Presentation on theme: "Quality Issues in Coagulation Laboratory"— Presentation transcript:
1 Quality Issues in Coagulation Laboratory Sukesh C NairCMCVelloreIndia
2 Laboratory issues in coagulation testing Pre analyticalAnalyticalPost analyticalBiological variation (inter and intra individual variability)
3 Pre analytical phase: Most issues are here:- Lack of standardized procedures forsample collectionpatient preparationspecimen acquisitionhandling and storageoften outside the control of the laboratory performing the test.
4 Patient preparation Testing for haemostasis should be avoided drugs interfering with the function of platelets and coagulation factorscloser to any transfusion of blood, blood products and factor concentrates.For factors like FXIII it needs to be avoided even upto 1 month.Biological variation in tests of haemostasis:Plasma clotting times - constant within and between subjects because proteins such as fibrinogen, clotting factors and antithrombin show a low biological variability.Fibrinolytic parameters such as PAI 1 and fibrinopeptide A show very high variability.
5 Proper CollectionUse a blood collection system that collects the specimen directly into a tube containing the anticoagulantOrder or draw for evacuated venous blood collection tube system the blood coagulation tube is always first when multiple containers are to be collected except when a blood culture too is needed.Mix by 3-4 inversionsProthrombin Time/Internal normalized ratio (PT/INR) and activated partial thromboplastin time (APTT) results are not affected if tested on the first tube drawnWhen using a winged blood collection set for venipuncture and a coagulation tube is the first tube to be drawn a discard tube should be drawn first.
6 Proper CollectionSyringe draws using a hypodermic needle/syringe may have increased risk of hemolysis and apparent safety issues.For this use a double-syringe technique, blood from the second syringe should be used for the coagulation specimen.Blood should be added to the appropriate volume of anticoagulant within one minute of completion of draw.All tubes should be inverted at least four times to mix. Excessive mixing can cause hemolysis and/or platelet activation, leading to erroneous results.
7 Scenario 1 Audit of sample collection at a large reputed hospital Main sample collection area, Wards, ICU etc.Main OPD sample collection area directly under control of the laboratoryThe hospital uses Evacuated tubes and the sample collection manual details out the procedure as has been mentioned in literaturePrepare the patient, put the needle into the needle holder, insert the needle and collect samples directly into the tubes connecting the tubes in the proper order and mixing it by inversions as mentioned in the manual.
13 Scenario 1Prepare the patient, put the needle into the needle holder, insert the needle and collect samples directly into the tubes connecting the tubes in the proper order and mixing it by inversions as mentioned in the manual.No issues in main sample collection area.
14 InpatientsSampled one ward (20), one ICU (4) and just to ensure an adequate sampling included accidents and emergency.All samples are collected into evacuated tubes.But not using the evacuated tube’s needle and needle holder system.All are collected by Syringe and needle and then transferred into the tubes.Mixing was variable among the staff.
15 Evacuated tube system Best system of sample collection Closed system – ensuring safety of the operator during pre-analytical, analytical and post-analytical phase.Blood comes directly in touch with the anticoagulant.The degree of vacuum ensures the exact volume – correct blood to anticoagulant ratio – CORRECT FILL.Pressure of the vacuum also ensures proper mixing as the blood is drawn in necessitating only a milder form of mixing 3-4 inversions (compared to 8-9) to avoid clot formation.
16 Evacuated tube system - Disadvantages CostThe tubes gets connected to the same needle whereby the contents of one tube can contaminate the otherClot activator contaminating Coagulation or cell countEDTA (Cell count) contaminating Coagulation or Clot tubeHeparin contaminating Coagulation, clot tube or cell countOrder of draw
18 Non Evacuated Tube System Blood out of the vein is not anticoagulatedRisk of volume being incorrect – overfilling and underfilling.Improper fill leads to abnormal results especially in coagulation tests – PT/INR/aPTT/Factor levels as all these tests are titrated to the correct blood anticoagulation mix.Risk of clot formingClot affects all haematology cell counts – Hb/RCC/WCC and more dangerously Platelet count.All coagulation tests will be abnormal
19 AuditDid this finding have an impact on the integrity of the primary sampleRisks are known??? Are the personnel doing phlebotomy awareSample rejection register – many rejections of samples received from inpatientsUnder/over filledClot in the sample
20 Root Cause Analysis Nurses are not aware but lab is aware Nurses training recordsSample collection is part of induction training1 & ½ hrsAll have attendedTraining contentOnly mentions the procedureNo mention of when to use syringe and needleWhen can this situation be changed to modified evacuated tube system – Luer adapterHow to transfer blood from syringe to tubes maintaining the integrity of the ETS and the blood sample.
21 Proper CollectionBlood should be added to the appropriate volume of anticoagulant within one minute of completion of draw.All tubes should be inverted at least four times to mix. Excessive mixing can cause hemolysis and/or platelet activation, leading to erroneous results.If the blood in the syringe is to be transferred to an evacuated tube system the rubber stopper of the evacuated tube is pierced with the needle. Use the same “order of draw” as for evacuated blood collection tube system.
22 Scenario 1b Large Lab Issues in samples received Leakage Hemolysis ClotNeedle stick injuries
23 Large LabThe samples are received from two major sources 1) Franchises with whom the lab has contract with and is fairly under their control. 2) The other is from another laboratory which could be processed sample.The sample collection of the franchisee’s are controlled by the laboratory by initial and frequent training and monitoring.They are provided sample collection manual (SCM).They also have instructions in a sample collection kit provided to them.This kit has 2 evacuated tubes.But for phlebotomy a syringe and needle is provided instead of:-the evacuated tube system(ETS)’s needle andneedle holder.
24 Large LabThe instruction also mentions to avoid opening the caps of the tubes.They pierce the tube to transfer the blood from the syringe into the tube with the same needle as used for phlebotomy:- this causesRisk of needle stick injury to the operator6 mL tube to which 4 mL blood has to be added, causinghemolysis while abruptly stopping the processSome of the franchisee’s are opening the tubes to add to avoid piercing.Recapping causes leakageAlso the instructions mentions mixing as shaking and not inversions.Clot in the sample
25 New Technology Full awareness Advantages vs Disadvantages Risks vs BenefitWhen can this situation be changed to modified evacuated tube system – Luer adapterHow to transfer blood from syringe to tubes maintaining the integrity of the ETS and the blood sample.Onus is more on the user than manufacturer
26 Collection from an in line. Step 1- Affix the luer adapter to the needle holder.Step 2- Open the cap just before you decide to draw blood.Needle Holder with a luer adapter and a 3 way line.Step 3- Blood can be drawn from a 3 way line or scalp vein needle directly into a vacutainer.Here Evacuated tubes can be used directly but follow the same order of drawWhen samples are collected in Syringe
31 Proper Collection105 to 109 mmol/L, 3.13% to 3.2% (commonly described as 3.2%) of the dihydrate form of trisodium citrate (Na3C6H5O7 • 2H2O), buffered or nonbuffered.The proportion of blood to the sodium citrate dihydrate anticoagulant volume is 9:1.Correction for hematocrit values above 0.55 L/L (55%).X mL = 60/(100-PCV) x Z mL
32 Issues related to sample collection Under filling - dilution of plasma resulting in underestimation of clotting factor levelsoverfilled standard vacuum tube will not give erroneous results until it is overfilled to more than 120%.Under mixing may affect tests downstream specialized hemostasis assays performed after some time.Vigorous mixing (shaking of tubes) might lead to hemolysis or spurious test activation and false shortening of test clotting times and even false elevation of clotting factor activity(factor VII).
33 Issues related to sample collection Not so uncommon error is the plasma may be a sample collected in EDTA.EDTA plasma may result in falsely prolonged plasma clotting times and will show an inhibitor effect and if the requested test is a Lupus anticoagulant (LA) this will cause a false positive result.Unseparated samples for VWF could lead to loss of high molecular weight multimers during transportation.Filtered plasma might produce spurious hemostasis tests results,false diagnosis of vWD could occur due to loss of factor VIII and vWF
34 Issues related to Storage Inadeqautely thawed samples would lead to inhomogeneous samplingCryoprecipitate portion would be selectively sampledvery high levels of FVIII:C, VWF, Fibrinogen or FXIII.Cryo poor partvery low levels of FVIII:C, VWF, Fibrinogen or FXIII.Frozen samples should be thawed and mixed at 370C waterbath for 5 minutes before testing.
35 Proper acquisition and Processing The whole blood specimen should be checked for clot formation by gentle inversion and observation.To obtain a plasma sample, the capped specimen tube should be centrifuged at a speed and time required to consistently produce platelet poor plasma (platelet count <10 x 109/L) (10,000/μL).This may be accomplished by centrifuging at 1,500 g for no less than 15 minutes at room temperature.
36 Proper acquisition and Processing Swing-out bucket rotor should be used to minimize remixing of the plasma and platelets, particularly with plasma removal.While it is crucial that an essentially platelet-free sample be obtained if the specimen will be frozen for subsequent testing, APTT, PT/INR, and TT performed on fresh plasma samples are not affected by platelet counts of at least up to 200 x 109/L (200,000/μL). Platelet counts >10 x 109/L are not acceptable for lupus anticoagulants, other phospholipid antibodies, and heparin monitoring.The reliability of the centrifugation procedure should be validated every six months or after modification of the centrifuge to ensure plasma platelet counts are within acceptable limits.Samples that have visible hemolysis should not be used because of possible clotting factor activation and end point measurement interference.
41 Scenario -2Sample sent for Thrombophilia markers in a patient with proximal DVT (Deep vein thrombosis)Sample collection – goodProcessing - goodPackaging – OKTransported to the laboratory at another locationCost to the patient – 35,000.
42 Scenario - 3Sample sent for Hemophilia assay in a patient with severe bleedingSample collection – goodProcessing - goodPackaging – OKTransported to the laboratory at another locationCost to the patient – 10,000.
43 Result Scenario – 2 :- Protein C and Protein S deficiency Scenario – 3 :- FVIII deficiency – Haemophilia AAction:-Scenario – 2 :- life long anticoagulationScenario – 3 :- treatment with FVIII concentrateEffect:-Scenario – 2 :- Developed bleedingScenario – 3 :- no response to FVIII concentrate and continued bleeding into joints leading to disuse and fixed flexion deformity – a lifelong affection
44 Follow-up Scenario – 2 :- Protein C and S were normal Scenario – 3 :- it was Hemophilia B – deficiency of FIX and not FVIII. Responded very well to FIX concentrate.
45 RCA Sample collection – OK Processing and Packaging – OK Transportation - ? OK claimed by the laboratoryProcess of transportationAt 40C within 15 hrsAll coagulation tests to be done within 4 hrs of collection – as factors are labileexcept PT –upto 24 hrs provided sample is at ambient temperature of C.But if sample are kept at 40C then all tests including PT to be done in 4 hrs – cold activation of factors (VII and IX) start after 4hrs leading to rapid loss of factors.
46 RCASuch samples should be frozen in the laboratory and transported in Dry-ice.The present transportation requirements are only to fulfill the feel of coldThe onus of the integrity of the primary sample belongs to the laboratoryShould not wait for the Accreditation body to audit and find the errorsLack expertise on doing thrombophilia workup – they do it as it is eminently possibleDoing activity based assay for Protein S – strong false positive
47 Solutions The laboratory should show evidence of Training and SOPsRisk analysis and Preventive action on all sample trails and as new developAudit the sample trails with sampling by data loggers and the necessary actions arising from findings.Records of this needs to be verifiedLaboratories having such tests should have staff who are competent to handle such testsTraining - awarenessExperience - interpretationCPDP
48 Proper Storage and Transportation APTT assays - for heparin kept at 2 to 4°C or 18 to 24°C should be centrifuged within one hour of collection and the plasma tested within four hours from time of specimen collection.Frozen at -200C for up to two weeks or -700C for up to six months.A frost-free freezer should not be used.If testing cannot be performed immediately, the specimen may be held for a maximum of two hours at 40C until tested.Cold activation at 2-40C is not known to occur in 4 hrs. Cold activation will result in activation of FVII and also FIX resulting in falsely decreasing the times in PT and APTT tests respectively.
50 IQC and EQA: Precision and Accuracy IQC is required to ensure results are precise. Consistent over time (from day to day etc)EQA is required to confirm that results are accurate. Results are in agreement with those in other centres.
51 QC materials Similar in properties to test sample All vials or aliquots identicalStable over period of use (lyophilised,frozen)
52 IQC target ranges Instrument and reagent dependent. May be lot/batch dependentVerify or establish locally
53 IQC target ranges Minimum 20 tests in at least 10 sessions Exclude statistical ( or visual) outliersNormal distributionMean +/- 2 sd includes 95.5%Mean +/- 3 sd includes 99.7%
54 Potential problems with IQC Control more than 2 std deviations from mean?False alarms more commonControl 3 std deviations from mean?Less false alarms but lower error detection
55 Multi rules - Westgard ? Advantages Less false alarms Better error detectionDisadvantagesGenerally used with 2 controls measured once or twice (ie 4 determinations) – multiple results to trigger some alarms – either additional IQC testing within run or delay before alert?Patient testing if single outlying QC not investigated?Suited to coagulation testing??
58 Test 2 levels One IQC out of target range? Suspend new patient testing and reporting of results since last QC result within limits.Re-test to exclude analytical error. Still out?
59 IQC out of target range?Suspend new patient testing and reporting of results since last QC result within limits.Re-test to exclude analytical error. Still out?Replace QC material and retest. Still out?
60 IQC out of target range?Suspend new patient testing and reporting of results since last QC result within limits.Re-test to exclude analytical error. Still out?Replace QC material and retest. Still out?Replace reagents and retest. Still out?
61 IQC out of target range?Suspend new patient testing and reporting of results since last QC result within limits.Re-test to exclude analytical error. Still out?Replace QC material and retest. Still out?Replace reagents and retest. Still out?Suspend method and switch to backup, and contact higher authority ( Manufacturer?)
62 ObjectivesAchieving quality in coagulation testing is the prime objective of a haemostasiologist.Many measures have been prescribed and applied succesfully.Only limited by resources.Reagents from reputed manufacturersQuality of reagentsControlsInstrumentationMaintananceCost limitationsLow shelf life of reagentsWhat indicators could be used to achieve quality in testing when these limitations exists.
63 Case - 4A major hospital that is a HTC. Its laboratory had a letter from WFH-IEQAS about persistent significant outlier for FVIII assay.
67 Root Cause Analysis Details of the Methodology One stage APTT based assay on – ThrombolyzerReagents – Commercial from reputed manufacturer:- Freeze dried FVIII deficient plasma; APTT reagent; CaCl2; Buffer; Freeze dried Calibrator.All within expiryControls within acceptable limitsAsked for the timings from the coagulometer and Manually Plotted on a Semi-log (Log Lin) graph.
77 Minimal requirement for 1 stage FVIII assay 1/10 Dilution for standard graph – 47 – 59 SecsDifferences in dilution – 5-10 secsSource for Standard GraphSource for FVIII Deficient plasma
78 EQA can identify: problems a laboratory has with a particular test problems with a particular methodproblems with reference plasmasproblems in diagnosis or interpretation of results
79 Case - 5Haematologist reports inability of his laboratory to detect inhibitors to FVIII.Many of them are positive when patient is referred to a larger centre with experience in handling patients with inhibitor.
80 Root Cause Analysis Details of the Methodology Bethesda Method FVIII:C - One stage APTT based assay on – Amax DestinyReagents – Commercial from reputed manufacturer:- Freeze dried FVIII deficient plasma; APTT reagent; CaCl2; Buffer; Freeze dried Calibrator.All within expiryAsked for raw data.
81 PNP+Buffer PNP+Undil test plasma 1:128 1:256 1:512 1:1024 1:2 1:4 1:8 1:161:321:641:1281:2561:5121:1024Incubate at 37 deg for 2 hours.Perform factor VIII assays on all.
83 RCA Loss of FVIII:C independent of inhibitor – Factor VIII:C assay in the 1st tube that was the source of FVIII (PNP) incubated reduced from 100% to 63%Missed inhibitor in this patient but also runs the risk of many false positives due to loss of factor VIII independent of inhibitorNijmegen modification – addresses thisImplementation of Nijmegan – resource restrained ---FVIII levels in the first tube
84 FVIII levels in the 1st tube be not less than 80% Minimal RequirementsFVIII levels in the 1st tube be not less than 80%
86 Issues Related to Skin Bleeding time Time taken by a Standard skin wound to stop bleedingStandard Skin woundDevicesAvaialbility
87 Modified Ivy’s method Standard Skin wound –3 mm deep and 1.5 mm wide Lancet tip is 3 mm long and 1.5 mm wide at the base
88 Bleeding Time (Modified Ivy’s Method) BP cuff to 40 mm HgSelect an area avoiding any vein or angiomasClean the Volar aspectStab confidently three times and start Stop watch at the end of the third wound.At least one wound is STANDARD
89 Disadvantages: Skin bleeding times Time consuming. Operator variability / subjectivity.Invasive.Difficult to standardise.Establishment of a normal range ????Relatively insensitive to mild defects (mild platelet dysfunction, mild VWD).No specificity (an abnormal result won’t diagnose a particular defect … further testing).
90 PFA-100 – Screening testWhole blood - 5 min platelet function test.High shear stress flow system (‘pseudo-physiological’).Blood added to reservoir – vacuum – drawn into a capillary.Capillary has a membrane with aperture.Membrane coated with CollagenActive platelets adhere to membrane ... platelet activation & release ... platelet aggregation … blocks capillary device … instrument detects this as a ‘closure time’ (CT).Assesses cessation of blood flow (closure time = CT).Two cartridge types (C/ADP & C/Epi) with differing sensitivities.
91 PFA-100: Advantages: Only requires small amount of blood. Very simple … with proper training - anyone can do it (but typically lab person).Quick test (5-10 min)No real operator variability / subjectivity.Able to standardise.Able to establishment a normal range.Very sensitive and specific
92 The PFA-100 closure time should be considered optional in the evaluation of platelet disorders and function, and its use in therapeutic monitoring of platelet function is currently best restricted to research studies and prospective clinical trials.Von Willebrand disease
93 Utility of BT in severe bleeding disorder Among 852 patients evaluated for primary haemostatic defect at our centre from 2004 to 2008The sensitivity of Ivy’s method was100% - Glanzmanns thrombasthenia,85% - Bernard Soulier syndrome,68% - platelet secretion defect63% - von Willebrand disease100% - VWD-352% - VWD-1Rodegheiro F, Ruiz-saez A, Bolton-Maggs PHB, Hayward CPM, Nair SC and Srivastava A.Laboratory issues in Bleeding disorders. Haemophilia (2008), 14, 93–103.
94 Utility of BT in severe bleeding disorder (developing countries) Majority of the patients - more severe defectsBT by Ivy method if done as per reference specifications can be a good screening test for primary haemostatic disorder.BT by modified Ivy’s method compares well with any Template bleeding time method provided is done by the described reference method.
95 Standardization of Ivy’s BT Site - Select a sitelateral one-third of the forearm,2 to 3 cm distal to the antecubital crease,in an area devoid of hair, scars, tattoos, bruises, surface veins, infected skin, moles, or other lesions.Direction of incision –perpendicular (vertical) orparallel (horizontal) to the antecubital crease (Recommended).One direction, horizontal to be used consistently.A horizontal incision gives a longer bleeding time when compared to a vertical incision. The vertical incision may produce less scarring. Both procedures have a similar degree of reproducibility. The horizontal incision is more sensitive to the effects of aspirin.Blot from the sideonly a fewer technologists and more so experienced ones to perform and 2-3 incision.
96 Case 6A query from the neurosurgeon that a PT, INR of 1.26 was reported on his patient going for meningioma surgery the next day.What was the cause of this?The patient gave no prior bleeding history or significant past history .To rule out pre-analytical variables a repeat sample was collected by a Senior phlebotomist .
97 54 year old /F posted for removal of Meningioma Lab IDPTAPTTCTINROriginal sample13’’16’’1.2631’’33’Repeat sample –in duplicate(semi-automated )15.5”1.2132’’Manual – in duplicate 117’’1.342
98 TroubleshootingDifferent vial of the Reagent X was tested (15.6/13, INR 1.22)Different lot number of Reagent X were triedSimilar resultsOn further analysing the data for the past week we noticed more samples with INR values between 1.2 & 1.5
99 Random Values of patients between 01/03/2013 and 06/03 /2013 Lab IDPTAPTTCTINR2513’’16’’1.2631’’3917’’1.3432’’1918’’1.43453833’’3529Patients who had come as outpatients posted for Surgery. No prior history of bleeding
100 Troubleshooting cont’d The machine was re-calibratedDispensing volume of the pipette was checked& was normalReagent refrigerator temperatures were rechecked & found to be normalAn order was placed for a different Reagent (Reagent Y)Tests repeated with Reagent Y gave normal results
101 Was there any way that this could have been picked up before the clinician complained ?
102 ThromboplastinA lot of manufacturers make PT reagent with Acetone dried extract of Rabbit brain.Most of the laboratories use Semi-automated coagulometer.These reagents require constant stirring to ensure homogeneity before a part is taken for the test.Most of these coagulomters do not have a reagent stirrer position and doing a manual PT there is significant variability in the mixing that could be done.There could be similar issues with APTT reagents that could use colloidal contact activators.
103 Minimal requirementsIndicators and minor technical modifications help overcome limitations that exists and will help improve quality in Coagulation testing.
104 Internal Quality Control (IQC) Assessment of ongoing assay performanceRegular use of control materialAims to ensure that assays are performing according to specificationsHelps to ensure results are accurate and reliablePredominantly measures precisionIQC must be run & analysed with each assay performed within the laboratory
105 Internal Quality Control (IQC) Generally includes ‘normal’ and ‘abnormal’ sample controlsFor controls, select material yielding results around the midpoints of normal/reference range & of pathological/therapeutic range respectivelyResults are plotted using a Levey-Jennings chart.Target is generally mean/median of repeated QC test data.Assessment of data around these targets – thus, IQC predominantly measures precision.
106 Internal Quality Control (IQC) Performed for each analyte (i.e., VWF:Ag, VWF:RCo, VWF:CB, FVIII:C, FIX, etc)Time-frame of testing should be suited to each analyte.For continuous test systems ICQ is perform periodically (e.g., every ‘2, 4 or so’ hours or every ‘10 or 40’ samples, etc).For tests performed in batches, perform at start of test runs, with middle and end for large runs.Run normal and ‘pathological’ controls
107 In the case of any unexplained abnormal coagulation test result, a new specimen should be obtained and the test repeated
108 Everybody talks about it, nobody understands it. Haemostasis = LoveEverybody talks about it, nobody understands it.