The International Society on Thrombosis and Haemostasis Scientific Subcommittee on Lupus Anticoagulant recommended two sensitive screening tests for lupus anticoagulants that assess different com- ponents of the coagulation pathway: clotting time- based assays, such as the dilute Russell viper venom time (DRVVT), and aPTT-based assays, such as kaolin clotting time and dilute prothrombin time (tissue thromboplastin inhibition test) [43]. Lupus anticoagu- lants prolong various phospholipid-dependent clotting times in the laboratory because they bind to phospho- lipid and thereby interfere with the ability of phospho- lipid to serve its essential co-factor function in the coagulation cascade. Lupus anticoagulant screening assays usually have a low concentration of phospho- lipid to enhance sensitivity. Any abnormal (prolonged) screening result typically requires a 1:1 mixing study in which the patient plasma is mixed with one equal vol- ume of normal plasma to demonstrate that the clotting time remains prolonged upon mixing. Confirmatory assays are performed if the screening assay remains
abnormal after the 1:1 mixing. Confirmatory assays typically demonstrate that upon addition of excess phospholipid, the clotting time shortens toward nor- mal. The platelet neutralization procedure is a confir- matory assay in which the source of the excess phospholipid is freezethawed platelets. The hexago- nal phospholipid neutralization procedure is also based on the same principle—that is, the clotting time becomes corrected after addition of phospholipid in hexagonal phase. Note that aPTT may or may not be prolonged, depending on the amount of phospholipid in the reagent.
In many lupus anticoagulant assays, heparin (including subcutaneous low-dose heparin) may cause false-positive lupus anticoagulant results. By enhanc- ing antithrombin activity, heparin inhibits activated factors II (thrombin), X, IX, XI, XII, and kallikrein.
Subsequently, clotting times such as PT and aPTT are prolonged and interfere with lupus anticoagulant assays. Lepirudin, danaparoid, and argatroban inhibit activated factor II and can also prolong clotting times.
Before coagulation testing, heparin may be removed or neutralized with polybrene in the coagulation labora- tory; however, residual heparin may continue to cause testing interferences [44]. Results for lupus anticoagu- lant assays can be interpreted correctly in patients on Coumadin. Table 19.2 summarizes important labora- tory coagulation errors due to various entities.
CASE STUDIES
Two case studies highlight the issues discussed in this chapter.
Case Study 1
A cardiac surgeon was following up on his patient during the first week of surgery. A CBC was done to assess current hematologic parameters. The hemoglo- bin level was acceptable at 12.5 g/dL. However, the RBC count was low at 2.9 million/mm3of blood. The values for MCV and MCH were also high. The sur- geon was naturally concerned with the low RBC count and found the values for hemoglobin and RBC count discrepant. He called the pathologist to discuss the findings. The pathologist reviewed the smear and found red cell agglutination. Red cell agglutination can be seen in cold hemagglutinin disease. Because hemo- globin levels are measured after lysing red cells, whether the red cells are agglutinated or not does not matter. However, with red cell agglutination, the total RBC count would be reduced. The MCV and MCH values would be falsely high. The high MCH value
should have been an indication for the lab technologist to preview the smear and to warm the blood prior to a repeat CBC on the hematology analyzer.
Case Study 2
A 52-year-old female who has long-standing rheu- matoid arthritis is under the care of an oncologist due to a recently diagnosed soft tissue sarcoma. The oncologist is concerned about chronic DIC and decides to evaluate her. Her CBC results show thrombocytope- nia. Her PT and aPTT results are within normal limits.
However, her D-dimer values are elevated. The
oncologist calls the pathologist to discuss the findings in this case. The pathologist reviews her peripheral smear and observes platelet clumping. The pathologist explains that this phenomenon may be seen especially with samples collected in EDTA tubes. Recollection in heparin or sodium citrate tubes should result in an accurate and higher platelet count. Rheumatoid factor is an example of a false-positive D-dimer test.
Ultimately, it was proven that this patient does not have DIC.
CONCLUSIONS
In this chapter, common tests performed in the hematology and coagulation section of the laboratory were discussed. Sources of errors can potentially include all steps in testing—collection of samples, transportation, storage, and methodology used—as well as intercurrent issues of the patients. It is impera- tive to follow procedures and protocols for all con- cerned to attempt to obtain meaningful, accurate values. Laboratory technologists and pathologists need to be aware of situations in which erroneous results may be obtained. Correlation with clinical information provided or from the medical records is required in certain situations. If aware of issues related to possible erroneous results, clinicians will also contribute to pro- viding appropriate interpretation of laboratory results.
In essence, it is a team effort of laboratory personnel and clinicians to provide an accurate interpretation of laboratory tests for better clinical decisions and patient management.
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TABLE 19.2 Sources of Laboratory Errors in Coagulation
Falsely prolonged clotting times Underfilling of citrate tube Polycythemia
Sample from indwelling catheters (dilution or contamination with anticoagulant)
Falsely shortened clotting times Overfilling of citrate tube Traumatic phlebotomy
Turbid plasma (e.g., hyperlipidemia, hyperbilirubinemia) in optical instrument
Falsely shortened aPTT in patients on heparin Delay in separation of plasma from platelets Elevated factor VIII (acute phase reactant) Falsely prolonged TT
Dysfibrinogenemia
Elevated levels of FDPs and paraproteins Amyloidosis
Heparin-like anticoagulants (in malignancy) Falsely high FDPs and D-dimer
Rheumatoid factor
Falsely abnormal platelet function Lipidemia
Hemolysis Thrombocytopenia Falsely low factor levels Heparin
Lepirudin Danaparoid Argatroban
False-positive results of lupus anticoagulant tests Heparin
Lepirudin Danaparoid Argatroban
aPTT, activated partial thromboplastin time; FDPs, fibrin/fibrinogen degradation products; TT, thrombin time.
313
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C H A P T E R
20
Challenges in Clinical Microbiology Testing
Laura Chandler
Philadelphia VA Medical Center and Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania
INTRODUCTION
Clinical microbiology is a discipline that encom- passes a broad range of testing methodologies, and it is complex in terms of organisms and methods used to isolate and identify them. Although significant improvements in testing methodologies have been made, clinical microbiology remains heavily reliant on culture-based methods and phenotypic methods for identification of culture organisms. The wide variety of pathogens and testing methods that are available makes microbiological testing challenging, and thus error detection and correction are important compo- nents of quality microbiology laboratory testing. Errors may occur at all stages of testing (pre-analytical, analyt- ical, and post-analytical), and an error in one stage of testing is likely to overlap with or lead to errors in other stages (e.g., incorrect specimen collection can lead to culture, identification, and reporting of organ- isms that are not involved in the disease process and to incorrect or unnecessary antimicrobial therapy as a result). In the clinical microbiology laboratory, as in every other discipline, the frequency of analytical errors has been reduced considerably with the imple- mentation of quality control and quality assurance pro- grams. Despite the improvements in microbiological testing, microorganisms remain a constant challenge, and errors do occasionally occur. This chapter dis- cusses some of the common interferences in the clinical microbiology laboratory.