Effects of Pre-analytical Factors Online Training

Identify the prevalence of pre-analytical errors and two key factors to be considered in the selection of collection tubes used for hemostasis testing.  State the impact of underfilled collection tubes on these tests and list three key factors affecting sample stability.  This clinical laboratory training qualifies for continuing education units (CEU).

Upon successful completion of this course, you will be able to: Identify the prevalence of errors in the pre-analytical phase of laboratory testing List two key factors in the selection of collection tubes used for hemostasis testing State the impact of under filled collection tubes on common hemostasis tests Identify three key factors affecting sample stability for hemostasis testing Select Next to continue. Welcome to the The Effects of Pre-analytical Factors Online Training course.  This course examines the effects of pre-analytical factors on coagulation testing. This course was developed by: Dr. Steve Kitchen, Sheffield Haemophilia and Thrombosis Centre Select Next to continue.   Lab section Clin Chem All labs Primary care Stat lab All labs Data period 1 yr 6 yr 6 month 3 month 3 years % of samples with errors 0.05%   0.1% 0.5% 0.6 % of errors occurring in Pre-analytical phase 32% 53% 56% 68% 75% Review of 5 studies (Bonini, Clin Chem, 2002)   A J Clin Path, 2003 3 large Italian labs - up to 3.5M samples per year Samples from inpatients   Number per 1000 tubes % Per week (n) Haemolysed samples   1.1 - 4.3 0.1 - 0.4% 300 Insufficient sample    1 - 1.3 0.1% 90 Plebani et al, 2006 WHO - Guidelines for plasma used for thromboplastin calibration   CLSI (USA) - Guidelines for blood specimens for coagulation tests   BOTH recommend 0.109M trisodium citrate (2H2O)  -  (3.2%)   But 3.8% continues to be used in some countries Dade® Innovin® (PT), Dade Actin® FS (APTT) / MLA   Normals PT and APTT 5  - 7% greater in 0.129M than 0.109M   24 Heparin APTTs 10% greater in 0.129M   6/33 INRs differed by > 0.7 (max difference 2.7 INR units)   Smaller or absent differences with insensitive reagents Adcock et al, 1997 PT of normals and OAC patients with 4 reagents (0.109M and 0.129M)   PTs of normals  1 – 5% greater in 0.129M   PTs of patients 10 – 23% greater in 0.129M   Effect is to reduce ISI in 0.129M relative to 0.109M by approx 10% Duncan et al,1994   Assign ISI with 0.129M and apply to samples in 0.109M —INRs would be too low   Assign ISI with 0.109M and apply to samples in 0.129M —INRs would be too high   An INR of 4.5 could be out by up to 20% in study of Chantarangkul (1998) Review scientific literature   Review manufacturer’s data   Assess locally, where necessary   Pay particular attention to screening tests   Effects may be reagent-specific   Expect differences unless evidence to contrary   Consider new local normal range—particularly screening tests Minimum fill volume    0.109M citrate PT (Innovin)                     60% APTT (Actin FS)              70% 0.129M citrate PT                                  70% APTT                              90% Adcock et al 1998 First sample – PT 104 seconds (properly filled 1 mL sample)   Second sample (no treatment) – PT 14 sec (properly filled 5mL sample)   Artifact? Wrong patient? Sheffield patient   Third sample – PT 14 seconds   First sample mixed 0.5 ml blood with 0.5 mL anticoagulant in a 5 mL tube this mix was added to a paediatric (1 mL) tube with further 0.1 mL anticoagulant   CITRATE excess prolonged PT from 14 sec to 104 seconds 100% 80% of target fill 60% Range from 56% to 79% full - mean 67%   PT -sec APTT -sec Fbg -g/L TT -sec Full 14.5 30.5 4.0 24.2 Under filled 17.4 35.6 3.75 26.5 P value 0.03 <0.001 <0.001 <0.001 Max differency 19 sec 14 sec 0.6 4.9 Unpublished Sheffield data Unpublished Sheffield data Adcock et al, Blood Coag. & Fib., 1998, 9, 6, 1-8 Blood samples (n = 30) collected into citrate APTT and anti Xa determined within 1 hour and after 4 hours mixing APTT (IL) - sec 70.6 52.6 APTT (AFS) - sec 53.9 38.2 Anti Xa - iu/mL 0.33 0.22 Unpublished Sheffield data APTT of 5 heparinised patients   Same sized container (holds 6.7 mL)   0.2 mL  0.11M citrate + 1.8 mL blood   or  0.5 mL + 4.5 mL   Mean APTT  120 sec (1.7 mL air space)   Mean APTT   79 sec  (4.7 mL air space)   PF4 in 2 mL 2x that in 5 mL   Under filling has similar effect Ray 1991 Citrate, Theophylline, Adenosine, Dipyridamole   Inhibit cAMP breakdown (Theo, Dipyr) or stimulate production (Aden) – elevate cAMP   Inhibits Platelet activation and release of PF4   Avoids loss of unfractionated heparin post collection Contant G et al., Thromb Res., 1983 van den Besselaar A et al., Thromb Heam., 1987     Anti Xa <1 hr Anti Xa 4 hrs AFS <1 hr AFS 4 hr APTT SP <1 hr APTT SP 4 hr Citrate 0.33 0.22 53.9 38.2 70.6 52.6 CTAD 4.5 mL 0.38 0.38 56.6 52.7 71.4 67.8 Unpublished Sheffield data 30 tubes from each of 2 batches of CTAD tube (Greiner) – 4.5 mL draw   Actual mean volume drawn 4.3 and 4.6 mL   Anticoagulant vol 0.44 and 0.43 mL (CV 1%)   Dipyridamole content 25 – 50% and 50 – 75% of stated concentration   “None of the 2 batches were within the range of specification required” Polack et al, 1997 15 g/L 6 g/L   1 g/L   control 34 consecutive haemolysed samples rejected for visible haemolysis Mini VIDAS   10 normal subjects   Freeze thaw lysed whole blood added back to plasma   D-Dimer higher by 7 - 8% (p < 0.05) in presence of 3% lysed cells (~5g/L plasma Hb)   Higher by 15 - 18% at 9% cell lysis   Variable between individuals Lippi et al, 2006   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) Unpublished Sheffield data *Not available for sale in the U.S.   Unpublished Sheffield data *Not available for sale in the U.S.   Unpublished Sheffield data *Not available for sale in the U.S. Unpublished Sheffield data *Not available for sale in the U.S.   Checks sample aliquot at 3 wavelengths   Checks for Haemolysis at 575 nm   Checks for Icterus at 405 nm   Checks for Lipaemia at 660 nm *Not available for sale in the U.S.   * Not available for sale in the U.S. ClinOleic 20% intravenous fat emulsion (Baxter) at 1:40 dilution in plasma     PT -sec APTT -sec Fbg -g/L TT -sec   No lipid Lipid No lipid Lipid No lipid Lipid No lipid Lipid CS2100i* 11.4 11.2 29.5 24.1 4.19 4.20 15.6 17.7 N = 20 20 20 17 20 20 20 20 Unpublished Sheffield data   Day 1 (lipaemic) Day 5 Trigrlycerides 7.4 umol/L 3.9 umol/L h (CA1500) 282 80 Reagent 1 INR 7.6 INR 3.9 Reagent 2 INR 5.3 INR 5.0 Unpublished Sheffield data Clotting assays except Clauss Fibrinogen Default = 660nm Sub-wavelength = 800nm Clauss Fibrinogen Default = 405nm Sub-wavelength = 660nm Wavelength switching (to 800 nm) if: Light level error Transmitted light error Turbidity level over error   Slight coagulation error   Frozen/stored –74°C Frozen –74°C Stored –24°C Frozen/Stored –24°C < 5% change   6 months 3 months 3 months < 10% change   18 months 6 months 3 months Woodhams et al 2001 Transport at 50m/min – no effect on haematology cell counting or routine coagulation (Kratz et al 2007)   Haemolyis can occur in some systems   30% loss of platelet function (system with travel at 8m/sec) Unpublished Sheffield data Unpublished Sheffield data Whole blood for INR determinations stable for up to 3 days (Baglin et al 1997)   Maximum of 6 hours between collection and testing for INR (Geest-Dallerop et al 1997)   Different test systems in use in the 2 studies   Practically all aspects of sample collection and processing! Collection Tube - anticoagulant and filling   Transport   Processing   Storage   Interfering substances - Haemolysis, lipids The final quality of haemostasis test results is totally dependent on the quality of the sample and its processing Anita Woolley Candy Hartshorne Hazel Carter Peter Cooper