Hemostasis and Coagulation Instrumentation (Rodak's Hematolo
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1
Q: What tests made up the traditional coagulation menu?
A: PT/INR, APTT, fibrinogen, thrombin time
2
Q: How did coagulation testing evolve with new instrumentation?
A: Specialized tests became routine, testing became standardized, consistent, and cost-effective
3
Q: What is still required despite automation?
A: Method validation, cognitive ability, and theoretical knowledge of hemostasis
4
Q: What are the six main end-point detection principles?
A: Mechanical, photo-optical, nephelometric, chromogenic, immunologic, viscoelastic
5
Q: How does electromechanical clot detection work?
A: Measures change in conductivity between two electrodes; clot completes the circuit and stops the timer
6
Q: What device used electrodes for clot detection?
A: BBL Fibrometer
7
Q: How do steel ball methods work?
A: A ball oscillates in plasma; fibrin increases viscosity and slows movement until timer stops
8
Q: Which analyzers use oscillating ball methodology?
A: Diagnostica Stago analyzers
9
Q: How does photo-optical detection work?
A: Measures changes in plasma optical density (light transmittance) during clotting
10
Q: What interferes with photo-optical methods?
A: Lipemia, icterus, hemolysis, or translucent clots
11
Q: How do modern instruments minimize interference?
A: Use multiple wavelengths to filter effects of icterus/lipemia
12
Q: What substrate is typically used in chromogenic assays?
A: Synthetic peptide substrate with para-nitroaniline (pNA)
13
Q: How does the chromogenic method measure enzyme activity?
A: Cleavage releases yellow pNA, OD at 405 nm is proportional to protease activity
14
Q: How does the chromogenic method measure enzyme activity?
A: Cleavage releases yellow pNA, OD at 405 nm is proportional to protease activity
15
Q: Difference between direct and indirect chromogenic assays?
A: Direct = OD proportional to analyte activity; Indirect = OD inversely proportional due to inhibition of a target enzyme
16
Q: How does nephelometry work?
A: Measures light scatter at 90° or forward-angle as fibrin polymers form
17
Q: What kind of result does nephelometry provide?
A: A clot curve or “signature” when continuous readings are taken
18
Q: What is immunologic end-point detection based on?
A: Antigen–antibody reactions with latex microparticles
19
Q: How is light absorbance used in immunologic assays?
A: Latex-coated microparticles agglutinate when bound to antigen, increasing light absorbance
20
Q: Example of analyte measured by immunologic methods?
A: D-dimer
21
Q: What does viscoelastic clot detection measure?
A: Time to clot, clot strength, clot kinetics, and fibrinolysis
22
Q: Which instruments use viscoelastic methods?
A: TEG (Thromboelastography) and ROTEM (Rotational Thromboelastometry)
23
Q: Advantage of mechanical clot detection?
A: Not affected by lipemia or icterus; detects weak clots
24
Q: Disadvantage of photo-optical detection?
A: Interference from lipemia, icterus, and hemolysis
25
Q: Advantage of chromogenic assays?
A: Measure proteins that do not clot; specific
26
Q: Disadvantage of immunologic methods?
A: High cost and limited automated tests available
27
Q: What are the three levels of coagulation instrumentation?
A: Manual, semiautomated, automated
28
Q: Describe manual coagulation instruments.
A: Reagents/specimens transferred manually; temperature via water bath/heat block; end-point detected visually by operator
29
Q: Examples of manual methods?
A: Tilt tube, wire loop
30
Q: Describe semiautomated coagulation instruments.
A: Manual transfer of reagents/specimens; internal 37°C temperature control; automatic timing and clot detection
31
Q: Examples of semiautomated analyzers?
A: Fibrometer, STart 4, Cascade M & M-4, BFT-II, KC1, KC4
32
Q: Examples of semiautomated analyzers?
A: Fibrometer, STart 4, Cascade M & M-4, BFT-II, KC1, KC4
33
Q: Describe automated coagulation instruments.
A: Reagents pipetted automatically; internal monitoring of 37°C; timers and clot detection automatic
34
Q: Examples of automated analyzers?
A: ACL TOP, STA-R Evolution, STA Compact, Sysmex CA-series, BCS XP
35
Q: Why were duplicate tests required in visual methods?
A: Coefficient of variation >20%
36
Q: What precision is achieved by automated analyzers?
A: CV <5%, sometimes <1%
37
Q: What is random access testing?
A: Ability to run multiple tests in any order on one/many specimens
38
Q: Advantages of random access testing?
A: Faster turnaround, reduced errors, reduced labor
39
Q: How have reagent and specimen volumes improved?
A: Tests now require half or one-quarter of traditional volumes
40
Q: Why are open reagent systems important?
A: Allow labs to choose reagents from different manufacturers
41
Q: What features support reagent tracking?
A: Lot/expiration records, volume monitoring, barcoding
42
Q: What is primary tube sampling?
A: Analyzer aspirates directly from centrifuged collection tube
43
Q: Benefits of closed-tube sampling?
A: Safer, reduces exposure, stabilizes pH, prevents crossover
44
Q: What quality step is needed with closed-tube sampling?
A: Visual check for fibrin strands/clots after centrifugation
45
Q: How do analyzers handle dilutions?
A: Perform automatic dilutions of specimens, calibrators, and controls
46
Q: What features do modern analyzer computers include?
A: Data storage/ retrieval, calibration curves, QC files, Westgard rules, automatic repeat testing
47
Q: What is bidirectional LIS interface?
A: Analyzer queries LIS for ordered tests and sends results back
48
Q: What is reflex testing?
A: Automatic additional testing/dilutions if results exceed preset limits
49
Q: What is a clot “signature”?
A: Graph of clot formation kinetics showing baseline, acceleration, deceleration, and end-point
50
Q: What does a “clotted” flag mean?
A: Shortened clotting times due to premature activation
51
Q: How does lipemia affect results?
A: Falsely prolongs clotting times on optical instruments
52
Q: How does hemolysis affect results?
A: Falsely shortens clotting times via factor/platelet activation
53
Q: How does icterus affect results?
A: Prolongs clotting times (factor deficiency) and interferes with OD instruments
54
Q: What does “no end-point detected” mean?
A: Instrument couldn’t detect clot; alternative method needed
55
Q: What factors should guide the selection of coagulation instruments?
A: Patient population needs, test menu, cost-effectiveness, and standardization
56
Q: What might be appropriate for a small hospital with low-volume esoteric testing?
A: A batch analyzer with high throughput for routine coagulation tests
57
Q: Name some criteria for selecting instruments (Box 42.3).
A: Cost, service response time, reliability, maintenance, ease of use, test menu breadth, LIS interface, reagent flexibility, training support
58
Q: What are examples of specialized features on current coagulometers (Table 42.3)?
A: Random access, primary tube sampling, cap piercing, barcoding, bidirectional LIS, flagging, liquid level sensing, onboard QC, stat capability, refrigeration, specimen storage, reflex testing, clot graphs
59
Q: What are POC coagulation instruments?
A: Handheld/ bedside devices for near-patient testing, surgeries, or self-testing
60
Q: Advantages of POC instruments?
A: Fast turnaround, portability, small specimen volume (useful in infants)
61
Q: What specimen is typically used in POC coagulation devices?
A: Capillary (fingerstick) whole blood
62
Q: How is testing usually performed on POC devices?
A: Blood applied to a cartridge with reagents, then inserted into the analyzer
63
Q: What was the first POC coagulation test introduced in 1966?
A: Activated clotting time (ACT) for heparin monitoring in cardiac surgery
64
Q: How is heparin therapy reversed after cardiac surgery?
A: With protamine, monitored by ACT
65
Q: What is the most common POC test today?
A: PT/INR for Coumadin (warfarin) monitoring
66
Q: Why must POC results be validated against central lab assays?
A: Because correlation can be weak due to differences in reagents, specimens, and instrumentation
67
Q: What INR results from POC testing require confirmation by central lab?
A: INR >6.0 or unexpected INR changes
68
Q: What are some POC instruments and their detection principles?
Abbott iSTAT: electrogenic
Alere INRatio: electrochemical
Helena Cascade POC: photo-mechanical
Accriva ProTime Micro: mechanical clot
Roche CoaguCheck XS: amperometric
Siemens Xprecia Stride: electrochemical (amperometric)
Alere INRatio: electrochemical
Helena Cascade POC: photo-mechanical
Accriva ProTime Micro: mechanical clot
Roche CoaguCheck XS: amperometric
Siemens Xprecia Stride: electrochemical (amperometric)
69
Q: What does global hemostasis assessment measure?
A: The entire kinetic process of whole blood clot formation (initiation, strength, fibrinolysis, hypercoagulability)
70
Q: What is the advantage of using whole blood in global assays?
A: Allows evaluation of platelets, RBCs, WBCs, coagulation factors, and proteins together
71
Q: When is thromboelastography (TEG) useful?
A: In hepatic disease, obstetrics, trauma, cardiac surgery, or liver transplant
72
Q: How does TEG work?
A: Whole blood in an oscillating cup (4.75°); fibrin links cup and stationary pin; pin torque transmitted to torsion wire → electrical signal → tracing
73
Q: How does ROTEM differ from TEG?
A: In ROTEM, the pin rotates while the cup is stationary; less vibration-sensitive, 4 channels, touch screen, automated pipettor
74
Q: What do TEG/ROTEM tracings show?
A: Clot initiation (R/CT), clot kinetics (K/CFT, α-angle), clot strength (MA/MCF), fibrinolysis (Ly30/CLI)
75
Q: Disadvantages of TEG/ROTEM?
A: Operator-dependent, require training, interpretation requires expertise
76
Q: Why is platelet function testing important?
A: To monitor antiplatelet therapy (aspirin, clopidogrel, GP IIb/IIIa inhibitors) and assess bleeding risk
77
Q: Why is bleeding time no longer used?
A: It’s technically demanding, operator-dependent, and not predictive of surgical bleeding
78
Q: What principle is used in classic platelet aggregometry?
A: Light transmission aggregation (LTA) – increased light transmission as platelets aggregate
79
Q: What parameters are measured in LTA?
A: Maximum % aggregation, area under the curve, velocity
80
Q: What is impedance aggregometry?
A: Measures platelet adhesion to electrodes in whole blood
81
Q: What is lumi-aggregation?
A: Uses luminescence to measure secretion + aggregation simultaneously
82
Q: What is the BioData PAP-8E?
A: An 8-channel platelet aggregometer using LTA, low sample volume (225 μL), with touch screen and barcode scanner
83
Q: What is the Chrono-Log 700 Lumi-Aggregation System?
A: Aggregometer for whole blood or PRP; uses impedance + luminescence detection
84
Q: What is the Multiplate Analyzer?
A: Whole blood impedance-based aggregometer; used for monitoring clopidogrel and GP IIb/IIIa inhibitor therapy
85
Q: What is the Helena AggRAM?
A: Modular platelet aggregometer with laser diode optics at 650 nm, 4 channels, microvolume testing
86
Q: What is the Siemens PFA-100?
A: Automated platelet function analyzer using whole blood through collagen/ADP or collagen/epinephrine membranes; closure time indicates function
87
Q: What conditions can the PFA-100 detect?
A: von Willebrand disease, aspirin effect
88
Q: What is the VerifyNow system?
A: Cartridge-based optical detection system using fibrinogen-coated beads and agonists; provides aspirin, GP IIb/IIIa, and P2Y12 assays
89
Q: What is Plateletworks?
A: Assay kit run on hematology cell counters; compares platelet counts before and after agonist activation
90
Q: What are advantages of flow cytometry for platelet function?
A: Whole blood testing, small volume (~5 μL), sensitive in thrombocytopenia, measures receptor upregulation, microparticles, platelet-leukocyte binding
91
Q: What platelet disorder can be diagnosed using flow cytometry?
A: Heparin-induced thrombocytopenia (HIT)
92
Q: What is molecular coagulation testing used for?
A: To detect thrombophilia-related mutations and inherited bleeding disorders
93
Q: What are the most common mutations tested in thrombophilia?
A: Factor V Leiden and Prothrombin G20210A
94
Q: Which gene mutation is also tested but has unclear clinical utility?
A: MTHFR (methylene-tetrahydrofolate reductase)
95
Q: What does ACMG recommend about MTHFR testing?
A: It should not be routinely performed in thrombophilia workups
96
Q: What is the most common molecular testing method?
A: PCR-based assays
97
Q: What mutations can PCR detect?
A: Point mutations and single-nucleotide polymorphisms
98
Q: What is RFLP analysis used for in coagulation testing?
A: To analyze PCR products, though not suitable for high-volume labs
99
Q: Name other PCR-based/non-PCR methods available.
A: ARMS (amplification refractory mutation system), LightCycler, array technology, Invader assays, ligand-based technologies
100
Q: What is an advantage of molecular testing compared to clot-based assays?
A: High sensitivity/specificity and no interference from anticoagulants/inhibitors
101
Q: What sequencing method has become the primary tool for detecting variants?
A: High-throughput sequencing (HTS)
102
Q: What are the advantages of HTS?
A: Parallel sequencing of millions of molecules, whole exome analysis, identifies mutations/variants
103
Q: What role does molecular testing play in hemophilia and VWD?
A: Identifies mutations in FVIII, FIX, and VWF genes; helps with diagnosis and classification
104
Q: What is a lateral flow assay?
A: A paper-based platform using antibody detection for rapid, low-cost results
105
Q: How does a lateral flow test strip work?
A: Specimen moves by capillary action through zones with reagents; antibody-conjugated particles bind target analyte and form visible lines
106
Q: What is a lab-on-a-chip (LOC)?
A: A microelectronic chip integrating fluid handling and protein assays using tiny blood volumes
107
Q: What is the advantage of lab-on-a-chip?
A: Simultaneous measurement of several proteins with increased sensitivity and rapid results
108
Q: How will future technologies help coagulation labs?
A: Provide low-cost, sensitive, user-friendly tools for diagnosing thrombotic/ bleeding disorders
109
Q: Which are examples of technology advances in coagulation labs?
A: Primary tube sampling, random access testing, reduced reagent/specimen volumes (all of the above)
110
Q: What is an advantage of mechanical end-point detection?
A: Not affected by lipemia
111
Q: What method of detection is used in both TEG and ROTEM?
A: Viscoelastic
112
Q: Which is a feature of semiautomated analyzers?
A: Manual addition of reagents/specimens; automatic timing/clot detection
113
Q: Which method is most susceptible to icterus interference?
A: Photo-optical clot detection
114
Q: Which platelet test method works for thrombocytopenia/pediatrics?
A: Flow cytometry
115
Q: Which is NOT a factor for analyzer selection?
A: Manufacturer’s home office location
116
Q: What method does the PFA-100 use?
A: Closure time as blood flows through aperture coated with platelet agonists
117
Q: Where is POC testing used in high volume?
A: Monitoring oral anticoagulant therapy
118
Q: Molecular testing can diagnose which disorders?
A: Factor V Leiden and Prothrombin G20210A mutations
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