# Presentation: 760705

## Blood Products Advisory Committee Meeting

- April 26, 2007
**Ben Marchlewicz, Ph.D.**
- PRISM R&D, Program Manager

## Abbott’s approach to Chagas Testing
PRISM Chagas
Description of the assay
Performance
Chagas Confirmatory
Description of the assay
Performance

**PRISM Chagas**
**Description of the assay**
**Performance**
**Chagas Confirmatory**
**Description of the assay**
**Performance**

**Notes:**
I want to thank the Committee for this opportunity to present some information on two assay under development at Abbott.  A screening assay for the PRISM instrument system, and an immunoblot confirmatory assay for specimens repeatedly reactive in the screening test.  An overview of each assay format along with preliminary test results will be presented.

## Abbott’s Strategy in Blood Screening for Chagas Disease

**Develop a recombinant antigen based assay for antibodies to *****T. cruzi***** on the PRISM platform**
- Well-characterized antigens
- Presenting multiple epitopes
- Non-biohazardous
**Develop a recombinant antigen based Confirmatory assay for resolution of PRISM Repeat Reactive specimens.**

**Notes:**
A key part of Abbott’s approach to Chagas testing is the use of well characterized recombinant peptides.  As you will see, this minimized the potential for cross reactivity with other parasitic diseases.  

## PRISM Chagas Assay

## PRISM Chagas Screening Assay Design Configuration

- Dual rAg Coated P, Single mAb Conjugate, Two–Step Assay

**Notes:**
This schematic shows the assay processing steps performed by PRISM instrument.   An aliquot of donor serum or plasma is mixed with Specimen Diluent and incubated with recombinant coated microparticles.  Following a wash step to remove unbound antibodies, an mouse anti-human conjugated is added.  Again following incubation and wash steps, an Activator solution is dispensed.   Positive specimens, that have bound the acridinium labeled conjugate, release photons of light in the presence of activator.   The amount of light released is proportional to the level of anti-T. cruzi antibody in the specimen.

## Multi-epitope Recombinant Antigens used in PRISM Chagas Assay

| Multi-epitope rAg | Amino acids | Mol. Wt. (Kda) |
| --- | --- | --- |
| TcF | 85 | 8.3  |
| FP3 | 693 | 76 |
| FP6 | 994 | 110 |
| FP10 | 722 | 78 |

**Multi-epitope Recombinant Antigens ****used in PRISM Chagas Assay**

**Notes:**
There are 4 peptides used to coat the microparticles, this is a brief overview of their relative sizes.

## Specificity

| Study | Total | Repeatedly Reactive | Confirmed Positive | Resolved Specificity |
| --- | --- | --- | --- | --- |
| In-House Feasibility Testing | 7,755 | 4 | 1 | 99.96% |
| Field Study | 4,100 | 8 | 3 | 99.88% |
| Total | 11,855 | 12 | 4 | 99.93% |

**Random Donors**

**Notes:**
As we have heard today, specificity is an important consideration for a Chagas screening assay.   This shows the performance of the PRISM assay on approximately 12,000 specimens.

The 4 confirmed positives were both Chagas Confirmatory and RIPA positive.

## Specificity

| Study | Total | Specificity |
| --- | --- | --- |
| Leishmania visceral
(sourced from India) | 30 | 100% |
| Leishmania cutaneous
(sourced from India) | 11 | 100% |
| Malaria | 10 | 100% |

**Cross Reactivity with other Parasitic Infections**

**Notes:**
In addition to specificity on general random donor populations, the performance on potentially cross reactive parasitic infections.   On a limited number of leismania and malarial samples the PRISM Chagas assay did not give any Reactive results.   To date we have not evaluated any Paracoccidioides brazilensis samples.  However, that specimen type will be tested during Validation studies.

**PRISM Chagas Assay Sensitivity****Chagas Positive Specimens (confirmed by RIPA)**

|  | RIPA Positive | RIPA Negative  |
| --- | --- | --- |
| PRISM Positive | 419 | 0 |
| PRISM Negative | 0 | 0 |

**Sensitivity**

**100% Sensitivity**

**Notes:**
Just as important as Specificity is the ability of an assay to detect as Repeatedly Reactive, true positive specimens.   T. Cruzi  anti-body positive samples collected from endemic areas of Central and South America, as well as some reactives from the US), were tested in the PRISM Chagas assay.  All 419 RIPA confirmed positive specimens were detected as Repeat Reactive.

## Chagas Confirmatory Assay

**Notes:**
Having a supplemental test to confirm Repeat Reactive specimens in a screening assay, is an important part of the issues presented so far this afternoon, and key to your discussions to follow.   In parallel with the development of the PRISM Chagas Screening assay, Abbott is developing an immunoblot confirmatory assay.

## Design of Chagas Confirmatory Immunostrip

- 			               Anti-hIgG
- 			     (Sample Addition CTL)
- 		     			             hIgG-H CTL
- 	    			           hIgG-L CTL
- 	   			             (Cut-off)
-  _4 rAgs for anti-__*T. cruzi*__ detection_
**       ****TcF **   **FP3 **   **FP6    FP10 **

-        ID#

- Test Bands

- On-board CTL bands

**Notes:**
This is a schematic of a representative Chagas Confirmatory Immunostrip.   There are 3 onboard controls, a High and a Low human IgG control.   The Low IgG control determines the positive cut-off of the assay.  Also, to assure sample addition and therefore minimize the chance for a false negative due to procedural error, there is an Anti-human IgG control.    The 4 remaining Test bands are specific for four recombinant T. Cruzi antigens.

## Slide 12

**Notes:**
Similar to the diagram shown earlier for the PRISM Chagas assay, this drawing indicates the processing steps of the Chagas Confirmatory assay.   In this case, the anti-human conjugate is labeled with Alkaline Phosphatase, and the enzyme substrate is a BCIP/NBT solution.  Positive presence of anti-T. cruzi  antibody is indicated by light gray to dark purple bands on the immunostrip.

## Visual Reading of Test Bands

| Intensity | Score |
| --- | --- |
| Absent | - |
|  Faint or < on-board hIgG-L CTL | +/- |
|   =  on-board hIgG-L CTL | 1+ |
|  Between on-board hIgG-L CTL and hIgG-H CTL | 2+ |
|   =  on-board hIgG-H CTL        | 3+ |
|   >  on-board hIgG-H CTL | 4+ |

**Notes:**
By definition, the on-board low IgG control is defined as a 1+ reaction, and the High IgG control is a 3+ reaction; the color intensity of the recombinant antigen  bands are scored based on relative intensity to those controls.   Although final criteria for determining positivity will be evaluated during Validation studies, currently the presence of 2 or more bands with at least 1 band having a score of > 1+, is considered positive.

## Sensitivity

**Sensitivity**

|  | RIPA Positive | RIPA Negative |
| --- | --- | --- |
| Immunoblot Chagas Confirmatory Positive | 410* | 0 |
| Immunoblot Chagas Confirmatory Negative | 0 | 0 |

**100% Sensitivity**

- * Also reactive in PRISM Assay & 2 reference ELISA’s

**Notes:**
Evaluating over 400 RIPA positive specimens, the Chagas Confirmatory assay correctly identified 100% as positive.

## Chagas Specimens Panel n = 410  (All are confirmed RIPA positive)

| Country of Origin | # of Samples |
| --- | --- |
| Argentina | 41 |
| Bolivia | 53 |
| Brazil | 176 |
| Chile | 5 |
| Colombia | 1 |
| Ecuador | 1 |
| El Salvador | 12 |
| Honduras | 4 |
| Mexico  | 33 |
| Nicaragua | 17 |
| Surinam | 1 |
| USA | 4 |
| Venezuela | 21 |
| Unknown | 41 |

**Chagas Specimens Panel**** ****n = 410****  **(All are confirmed RIPA positive)

**Notes:**
This slide shows a breakdown of the countries of origin of the samples tested.  As you can see, the specimens span the most commonly recognized endemic areas of Chagas infections.

## Specificity

**Specificity**

|  | RIPA 
Positive | PRISM
Chagas Assay Non-reactive  |
| --- | --- | --- |
| Immunoblot Chagas Confirmatory Positive | 1* | 0 |
| Immunoblot Chagas Confirmatory Negative | 0 | 500 |

**100% Resolved Specificity**

**Unscreened Random Blood Donors**

- * Also reactive in PRISM Assay & 2 reference ELISA’s

**Notes:**
Although the Chagas Confirmatory assay will be used to confirm samples that test Repeatedly reactive in the PRISM Chagas assay, it is important to evaluate the specificity of assay with presumptively negative random blood donor samples.   This clearly shows that on 500  PRISM Chagas negative samples, the Chagas confirmatory assay was also negative.   The one sample the tested positive, in this study was also RIPA positive and 

## Specificity

**Specificity**

|  | RIPA 
Positive | PRISM
Chagas Assay Non-reactive  |
| --- | --- | --- |
| Immunoblot Chagas Confirmatory Positive | 2* | 0 |
| Immunoblot Chagas Confirmatory Negative | 0 | 316 |

**100% Resolved Specificity**

**Unrelated Medical Conditions**

- * Also reactive in PRISM Assay & 2 reference ELISA’s

**Notes:**
In addition to the negative Random Donor population, specimens from specific disease states including CMV, Toxo, Rubella Syphilis, HBV, HSV, HAV, HIV, VSV, EBV, Yeast, Leprosy, intestinal protozoans, TB, hymolysis, bilirubin, trigs, RF, HAMA,  hyper IgG & IgM, Flu vaccine, sclerosis, muliple myeloma,  Leishmania (30) , malaria (13), SLE (11)

## Specificity

| Study | Total | Specificity |
| --- | --- | --- |
| Leishmania visceral
(sourced from India) | 30 | 100% |
| Leishmania cutaneous
(sourced from India) | 11 | 100% |
| Malaria | 10 | 100% |

**Cross Reactivity with other Parasitic Infections**

**Notes:**
Similar to the PRISM Chagas results shown above, the same Leishmainia and Malaria samples were tested in the Chagas Confirmatory assay and found to be negative.

## Summary

- 	A fully automated blood screening assay for antibodies to *T. Cruzi, *on the PRISM system,* *is under development;
- 	Along with an Immunoblot Confirmatory assay, that can be performed on site, to confirm Repeat Reactive specimens to aid in donor management.

**Notes:**
Finally, we wanted to present to the committee, this brief overview of an additional pair of assays for the screening and confirmation of blood donors for the presence of anti-T. cruzi antibodies.  A key part of this strategy is an FDA licensed Confirmatory kit that can be performed on-site at blood bank testing facilities affording a rapid turn around of supplemental test results for better donor management.
There are currently over 55 PRISM instruments installed in 30 Blood Bank and Plasma centers across the country.  Screening millions of blood units annually.   A T. Cruzi antibody assay on that platform can be implemented seamlessly into that testing environment.   

THANK YOU,