation laboratory. 14 An upcoming article on point-ofcare coagulation testing describes in detail the necessary studies to ensure that results from all areas are properly interpreted. 15 l References 1. Quick AJ, Stanly-Brown M, Bancroft FW. A study of the coagulation defect in hemophilia and in jaundice. Am J Med Sci. 1935;190: Koepke JA, Klee GG. Automated coagulation detection systems. Clin Lab Haematol. 1979;21: Heins M, Reinauer H. Automation in coagulation testing. J Int Fed Clin Chem. 1996;8: Drewinko B, Roe E, Hasler D, et al. Evaluation of automatic and semiautomatic coagulation assay instruments. Clin Lab Haematol. 1980;2: Italian Cismel Study Group. Multicentre comparison of nine coagulometers and manual tilt-tube methods for prothrombin time performance. Clin Lab Haematol. 1983;5: Goyzueta FG, Billet HH. Evaluation of an automated sampler for routine coagulation testing. Lab Med. 1992;23: Participant Summary. Coagulation (Limited) Survey Set CG1-A. Northfield, IL: College of American Pathologists; Coagulation analyzers. CAP Today. January 1999;13: Aller R, Sheridan B. Coagulation analyzers: service above all [editorial]. CAP Today. January 1999;13: College of American Pathologists Conference XXXI: laboratory monitoring of anticoagulant therapy. Arch Pathol Lab Med. 1998;122: Reneke J, Etzell J, Leslie S, et al. Prolonged prothrombin time and activated partial thromboplastin time due to underfilled specimen tubes with 109 mmol/l (3.2%) citrate anticoagulant. Am J Clin Pathol. 1998;109: NCCLS. Collection, Transport and Processing of Blood Specimens for Coagulation Testing and General Performance of Coagulation Assays; Approved Guideline, 3rd Edition; H21- A3. Wayne, Pa: NCCLS; Arkin CF, Bovill EG, Brandt JT, et al. Factors affecting the performance of factor VIII coagulant activity. Arch Pathol Lab Med. 1992;116: Rose VL, Dermott SC, Murray BF, et al. Decentralized testing for prothrombin time and activated partial thromboplastin time using a dry chemistry portable analyzer. Arch Pathol Lab Med. 1993;117: Koepke JA. Point-of-care coagulation testing. Lab Med. In press. Please let us know your opinion of the Immunology (001) series. Place an X in one box for each question. Return this form (or a photocopy) by fax to: (312) ; or, mail to: ASCP Press Administration, 2100 W Harrison St, Chicago, IL Thank you for your input. 1 Defici Excell The series met the objectives stat 2 The series provided useful technical d 3 The information provided in the series was new and tim 4 Technical points were explained clearly and were easy 5 The text was organized 6. Illustrations, charts, and tables helped explain text Comments: (Attach additional pages, if necessary.) Section 4 Scientific Communications APRIL 2000 VOLUME 31, NUMBER 4 LABORATORY MEDICINE 2 1 5

6 First Name Address City State Zip - Country CONTINUING EDUCATION UPDATE EXAM Instrumentation (002) Signature (Required for all submissions) Last Name Telephone Number ( ) - Date Completed (Required) / / To earn four CMLE credit hours, complete this exam form (or a photocopy) with your credit card information authorizing a $25 processing charge and fax it to (312) Mailed requests must be accompanied by a $30 processing fee, payable by credit card or check, and forwarded to ASCP, Dept , Chicago, IL Payment must be included with your examination. After processing this form, ASCP will mail you a certificate of participation and the answer key. NOTE: This examination must be received by June 30, CE Update is approved to meet licensure requirements for California, Florida, and other states. If you have questions regarding this exam, call us toll free at (877) ASCP PRESS [(877) ; in Illinois, (312) , ext 1292]. Please print carefully; this form will be read by a computer. PaymentCredit Card ($25) [$30 if requesting by mail] Check ($30, payable to ASCP) Visa MasterCard Check # Credit Card Number Exp Date / Answers: Please select the one best answer for each item by placing an X in the box A B C D E Multiple-Choice Questions 1. The consumer use of glucose meters was made possible by the development of A. dry reagent immunologic tests. B. end-point methods. C. low-cost digital processing. D. quantitative reference methods. 2. A common application of automated flat method staining by the histology laboratory is A. periodic acid Schiff. B. immunohistochemistry. C. Gram s stain. D. frozen-section staining. 3. Most hematology analyzers measure hemoglobin concentration A. spectrophotometrically. B. by impedance. C. by flow cytometry. D. with light scatter. 4. Typical chemical reactions occurring in dry diagnostic reagent strips can be grouped as A. dye binding, enzymatic, and immunologic. B. immunologic and polymerase. C. dye binding, enzymatic, immunologic, and redox catalysis. D. dye binding, immunologic, and polymerase. 5. Which of the following is the preferred anticoagulant for prothrombin time, activated partial thromboplastin time, and fibrinogen testing? A. Sodium citrate (3.2%) B. Sodium oxalate C. Ethylenediaminetetraacetic acid D. Heparin 6. Some large hematology analyzers have expert systems, computer-based programs that A. automate instrument maintenance. B. store patient results. C. analyze and store quality control results. D. automate review criteria for reflex testing. 7. Approximately how many different coagulation instruments are currently marketed in the United States? A. 18 B. 27 C. 34 D Which of the following is least likely to be a factor when considering an autostainer? A. Available plumbing B. Lighting C. Workload D. Space 9. Immunochromatography-based diagnostic test strips generate color response in which of the following reagent areas? A. Sample application B. Absorbent C. RBC separation D. Capture zone 10. A coagulation system is defined as which of the following? A. Method and reagent B. Blood specimen and anticoagulant C. Instrument and reagent D. Reagent and control plasma LABORATORY MEDICINE VOLUME 31, NUMBER 4 APRIL 2000