1

Dr. Kabita Chatterjee

Blood Bank

• # 2700

Blood Collection

• 5.59 million

Total Population

• 1.1 billion

Voluntary Donation

= 3.08 million (55.1%)

Replacement Donation = 2.51 million

(44.9%)

Out of 5.59

million Blood

collection:2

General Population

HIV-1

0.36%

HCV

0.9%

HBV

2.5 – 4%

Blood Donors

HIV-1

0.3%

HCV

0.7%

HBV

1.4%

* NACO Report

3

HBV

43 Million

HCV

10 Million

HIV-1

5 Million

* NACO Report

Carriers of HIV-1, HCV & HBV in India

4

Nucleic Acid Testing

Nucleic Acid testing (NAT) for HIV, HCV and HBV became available in the latter 1990’s and is now routinely used in many countries

Technological advance in blood screening that enables further reduction of window period for HIV, HBV and HCV

Detects very low levels of viral RNA or DNA that may be present in donated blood

Highly sensitive & specific — targets specific viral nucleic acid sequences

Reduces window period through direct detection of viral nucleic acid sequences

Prevents transfusion of infected blood components and therefore exposure to life-threatening transfusion-transmitted disease

Provides additional layer of safety to the world’s blood supply

Detection of Infectivity in blood

T = 0

high medium low

ID NAT detection limit

antibody

virus

relative

concentration

infectivity

neutralized

infectivity threshold

infectivity threshold

loweclipse

NAT

serologyantigen antibody

antigen detection limit

MP NAT detection limit

eclipse

Why Nucleic Acid Testing? Reduction of Window Period

7

Busch MP et al. Transfusion 2005;45:254-264, Assal A et al. Transfusion 2009;49:289-300, Weusten J et al, Transfusion 2011;51:203-15

•Higher sensitivity

•Pooled testing requires deconvolution to identify

the single positive unit

•This process requires an additional step of

handling, additional time for testing and hence delay

in release of units

•In High prevalence situation high number of pools

will be positive resulting in deconvolution of several

pools

•In high prevalence situation additional

manipulations can increase the chances

Benefits of Individual Donor Testing (IDT)

8

•The PROCLEIX® ULTRIO Screening Assay is

an in vitro nucleic acid amplification test for the

simultaneous detection of HIV-1 RNA, HCV

RNA and HBV DNA in human plasma

•Unique two region target of HIV-1 genome

•The assay yields QUALITATIVE results as

Reactive or Non-Reactive for the three targets

•The maximum number of tests are in a batch of

100 tubes

THE Procleix Ultrio NAT Screening ASSAY

9

Sample Collection

10

11

Sample

Pipetting

Target

CaptureAmplification Detection Results

ASSAY PROCEDURE- OVERVIEW

12

• ULTRIO Assay involves three

main steps that take place in a

single tube:

1. Target capture effects Nucleic

acid isolation from 0.5mL

sample

2. Amplification by Transcription

Mediated Amplification (TMA)

3. Detection by Dual Kinetic Assay

(measures flasher and glower

light simultaneously).

THE ASSAY Steps

13

o When a NAT lab is being set up or a new assay is being

brought on-board three consecutive runs should have less

than 3% invalid results.

o Valid run - is a run were internal parameters perform

according to specification there by assuring validity of the

result. Percentage invalid should be below10%.

o If the invalid results exceed 10%, the protocol requires a

repeat Run, and the final decision rests on HOD or on an

authorised person.

Validation of NAT Test

14

•Internal control:

An internal control RNA added to the sample assures

fool proof system check for the entire procedure.

•Dual Kinetic Assay monitoring (DKA):

Flasher light emission by the internal control

authenticates the operator’s performance and

the Glower light emission by the viral amplicons

indicates the presence of viruses in the specimen.

Quality Control for the ASSAY & SYSTEM

15

•Calibrators:

Each run of 100 maximum tests includes several tests

dedicated to Positive and Negative Calibrators. The

positive calibrators are for each of the three targets.

The result for the calibrators has to meet the expected

criterion for the test results to be valid.

Quality Control for the Run & SYSTEM

16

Specimens found to be reactive in the ULTRIO Screening

Assay are tested by PROCLEIX® Discriminatory Assays

(dHIV-1, dHCV, and dHBV) to determine if they are

reactive for HIV-1, HCV, HBV or any combination of

these three.

The PROCLEIX® HIV-1, HCV, and HBV Discriminatory

Assays utilize the same Assay reagents with one

difference: HIV-1-specific, HCV-specific, or HBV-

specific probe reagents are used in place of the ULTRIO

Assay Probe Reagent.

THE Discriminatory ASSAY &

SYSTEM

17

Ultrio

Non-Reactive

Testing

complete

Ultrio Reactive

Repeat Ultrio

Discriminatory

If NR If R

Testing Testing

Repeat Complete

Store the plasma for ALT

NAT

Ultrio Invalid

Repeat Ultrio

If Non-Reactive

Testing Complete

If Reactive Repeat

Ultrio

Testing Algorithm for PROCLEIX Ultrio

18

Conducted ID-NAT Evaluation in 2009 byProcleix Ultrio Assay

Routine ID NAT for HIV-1, HCV and HBVstarted from July 2010 by Procleix Ultrio Assay

Current annual donation – approx 40,000

19

TTI Marker Kit Detail Prevalence

HIVBIO-RAD 4th generation

Genscreen Ultra HIV Ag-Ab0.23%

HCV BioMerieux Hepanostika

HCV Ultra 3rd Generation kits0.48%

HBVBioMerieux Hepanostika

HBsAg Ultra 3rd Generation

kits1.36%

20

NAT Reactive:

A sample that isUltrio Reactiveirrespective of theSerology result isconsidered UltrioNAT reactive. Thissample is tested by arepeat screening testand discriminatorytest.

NAT Yield:

A sample that isUltrio Reactiveand SerologyNegative isconsidered as NATYield. WithDiscriminatoryresult it is called aYield for thespecific target.

Yield Rate:

Number of Sampletested /Number ofNAT Yield.

21

40672

samples

tested HIV-1 HCV HBV

Co

Infection

Combined

Total

NAT

Reactive

44

(0.11%)

96

(0.24%)

412

(1.01%)

37*

(0.09%)

589

(1.45%)

NAT Yield# 00 26 39 3** 68

Yield Rate 00 1/ 1564 1/ 1043 1/13557 1/598

* Co Infection: HIV-1-HBV: 6, HBV-HCV: 28, HIV-1-HCV:1, HIV-1-HCV-HBV:2.

** Co Infection Yield: 3 HBV-HCV 22

AIIMS NAT Experience

AIIMS NAT Experience

• More than Forty Thousand samples tested with routine Nat screening

• 68 samples were reactive for NAT but Non reactive with Serology

• NAT yield rate is 1 in 598 for all the three viruses

AIIMS ULTRIO PLUS*

STUDY

• 2nd Generation NAT assay for HIV-1,HCV

and HBV

• Brings down the HBV window period to

only 15 days

• New Target Enhancer reagent provides

optimal virus disruption, amplification and

detection of HBV DNA

• Excellent Sensitivity for HIV-1 and HCV

• Unique 2 region target for HIV-1 genome

* Product not Registered in India and not available for commercial use

AIIMS Ultrio Plus Experience

Samples tested:

2532HIV-1 HCV HBV Combined Total

Ultrio Plus

Reactive /

Discriminated

0

(0%)

3

(0.12%)

30

(1.18%)

33

(1.30%)

Ultrio Plus

Yield

0

(0%)

1

(0.04%)

7

(0.28%)

8

(0.32%)

Ultrio Plus

Yield Rate0 1 in 2532 1 in 362 1 in 317

Ultrio Plus Yield: Samples Reactive for Ultrio Plus but Non Reactive for Ultrio &

Serology

• Among 2532 samples tested Ultrio Plus

yield rate is 1 in 317

• Compared to our Routine NAT with Ultrio

Yield rate is 1 in 598.

• 8 samples (1 HCV and 7 HBV) were

detected by Ultrio Plus which are non

reactive by Ultrio and Serology

• Proves the better sensitivity and window

period reduction of the assay.

AIIMS Ultrio Plus Experience

1. Reddy R, Vermeulen M. “ Sensitivity of NAT Options: The SANBS Experience,” Novartis Symposium ISBT Lisbon, Portugal, June 2011.

Sensitivity shown when testing 23 HIV-1 low viral load, IDT window period yield samples.

Greater clinical sensitivity in IDT than MP6.

Both Ultrio and Ultrio Plus in MP8 were comparable to MPX in MP6 for HIV-1.

94%

76%

61%

42%

94%

76%

66%

52%

91%

62%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

IDT MP4 MP6 MP8 MP16

Ultrio

Ultrio Plus

MPX

% R

eacti

ve

The South African Experience with NAT

Ultrio and Ultrio Plus in IDT Detected More HIV-1 Positive Samples

than did MPX in MP61

% R

ea

cti

ve

62%

77%

39%

53%47%

31%

41%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Ultrio Ultrio Plus MPX

IDT

MP4

MP6

MP8

1. Reddy R, Vermeulen M. “ Sensitivity of NAT Options: The SANBS Experience,” Novartis Symposium ISBT Lisbon, Portugal, June 2011.

The South African Experience with NAT

Ultrio and Ultrio Plus in IDT Detected More HBV Positive Samples

than did MPX in MP61

Sensitivity shown when testing 107 Ultrio HBV IDT NAT yield samples.

Greater clinical sensitivity in IDT than MP6

Both Ultrio and Ultrio Plus in MP4 were comparable to MPX in MP6 for HBV.

NAT Testing on Buffy Coat Platelet

ID-NAT screened

Pooling

Platelet Concentrate

ID-NAT screened ID-NAT screened ID-NAT screened

29

Cost dynamics of ID NAT

o For a high volume blood bank doing 100% component

preparations Procleix assay is very cost effective as the

charging is cost per reportable result, which gets divided

across the components issued.

o Cost effective in the long run by preventing window

period infection thus eliminating the treatment cost and

burden on Health care and relief from infection to an

individual, the family and the society.

30

.

.

.

.

After implementing ID-NAT, Anti-HBc screening was

discontinued, thus reducing our discard rate from 16-20%

to 2-4%.

It helped in improving our inventory status for Blood

and Blood products.

Reduced discard rate meant Less requirement to

targeting higher number of donation.

Saving manpower and cost (which was earlier used for

Anti-HBc testing).

31

For a blood bank which is making 100% component from

a single unit of blood for e.g.

(a) Packed RBC (b) Platelet concentrate (c) FFP

For AIIMS Main Blood Bank, NAT yield rate is 1 in 598

donations (all three viruses)

3 lives are saved by testing one unit whole blood.

32

Serology tests will fail to detect Window Periodcases, use of NAT is an additional layer of safety tothe supply of blood and blood products.

Blood safety is a greater challenge in India becauseof the high Sero-prevalence of HIV (0.3%), HCV(0.7%), and HBV (1.4%) in blood donor populationand relatively low percentage (55.1%) of voluntarydonors.

In continuing the efforts to make the blood Supplysafer AIIMS main Blood Bank is evaluating anupgraded version of the Ultrio Assay (Ultrio Plus),in hopes of implementing this when the benefits arerealized.

33

34