Pre-analytical Challenges in Hemostasis Testingcamlt.org/wp-content/uploads/2018/09/hemo-preanalytical-challenges-FINAL.pdfObjectives • Elucidate the effect common pre-analytical

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)

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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

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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.

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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

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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

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5n

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57

0n

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67

1n

m

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


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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


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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


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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


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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


Standardization—Methods

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Standardization—Results

5,060 samples

124 rejected using manual

method

70 unnecessary redraws using

manual method

54 rejected with automation

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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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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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Pre-analytical Challenges in Hemostasis Testingcamlt.org/wp-content/uploads/2018/09/hemo-preanalytical-challenges-FINAL.pdfObjectives • Elucidate the effect common pre-analytical

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