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Pre-analytical Challenges in
Hemostasis Testing
Larry Darnell, BS
Applications Consultant
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Objectives
• Elucidate the effect common pre-analytical errors have on
hemostasis assays
• Present correct collection and processing conditions for
hemostasis samples
• Review new technologies to standardize unacceptable
sample flagging and document compliance with quality
standards (ISO 15189:2012)
2
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What is Laboratory Quality?
Accuracy Reliability Timeliness QUALITY
www.who.int/ihr/training/laboratory_quality/1_b_content_introduction.pdf3
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Frequency of Laboratory Errors
63%15%
22%
Pre-analytical
Analytical
Post-analytical
Carraro P, Plebani M. Errors in a Stat Laboratory: Types and Frequencies 10 years Later. Clin Chem. 2007;63(7):1338-42.4
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Frequency of Pre-Analytical
Laboratory Errors
63%15%
22%
Pre-analytical
Analytical
Post-analytical
#1 Incorrectly filled tubes
for hemostasis testing
#2 Wrong ID
#3 Wrong tube
5
Carraro P, Plebani M. Errors in a Stat Laboratory: Types and Frequencies 10 years Later. Clin Chem. 2007;63(7):1338-42.
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Pre-analytical Variables in Coagulation Testing
Associated with Diagnostic Errors in Hemostasis
Overall, a significant impact of patient care arising from diagnostic errors has been estimated to arise in
around 9% to 15% of errors, while the likelihood of inappropriate care has been described in 2% to 7% of
such cases. Many of these errors will originate due to the inappropriate collection, handling, or processing of
samples referred for testing and sometimes because testing has been initiated on the wrong patient or at the
wrong timepoint. In these instances, test results will accurately reflect the status of the test sample, but
conversely they will not accurately reflect the clinical status of the patient being investigated. These issues are
Favaloro EJ, Funk DM, Lippi J. Lab Med. 2012;43(2):1-10.
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• 28-yr old male hospitalized for appendectomy
• Admission results:
- PT = 12.5 secs (RI 10–14 secs)
- APTT = 35 secs (RI 24–36 secs)
- Fibrinogen = 300 mg/dL (RI 180–480mg/dL)
Case of the Prolonged APTT
http://www.medicinenet.com/appendicitis_appendectomy_pictures_slideshow/article.htm7
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24-Hours Post-Op
• Test results:
- PT = 13.1 sec (RI 10–14 secs)
- APTT = 38 sec (RI 24–36 secs)
• Question: What is appropriate action for the lab?
- Lab work-up for abnormal APTT
- Recollect
Case of the Prolonged APTT (cont’d)
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Why is the APTT Prolonged?
Pre-analytical
Collection artifact
Processing or storage error
Analytical
Reagent integrity
Instrument integrity
Physiologically True
Decrease in factor levels
Anticoagulant
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• All analytical parameters
and QC were within
acceptable limits
• The tube appeared to be
under-filled but tech was
busy and didn’t screen
for proper fill volume
Observations
Over-filled and under-filled
tubes cannot be tested.
Midway between label and lid
Upper Limit
Tube must be filled within the
green zone to be tested.
Frosted band on tube
Lower Limit
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• Plasma in under-filled blue-top tubes (<90% fill volume)
contains excess citrate
• Citrate reversibly binds Ca2+, preventing clotting until
Ca2+ is added during testing
• Excess citrate causes prolongation of the clotting time
due to excess Ca2+ binding in assay
Importance of Proper Fill Volume
CLSI Guideline H21-A5, 2008
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Effect of Under-filled Tubes on PT and APTT
• Not all assays are affected equally
• PT is more forgiving of under-filling than aPTT
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.
12
100 89 78 67
Tube filling (%)
100 89 78 67
Tube filling (%)
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Effect of High Hematocrit
• Citrate volume must be adjusted
when Hematocrit >55% to
maintain proper citrate
concentration in plasma
• In a 5mL tube (4.5mL blood +
0.5mL citrate), remove 0.1mL
citrate prior to collection
• C = (1.85x10-3)(100-HCT)(V)
- C = volume of citrate remaining
- V = volume of blood added
http://jtd.amegroups.com/article/viewFile/249/html/1817
CLSI. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis
Assays; Approved Guideline—Fifth Edition. CLSI document H21-A5. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.13
Patient
Hct=0.75
0.4mL
0.9mL
2.7mL
0.4mL
2.1mL
1.5mL
Normal
Hct=0.40
Citrate anticoagulant
Plasma
RBC
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Another Cause of Prolonged PT and APTT
50%
+= Double citrate (4.5:1)
50%
• Two halves do NOT make a whole
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• Clotted samples MUST be rejected
- Any fibrin, even small strands
• Prolonged PT/APTT/TT
- if fibrinogen is consumed by clot
• Shortened APTT
- if sample is activated but fibrinogen
still present
• Elevated D-dimer
• Frequency in a large reference
laboratory:
- 0.4–0.7% of samples were clotted
(approx. 80-100/month)
Clotted Samples
Funk DM, et.al. Quality Standards for Sample Processing, Transportation, and Storage in Hemostasis Testing. Semin Thromb Hemost. 2012;38:576-85.15
Fibrin clots in plasma
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Spectra of Chromatic Substances
• Demonstrates the relationship between hemolysis,
icterus, and lipemia
16
Hbg
Icterus
Lipemia
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Spectra of Chromatic Substances (cont’d)
• Today’s optical analyzers reliably measure at
wavelengths > 671nm due to LED light technology
17
40
5n
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57
0n
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67
1n
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Hbg
Icterus
Lipemia
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Hemolysis
• Traditionally graded with “+” system
• What degree of hemolysis is acceptable in
hemostasis testing?
0 50 150 250 525 mg/dL
CLSI: Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis;
Approved Guideline. CLSI document C56-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. 18
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• In vitro- Difficult venipuncture
- Small-bore needle
- Freezing whole blood sample
• In vivo - Hemolytic anemia
- Severe infections
- DIC
- Transfusion reactions
• Up to 3% of routine hemostasis samples are hemolyzed
• Hemolysis accounts for up to 70% of rejected samples
Causes of Hemolysis
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.19
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Effect of Hemolysis on Hemostasis Assays
Assay Effect
PT
APTT
Fibrinogen
D-dimer
Antithrombin
Favoloro EJ, et.al. Pre-analytical Variables in Coagulation Testing Associated with Diagnostic Errors in Hemostasis. Lab Med. 2012;43(2):1-10.
• While instruments utilizing mechanical clot detection
may not show interference, results may be inaccurate
because cell lysis products include tissue factors that
can activate coagulation.
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• Elevated bilirubin
• In vivo condition
• Usually present in severe liver disease
- Most hemostatic proteins are produced in the liver
- Many hemostasis assays are prolonged
• Optical interference not seen in assays read at >550nm
Icterus
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.
0 1.7 6.6 16 30 mg/dL
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• Traditionally graded with “+” system
• How does this compare to package insert limits for assays?
Lipemia
CLSI: Hemolysis, Icterus, and Lipemia/Turbidity Indices as Indicators of Interference in Clinical Laboratory Analysis;
Approved Guideline. CLSI document C56-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. 22
+ ++ +++ ++++-
0 125 250 500 1000 mg/dL
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• Includes a variety of in vivo lipid forms
- Intralipid®, traditionally used for analytical interference studies
does not fully mimic sample lipemia
• Genetic hyperlipidemia
• Post-prandial collection
- After fatty meal
Lipemia
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Effect of Lipemia on Hemostasis Assays
• Analytical effect- May cause high baseline readings if the assay is read at 405nm
- Not significant with wavelengths >600nm
• Biological effect- Elevation of FVII activity
- Acute effect on platelet function
- Decrease of some clotting factor activities (i.e., FII, FIX, FX)
Best approach:
Recollect when fasting
Favoloro EJ, et.al. Pre-analytical Variables in Coagulation Testing Associated with Diagnostic Errors in Hemostasis. Lab Med. 2012;43(2):1-10.Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.
24
Hbg
Icterus
Lipemia
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• Cold activation of FVII occurs if samples are
transported on ice or refrigerated
• Refrigeration of whole blood causes a decrease in
high-molecular-weight multimers of VWF
• Frozen plasma samples must be thawed at 37°C
- Decrease in fibrinogen, FVIII and VWF if thawed at room
temperature
• Many coagulation parameters are not stable
through more than one freeze/thaw cycle.
- Validate internally if more are required
Effect of Temperature on Hemostasis Samples
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.25
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• Physical stress (exercise)
- Platelet aggregation to ADP and EPI is enhanced
- FVIII and VWF increase 2.5x for up to 10 hours
• Mental stress
- Acute stress increase FVIII, VWF, Fibrinogen and tissue
plasminogen activator (t-PA)
• Pregnancy
- Modest increase in FVII and FX
- FVIII, VWF and Fibrinogen increase 2-3x
- Free Protein S and APCR ratios decrease
- Plasminogen Activator Inhibitor-1 and PAI-2, D-dimer all increase
Effect of Physiological Conditions on
Hemostasis
Blombäck M, Konkle BA, Manco-Johnson MJ, Bremme K, Hellgren M, Kaaja R, on behalf of the ISTH SSC Subcommittee on Women’s Health
Issues. Preanalytical conditions that affect coagulation testing, including hormonal status and therapy. J Thromb Haemost. 2007;5:855-8.
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Circadian Variation
• AM Peak Platelet Aggregation
FVIII
FVIIa
Protein C
Protein S
PAI-1
• PM Peak Antithrombin
Global fibrinolysis (t-PA)
27
Blombäck M, Konkle BA, Manco-Johnson MJ, Bremme K, Hellgren M, Kaaja R, on behalf of the ISTH SSC Subcommittee on Women’s Health
Issues. Preanalytical conditions that affect coagulation testing, including hormonal status and therapy. J Thromb Haemost. 2007;5:855-8.
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Regulatory Requirements
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• International guideline to improve quality, specifically in
clinical laboratories
• Total quality management plan can reduce the frequency
of medical errors
• Focuses on two areas
- Administrative
- Technical
• Pre-analytical
• Analytical
• Post-analytical
International Quality Standards for the
Clinical Lab (ISO 15189:2012)
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• ISO 5.5.3 “Documentation of examination
procedures”…interferences (e.g., lipaemia, haemolysis,
bilirubinemia, drugs) and cross reactions
• ISO 5.8.2 “Report attributes”…comments on sample quality
that might compromise examination results
• ISO 5.9.2 “If the laboratory implements a system for
automated selection and reporting of results, it shall establish
a documented procedure to indicate the presence of
sample interferences (e.g., hemolysis, icterus, lipemia) that
may alter the results of the examination”
Pre-analytical Requirements
(ISO 15189:2012)
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CLSI Guideline for Hemostasis Specimens
(H21-A5)
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Citrated Whole Blood
(CLSI H21-A5)
Assay RT Refrig. Frozen
PT Up to 24 hrs Unacceptable Unacceptable
APTT Up to 4 hrs Unknown Unacceptable
APTT
(UFH)1 hr Unknown Unacceptable
APTT
(VWF,
FVIII)
4 hr Unacceptable Unacceptable
Other 4 hr Unknown Unacceptable
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Platelet Poor Plasma
(CLSI H21-A5)
Assay RT Refrig. -20°C -70 °C
PTUp to
24 hrsUnacceptable 2 weeks 12 mo.
APTT 4 hrs 4 hrs 2 weeks 12 mo.
APTT
(UFH)4 hrs 4 hrs 2 weeks Unknown
APTT
(VWF,
FVIII)
4 hrs 4 hrs 2 weeks 6 mo.
Other 4 hrs 4 hrs Analyte-dependent
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D-dimer
• D-dimer is a stable measure
- Up to 24 hours at room temperature or refrigerated
- Up to 24 months frozen (-24 to -74°C)
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Proper Collection Steps
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• Patient identification
- At least two identifiers
- Pre-print tube labels and verbal verification with patient
• Patient:
- Fasts 8-12 hours
- Sits up 10-15 minutes
- Avoids strenuous exercise for 24 hours
- Avoids smoking immediately before venipuncture
• Record anticoagulant and antiplatelet therapies
Patient Preparation
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.36
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• 3.2% Sodium Citrate
• Collect the blue-top tube before tubes with additives
• Routine collection of discard tube is unnecessary
- Only needed with line draw or butterfly needle
• Remove tourniquet after <1 minute
• No fist-pumping – may cause hemolysis
• 19-21g straight needles best
Collect Correct Tube Without Stasis
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75. 37
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Mix with Anticoagulant
• Gently invert 3-6x or according to the tube-
manufacturer’s recommendations
• Syringe draws
- 3–5mL syringes preferred
- Transfer to blue-top tube immediately
- Allow blood to fill the evacuated tube without pressure
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.38
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Transport to Laboratory
• Promptly transport to lab (CLSI H21-A5 guidelines)- Test within 1 hour of collection if patient on unfractionated heparin
• Room temperature- Do not ice; activates FVII
• Pneumatic-tube system- Verify that system does not activate platelet assays
• Separated plasma samples can be shipped frozen- Use Na+, K+ and Ca2+ levels to verify that sample is citrated
plasma
Lippi G, et.al. Quality Standards for Sample Collection in Coagulation Testing. Semin Thromb Hemost. 2012;38:565-75.39
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Centrifugation
• Centrifuge to achieve platelet-poor plasma- <10,000 platelets/μL
- Confirm and document according to quality plan
- 1,500g for 15 minutes
- No brake
- Especially critical when preparing plasma to be frozen for:
• Lupus anticoagulant testing
• Unfractionated heparin testing
• PT, APTT, and TT not affected by platelets < 200,000 platelets/μL if tested on fresh plasma- 1,500g for <15 minutes if sample is not used for further testing or does
not require frozen plasma
• STAT spin- 11,000g for 2 minutes
- Test within 10 minutes to prevent platelets from returning to plasma
Funk DM, et.al. Quality Standards for Sample Processing, Transportation, and Storage in Hemostasis Testing. Semin Thromb Hemost. 2012;38:576-85.40
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Proper Collection Steps Summary
ID patient
Collect the sample
Mix with citrate
Label
Transport
Centrifuge
• Check ID on wristband
• Query patient for identifying information
• Use two identifiers
• Remove tourniquet to prevent stasis
• Achieve >90% fill volume
• Draw blue-top tubes after any blood cultures – correct order of draw
• Mix immediately with anticoagulant
• 3-6 inversions recommended
• Label tube before leaving patient
• Patient verifies correct label
• Pneumatic-tube system
• No ice
• Achieve PPP
• Double spin for certain special coagulation tests
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New Technology for
Standardized Pre-Analytical
Variables Flagging
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Automatic Detection of Under-filled Tubes
• Alerts operator to possible
sample-collection errors
• Ensures appropriate
sample:anticoagulant ratio
• Verifies sample-draw
quality
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Abnormal Sample Aspiration
• Alerts operator to possible clogs in plasma
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Assay-Specific Interfering Substance DetectionHemolysis, Icterus and Lipemia
• Values exceeding assay-specific thresholds are flagged
Icterus (mg/dL)
Lipemia (mAbs)
Hemoglobin (mg/dL)
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Assay-Specific Interfering Substance DetectionHemolysis, Icterus and Lipemia
• Assay-specific thresholds are best
• Base thresholds on real test results
• Same interference substance readings can flag
results for one assay, but not another
PT with no flag Fibrinogen with flag
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Icterus (mg/dL)
Lipemia (mAbs)
Hemoglobin (mg/dL)
Icterus (mg/dL)
Lipemia (mAbs)
Hemoglobin (mg/dL)
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• HIL check performed during the lag phase of routine
coagulation assays or as a separate reaction
• Technology varies by manufacturer
• OD at multiple wavelengths is read to calculate HIL
Interfering Levels of Hemolysis, Icterus and
Lipemia
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• 600-bed hospital
• 146,000 routine hemostasis samples annually
• Study design
- 5,060 samples checked manually, per lab protocol
• Tube-fill volume
• Presence of clots
• Hemolysis, Icterus, and Lipemia
- Samples “Accepted” or “Rejected”
Case Study: Impact of Pre-analytical Check
Standardization
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• All 5,060 samples tested for PT, APTT, Fibrinogen
and D-dimer
• Automated checks and flags for:
- Tube-fill height
- Clog detection
- HIL interference
Case Study: Impact of Pre-analytical Check
Standardization—Methods
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Case Study: Impact of Pre-analytical Check
Standardization—Results
5,060 samples
124 rejected using manual
method
70 unnecessary redraws using
manual method
54 rejected with automation
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Case Study: Impact of Pre-analytical Check
Standardization—Results (cont’d)
4,936 samples
4,936 accepted with no visible
clots using manual method
5 samples with micro-clots
reported with automation
5 rejected for clogs with
automation
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Case Study: Impact of Pre-analytical Check
Standardization—Results (cont’d)
• 8.5% of samples with pre-analytical issues were not
detected visually
• 52% reduction in sample rejections
• 70% reduction in staff time vs. visual inspection
Annualized Data
Samples
Recollected
Associated costs
($)
Manual 3,529 20,284
Automated 1,679 9,571
1,850 fewer 10,713 saved12,054
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Conclusions
• Strict criteria must be consistently followed when collecting
and transporting samples for coagulation testing to ensure
accurate results
• Standardized flagging of sample-quality issues lead to
fewer incorrect results
• Three healthcare goals achieved
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