Quality Issues in Coagulation
Laboratory
Sukesh C Nair
CMC
Vellore
India
Laboratory issues in coagulation
testing
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•
Pre analytical
Analytical
Post analytical
Biological variation (inter and intra individual
variability)
Pre analytical phase:
• Most issues are here:• Lack of standardized procedures for
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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
8
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.
9
Vacuum collection device
10
Venipuncture
11
Venipuncture
12
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
17
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
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Large Lab
Issues in samples received
Leakage
Hemolysis
Clot
Needle stick injuries
22
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.
23
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
28
Blood transfer device
Step- 1
Step- 2
Step- 3
Step- 4
Blood Transfer Device
30
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
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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
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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 18-240C.
• 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.
http://www.biochemia-medica.com/
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
out
in
QC 1
material
In
Out
In
out
QC 2
material
out
out
in
in
PT reagent
In
In
Out
Out
APTT reagent
Out
Out
Out
Out
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
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
Dilution
Cal Secs
Test Secs
80
1/10 (100%)
71.0
85.5
70
1/20 (50%)
84.5
100.0
1/40 (25%)
98.5
113.5
60
50
WFH IEQAS Report – CMC Vellore
100
90
80
Dilution
40 PNPSecs
Test Secs
1/5 (200%)
47.0
1/10 (100%)
53.0
64.0
1/20 (50%)
58.0
69.0
1/40 (25%)
64.0
75.5
70
60
40 PNP has 111 u/dL FVIII:C. FVIII:C on sample W09/14 is 26.4 x 1.11 = 29.3)
50
40
Comparison
Comparison
100% timings very high
FVIII in the Calibrator has
deteriorated
Difference between dilutions
higher
FVIII deficient plasma has
deteriorated
Deterioration of Reagents
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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
40 PNP
5 PNP
3 PNP - a
3 PNP - b
FVIII:C
107.9
119.8
111.8
111.8
FIX
104.0
115.0
104.6
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
1:2
PNP+Undil test plasma
1:4
1:64
1:128
1:8
1:256
Incubate at 37 deg for 2 hours.
Perform factor VIII assays on all.
1:16
1:512
1:32
1:1024
• PNP:B – 63%
• PNP: Neat – 48%
• 1:2 – 65%
1:4 – 68%
• 1:8 – 67%
1:32 – 63%
• 1:64 – 69%
• 1:128 – 68%
• 1:256 – 67%
1:512 – 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
Minimal Requirements
FVIII levels in the 1st tube be not less
than 80%
Evaluation of Primary haemostasis
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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
Skin bleeding times
• Disadvantages:
 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
PT
APTT
Lab ID
C
T
INR
C
T
Original sample
13’’
16’’
1.26
31’’
33’
Repeat sample –in
duplicate
(semi-automated )
Manual – in
duplicate 1
13’’
16’’
15.5”
1.26
1.21
31’’
32’’
13’’
17’’
1.34
31’’
32’’
13’’
16’’
1.26
31’’
33’
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
PT
APTT
Lab ID
C
T
INR
C
T
25
13’’
16’’
1.26
31’’
31’’
39
13’’
17’’
1.34
31’’
32’’
19
13’’
18’’
1.43
31’’
31’’
45
13’’
17’’
1.34
31’’
31’’
38
13’’
16’’
1.26
31’’
33’’
35
13’’
17’’
1.34
31’’
31’’
29
13’’
17’’
1.34
31’’
31’’
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
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Internal Quality Control (IQC)
 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.
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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
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• In the case of any unexplained abnormal
coagulation test result, a new specimen
should be obtained and the test repeated
Haemostasis = Love
Everybody talks about it, nobody
understands it.
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Thank You