reapan micro

© 2008 Reametrix, Inc. All rights reserved.ReaMetrix Confidential

: R e a P a n M ic r oPresence/Absence Test for Bacteria in Urine

2© 2008 Reametrix, Inc. All rights reserved.ReaMetrix Confidential

?Wh a t is R e a P a n M ic r oA diagnostic test to c o n f ir m the absence of bacteria in urine, based on the principle of nucleic acid detection in bacteria on a fluorescence event detector such as a flow cytometer

Wh a t is t h e in f o rm a t io n t h a t t h e

?t e s t p r o v id e s• Provides a doctor information that

r u le s o u t bacterial Infection

K e y f e a t u r e s o f t h e t e s t• High sensitivity ( 1 0 0 % N e a r ly

N e g a t iv e P r e d ic t iv e Va lu e )• Time to result: Within 1 5 m in of sample reaching a flow cytometer • Easy to use, simple assay workflow

B y C u lt u r e

119

43

TN

99.2NPV

67.7%PPV

1Negative by Flow

90Positive by Flow

TPO v e r a ll


In it ia l S t u d ie s o n C lin ic a l U r in eS a m p le s


: E x p e r im e n t a l D e s ig n S t a in in g o f C lin ic a l U r in e S a m p le s

Tr a in in gS e t6 culture negative (<10,000 CFU/mL) clinical urine samples6 highly positive (>100,000 CFU/mL) clinical urine samples

These 12 samples were used to determine the ideal gating strategy

S t a in in g P r o t o c o l u s in gR e a g e n t s

Filter 1 mL urine

sample through a

11 um Syringe

Filter into a 1.5 mL

eppendorf tube

Add900 uL of the

filtered urine to FACStube filled

with 50 uL of nucleic acid

stain and 10,000 volume control beads. Add 50 uL of

PFA to fix

Vortex and

incubate for 5 min

Run the sample on the

Flow Cytometer

using pre-set template

Filtering is done only to remove any uric acid crystals, and “debris”


: R u n n in g t h e Te s t Wo r k f lo w E n v is a g e d

ID’ing FACSTube with dried down reagent:

1 min

Filtering 900 uL of urine sample into FACStube using enclosed syringe

filter2 min

Vortex and incubate sample at RT

5 min

Run MilliQ water tube thru FACScan

1 min

Acquire data on FACScan with vortexed patient sample

2 min

Time to Result:

Within 15 min of sample reaching

instrument


Calibration Beads

Before the gain on the PMT’s are adjusted After the gain on the PMT’s are adjusted

: S e t t in g u p t h e t e m p la t e In t e n s it y C o n t r o l B e a d s D e t e rm in e Ap p r o p r ia t e G a in S e t t in g s

o n t h e In s t r u m e n t

Gate where bacterial events are collected

Gate where bacterial events are collected


1 2 G a t in g S t r a t e g y O p t im iz e d u s in g Tr a in in g S e t S a m p le s

Counts (Arb itrary Units)

Culture Negative (Less than 10,000 CFU/mL)

Culture Positive (More than 100,000 CFU/mL)

Region R6 gate has been applied

ReaMetrix Confidential

8 5 .2 4 c o u n t s p e r u n it v o lu m e

4 0 0 8 3 c o u n t s p e r u n it v o lu m e

Counts (Arb itrary Units)


S t a t is t ic a l An a ly s is t o D e t e rm in e

D e c is io n Th r e s h o ld = 2 5 3 u s in g N s a m p le s

Counts

S c a t t e r P lo t s o f B a c t e r ia l C o u n t s

f r o m F lo w C y t o m e t r yN = 91N = 162

N = 91N = 162

Counts M e a n

Counts

N = 91


= S t a t is t ic a l An a ly s is t o D e t e rm in e D e c is io n Th r e s h o ld u s in g N2 5 3 s a m p le s

: Z o o m in g in E x c e p t io n An a ly s is a n d Id e n t if y in g t h e G r a y R e g io nThis definitely could not have been negative by culture. We will look at other clinical investigations that were done on these samples

= 2 9N(1 8 % o fn e g a t iv e s)

= 6 8 N(7 5 % o fp o s it iv e s)

Counts

Gray Region

= 1 7 N(1 1% o fn e g a t iv e

)s

= 2 2 N(2 4 % o fp o s it iv e

)s

Could these samples have all been similar in some respect?

0-3000 counts

3000-10000 counts

> 10000 counts

= 1 1 6 N(7 2 % o fn e g a t iv e

)s

= 1 N(1% o fp o s it iv e

)s


2 5 3 S t a t is t ic a l A n a ly s is o f C lin ic a l S a m p le s t e s t e d t h u s f a r

Threshold defined using the training set. Threshold was set to ensure that NPV was 100%

TP = True Positive at that level using culture as Gold Standard

TN = True Negative at that level using culture as Gold Standard

B yC u lt u r e

B yF lo w

/C FU m L Ar b

U n it s

N e g a t iv e < 10000 < 3,000

P o s it iv e > 100000 > 3,000

PPV - Positive Predictive Value

NPV - Negative Predictive Value

B y C u lt u r e

119

43

TN

99.2NPV

67.7%PPV

1Negative by Flow

90Positive by Flow

TPO v e r a ll


S a m p le s S t a in e d w it h ju s t TO w it h o u t f o rm a ld e h y d e a n d

4 ( =2 2 )s t o r e d a t C o v e r n ig h t N s a m p le s :Y Ax is Counts measured on

samples immediately after staining

:X Ax is Counts measured on samples stained and kept at 4 C for 24 hrs. No fixative was used

: R e d S q u a r e P o in t s Indicative of False Positive samples as per culture results

: F a ls e P o s it iv e Z o n eSamples that lie in this rectangle refer to those that show high counts deeming them positive, 24 hrs later, but were negative when counted immediately after staining

: F a ls e N e g a t iv e Z o n eSamples that lie in this rectangle refer to those that show low counts deeming them negative, 24 hrs later, but were positive when counted immediately after staining

We find no samples in the False Positive or False Negative Zones. Th u s t h e s t a in e d

s a m p le s c a n b e t r a n s p o r t e d a f t e rf ix in g


Tr a n s f e r r in g t h e a s s a y t o : D ia g n o s t ic La b

In s t r u m e n t S e t U p P r o t o c o l u s in g R e a TC o u n t B e a d s

User will open SSC, FL1, FL2, FL3 histograms in a template and set the gains on these channels to get channel numbers within the specifications given below.

On 5 different days the maximum variation seen for the same gain is no more than 2 channel numbers on any of the channels

We ran experiments to determine , what is the variation that is tolerable (that does not impact counts by more than 10%)

- 3 We h a v e f o u n d t h a t u p t o c h a n n e l n u m b e r s v a r ia t io n a b o u t e it h e r s id e o f t h e

m e a n d o e s n o t im p a c t t h e c o u n t s s e e n in u r in e s a m p le s

, If t h e s e t u p is w it h in s p e c t h e t h r e s h o ld 1 2 1 5is t h e n s e t o n F L a t c h a n n e l n u m b e r


Q u a n t if y in g t h e M a x im u m Va r ia b il i t y in C o u n t s S e e n w it h A s s a y R e p e a t s

( A s s e s s in g t h e r o b u s t n e s s o f t h e f in a l iz e d )a s s a y p r o t o c o l

6 culture positive and 4 culture negative samples were filtered and stained in triplicate, to determine the assay variability seen with the finalized protocol.The assay variability is typically less than 15%. Thus the gray region for the threshold of 3000 is nearly +/- 450 counts


ReaTCount Volume Control Beads

3 – FL LD S

s t a in e d c e l ls a r e

g a t e d o n9R

R9 gate is applied on FL1 to get counts using M1

– P r o o f o f c o n c e p t s e x p e r im e n t s D u a l S t a in in g o f B a c t e r ia u s in g LD S a n d

Th ia z o le O r a n g e N EG ATIVE U R IN E

S AM P LES P O S ITIVE U R IN E

S AM P LES


C o n c lu s io n

We anticipate ReaPanMicro to enable the doctor to rule out bacterial infection quickly – without the need for results of culture (that typically takes 24-48 hrs)

The assay has been validated with Anand Diagnostics at Bangalore


Manufactured by ReaMetrix India Pvt. Ltd.50-B, II Phase, Peenya Industrial Area

Peenya, Bangalore 560058, IndiaPh: +91-80-28378693/5, Fax: +91-80-41172451

E-mail: [email protected]

:F u r t h e r d e t a ils

reapan micro

Health & Medicine