ENHANCING BLOOD SAFETY THROUGH NAT: THE FIRST ......NAT : Indian Scenario Study Sample s tested Type of NAT Yield Remark Makroo et al, 2008 12,224 ID 8 (1/1108) 1 HIV, 1 HIV-HCV, 6

ENHANCING BLOOD SAFETY THROUGH NAT: THE FIRST BLOOD BANK EXPERIENCE FROM EASTERN INDIA

Dr. Ritam Chakrabarty, MD

Department of Transfusion Medicine

Apollo Gleneagles Hospitals, Kolkata

TRANSMEDCON 2016

Introduction

• Transfusion-transmitted infections are a major problem with blood

transfusion

• The benefit of Nucleic acid amplification test (NAT) over serology

has already been discussed elaborately by previous workers

• NAT has been able to detect infectious donors who were

otherwise designated normal serologically

• Here we share our experience of using NAT to screen blood

donors in Eastern India

Material and Methods • All blood donations between 23rd November 2013 and 31st August 2016

were included.

• Donors non-reactive for HBsAg, anti-HIV 1 & 2 and anti-HCV by CLIA

(VITROS, ECiQ, OCD) and negative for Syphilis and Malaria, were

subjected to NAT.

• NAT for HBV-DNA, HCV-RNA and HIV-RNA in the minipool of 6 samples

was performed using the Roche Cobas TaqMan MPX assay

• Individual sample of each positive pool was tested subsequently in the

same platform.

Material and Methods:

• Each positive sample was then investigated for viral

discrimination and viral quantitation from reference

laboratory.

• Sample positive for hepatitis- B virus (HBV) DNA was

further screened for anti-HBc antibody & antibody to

HBsAg (anti-HBs) by CLIA (VITROS, ECiQ, OCD).

• All results were documented & recorded as per the SOP.

Results • Of the total 24799 blood donations, HBsAg, anti-HCV, and anti HIV

by CLIA were detected in 168 (0.677%), 148 (0.596%) and 58 (0.233%)

donors respectively (Table 1)

• 172 donors (0.694%) tested positive for VDRL

• Out of 24253 samples tested for NAT, 9 (0.037%) were positive for

HBV DNA and 1 (0.004%) for HIV RNA

• The NAT yield was observed to be 1 in 2425 donations

• 7 NAT positive donors were sero-reactive for anti-HBc and 6

reactive for anti-HBs antibodies

• Viral load in each sample was < 6IU/ml (Figures)

Donor screening by NAT

Total blood donation

N = 24799

Serology / CLIA non reactive: 24253 Serology / CLIA reactive: 374

HIV 1& 2

N = 58

(0.233%)

HbsAg

N = 168

(0.677%)

HCV

N = 148

(0.596%)

NAT positive: 10 (yield = 1 / 2425)

HIV 1& 2: 1 HBV: 9 HCV: 0

Anti-HBc reactive: 7 Anti-HBs reactive: 6Viral load (N = 9): < 6IU/ml

VDRL +ve: 172

Viral discrimination & quantitation

Viral discrimination & quantitation

Viral discrimination & quantitation

DISCUSSION

HIV

HBVHCV

1996199419921990198819861984

1:100

1:1000

1:10 000

1:100 000

1:1 000 000

1998 2005

Ris

k p

er u

nit

Modified from Busch et al. JAMA 2013; 289: 959-62

2012

Evolution of Transfusion Risks near “eradication” of major viruses

Blood Safety in IndiaOut of 8 million annual donations, majority are first time donors

Heterogeneous structure of blood banking: Government / Private

/ NGO / Stand Alone

Marker General Population Blood Donor

HIV 0.36 % 0.3%

HBV 2.5 – 4% 1.4%

HCV 0.9% 0.7%

Marker

HIV

HBV

HCV

Antibody

Genome

Time of

infection

Time

Months/years

Antigen

Generalized course of TTI

30-70 days 3rd Gen ELISA

8-14 days

NAT

25-60 days 4th Gen ELISA/CLIA

Mandatory

Rs 100 - Rs 200

Additional Rs 200 - 300

Additional Rs 700 - 1000

Who will bear the economic burden?

What is NAT?• Nucleic Acid Technology (Nucleic Acid

Amplification Testing)

• All involve extraction or capture of nucleic acid,

amplification and detection

Commonly used systems are

PCR-based assays – NA amplification by

denaturation, annealing & primer extension

Transcription mediated amplification (TMA) assay

- Isothermal NA amplification in the same tube

Benefits of Implementing NAT

Decreases WP infection unlike serological assays

Increased sensitivity and specificity

Detects viral mutants and immunosilent carriers

Earlier donor counseling and patient care

Characterization of genotype is possible.

Future application may include additional viruses e.g.

CMV, Dengue etc

Genotype coverage in NAT

HIV-1 Group M, subtypes A, B, C, D, F, G, H

HIV-1 Group O (constitute 1-5% of all HIV)

HIV-2 (Increasing prevalence in India)

HBV, genotypes A, B, C, D, E, F, G

HCV, genotypes 1a, 1b, 2 a, 2b, 3a, 4a, 5a, 6a

NAT : Indian ScenarioStudy Sample

s testedType

of NATYield Remark

Makroo et al, 2008

12,224 ID 8 (1/1108)

1 HIV, 1 HIV-HCV, 6 HBV

Singh et al 2009

20,256 MP 11 (1/2000)

All HBV

Jain et al, 2012

23,779 MP 8 (1/2972)

8 HBV

Agarwal et al, 2013

73,898 ID 121 (1/610)

1 HIV, 37 HCV, 73 HBV, 10

co-infKoshy et al 2013

52,083 ID 47 (1/1108)

43 HBV, 3 HCV, 1 HIV

Cobas® TaqScreen MPX Test, v2.0 Simultaneous detection of multiple targets

FAMHIV-1-M, O, 2485/520 nm

HEXHBV

540/575 nm

JA270HCV

610/640 nm

CY5.5IC680/715 nm

Ch. 1

Ch. 3 Ch. 2

Ch. 4

Dyes and Filters

Real time multi-channel detection

Analytical SensitivityComparative analysis

Markers ProcleixUltrioElite

CobasTaqScreenMPX v2.0

Comments

HIV M Group 18 IU/mL 46.2 IU/mL Doubling Time of HIV (17 hrs) & HCV (10.8 hrs) virus is short& also viral load in window period is high for these RNA viruses, so lower sensitivity for HIV & HCV shall not miss any window period infections.

HIV O Group No claim 18.3 copies/mLHIV-2 10.4 IU/mL 7.9 IU/mLHCV 3.0 IU/mL 6.8 IU/mL

HBV 4.3 IU/mL 2.3 IU/mL Doubling time of HBV is long (2.6 days) & viral load in initial window period infection is not very high so probability of missing out HBV with a less sensitive assay is high

Viral load (copies/ul)HIV: upto 107

HCV:upto 107

HBV: 10 – 104

Conclusion • Introduction of NAT has successfully identified infectious donors

• Despite its cost effectiveness issues NAT will be a standard of

blood donor screening in the future

• With the implementation of NAT in our blood centre we could save

30 patients in 33 months who would have otherwise received the

infected blood components

• NAT could detect 9 occult hepatitis in otherwise healthy donors

who were then referred to gastroenterology for management

• 1 HIV donor was detected in window period

• Restraining these donors from further donations saved more lives

• Blood bank without NAT facility may start routine anti-HBc testing

Thank [email protected]