primary sample collection systems: how useful are they in

Primary sample collection systems: How useful are they in reducingHow useful are they in reducing

Preanalytical variability?

Stephen Church pBD Diagnostics - Preanalytical Systems,

Oxford, UK

How good is your sample quality?

• Appropriate Sample Identification?Appropriate Sample Identification?


• Haemolysis?Haemolysis?


• Fibrin/Clotting Plasma?g


• Latent Clotting Serum?


• Incorrect filling/Insufficient Quantity?

1. Petersonn, P & Gottfired, E. The Effect of Inaccurate Blood Volume Draw on Prothrombin Time (PT) & Activated Partial Thromoplastin Time, Thrombosis Haemostasis, 47(2)101 -103, 1982


• Poor Barrier Formation? 1

2

1 Spirit s T Iodinated Contrast Media Interfere ith Gel1. Spiritus,T. Iodinated Contrast Media Interfere with Gel Barrier Formation in Plasma and Serum Separator Tubes.Clin. Chem., Jul 2003; 49: 1187 - 1189.

2. van den Ouweland. High Total Protein Impairs Appropriate Gel Barrier Formation in BD Vacutainer Blood Collection Tubes Clin. Chem., Feb 2007; 53: 364 - 365.


• Good or Bad Sample?Preanalytical error:• long tourniquet time

• wrong order of draw

• storage at 4°C for hours before centrifugation

• patient came by bicycle and ran up the stairs because he was late, blood collection was donebecause he was late, blood collection was done immediately

• tube shall be used for monitoring test of tricyclic antidepressents

• tube has been forgotten over night on the ward• tube has been forgotten over night on the ward before transport

Consequences:elevated proteins• elevated proteins

• carry over of additive, e.g. K

• elevated K because RBC Na/K-ATPase was inhibited

• incorrect lab results because of stress & activity (Glucose, lactate, creatinin, ...)

• falsely low TDM results

i t l b lt K• incorrect lab results, e.g.K

Analytical trends

Analyte Concentration

mmol/L

pmol/L

Does it matter?

1. Foubister, Vida. Cap Today Bench press: The Technologist/technician shortfall is putting the squeeze on laboratories nationwide; September 20002. Datta, P. Resolving Discordant Samples. Advance for the Administrators of the Laboratories; July 2005: p.60.

Reducing Variability in the PA Phase

PATIENT PHLEBOTOMY SPECIMEN TRANSPORT PROCESSING ANALYSIS SPECIMEN

STORAGE

• Patient ID• In Vivo Hemolysis

due to patient factorsMetabolic

• Catheter, IV Collection

• Capillary CollectionNeedle Gauge

• Origin of Specimen Maternity, Emergency & Intensive Care

• Origin of Specimen

• Verify Tube with Request

• Generate Laboratory Barcode

• Long Time after Centrifugation

• Serum vs. Plasma vs. Whole BloodTube mixed prior

• Re-Centrifugation Add-On

• Post-Analysis Storage Temperature• Metabolic

Disorders (eg. Liver disease)

• Chemical Agents (eg. Medication)

• Physical Agents (eg. Mechanical heart valves)

• Needle Gauge• Position of Arm• Location of

Venipuncture• Antiseptic Used for

Phlebotomy• Tourniquet Time

Origin of Specimen In-patient

• Origin of Specimen Physician Office Lab

• Origin of Specimen Out-patient

• Tubes Transported

Barcode• Time between

Collection and Centrifugation

• Type of Centrifuge• Centrifuge

Calibrated• Centrifuge

• Tube mixed prior to analysis

• Re-run Specimen (Same Day)

• Verify Instrument Cal & Controls

• Identify Instrument Used for Testing

Temperature• Duration of

Storage

heart valves)• Infectious Agents

(eg. Bacteria)• Traumatic Draw• Fist Clenching• Tube Type

Collected• Tube Under Filled• Order of Draw

Tubes Transported Vertical or Horizontal

• Transport by Pneumatic Tube

• Courier Transport• Transport Duration• Pre Centrifugation

• Centrifuge Temperature Extremes

• Speed of Centrifuge

• Duration of Centrifugation

• Poor Separator

Used for Testing• Identity Tech

Performing Testing• Verify Report Value

• Vigorous Mixing• No Mixing• Syringe Transfer

• Pre-Centrifugation and Transport Temperature

• Poor Separator Barrier Integrity

• Cells on Stopper• Automated

Decapping• Specimen Re-

CentrifugationAli t L b li• Aliquot Labeling

• Specimen Aliquoted

The Sample Collection System

Cl

Vascular Access Device + Sample Container

ClosureRubber Stopper

Cannula materialL b i i

Tube MaterialLubricationCannula gaugeFluid Path

AdditivesLabel

EvacuationSterilization

Reducing PAV: Sample ID

CAP Q P b St d t *CAP-Q Probes Study outcomes *

# of labs included in the study 120Total # of errors recorded in the survey period 6.705

Total # of errors for which it has been possible to identify a precise cause 4.852 (72%)

Cause of the error # IncidenceCause of the error # IncidenceWrong identification (labeling) of primary samples 2.691 55,5 %Wrong Lab check-in (prescription) 1.088 22,4 %Data transcription 604 12,4 %Wrong identification (labeling) of secondary tubes 184 3,8 %Test results in-put 80 1,7 %Test results in put 80 1,7 %Others (miscellaneous) 205 4,2 %

1. CAP-Q Probes Study (Valenstein PN, et al. Identification Errors Involving Clinical Laboratories Arch Pathol Lab Med 2006;130:1106-1113)

Reducing PAV: Sample Collection

• Ensuring the correct sample ID:• Minimum requirements for patient identification are defined:• Minimum requirements for patient identification are defined:

– Patient full name, address, Patient ID, Patient DOB1

– Solutions:

• Accurate label placement is key for barcodes– Solutions:

1. Clinical and Laboratory Standards Institute, Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. 6th ed. H3-A6, Wayne, PA: CLSI; 2007


• Choosing the Serum non gel

Other CLSI/ISO1,2

gcorrect sample type

• Solution: Use of colour codes

g

Serum Separator*

colour codes HeparinHeparin Separator*

EDTA

Citrate

Glucose

ESR

* Consult Manufacturer

1. Clinical and Laboratory Standards Institute, Tubes and Additives for Venous Blood Specimen Collection; Approved Standard. 5th ed. H1-A5, Wayne, PA: CLSI; 2003

2. ISO 6710, Single use containers for venous blood specimen collection


Blood Cultures• Additive Carry

Sodium Citrate

ESRDraw tubes

Blood CulturesyOver

• Carry-over is extremely unlikely

Serum

ESRin

descending

y ywith vacuum blood collection systems, but cannot be

EDTA

Lithium Heparindescending

orderexcluded completely 1,2

• Solution: Order of

Glucose

Trace element

draw definition

Trace element

1. Sharratt CL, Gilbert CJ, Cornes MC, Ford C, Gama R. EDTA sample contamination is common and often undetected, putting patients at unnecessary risk of harm. Int J Clin Pract 2009; Vol. 63: 1259-62.

2. Davidson DF. Effects of contamination of blood specimens with liquid potassium EDTA anticoagulant. Ann Clin Biochem. 2002, Vol. 39, 273-280.


• Collection induced haemolysis

• Solution range of partial draw tubes

1. Sixsmith DM, Weinbaum F, Ann Chan SY, Nussabaum M & Magdich K. Reduction of Hemolysis of blood specimens drawn from ED patients for routine chemistry tests by use of low vacuum collection tubes. Academic Emergency Medicine 2000; 7(5): 524

Reducing PAV: Sample CollectionÖrebro Hospital Emergency Department

Falun Hospital Emergency DepartmentNew blood collection system introduced

8%10%12%14%16%18%

erce

nt

2 53

New blood collection system introduced

0%2%4%6%

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35

w eek

pe

00.5

11.5

22.5

perc

ent

Percentage of Potassium Results not reported because of haemolysis (n=approx 400 samples/week) Ortho Diagnostics Vitros 5.1 Biochemistry

01 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43

week

Percentage of Potassium Results Not Reported because Mean 4% before conversion 10% after

The author hypothesizes that increase in haemolysis may be attributed to increased

d f th bl d fl i t th t b (T b

Of Haemolysis (n=approx 300 samples/week)Abbott ci8200

conversion

Mean 0.3%% before conversion 1.5% after conversionspeed of the blood flow into the tube (Tube

Fill Rates) noted (0.8mL/sec) 4ml versus (0.6mL/sec) 3.5ml

conversion

1. Swedish Society for Clinical Chemistry’s Members Magazine, Klinisk Kemi, No 2, 2009

Reducing PAV: Sample Collection• Collection induced haemolysis• Solution: Selection of tube type & trainingyp g

1. A Monitoring Process To Assess The Impact Of Preanalytical Variables On Sample Quality Following A Change In Blood Collection System, Muller C et al, Ahead of Publication Euromedlab 2011

Reducing PAV: Transportation & Stability

• Sample degradationSample degradation during transport

• Glucose, K, LDH < 2 , ,hours contact whole blood 1

• Separation of supernatant from

ll lcellular mass• Solution: gel based

separator tubesseparator tubes

1. Nadja N. Rehak and Betty T. Chlang. Storage of Whole Blood: Effectof Temperature on the Measured Concentrationof Analytes inSerum Clin Chem 1985;31:2005-6.

2. BD White Paper: Comparison of BD Vacutainer™ SST™ Plus Tubes with SST™ II Plus Tubes Common Analytes on the Toshiba/Abbott Aeroset, VS5780, 2001

3. BD White Paper: Gel Barrier Stability Comparison of BD Vacutainer™ SST™ II PLUS,SST™ PLUS and SST™ PLUS Transport Tubes in Post Centrifugation Transport, VS5824, 2001


• Sample degradation during transport• Solutions: Control & monitoring of time and temperature• Solutions: Control & monitoring of time and temperature


• Sample degradation during transport• Stability APTT citrate <1 hour 1Stability APTT citrate <1 hour • Solutions: CTAD provides stability for up 4 hours

at RT 2,3at RT

1. Collection, Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline - Fifth Edition. CLSI document H21-A5. 2008

2. G. Contant: Use of CTAD versus sodium citrate in different haemostasis assays. Serbio, March 1995.

3. van den Besselaar: Photochemical decomposition of dipyridamole in aqueous solution and the utilisation of CTAD for monitoring heparin, Int. Jnl. Lab. Hem. 2010(32):265-267


• Sample degradation during transport• Stability of GlucoseStability of Glucose • Solutions: Glycolytic inhibitors

1. Guder WG, Narayanan S, Wisser H, Zawta B. Samples: From the Patient to the Laboratory. Wiley-VCH 2003.

2. BD White Paper: A Comparative Evaluation of BD Vacutainer® Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation After 24 Hours Storage at Room Temperature, VS7594, 2007

3. Gambino, R. Acidification of Blood Is Superior to Sodium Fluoride Alone as an Inhibitor of Glycolysis, Clinical Chemistry 55(5)1019–1021 (2009)

Reducing PAV: Sample Processing• Incorrect centrifugation results in poor separation & sample

instabilityS l ti G l t b ith b d f t if ti diti• Solutions: Gel tubes with a broad range of centrifugation conditions

• Solutions: Coagulation plasma centrifuged at a speed and time to consistently product platelet-poor plasma (platelet count <consistently product platelet poor plasma (platelet count 10,000/µL)

1. BD White Paper: A Comparative Evaluation of BD Vacutainer® Plus Fluoride/EDTA Tubes for Glucose & HbA1c Preservation After 24 Hours Storage at Room Temperature, VS7594, 2007

2. Collection, Transport and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline - Fifth Edition. CLSI document H21-A5. 2008

Reducing PAV: Sample Processing

• Latent Fibrin/Fibrin Mass Formation:• Solutions: To clot or not to clot? • Anticoagulant Lithium Heparin • Rapid collecting additive such as thrombin or heparin neutralisers

h t isuch as protamine

Visible clotting was achieved rapidly in RST specimensrapidly in RST specimens within 5 min where participants had received a total of 0to 5000 units of heparin) butto 5000 units of heparin) but not from participants whoreceived a total of 7000 units of heparin (APTT >150 s). 2p ( )

1. BD internal data to be published2. Dimeski et al. Evaluation of the Becton-Dickinson rapid

serum tube: does it provide a suitable alternative to lithium heparin plasma tubes? Clin Chem Lab Med 2010;48(5)

Reducing PAV: Analysis

Reducing PAV: Analysis• Assay & sample compatability

• TDM & Immunoassays with gel separators

Factors Influencing Analyte Stability – TDM & Special ChemistryManufacturer Control User Control No Control

• Chemical nature of resin• Surface area of the gel

i t t if ti

• Duration of gel:sample contact both before and after centrifugation• Storage temperature

• Chemical nature of the analyte or drugprior to centrifugation • Actual volume of sample on the gel

• Tube orientation before centrifugation• Surface area of gel after centrifugation

drug

1. Extracted from Quality of Diagnostic Samples: Recommendations of the Working Group on Preanalytical Quality of the German Society for Clinical Chemistry and Laboratory Medicine, 2010

Reducing PAV: Analysis• Solutions:• Monitoring of quality critical parameters during the manufacturing

processes• Cooperation with IC during assay development• Demonstrate analyte compatibility across a broad spectrum of sample• Demonstrate analyte compatibility across a broad spectrum of sample

types, pathologies & instrumentation platforms:

1. BD White Paper: Therapeutic Drug Compatibility in BD Vacutainer® SST™ II Plus Tubes, VS7050, 2004

2. BD White Paper: Performance of BD Vacutainer® SST™ II Plus Tubes for Special Chemistry Testing, VS7051, 2004

Reducing PAV: Analysis

Reducing PAV: Post Analysis• Stability - Freezing primary sample • Often used for blood banking and sometimes for ‘add-on’ tests

No sample aliquoting– No sample aliquoting– Reduced identification errors– Reduce biohazard risk

R d d t– Reduced cost• Solutions:

– Airflow around the tube, ensure that the samples are in wire racks that 1allow airflow rather than EPS/styrofoam trays.1

– Gradual freezing of the tubes, refrigerated 4-8C for 4 hours, then -20C for 24 hours then final temperature. 1

2– Most analytes in aliquoted serum are stable when frozen (≤ −20°C).2– Even repeat freeze/thaw cycles at -20°C or -70°C seem to have little

effect on many analytes. 3M j i f i h i i bl i b 1– Majority of routine chemistries are stable in serum separator tubes. 1

1. A Jaap Bakker et al, An Evaluation Of The Integrity of BD Vacutainer® SST™ II and Analyte Stability when Subject to Freezing at -20°C.,EUREGIO, 2003.g , ,

2. Heins Met al. Storage of serum or whole blood samples? Effects of time and temperature on 22 serum analytes. Eur J Clin Chem Biochem. 1995; 33:231-38.

3. DiMagno EP et al, Effect of long-term freezer storage, thawing and refreezing on selected constituents in serum. Mayo Clinic Proc 1989; 64:1226-34.

Reducing PAV: The Future

• Reducing sample collection volumes - Why• Advances in instrument technology should be reflected in theAdvances in instrument technology should be reflected in the

collection requirements • Blood collection 45 times greater than the volume required (Range

2 102 ti ) 12-102 times) 1

• Patients in Intensive Care Units (ICU) are phlebotomised three times as often as patients on the wards. 2

• 5% of the patients undergoing intensive care, the volume of blood collected for laboratory testing was >200 mL and for 0.7% was >600 mL during their hospital stay.3

• Complications: Investigational anaemia, neonatal anaemia

• Solution instrumentation compatible low draw blood collection• Solution instrumentation compatible low draw blood collection tubes 0.5 – 2 mL

1 Dale JC Pruett SK Phlebotomy - a minimalist approach MayoClin1. Dale JC, Pruett SK. Phlebotomy a minimalist approach. MayoClin Proc. 1993; 68: 249-255.

2. Smoller BR, Kruskall MS. Phlebotomy for diagnostic tests in adults: pattern of use and effect on transfusion requirements. N Engl J Med. 1986; 314: 1233-1235

3. Wisser D, Van Ackern K, Knoll E, et al. Blood loss from laboratory tests. Clin. Chem. 2003;49:1651-1655


• Alternatives to gel separators• Provide improved sample purity analyte stability• Provide improved sample purity, analyte stability,

eliminate analyte adsorption

1. BD Data on file

Reducing PAV: The Future• Proteomic: Protein stabilisation• Solution: P100 Anticoagulant: K2EDTA OptimizedSolution: P100, Anticoagulant: K2EDTA, Optimized

protease inhibitor mix & Mechanical plasma separatorEDTA-0\0_K1\1\1SRef4x10

a.u.

]

P100-0\0_K4\1\1SRef4x10

a.u.

]

1537.001 1896.3121060.802

1465.9651211.958 1348.998882.674 2021.3881778.279

1606.140

1

2

3

4

Inte

ns. [

a

863.7091536.964

1865.2272021.312

1465.9411166.010 1778.2191051.964

1

2

3

4

Inte

ns. [

a

EDTATime “0”

P100

0

1537.067

1060.9211896.428

882.944 1166.174 2021.534

EDTA-15m\0_K2\1\1SRef

1

2

3

4

4x10

Inte

ns. [

a.u.

] 0

1536.936

863.689 1865193 202129

P100-15m\0_K5\1\1SRef

1

2

3

4

4x10

Inte

ns. [

a.u.

]

15 min1349.065 1617.213 1778.377

01466.285

1896.644

1061.1041537.347

EDTA-2h\0_L1\1\1SRef

2

3

4

4x10

Inte

ns. [

a.u.

]

1865.1931465.891 2021.2951060.803 1778.1621350.893

0

1537.076

1466.022

P100-2h\0_L4\1\1SRef

2

3

44x10

Inte

ns. [

a.u.

]

2 hr1207.183882.994

1351.262 2021.7561762.781

0

1

800 1000 1200 1400 1600 1800 2000 2200m/z

1896.470

1350.950863.790 2021.568948.964 1206.896

0

1

800 1000 1200 1400 1600 1800 2000 2200m/z

1. Jizu Yi,* Changki Kim, and Craig A. GelfandInhibition of Intrinsic Proteolytic Activities Moderates Preanalytical Variability and Instability of Human PlasmaJournal of Proteome Research 2007, 6, 1768-1781


• Molecullar: Stabilisation against gene induction and degradationS l ti PAX TM f i l ti f ll l RNA f h l bl d• Solution: PAXgeneTM for isolation of cellular RNA from whole blood -RNA is stabilized immediately at blood collection and remains stable for days at room temperature

Gene InductionGene Induction

PAXgeneTM

EDTA

1. Lynne Rainen,et al Stabilization of mRNA Expression in Whole Blood Samples Clin. Chem., Nov 2002; 48: 1883 - 1890.

Reducing PAV: Conclusions

• Selection of the appropriate sample collection systemSelection of the appropriate sample collection system• Together with correct handling and procedures• Minimises Preanalytical variabilityMinimises Preanalytical variability• Optimum sample quality• Enable the generation of the true in-vivo result• Enable the generation of the true in-vivo result• Maximise laboratory efficiency• Reduce costs• Reduce costs• Assure patient treatment and care

primary sample collection systems: how useful are they in

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