Workflow Benefits of Test-specific thresholds on Sysmex CS analysers Steve Kitchen, PhD Lead Clinical Scientist Sheffield Haemophilia & Thrombosis Centre Sheffield, UK

Causes of errors in laboratory medicine Review of 5 studies Lab section Clin Chem All labs Primary care Stat lab Data period 1 yrs 6 yrs 6 months 3 months 3 yrs % of samples with errors 0.05 0.1 0.5 0.6 % of errors occurring in pre- analytical phase 32 53 56 68 75 (Bonini, Clin Chem 2002)

Causes of sample rejection Clotted Under-fill Labelling Haemolysis 2010 411 356 (0.6%) 327 119 (0.2%) 2011 439 349 255 247 (0.4%) 2012 204 434 (0.7%) 141 650 (1.1%) 2013 (Accident and emergency) 0.9% 2.6%

Are rejected samples replaced? 1077 samples rejected in a 3 month period (2014) (~3% of all samples) (48%) not replaced within 24 hours (including 106 INRs) (29%) not replaced within 7 days (including 24 INRs)

Haemolysis Plasma Haemoglobin 15 g/l 6 g/l 1 g/l control

What level of haemolysis is routinely encountered? 34 consecutive haemolysed samples rejected for visible haemolysis n Plasma Hb g/l

Effect of Haemolysis Freeze/thaw/artificial lysis Citrated whole blood frozen Lysed cells added to plasma from same subject Different mixtures prepared

FREEZE/THAW (spiked normal plasma) 1000 mg/dl = 10 g/l

FREEZE/THAW (spiked normal plasma)

FREEZE/THAW 3

Effect of Haemolysis – freeze/thaw/artificial lysis 6 normal subjects Vacutainer 0.109M trisodium citrate (3.2%) Whole blood frozen Lysed cells added to plasma from same subject 12 different mixtures prepared Mean plasma haemoglobin 0 – 20 g/l (10-15% haemolysis)

Haemolysis in real patient samples Samples with haemolysis that failed local visual inspection assessment (observer variable?) Free haemoglobin in plasma samples with haemolysis ranged from 0.5 to 9 g/l. Matched samples from same patients which were Free of visible haemolysis Collected within 4 hours of haemolysed sample

Samples with > 4 sec difference in APTT with AFS (normal <30 Samples with > 4 sec difference in APTT with AFS (normal <30.5 sec) n= 5/56 Sample code Haemolysed APTT (sec) Clear Plasma Hb (g/l) 3 35.7 30.8 30 34 38.8 1 33 33.2* 27.2 35 19.3 24.1 41 58 47 0.5 * False abnormal

Not significant. Mean difference 0.05

False abnormal PT

Samples falsely raised above D-Dimer > VTE cut-off (0.5 µg/ml) Haemolysed D-Dimer (µg/ml) Clear Plasma Hb (g/l) 0.95 0.19 0.5 1.53 0.24 1 2.7 0.35 2.98 0.48 5 Potentially unnecessary ultrasounds

In Vivo haemolysis (Sickle cell anaemia)

HIL check – Sysmex CS series Checks daughter plasma aliquot at 3 wavelengths Checks for Haemolysis at 575 nm Checks for Icterus at 405 nm Checks for Lipaemia at 660 nm Flag levels 1 (low) to 5 (high) Some models - test specific thresholds/flags

Haemolysis Conclusions Level of haemolysis that causes diagnostic errors is variable and test dependant Effects are not always related to the degree of haemolysis when present Therefore any in vitro hemolysis – reject for APTT PT/INR is less affected

Tracking for coagulation analysers? Combined with chemistry samples _ sample numbers, Centrifugation conditions Detection of Haemolysis and underfilling (visual vs automated Auto-validation of selected results– automated pre-analytics

RHH – Old layout

RHH – New layout

Work in progress

Temporary home!

Sample Transport Simplicity and reliability with belt free technology Sample transport via individually powered CARS Max. throughput of 6000 tubes / hour NFC tech for sample identification Scalable, Modular, Flexible design Talk through GLP Sample delivery system and benefits Speed Efficiency

Taking shape

Recommendations for flag levels Test Flag level Plasma Hb APTT – AFS 1 0.4 g/dl APTT - FSL DDimer FXIII Antithrombin 3 1.8 g/dl PT /INR Innovin 5 (4 with derived fib) 3.0 (2.4) g/dl Protein C 5 3.0 g/dl PT/INR – Thromborel S Fibrinogen (clauss) Thrombin Time Anita Woolley et al poster ISTH 2015

Anita Woolley Yuka Tabuchi Acknowledgements Anita Woolley Yuka Tabuchi

Wavelengths used for result reporting Clotting assays except Clauss Fibrinogen Default = 660nm Sub-wavelength = 800nm Clauss Fibrinogen Default = 405nm Sub-wavelength = 660nm

Wavelength Switching for lipaemic samples Wavelength switching (to 800 nm) if: Light level error Transmitted light error Turbidity level over error Slight Coagulation error