Quality Issues in Coagulation Laboratory

Quality Issues in Coagulation Laboratory
Sukesh C Nair CMC Vellore India

Laboratory issues in coagulation testing
Pre analytical Analytical Post analytical Biological variation (inter and intra individual variability)

Pre analytical phase: Most issues are here:-
Lack of standardized procedures for sample collection patient preparation specimen acquisition handling and storage often outside the control of the laboratory performing the test.

Patient preparation Testing for haemostasis should be avoided
drugs interfering with the function of platelets and coagulation factors closer 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.

Proper Collection Use a blood collection system that collects the specimen directly into a tube containing the anticoagulant Order 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 inversions Prothrombin Time/Internal normalized ratio (PT/INR) and activated partial thromboplastin time (APTT) results are not affected if tested on the first tube drawn When 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.

Proper Collection Syringe 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.

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 laboratory The hospital uses Evacuated tubes and the sample collection manual details out the procedure as has been mentioned in literature Prepare 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.

Tying the Tourniquet

Precautions Tourniquet application shall not exceed one minute.
If the tourniquet is applied to select the best vein it must be released and redone after two minutes.

Vacuum collection device

Venipuncture

Scenario 1 Prepare 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.

Inpatients Sampled 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.

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.

Evacuated tube system - Disadvantages
Cost The tubes gets connected to the same needle whereby the contents of one tube can contaminate the other Clot activator contaminating Coagulation or cell count EDTA (Cell count) contaminating Coagulation or Clot tube Heparin contaminating Coagulation, clot tube or cell count Order of draw

Order of Draw

Non Evacuated Tube System
Blood out of the vein is not anticoagulated Risk 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 forming Clot affects all haematology cell counts – Hb/RCC/WCC and more dangerously Platelet count. All coagulation tests will be abnormal

Audit Did this finding have an impact on the integrity of the primary sample Risks are known ??? Are the personnel doing phlebotomy aware Sample rejection register – many rejections of samples received from inpatients Under/over filled Clot in the sample

Root Cause Analysis Nurses are not aware but lab is aware
Nurses training records Sample collection is part of induction training 1 & ½ hrs All have attended Training content Only mentions the procedure No mention of when to use syringe and needle When can this situation be changed to modified evacuated tube system – Luer adapter How to transfer blood from syringe to tubes maintaining the integrity of the ETS and the blood sample.

Proper Collection 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. 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.

Scenario 1b Large Lab Issues in samples received Leakage Hemolysis
Clot Needle stick injuries

Large Lab The 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 and needle holder.

Large Lab The 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 causes Risk of needle stick injury to the operator 6 mL tube to which 4 mL blood has to be added, causing hemolysis while abruptly stopping the process Some of the franchisee’s are opening the tubes to add to avoid piercing. Recapping causes leakage Also the instructions mentions mixing as shaking and not inversions. Clot in the sample

New Technology Full awareness Advantages vs Disadvantages
Risks vs Benefit When can this situation be changed to modified evacuated tube system – Luer adapter How 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

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 draw When samples are collected in Syringe

Blood transfer device Blood Transfer device

Blood transfer device Step- 1 Step- 2 Step- 3 Step- 4

Blood Transfer Device

Proper Collection 105 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

Issues related to sample collection
Under filling - dilution of plasma resulting in underestimation of clotting factor levels overfilled 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).

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

Issues related to Storage
Inadeqautely thawed samples would lead to inhomogeneous sampling Cryoprecipitate portion would be selectively sampled very high levels of FVIII:C, VWF, Fibrinogen or FXIII. Cryo poor part very low levels of FVIII:C, VWF, Fibrinogen or FXIII. Frozen samples should be thawed and mixed at 370C waterbath for 5 minutes before testing.

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.

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.

Preanalytical errors

Scenario -2 Sample sent for Thrombophilia markers in a patient with proximal DVT (Deep vein thrombosis) Sample collection – good Processing - good Packaging – OK Transported to the laboratory at another location Cost to the patient – 35,000.

Scenario - 3 Sample sent for Hemophilia assay in a patient with severe bleeding Sample collection – good Processing - good Packaging – OK Transported to the laboratory at another location Cost to the patient – 10,000.

Result Scenario – 2 :- Protein C and Protein S deficiency
Scenario – 3 :- FVIII deficiency – Haemophilia A Action:- Scenario – 2 :- life long anticoagulation Scenario – 3 :- treatment with FVIII concentrate Effect:- Scenario – 2 :- Developed bleeding Scenario – 3 :- no response to FVIII concentrate and continued bleeding into joints leading to disuse and fixed flexion deformity – a lifelong affection

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.

RCA Sample collection – OK Processing and Packaging – OK
Transportation - ? OK claimed by the laboratory Process of transportation At 40C within 15 hrs All coagulation tests to be done within 4 hrs of collection – as factors are labile except 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.

RCA Such samples should be frozen in the laboratory and transported in Dry-ice. The present transportation requirements are only to fulfill the feel of cold The onus of the integrity of the primary sample belongs to the laboratory Should not wait for the Accreditation body to audit and find the errors Lack expertise on doing thrombophilia workup – they do it as it is eminently possible Doing activity based assay for Protein S – strong false positive

Solutions The laboratory should show evidence of
Training and SOPs Risk analysis and Preventive action on all sample trails and as new develop Audit the sample trails with sampling by data loggers and the necessary actions arising from findings. Records of this needs to be verified Laboratories having such tests should have staff who are competent to handle such tests Training - awareness Experience - interpretation CPDP

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.

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.

QC materials Similar in properties to test sample
All vials or aliquots identical Stable over period of use (lyophilised,frozen)

IQC target ranges Instrument and reagent dependent.
May be lot/batch dependent Verify or establish locally

IQC target ranges Minimum 20 tests in at least 10 sessions
Exclude statistical ( or visual) outliers Normal distribution Mean +/- 2 sd includes 95.5% Mean +/- 3 sd includes 99.7%

Potential problems with IQC
Control more than 2 std deviations from mean? False alarms more common Control 3 std deviations from mean? Less false alarms but lower error detection

Multi rules - Westgard ? Advantages Less false alarms
Better error detection Disadvantages Generally 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??

… in my lab

Troubleshooting IQC Why 2 levels?
PT 1 PT 2 APTT 1 APTT 2 problem out in QC 1 material In Out QC 2 PT reagent APTT reagent Instrument

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?

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?

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?

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?)

Objectives Achieving 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 manufacturers Quality of reagents Controls Instrumentation Maintanance Cost limitations Low shelf life of reagents What indicators could be used to achieve quality in testing when these limitations exists.

Case - 4 A major hospital that is a HTC. Its laboratory had a letter from WFH-IEQAS about persistent significant outlier for FVIII assay.

WFH IEQAS Report

WFH IEQAS Performance evaluation

Root Cause Analysis Details of the Methodology
One stage APTT based assay on – Thrombolyzer Reagents – Commercial from reputed manufacturer:- Freeze dried FVIII deficient plasma; APTT reagent; CaCl2; Buffer; Freeze dried Calibrator. All within expiry Controls within acceptable limits Asked for the timings from the coagulometer and Manually Plotted on a Semi-log (Log Lin) graph.

110 100 90 80 Dilution Cal - Secs Test - Secs 1/10 (100%) 71.0 85.5 1/20 (50%) 84.5 100.0 1/40 (25%) 98.5 113.5 70 60 50

WFH IEQAS Report – CMC Vellore

100 90 80 70 Dilution 40 PNP- Secs Test - Secs 1/5 (200%) 47.0
1/5 (200%) 47.0 1/10 (100%) 53.0 64.0 1/20 (50%) 58.0 69.0 1/40 (25%) 75.5 60 50 40 40 PNP has 111 u/dL FVIII:C. FVIII:C on sample W09/14 is 26.4 x 1.11 = 29.3)

Comparison

Comparison 100% timings very high Difference between dilutions higher
FVIII in the Calibrator has deteriorated FVIII deficient plasma has deteriorated

Deterioration of Reagents
Reputed manufacturer Not directly represented Through a distributor Transportation and storage compromised

Proposed Corrective actions
Get fresh reagent Similar risk Make in-house - fresh FVIII Deficient plasma Harvesting from a Severe Hemophilia A patient without inhibitor Calibrator PNP ???

Pooled Normal Plasma Source FVIII:C FIX 40 PNP 107.9 104.0 5 PNP 119.8
115.0 3 PNP - a 111.8 104.6 3 PNP - b 115.4

Corrective action effective
20 PNP and in-house FVIII deficient plasma

Minimal requirement for 1 stage FVIII assay
1/10 Dilution for standard graph – 47 – 59 Secs Differences in dilution – 5-10 secs Source for Standard Graph Source for FVIII Deficient plasma

EQA can identify: problems a laboratory has with a particular test
problems with a particular method problems with reference plasmas problems in diagnosis or interpretation of results

Case - 5 Haematologist 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.

Root Cause Analysis Details of the Methodology Bethesda Method
FVIII:C - One stage APTT based assay on – Amax Destiny Reagents – Commercial from reputed manufacturer:- Freeze dried FVIII deficient plasma; APTT reagent; CaCl2; Buffer; Freeze dried Calibrator. All within expiry Asked for raw data.

PNP+Buffer PNP+Undil test plasma 1:128 1:256 1:512 1:1024 1:2 1:4 1:8
1:16 1:32 1:64 1:128 1:256 1:512 1:1024 Incubate at 37 deg for 2 hours. Perform factor VIII assays on all.

PNP:B – 63% PNP: Neat – 48% 1:2 – 65% :4 – 68% 1:8 – 67% :32 – 63% 1:64 – 69% 1:128 – 68% 1:256 – 67% :512 – 72%

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 inhibitor Nijmegen modification – addresses this Implementation of Nijmegan – resource restrained --- FVIII levels in the first tube

FVIII levels in the 1st tube be not less than 80%
Minimal Requirements FVIII levels in the 1st tube be not less than 80%

Evaluation of Primary haemostasis
Skin Bleeding time PFA LTA – Aggregometry Impedence methods

Issues Related to Skin Bleeding time
Time taken by a Standard skin wound to stop bleeding Standard Skin wound Devices Avaialbility

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

Bleeding Time (Modified Ivy’s Method)
BP cuff to 40 mm Hg Select an area avoiding any vein or angiomas Clean the Volar aspect Stab confidently three times and start Stop watch at the end of the third wound. At least one wound is STANDARD

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).

PFA-100 – Screening test Whole 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 Collagen Active 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.

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

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

Utility of BT in severe bleeding disorder
Among 852 patients evaluated for primary haemostatic defect at our centre from 2004 to 2008 The sensitivity of Ivy’s method was 100% - Glanzmanns thrombasthenia, 85% - Bernard Soulier syndrome, 68% - platelet secretion defect 63% - von Willebrand disease 100% - VWD-3 52% - VWD-1 Rodegheiro F, Ruiz-saez A, Bolton-Maggs PHB, Hayward CPM, Nair SC and Srivastava A. Laboratory issues in Bleeding disorders. Haemophilia (2008), 14, 93–103.

Utility of BT in severe bleeding disorder (developing countries)
Majority of the patients - more severe defects BT 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.

Standardization of Ivy’s BT
Site - Select a site lateral 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) or parallel (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 side only a fewer technologists and more so experienced ones to perform and 2-3 incision.

Case 6 A 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 .

54 year old /F posted for removal of Meningioma
Lab ID PT APTT C T INR Original sample 13’’ 16’’ 1.26 31’’ 33’ Repeat sample –in duplicate (semi-automated ) 15.5” 1.21 32’’ Manual – in duplicate 1 17’’ 1.34 2

Troubleshooting Different vial of the Reagent X was tested (15.6/13, INR 1.22) Different lot number of Reagent X were tried Similar results On further analysing the data for the past week we noticed more samples with INR values between 1.2 & 1.5

Random Values of patients between 01/03/2013 and 06/03 /2013
Lab ID PT APTT C T INR 25 13’’ 16’’ 1.26 31’’ 39 17’’ 1.34 32’’ 19 18’’ 1.43 45 38 33’’ 35 29 Patients who had come as outpatients posted for Surgery. No prior history of bleeding

Troubleshooting cont’d
The machine was re-calibrated Dispensing volume of the pipette was checked & was normal Reagent refrigerator temperatures were rechecked & found to be normal An order was placed for a different Reagent (Reagent Y) Tests repeated with Reagent Y gave normal results

Was there any way that this could have been picked up before the clinician complained ?

Thromboplastin A 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.

Minimal requirements Indicators and minor technical modifications help overcome limitations that exists and will help improve quality in Coagulation testing.

Internal Quality Control (IQC)
Assessment of ongoing assay performance Regular use of control material Aims to ensure that assays are performing according to specifications Helps to ensure results are accurate and reliable Predominantly measures precision IQC must be run & analysed with each assay performed within the laboratory

Generally includes ‘normal’ and ‘abnormal’ sample controls For controls, select material yielding results around the midpoints of normal/reference range & of pathological/therapeutic range respectively Results 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.

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

In the case of any unexplained abnormal coagulation test result, a new specimen should be obtained and the test repeated

Everybody talks about it, nobody understands it.
Haemostasis = Love Everybody talks about it, nobody understands it.

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Quality Issues in Coagulation Laboratory

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