residual protein testing - deconidi.ie · residual protein testing david perrett. professor of...

Residual Protein Testing

David PerrettProfessor of Bioanalytical Science

Barts & the London School of Medicine & DentistryQueen Mary University of London

IDI Cork November 2015

vCJD caused the UK Dept of Health to spend over £20 million on decontamination research

Instrument (n=4) Mass (mg) of Tissue addedKidney Brain

(wet) (dry) (wet) (dry)Surgical Blade 5.7±2.4 2.0±0.8 48±8 12±1

Forceps 60±45 8.6±13.4 33±32 15.8±15

Tweezers 12±6 1.9±0.9 43±16 14.3±5

The PROBLEM

Mass (µg) of Protein equivalentBlades 1140±120 2000±800

Forceps 3000±2250 3300±320

Tweezers 600±300 4300±1600

Total Residual ProteinSome Barts data

µg/instrument

Hospital A Hospital B Hospital C Hospital D Hospital E0 0 0 10 18 0 0 18 6

12 2 3 35 1216 10 5 41 1448 18 8 88 1456 42 8 93 9460 138 13 17386 302 30 213

260 53 432168 488

© Professor David Perrett

Sheet1

Protein levels on instruments sets

HospitalSetInstrumentWashingProtein concentrationTotal protein removedNotesAppearanceFiltered insoluble residues

CodeVolumeremovedweight filterdepositinstrument - control

Numbermlµg/mlµgµg

Royal PrestonAblank204.70

a1205.108stained on joint93.363560

a24004.700stained on joint93.884080

a320lost in sonic bath

a4605.7060small cuttersmall cutter with sellotape97.347540

a5209.0086curved scissorsflithy behind hinge93.343540

a6205.5016forcepsdeposit in serrations93.353550

a7405.9048ball and socket forcepsclean95.485680

a8207.5056scarpelgrubby92.843040

a91030.70260tweezerobvious lump of tissue92.262460

a10205.3012forcepsclean93.63800

RLHBblank0.0091.5186.834680

b1600.7042snarev.clean93.4787.3761001420

b2200.102forcepsv.clean91.4685.7257401060

b3088.2

b4255.50138bent forcepsstaining in channel9588.1568502170

b5250.4010forcepsv.clean95.3188.8264901810

b6200.000scissorswater marked94.788.7259801300

b71323.20302snarev.clean93.9688.45560880

b8250.7018bent forcepsv.clean94.1687.9262401560

Mean dry filter87.79

AddenbrooksCblank0.3089.8

c1250.608gold handle scissorsstaining on hinge90.66860

c2252.4053mirrorstain on the glass92.192390

c31313.20168snarev.clean91.671870

c4251.5030tweezersv.clean91.021220

c5250.505split stem instrumentclean135452

c6250.608forcepsbrown deposit on hinge92.22400

c7250.20-3scissorsv.clean but damaged

c8250.8013forcepsv.clean92.42600

c9250.403long scissorswater marked91.51700

c10250.10-5u-ended forcepsv.clean91.71900

??Dblank0.0089.62

d1450.9041forcepsclean

d22519.50488spencer wellsstain/deposit on hinge91.611810

d3250.7018small forcepsclean91.121320

d4256.90173curved scissorsstain/deposit on hinge91.651850

d5250.4010forcepsclean89.9100

d6251.4035styluswater marked91.291490

d7258.50213small scissorsdeposit on blade91.291490

d8253.5088forcepsclean91.021220

d92401.80432complex forcepmulti deposits91.681880

d10253.7093plastic covered forcepsdirty at plastic/ ss interface91.41600

United BristolEblank8.3289.48

E/Ae14010.6794Part of ocular setStaining behind screws that could be scrapped off!88.68-800

e2109.5012very tiny forcepsclean90.521040

e3159.2714fine scissorssome staining89.95470

E/Be4209.0014Spencer Wellsclean

e5201.09Spencer Wellsfell over in bath89.8320

e6208.636Spencer Wellsclean90.25770

e7208.381Spencer Wellswater-marked90.29810

Sheet2

Hospital AHospital BHospital CHospital DHospital E

000101

800186

12233512

161054114

481888814

564289394

6013813173

8630230213

26053432

168488

Sheet3

Hospital AHospital BHospital CHospital DHospital E

356014208601810-79

40801060239013201040

7540217018701850470

354018101220100

35501300452001490320

568088024001490770

3040156026001220810

246017001880

380019001600

Sheet4

Total ProteinDry MaterialTotal

µgµgµg

835603568

040804080

6075407600

8635403626

1635503566

4856805728

5630403096

26024602720

1238003812

4214201462

210601062

13821702307

1018101820

013001300

3028801182

1815601577

8860867

5323902443

16818702038

3012201250

5452457

824002408

1326002613

317001703

-519001895

48818102298

1813201338

17318502023

10100110

3514901525

21314901703

8812201308

43218802312

9316001693

94-800-706

1210401052

14470484

6770776

1810811

Sheet4

Total Material µg v Protein µg

Total Protein µg

Total Material µg

Sheet5

Protein released from SSD visually clean and QC failed instruments

Passed Mean mass on Instrument = 40.1µg

Failed Mean mass on Instrument = 49.2µg

N =20 in each group

Visually clean does not mean protein-free

So why the failings:

If guidelines were being followed?

Human Serum Albumin

550 amino acids Molecular weight 66000da Proteins are very big with many binding sites!

ISO / EU protein test chemistries


ISO 15883-1:2009 annex CTest methods for the detection and assessment of residual proteinaceous contamination

Methods are

By swabbing then check swab with e.g. Ninhydrin

Biuret as BCA

By solubilisation of “all” residual protein in a detergent (SDS) solution

Followed by OPA/thiol (DMMEA) reactionwith spectrophotometric measurement

Off-instrument testing


ISO 15883-1:2009 part C.3.3.1.2 requires that about 10 cm2 of an instrument is swabbed with wetted swabs prior to testing with recommended chemical tests

Swabbing 10 cm2 in itself is a challenge!

Swab with what?

For how long?

Tend to swab visually dirty area not necessarily protein

Why a choice of tests offered

Efficiency of removing proteins with Rayon swabs wetted with two different solutions

Water 0.5% Triton X-100

Swabbing

Bovine Serum Albumin (BSA)A water soluble protein

32 ±4%

remaining

20 ± 3%remaining

10-15 strokes

FibrinogenA hydrophobic protein

61 ±5%

remaining

24 ± 3% remaining

10-15 strokes

n = 6


We swabbed where we knew we had placed the protein!

Nayuni N, et al. (2013) A critical evaluation of ninhydrin as a protein detection method J Hospital Infection 84, 97-102

Common biochemical methods for protein

determination


NinhydrinA currently recommended method of protein detection

Reacts with primary amines and amino acids forming Ruhemann’s purple


Positive control is not a Protein!


Ninhydrin Test - Different Proteins and Bio-fluids


Nayuni N, et al. (2013) A critical evaluation of ninhydrin as a protein detection method J Hospital Infection 84, 97-102

Single use neuro-instrument

Wet weight of adhered rat brain tissue

Total protein adhered

Visual score after washing(N=12 observers 5 is worst)

Ninhydrin test

(mg) (mg) Mean SDC5 146 5.88 4.9 0.3 negativeA8 238 9.59 2.9 1.0 negativeE29 235 9.47 2.7 1.3 negativeE27 229 9.22 2.2 1.4 negativeB12 209 8.42 1.7 0.8 negativeE16 50 2.01 2.4 0.7 negativeB13 191 7.70 1.6 0.7 negativeD40 173 6.97 4.6 0.7 negativeD43 243 9.79 4.2 0.9 negativeD37 239 9.63 4.5 0.8 negativeE26 193 7.78 1.9 1.0 negativeD35 220 8.86 3.0 1.2 negativeP2 53 2.14 1.7 0.8 negative

Mean ± SDArginine

184 ± 61 7.4 ± 2.5 2.95 ± 1.20 Positive

Brain is much stickier than other tissuesNinhydrin response did not correlate to visual scores


Comparison of visual scoring and ninhydrin

Summary - 1Swabbing with water does not efficiently remove residual proteins especially hydrophobic proteins

Few workers swab 10cm2 of an instrument!

Ninhydrin procedures do not work with proteins with any acceptable sensitivity


• Even in the research laboratory it is difficult to get accurate measurements with standard protein assays.

• Most protein assays are expressed as Bovine Serum Albumin (BSA) equivalents

• Each method has its limitations & advantages


Other Simple Protein assays

Biuret MethodReaction of peptide bonds in proteins with copper ions in alkaline solution

Sensitivity 1000 µg / mL© Professor David Perrett

Bicinchoninic (BCA) Assay• Developed by Pierce Co. in 1977 based on Lowry assay (1951)

• Protein reacts with Biuret reagent followed by colorimetric measurement of Copper

• Proteins reduce Cu (II) to Cu (I) and BCA is a specific chromogenic reagent for Cu (I)

• Incubate 60°C for 30 minutes (working range = 5 - 250μg/mL)

• Measure @ 562nm


BCA Assay – colour change

Max.Sensitivity 5 µg / mL

Different proteins respond differently

BCA direct reaction with 1000 µg BSANegative control


Negative control

I mg BSA

@ 2 min

@ 30 min

@ 8 min Negative control

I mg BSA

I mg BSA

BCA TestBrain homogenate dried for 48 h @ RT, swabbed

with water and then placed in the test vial

Can just see a spot

ProTest-Q© Professor David Perrett

• Published by Bradford in 1976 • Popular in research labs (simple, rapid, inexpensive)• Coomassie Brilliant Blue G-250 dye (CBBG) binds to protein with

a colour change Free CBBG (λmax 475 nm) CBBG - protein complex (λmax 595 nm)

• Different proteins respond differently


Sensitivity 1 µg / mL

Bradford Assay

Bradford Assay is used in

Valisafe Denta-check

Also used in a Miele product



Pyrogallol Red + Molybdate reaction

Reaction reported by Fujita et al. 1983. Introduced as a urinary protein assay (Watanabe et al 1986)

The binding reaction requires 5 min at pH 2.5

Colour change from 460nm to 600nm (blue)

Max.Sensitivity 5 µg / mL

After 15 minutes with the swabs taken out


- 8 4 2 0.8 µg

Pereg test

ATP : SOME FACTS - 1 ATP stands for Adenosine tri-phosphate

It is the energy molecule in LIVING CELLS

ATP is a PURINE NUCLEOTIDE not a protein!

ATP degrades to ADP, AMP & HYPOXANTHINE with heat, tissue ischaemia and pH

ATP does not bind strongly to stainless steel


Molecular weight = 507

Commercial test kits for residual proteins available in U.K.Chemistry Mode Trade Name Supplier Sensitivity*

(µg BSA spot)Copper binding + complexation

BCA Pro-Test-Q Medisafe 1

BCA Clean-Trace protein 3-M 3 – 50 Depending on

temperature and time

BCA Medi-check residual protein test

Hygiena 1 – 10 Depending on

temperature and time

Dye binding Pyrogallol Red + Molybdate

Pyromol Test Pereg GmbH 1

Coomassie Brilliant Blue(Bradford Assay)

Scope Check Valisafe/Medisafe 1

Denta Check Valisafe/Medisafe 1

Ninhydrin Ninhydrin Ninhydrin Protein Detection Test

Browne (Steris) !!!

Haemoglobin Haemostik na

Bioluminescence ATP na

* Sensitivity taken from manufacturer’s publications

Validation of AWDs in UKA qualified test engineer applies a test soil to some instruments, then runs a washer cycle and then checks for residual protein

Browne’s soil

Add water to powder, shake vigorously and apply to the test load with a brush

Dry for 30 min at RT, wash with normal procedures then inspected for residual soil. The soil’s bright red colour allows easy identification of areas not properly cleaned

Non-toxic; contains no blood products

Edinburgh soil

100ml Fresh egg yolk10ml Defibrinated horse blood

2g Dehydrated hog mucin

Dry on, wash and then test with…

ninhydrin


Summary - 2• Biuret methods are very insensitive

• BCA need careful timings of incubation for true colour formation

• CBB methods are relatively insensitive and need examination of the swabbed tip

• ATP is a biochemically inappropriate assay

• Commercial test kits often show poorunderstanding of the underlying chemistry


DH (England) working group on Decontamination of Reusable Surgical Instruments

concluded that the present methods for protein detection on reusable instruments are not fit for purpose.

Methods need to be some 100x more sensitive and detect residual proteins on the total instrument

Realistically this is only possible using fluorescence

FluorescenceCompared to colorimetric measurements

is many fold more specific

is more 1000x more sensitive

but it needs intense light sources often lasers


In situ protein detection technologies funded by DH (England) in 2010

University. Clinical Centre

Chemistry(Technology)

Sensitivity Notes.

BartsQueen Mary University of London

GOSH and UCLH(London).

OPA/NAC(CCDimaging)

Detection at levels

‘In situ’ detection – My idealSimple – suitable for the SSD environment: Giving a permanent recordSensitive – need 100 fold better than present methods

Protein specific

Fast – capable of high throughputInstrumentation - Readily available, should not use lasers

Stable reagents giving stable fluorophores

Non-toxic – Safe reagent: Protein products can be readily removed

Can reveal proteins residues - on “whole” instruments

Low cost - both capital and running


My system at Barts combines

OPA/N-acetyl-cysteine (a fluorescent reagent) with

CCD visualisationin

2-D & 3-D with quantitationto

detect & quantify residual proteins in situ


OPA/NAC Reagent for Proteins


o

o+HH

OPA

NH

Denatured protein

with fluorescent

Isoindoles

+

NH2

-S-S-

-S-S- NH2

NH2

NH2

+

OH

O

HNHS

O

SH

SHSH

SH

+ DTT

+ Triton-X100

OHO

NHS

O

N

OHO

NHHS

O

N

OHO

NHS

O

N

OHO

NHS

O

Intact protein

N-acetyl-L-cysteine

Denatured protein

SH

SHSHSH

H2N

+

NH2NH2

NH2

NH2

Fluorescence Ex 350nm Em 450nm

The Spray Reagent• OPA/NAC was reformulated with improved

chemistries

• It is now stable for six months

• It retains its sensitivity

• The revealed fluorescent residues are very stable.

• The reagent can be completely washed off

• BSA protein standard is stable


U.K. Patented by QMUL

The Proteomic based system

G-Box from Syngene Cambridge © Professor David Perrett

G-Box system in 2008

Cooled CCD Camera

Platformwith sheet

of black paper

Lamps – White lightMercury 338nm

Optimum Emission filters


480 ng protein

60ng

Imaged data processed using D-Plot


U.K. Patented by Synoptics and QMUL

• Stainless steel (316 grade) tags are contaminated with brain homogenate.

• A total of 750ug protein is loaded on to each tag

• Air dried (RT for 48 hours) or baked (75oC for 6 hours)

• Washed in validated AWDs using SSD defined procedures

• Spray with OPA/NAC reagent

• Quantify using G-box

Quantitative Experimental Procedures


Typical results after an alkaline wash in an AWD

48 H AIR DRIED post washBrain spotted tags pre-wash

Typical result – moist studiesAlkaline detergent

48 h

3 h

2 h

1 h

0 min

BSA (2 – 10 µg) Moist controlSpray control

Enzymatic wash

48 h

3 h

2 h

1 h

0 min


ProReveal - 2015

Typical BSA protein standard calibranty = 4E+07x + 209210

R² = 0.9912

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

0 0.2 0.4 0.6 0.8 1

RFU


ALKALINE (Dry)

ENZYMATIC (Dry) ENZYMATIC (Moist)

ALKALINE (Moist)


Neuro-instrument studies Enzymatic (moist)Enzymatic (dry)Alkaline (moist)Alkaline (dry)


Washed in SSD A Washed in SSD C

Titanium Eye Set

Total Protein shown = 158 µg Total Protein shown = 0.8 µg


Setting limitsEye set (dry µg/side)


0

10

20

30

40

50

60

701 4 7 10 13 16 19 22 25 28 31 34 37 40 43 46 49 52 55 58 61 64 67 70 73 76 79 82 85 88 91 94 97 100

103

106

109

112

115

118

121

124

127

130

133

136

139

142

145

148

µg p

rote

in /s

ide

Total Residual Protein on 150 instruments from 6 hospitals

Optimisation of Automatic Washer Study


PresenterPresentation NotesThree years ago the Department of Health funded myself and colleagues to investigate the performance of detergents and AWDs from first principles.

Protein TagCleaned, polished 316L stainless steel tag

Size of standard microscope slide i.e. 26 x 76mm

Accurately pipetted 80 µL spots of 10% brain homogenate in PBS(equivalent 40 µg protein or 1 µg/mm2)

Proteins annealed at 75oC for 1 hour

Q.C. tested


The system is designed so that a wash in RO water in a laboratory sonic bath removes about 50% of the annealed protein

PresenterPresentation NotesFirst I had to invent a simple system that could be used to quantitatively measure the performance of the test conditions. So the Pro-Tag system was developed. It is a simple device the size of a microscope slide to which we anneal spots of brain homogenate. I have some with me.

Summary of washing efficiency

% residual protein

Water alone 1.14 ± 2.8

Alkali 0.63 ± 0.6

Non-ionic detergent 0.44 ± 0.88

Enzyme 0.44 ± 0.48

Data is mean ± SD for a whole group (n = 60 instruments) in each study

Randomly chosen images of optimally washed instruments

Alkali washed forceps 4A

Non-ionic washed forceps 8AEnzyme washed forceps 17A

Water washed forceps 4A

How clean is clean?DH committee considered this question and updated the UK Decontamination Guidelines in May 2015www.gov.uk/government/uploads/system/uploads/attachment_data/file/427855/Annex_C_v3.0.pdf

Maximum total residual protein = 5µg / total instrument side

Units are BSA equivalent

Lower limits are needed for neurosurgical instruments

SSDs can develop their own QC based on a cohort of

some 20 instruments regularly tested

Potential time post-operation and pre-washing should not

exceed 1 hour

Now they have to be adopted by hospitals DH will publish instructional guidance in spring 2016

Summary• New protein detection tests make progress

possible

• SSDs can routinely produce very clean instruments

• General upper limit of 5µg per instrument side is likely to give meaningful risk reduction

Applications of ProReveal• Routine QC and QA in SSDs

• Performance testing of AWDs and/or detergents before purchase

• Validation of AWDs in routine operation

• AWDs or detergent manufacturers can use ProReveal for developing improved systems and chemistries

• Surgical instrument manufacturers to improve their designs

• General research applications


Conclusions• A simple, high sensitivity in situ system to visualise and

quantify residual proteins useable in SSDs is possible

• Keeping instruments moist and washing with enzymatic detergent is our best current advice.

• However all actual agents need testing, AWD operation needs better validation, new procedures are not necessarily improvements

• The 5ug level can be achieved after optimisation

• Very clean instruments can be achieved


The late Prof Don Jeffries (Chair of DH Decon Group)

All members of the DH Decon Working Group

The teams at the various SSDs we’ve worked with

Alasdair at Synoptic Health for his programming

DH for funding all this

Thanks to

Thanks to you for listeningDo look at the system. It is on display in the exhibition

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Efficiency of removing proteins with Rayon swabs wetted with two different solutionsSlide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32In situ protein detection technologies funded by DH (England) in 2010Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Typical result – moist studiesSlide Number 44Typical BSA protein standard calibrantSlide Number 46Neuro-instrument studies Slide Number 48Setting limits�Eye set (dry µg/side)Slide Number 50Slide Number 51Slide Number 52Slide Number 53Slide Number 54Slide Number 55SummaryApplications of ProRevealConclusionsSlide Number 59

residual protein testing - deconidi.ie · residual protein testing david perrett. professor of...

Documents