Feasibility study outline (final)

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INDIA OFFICE: C-33A SHASTRI NAGAR, GHAZIABAD-201002 (UP) INDIA

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Index

S.No Description Page No.

1 Introduction to SMARTubeTM

3

2 Back Ground 4

3 Purpose and Scope of the Study 4

4 Study Design 5

5 Expected results 7

6 Outcome of studies 7

7 Benefits to the participating organization 8

8 Research Procedures 9

9 Methodology 10

10 Comments & Notes 12

Administrative and Financial Issues

11 Economic/ Financial Implications 14

12 Equipment and Material Required 15

13 Defining of Responsibilities 15

Additional Inputs for Clinical Trial

14 Instructions for Use 18

15 Guidelines for Running a laboratory evaluation 19

16 Notes & Tips 20

17 Trouble Shooting 25

18 Extracts of Clinical Trials in different parts of the word 27

19 Case Histories – Applications & Benefits 30

20 Follow up questions and clarifications 33


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Introduction to SMARTubeTM

SMARTubeTM

being introduced for the first time in India – is a first of its kind, innovative medical

diagnostic product that will revolutionize HIV & HCV detection. Stimulating Maximal Antibody

Response Tube – SMARTube™ enables earlier, better and complete detection of HIV/HCV just a

week after exposure. SMARTube™ not only enables the detection of all the patients who are

diagnosed in the conventional testing - but also enables detection in additional patients that are

infected, but otherwise would have gone undetected at that testing time. As a cost effective

method that increases the SENSITIVITY and SPECIFICITY of other known HIV & HCV detection

devises—with very little additional training or cost input, it will help in saving millions of lives.

SMARTube™ is manufactured under strict ISO 9001:2000 and ISO 13485:2003 regulations and the

highest global Quality Control, R&D and professional standards. SMARTube™ has been awarded--

CE Mark—the regulatory stamp of approval in the whole of Europe (the EU countries) and is

certified for public and individual use in Germany, Russian Federation, South Africa, Israel,

Romania, Nigeria, and Turkey. It is being used in these countries in hospitals, diagnostic labs,

blood banks, health or life insurance uses—anywhere blood samples need to be tested for HIV.

SMARTube™ Benefits include:

• Enables early detection than any other existing methods, within days of exposure

• Simple, affordable and reliable

• Requires no changes to the existing testing procedure

• Saves lives and suffering

• Proven effective

• Increased sensitivity

• Increased specificity

• Cost effective – Saves: Money, Time, Resources

SMARTube™ has been tested in controlled clinical trials on over 10,000 patients/individuals in

several countries like China, Israel, Kenya, Mexico, Romania and South Africa. Most of these

clinical trials and tests were done by reputed government agencies, blood banks, reference

laboratories, academic and professional bodies.


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Background:

The window period of HIV and HCV infection is a major concern for the governments, health

authorities and professionals, blood banks, vaccine and drug developers all over the world, as

many infected individuals test negative for HIV or HCV antibodies, and are thus misdiagnosed.

A simple process, called Stimmunology, for stimulating the antibody production in-vitro in the

blood sample, prior to testing it for HIV (and/or HCV) antibodies, has been developed, to solve the

window period. This has been implemented in the SMARTube™ HIV&HCV, a blood pre-treatment

device, which enables the detection of HIV and HCV infections (during the window period) by

overcoming, in vitro, the specific immune suppression exerted by the virus, and which is the cause

for the window period.

This process involves placing 1ml of blood sample inside the SMARTube™ for a 3-5 day incubation

period, leading to the formation of HIV and/or HCV antibodies in detectable levels in all those

infected, including those in the window period. The detection of the antibodies is done by the

current serological assays and antibody detection kits (ELISA, WB), following the same procedures

and algorithms, just with an improved sample – the SMART-plasma.

1. Purpose & Scope: To evaluate the effectiveness of SMARTubeTM

under Indian conditions.

1.1 The purpose of this study is to evaluate the feasibility of using SMARTube™ in India, in

different settings such as hospitals, clinical and diagnostic laboratories, VCT clinics, blood

transfusion centers, AIDS centers, epidemiological and research institutes by conducting

short term studies validating the usefulness of the SMARTube™ in Indian Conditions.

1.2 By participating in the study, the investigators will be able to have early access to on an

innovative technology, and collect data within the Indian health, scientific, and public

settings, gaining important scientific insight into the early stages of infection, confirmed

diagnosis, true measure of prevalence and incidence rate, and more.

1.3 Based on the results obtained in this study, the participating investigators will be able to

evaluate the feasibility of using the SMARTube™ technology in the Indian settings,

become opinion leaders on this issue for curtailing the HIV and HCV epidemics in India.


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2. Study Design:

2.1 - Design Rational

Previous testing of thousands of individuals and blood donors in different countries have

shown that pre-treating the blood sample using SMARTube™ HIV&HCV (a technology

which drives primed HIV and/or HCV immune cells to complete their proliferation and

differentiation and secrete HIV and /or HCV specific antibodies in culture, at levels

detectable by current antibody detection assays and kits) leads to the detection of

additional antibody positives, currently missed by regular serology. [All positive results

confirmed by the local algorithms using the locally used kits, cutoff points, controls,

practices, and guidelines. These seronegative yet infected individuals are infected, yet

missed by current serology, as they are at the very early stages of the infection, when they

are potentially infectious and undetectable (i.e. in the window period).

The rate of additional positives is dependent on several factors including:

• Incidence in the regular population,

• Incidence in the donor population (if different from the above due to donor

• selection, where applicable),

• Length of the window period in that population.

• Length of the asymptomatic period in that population.

These factors are not always known, and thus estimates can be done based on the

seroprevalence and the known (if any information available and provided) dynamics of the

spread of the HIV and/or HCV epidemic in that population/area/nation.

The aim of this study is to test the feasibility of using Stimmunology (as embodied in the

SMARTube™) for use in routine settings of HIV and/or HCV testing, and thus enabling the

detection of HIV and/or HCV infections among the seronegative individuals prior to the

appearance of anti-HIV or anti-HCV antibodies in the serum, in different settings in India.

The end point is either the detection of an additional HIV (and/or HCV) carrier, or the

‘routine’ implementation of the SMARTube™ incubation step in the testing center’s setting

for 1-3 months, testing 500-2000 samples.


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The study shall include ‘all’ blood samples, collected on the site, and brought to the

laboratory for testing, for a set time (1-3 months). A small heparin tube (or blood collected

in heparin washed syringe) will be collected from each individual (or donor) to be tested for

HIV and/or HCV antibodies. The blood sample will be sent to the laboratory at room

temperature. In the laboratory, one ml of blood will be aseptically transferred into a

labeled SMARTube™ and incubated in a humidified 5% CO2 incubator set at 370

C for 5

days (3 days in blood banks). The rest of the plasma will be stored after the routine testing

for HIV and/or HCV antibodies.

Following the incubation step, the supernatant = SMART-plasma will be collected and also

tested for HIV and/or HCV antibodies, using the same kits and algorithms as the regular

plasma is/was tested. The remaining volume of SMART-plasma is stored for repeat or

confirmatory testing and for future research work.

The results for HIV (and HCV) antibodies in plasma and in SMART-plasma will be compared.

2.2 - Population size, basic outline, and end point.

The study will have two types of end points:

I. Completion of a 1-3 months validation of implementation protocol for entering it into

the center’s/ laboratory’s routine testing.

II. Having an initial estimate of the rate of hidden / missed infection among the blood

donors achieved by the detection of one or more additional positive (defined as

routine plasma negative, SMART-plasma positive), or greyzone/ indeterminate

/discrepant readings in plasma that turn clear positive in the SMARTplasma.


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3. Expected results:

• All seropositive samples will also be SMART-plasma positive. – No loss of sensitivity.

• There might be (pending sample size and incidence rate in the tested populations) – additional

positives, i.e. seronegative yet antibody positive in the SMART-plasma –

� Increase in sensitivity,

� More confirmed diagnosis

� Marked improvement of blood safety,

� More true prevalence rates,

� A good measure of incidence rates.

• There might be (pending sample size and incidence rate in the tested populations) –

Seropositives, which will have increased levels of antibodies in the SMART-plasma. These

individuals are recent sero-converters. - A measure of the incidence levels.

• Some low positives or borderline/gray-zone positives might become clear positives (or clear

negatives) after the incubation in the SMARTube™. – Increase in sensitivity and specificity.

• Some plasma false positives might be clear negative in the SMART-plasma, reducing the false

positive rate. -Increase in specificity.

Note: General sensitivity and specificity of the results are dependent on the antibody testing kits

used and their intrinsic qualities.

4. Outcome of studies:

Following validation and implementation steps, the criteria for diagnosing an HIV or HCV infection,

based on antibodies, or for releasing a blood unit based on SMART-Plasma results should follow

the same guidelines as those for plasma results for HIV and/or HCV antibody screening – leading to

a healthier society and safer blood supply.

The studies will be the initiation of an implementation plan, initially in the participating institutes

and eventually in others in the city, district and eventually the country, with the first users offering

leadership and guidance to others.


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5. Benefits to the participating organization:

• Thought Leadership-- Prestige of being part of a handful of organizations in different

regions across India

• First mover Advantage: The organization can claim credit, if so desired, for being involved

in trying out a new technology for the first time in the country

• Corporate Social Responsibility & Contribution for a noble cause: Any organization which

volunteers to participate in this research is obviously contributing towards the noble cause

of understanding and removing the barriers in the eradication/containment of lethal HIV-

HCV combine.

• Contribution to medical research and sharing of information for better and complete

diagnosis of HIV & HCV

• Credit for international collaborative research at a justifiable yet negligible cost, effort,

manpower and material expense

• Valuable experience for the staff, research associates, students and technicians towards

tackling the window period

• Key Opinion Leaders: After the completion of the study select individuals in different

organizations will be identified as key opinion leaders for this new technology for

participation as special invitees in various national-international medical forums and

conferences and if they do so agree their interviews/ opinions will be published in national-

international media besides assisting them in getting their study findings published in

reputed medical journals and websites.


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6. Research Procedures:

All procedures are performed at room temperature except for the incubation of the SMARTube™

with the blood samples performed at 36.5-37.5°C.

6.1 Work Instructions (using regular SMARTube™)

At Site:

6.1.1 Mark a (Lithium) Heparin coated vacuum tube with the donor code. Record date

and time. [If syringes are used, rinse the syringe with heparin]

6.1.2 Fill, aseptically, the labeled vacuum tube, (best, using a G21 sterile needle). Mix the

blood by inverting gently the tube 8 times.

6.1.3 If using other means for blood collection, use heparin as the anti-coagulant.

Note: Blood should be transferred to the SMARTube™ and put into incubation the same

day (within 24 hours, at room temperature).

In the Laboratory:

6.1.4 Record the incoming blood samples in Blood Sample Log-in Book.

6.1.5 Label the SMARTube™ accordingly.

6.1.6 Set up the culture in the SMARTube™ by mixing the blood and then, aseptically,

adding 1 ml blood to SMARTube™ with a sterile disposable pipette. Re-cap the

SMARTube™ lightly (’one click’ not two)

6.1.7 Incubate SMARTube™ with blood in 36.5-37.5C, 5% CO2 humidified incubator for

3-4 days as described in Table 1.

Note: Be careful not to close the cap tightly, in order to allow gas exchange.

Note: Record draw time and incubation time in “Laboratory Sheet”.


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6.1.8 Spin down, or let the RBC settle to the bottom of the tube and then transfer some

of the plasma (~1 ml) into an eppendorf tube, mark it accordingly and keep for future

testing (refrigerated for several days or frozen <-20oC for longer periods.).

Use the rest of the plasma for testing for HIV and/or HCV antibodies using the current

routine and testing algorithms (ELISA, WB).

6.1.9 At the end of the incubation period of the sample in the SMARTube™ (5 days, with

minimum 3 days in a blood bank) aspirate the upper phase (ST supernatant fluid with

plasma = “SMART-plasma”) and transfer to sterile, screw cap, Eppendorf tubes (or other

small test tubes), mark accordingly.

6.1.10 Write the incubation stop date and time in the “Laboratory Sheet”*.

6.1.11 Test the SMART-plasma and the plasma for HIV (and/or HCV) antibodies using the

routine ELISA kits (compensate for the dilution – see "instructions for use", and “notes and

tips”). If positive, confirm result by repeat ELISA testing (on a different kit?), and WB where

relevant.

6.1.12 Freeze all the remaining SMART-plasma immediately at -80C (or > 20C), for future

testing.

6.1.13 The Optical Density (OD) results, the Cut Off (CO), the positive control value(s), the

kit’s name, and the plate number will be recorded together with the OD of the plasma and

/or the SMART-Plasma.

6.2 Methodology to be adopted:

6.2.1 HIV and/or HCV antibodies detection:

6.2.1.1 The level of HIV and/or HCV antibodies in plasma and in SMART-Plasma after

incubation of blood in SMARTube™ will be detected using the same ELISA kits used

routinely by the laboratory and/or blood bank. The loading conditions of the samples will

be modified to achieve a final plasma concentration as recommended in the kit. To this

end, when possible, 5 times the recommended volume of ST-Plasma will be loaded on the

plates. Diluent should be adjusted to obtain the same final volume, as mentioned in the

instructions for use.


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6.2.1.2 The volume of SMART-Plasma used on the ELISA will be adjusted as described in

6.2.1.1. Examples for correction for the dilution of the plasma in the SMARTube™,

depending on the kit used and the volume of plasma it requires:

Full compensation:

10ul plasma + 100ul diluent = 50ul ST-Plasma + 60ul diluent


Partial compensation



No compensation

100ul plasma = 100ul ST-Plasma

Stimmunology has been tried using different ELISA kits and different plasma volumes required. In

all settings, plasma positives were also positive after the SMARTube™.

6.3 Culture Incubation days

Day in Day out (day 3) Day Out (day 4)

Sunday Wednesday Thursday

Monday Thursday Friday

Tuesday Friday Saturday

Wednesday Saturday Sunday

Thursday Sunday Monday

Friday Monday Tuesday

Saturday Tuesday Wednesday

6.4 Interpretation of results:

Any sample, with or without the SMARTube™ pre-treatment, which tests positive for HIV or HCV

antibodies according the site’s algorithm, is positive for HIV or HCV infection respectively.

Sensitivity and specificity of the results are dependent on the kit itself and its intrinsic qualities.


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7. Comments and notes:

1. The SMARTube™ is simple to use and no complex training is required. A CO2 incubator is a

must as the incubation in the SMARTube™ is a tissue culture step requiring “body-like”

conditions for the proliferation and differentiation of the relevant lymphocytes leading to

antibody production in-vitro, against the HIV and/or the HCV virus which was encountered in-

vivo.

2. The above outline is a minimal one. Based on the interests of the principal investigator(s)

many additional questions can be addressed, both immunological and epidemiological. These

can be incorporated into the basic initial study, or be designed as a complementary or

continuation study. Many questions can be asked using the same initial blood draw, with

specific tests run either in parallel or following the initial study. Others might require follow-

up samples, on later dates, parallel to and following the conclusion of the initial study.


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Administrative and Financial Issues


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8. Economic/ Financial Implications

Since the SMARTube™ is implemented within the existing testing and laboratory set-up, the

additional expenses are only:

1. Pipettes for 1ml blood transfer.

2. Test tubes for storing SMART-plasma for future testing and research.

3. The additional testing of SMART-plasma, (as it is done parallel to the regular plasma

testing) for HIV and/or HCV antibodies.

• Each site should have a (5%) CO2 incubator set to 37oC for the culture step in the

SMARTube™.

• Training is minimal and the additional technician time is very small and can be calculated

as per the additional work listed above.

• The total additional cost, per sample, can be calculated based on the local cost of testing

kits, with the additional (minimal) expenses for a pipette and two test-tubes.

Scope of study –

8.1 Basic Study - testing, for antibodies only for HIV and/or HCV. (Sample size 1-200)

8.2 Medium Study – testing, for antibodies only for HIV and/or HCV. (Sample size 201-500)

8.3 High Level Study – testing, for antibodies for HIV and/or HCV. (Sample size 501-above)


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9. Equipment and Materials Required:

9.1 ELISA kits for HIV and/or HCV.

9.2 SMARTube™ HIV&HCV (stored refrigerated, brought to room temp prior to use)

9.3 Humidified CO2 incubator set at 370C and 5% CO2

9.4 Sterile plastic Pasteur pipettes (1 ml)

9.5 Sterile Eppendorf tubes (or other small test tubes), screw cap, for repeat testing,

confirmation testing, and for freezing samples for future testing.

10. Sharing of Responsibilities

10.1 What we Eternal Well Foundation will provide?

• Eternal Health & Wellness Foundation will provide technical support to the research site for

data analysis and use of SMARTubeTM

• Eternal Health & Wellness Foundation will facilitate the research site wherever possible with

information/ research and scientific insight from the experiences gained in the other

countries

• Eternal Health & Wellness Foundation will not fund the research/ efficacy study but will be

happy to provide SMARTubeTM

(s)

required for the research at the following highly

subsidized and special concessional rates:

• Basic Study (Sample size 1-200)

o Eternal Health & Wellness Foundation will provide 75 SMARTubeTM

s FREE

• Medium Level Study (Sample Size 201-500)


s FREE

• High Level Study (Sample Size 501 – Upwards)


s FREE

• Additional number of SMARTubeTM

s required for the trails/ efficacy studies will be provided

at the rate of 3 Euro per SMARTubeTM


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10.2 What is expected of you (Research Site)?

• To conduct feasibility studies for implementation of SMARTube™ in India, and to measure

the rate of new infections in the studied populations using SMARTube™ HIV&HCV for

indentifying the HIV and HCV infections during the window-period leading to a healthier

society and a safer blood supply in India.

• The site will be responsible for the collection of the blood, the incubation in the

SMARTube™, and the antibody testing as per mutually agreed time line for initiation of the

study, its completion, conclusions, and future recommendations.

• The site will be responsible for the data management, as per the agreed protocols and

format, and for the storage of the samples for future use

• The site will be responsible for timely completion of the study as per agreed schedule.

• All data will be shared in an open and cooperative form. Eternal Health & Wellness

Foundation shall have access to information about the progress of the study and its

direction.

• The research institute will provide copy of the data, final report and recommendations to

Eternal Health & Wellness Foundation before releasing it to any third party.

• The final report and recommendation will be published only in consultation and with the

consent of Eternal Health & Wellness Foundation.


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Additional Inputs for Clinical Trial


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11. Instructions for Use:

For using SMARTube™ HIV&HCV as a blood pre-treatment prior to HIV or HCV antibody testing

of a blood sample.

1. Limit exposure of SMART media to fluorescent lights (refrigerators for SMARTubeTM store

should not be with glass doors).

2. Blood should be collected into heparin and transferred to the laboratory within 20 hours at

room temperature (RT) (15-25oC).

3. Transfer 1ml of blood (mixed well) into a properly marked SMARTube™ at RT under

aseptic/clean conditions. (See “Notes and Tips” #6).

4. Re-cap the SMARTube™ in the loose position (one click instead of two), and put into 5% CO2

incubator, at 37 oC (CO2 gas phase in the incubator can be anywhere between 5% and 7%, best

set at 5%). Write the date on the tray. (You might want to already mark the planned “out” date

also on it so it is easier to follow).

5. After 3-5 days (best results are obtained on day 5), take the SMARTube™ samples out of the

incubator, and use the supernatant as the sample on the ELISA antibody test used in the lab.

6. Keep the remaining sample volume refrigerated for repeat or further testing within that day. For

later repeat testing, transfer the supernatant fluid to another, properly marked sterile test tube to

prevent hemolysis, and keep refrigerated. For future reference and long term storage, freeze at -

20oC.

7. Since the plasma has been diluted in the SMARTube™ (1ml of blood = ~0.5 ml of plasma, was

put into 2ml of SMART medium), it is best, when possible, to Compensate for this dilution (x5) in

the sample volume used on the ELISA (See “Notes and Tips” #8).


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12. Guidelines for running a laboratory evaluation:


of a blood sample.

1. Clear objectives should be set. These may be either an increase in detection level, or

reduction of false positive/noise, in the HIV and / or HCV antibody testing at the site.

2. Based on the objectives, the scope of the testing to be done, and your time frame, chose

the populations/samples to be tested for evaluation, and number of samples to be run.

3. SMARTube™ enhanced blood samples will be the only samples tested for HIV and/or HCV

antibodies once SMARTube™ is introduced into the routine work of the laboratory, thus

no additional ELISA assays will need to be purchased or preformed. However, during the

evaluation period each blood sample will get tested twice, on the same ELISA -- once with

untreated plasma and once with the SMARTube™ enhanced sample.

4. The key points one could look at during an evaluation:

a. All (true) seropositives are positive also after the SMARTube™.

b. In a few seropositives, elevated antibody levels might be measured after the

SMARTube™ enhancement step (due to early seroconversion status of the

donor/patient). Conclusion: Increased sensitivity of your assay and enabling

evaluation of incidence levels.

c. In a population with a high incidence level, and with the right population size to be

tested, additional antibody positive(s) will be detected. Conclusion: Increased

sensitivity of your assay by detecting those previously missed, enabling the detection

of those in the window period, and giving a more true indication of the current state

of the epidemic, for both prevalence and incidence information.

d. When using an assay system with a high false positive rate, some false positives

(mostly those due to noise or interfering materials), will test negative after the

SMARTube™ enhancement step, thus increasing specificity of your assays.

REMEMBER:

• Every infection missed, means tens of lives risked.

• Every infection diagnosed means saving the life of just as many, if not more.


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13. Notes and Tips:


of a blood sample.

The CO2 incubator:

1. Use a SMARTube™ (with no blood in it) constantly monitor CO2 levels in incubator by

observing the color daily (color should correspond to 6.6-7.0).

2. Use a thermometer in the incubator to monitor the temperature.

3. A CO2 cylinder of 27kg should last for 4-6 months. It is best to have a backup cylinder.

4. Since the osmolality of culture media can rise as a result of evaporation CO2 incubators

must be well humidified in order to prevent evaporation from SMARTube™.

Transferring blood into the SMARTube™:

5. Take out of the refrigerator the SMARTube™(s) to be used during the day, and keep at

Room Temperature (RT 18-30oC), as SMARTube must be at RT before adding the blood.

6. The blood transfer should be done aseptically.

a. Use only fresh blood drawn within the last 20 hours into heparin, and kept at RT.

b. Do not open the SMARTube™ in an unclean environment. This could lead to

contamination, which could affect the results.

c. If a hood is not available and work is done at the bench then:

� Clean and disinfect the area/bench/table which you plan to work on, and your

gloves/hands.

� Lite a flame (gas Bunsen, spirit flame, or candles) to provide semi-sterile

environment for the transfer of the blood into the SMARTube™. Work close to the

flame.


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d. Before adding blood to the SMARTube™ mixes the sample well and check that there is NO

coagulation.

e. Use a sterile pipette/tip for blood transfer, and a new pipette for each blood sample.

f. Immediately after transferring the blood of all samples into the SMARTubes™, incubate the

SMARTube™ with cap in the loose / ventilating position (one “click” of the cap and not two).

Using the SMARTube™ enhanced sample for HIV or HCV antibody testing:

7. At the end of the incubation (day 5) the ST-supernatant is tested for antibodies. The tubes

can be kept in the cold for immediate testing and repeat testing. It is recommended that

you keep the samples for additional testing you might want to perform in the future. For

that you should collect the ST-supernatants to a sterile test tube, seal, and freeze at -20oC

or -80oC (best). Once the samples inside the SMARTube™ are out of the incubator, it is best

to close the cap.

8. To test for the antibodies in the sample after the SMARTube™ incubation step

(STsupernatant), follow the instructions of the ELISA kit. However, when applying the

sample to the ELISA one should take into account the 1:4 = X5 dilution of the plasma which

has already occurred when 1ml of blood, i.e. ~0.5 ml of plasma, was put into 2ml of

SMARTmedium.

Full compensation:



Partial compensation



No compensation

100ul plasma = 100ul ST-Plasma

The SMARTube™ has been tested using different ELISA and different plasma volumes

required. In all settings, plasma positives were also positive using the SMARTube™.


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9. The SMARTube™ should become the blood pre-treatment of choice in testing for HIV and

HCV to enable more complete detection of HIV and HCV infected individuals.

10. However, should you choose to run the two assays in parallel, these are the results you can

expect to see:

� All true plasma positives will be SMARTube™ positives.

� Some of the plasma positive samples will have higher antibody levels following the

SMARTube™ incubation (= earlier months after seroconversion).

� Seeing a higher O.D. reading will depend on the level of compensation for the

dilution of 1:4 (=5x) when putting sample on the ELISA.

� Some plasma negatives will be SMARTube™ positive (= very early infection, still in

the window period).

� Most plasma-negatives will also be SMARTube™ negatives (that is, not-infected).

� Some samples which fall into the “gray zone” reading would be cleared in the

SMARTube™ sample as either a true positive (reading above the “grey zone”) or a clear

(low) negative. Also some false positive plasma readings (which will not be repeat

positive) could read negative from the start in the ST-supernatant This is because using

SMARTube™ reduces the false positives reported by some kits).


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Table 1. O.D. HIV readings with FULL dilution compensation.

Table of O.D. readings as example of SMARTube™ enabled results using an HIV antibody ELISA

with full compensation for the dilution by using a larger volume of sample on the ELISA. The cutoff

was 0.140

Sample # (for

ref. only)

Plasma O.D. (10ul+

90ul diluents)

SMARTube™ O.D.

(50ul+50ul diluents)

Diagnosis & [comment]

001 0.012 0.016 Negative

002 0.090 0.020 Negative [less noise]

003 0.138 0.026 Negative [an almost false positive–

cleared]

004 0.142 0.030 Negative [false positive due to

noise – cleared]

005 0.157 0.280 Positive [increase in antibody

levels to a clear positive-> early

seroconversion]


levels -> early seroconversion]





008 2.678 2.640 Positive [ no increase in antibody

levels -> later seroconversion]

009 2.898 2.752 Positive [ no increase in antibody

levels -> later seroconversion]

010 1.703 1.112 Positive [ decrease in antibody

levels -> immuno-deficient state]

011 0.087 0.178 Positive [window period]



014 0.077 0.822 Positive [window period,

probably close to seroconversion]


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Table 2. O.D. HIV readings WITHOUT full dilution compensation.

Table of OD readings as example of SMARTube™ enabled results using an HIV antibody ELISA with

no complete compensation for the dilution by using a larger volume of sample on the ELISA. The

cutoff was 0.142

Sample # (for ref. only)

Plasma O.D. (100ul

+ no diluent)

SMARTube™ O.D.

(100ul+no diluent)

Diagnosis & [comment]

001 0.012 0.016 Negative

002 0.090 0.020 Negative [less noise]

003 0.138 0.026 Negative [an almost false positive – cleared]

004 0.142 0.030 Negative [false positive due to noise – cleared]

005 0.157 0.152 Low Positive [increase in antibody levels most

probably hidden in the X5 dilution factor.->

early seroconversion]

006 0.508 0.370 Positive [increase in antibody levels most

probably hidden in the X5 dilution factor ->

early seroconversion]

006a 0.508 0.502 Positive [increase in antibody levels hidden in

the X5 dilution factor -> early seroconversion]

007 1.882 1.701 Positive [increase in antibody levels hidden in

the X5 dilution factor->probably early

seroconversion]


levels might be hidden in the X5 dilution factor

]

008 2.678 2.400 Positive [no info on stage of

seroconversion]

009 2.898 2.598 Positive [no info on stage of

seroconversion]

010 1.703 1.033 Positive [decrease in antibody levels -> either

late seroconversion or early immunodeficient

state]




014 0.077 0.377 Positive [window period, maybe

close to seroconversion]


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14. Trouble-shooting:


of a blood sample.

1. Blood sample is (even partially) coagulated. This occurs when the tube has not been mixed

well at the collection site, or not enough heparin was used. Do not use such blood.

2. Blood sample is very hemolytic. It can be used, but severe hemolysis may affect the results.

3. Blood sample collected in an anticoagulant other then heparin. The current formulation

only accepts blood in heparin. If this is problem at your facility, please inquire about other

versions of the SMARTube™.

4. Medium in SMARTube™ is not clear. Do not use.

5. After incubation the supernatant is not clear. Can use with caution as it indicates either a

high lipid content in the plasma or contamination; the latter may affect results.

6. After incubation there are white aggregates close to the red cells but supernatant is clear:

no problem.

7. Longer than 5 days incubation. Supernatant from the SMARTube™ should be tested after 3-

5 days (best on day 5). If cannot be read on day 5, remove the supernatant from

SMARTube™ and put into a sterile, empty test tube, seal and store refrigerated at 2-10oC.

To store more than a few days, freeze at -20oC. Incubation of over 5 days may lead to

reduction in the antibody levels in the sample.

8. When comparing plasma and SMARTube™ results, no additional positives were detected.

The prevalence of individuals in the window period depends on the incidence and the

length of the window period in the tested population. (See #10, below.)

9. When comparing plasma and SMARTube™ results, antibody levels in the SMARTube™ are

the same or lower(and not higher) than in the plasma. An increase in antibody levels


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following stimulation in the SMARTube™ is observed only when antibody production in the

body is not at its full capacity. This is so especially during the early stages of the

seropositive state, or in a state of partial tolerance or suppression of the immune response.

The prevalence of individuals in the early sero-conversion period depends on the incidence

and prevalence of the HIV or HCV infection in the tested population. (See # 10, below).

10. Factors that affect the performance of the SMARTube™: While the instructions for use of

SMARTube™ are relatively simple and should lead to expected results, some factors that

could interfere with the performance of the SMARTube™ and affect the results include:

• Contamination of the sample.

• Strong hemolysis of red blood cells.

• Blood sample conditions from collection to SMARTube™ were longer than 24 hours at

room temperature (RT) [15-30oC], or were too hot or too cold.

• Blood sample not mixed well before collecting 1ml for adding to SMARTube™.

• Blood too “old” prior to incubation in the SMARTube™.

• SMARTube™ was not at RT when blood was added.

• Incubation time was outside the recommended range in the SMARTube™ (less than 3

days or longer than 5 days).

• CO2 levels were outside the recommended range (too high or too low).

• SMARTube™ cap was not in half closed (ventilation) position during incubation.

• Freeze-thaw cycles reduce the levels of antibodies in the sample.

• Storage of the SMART supernatant in the SMARTube™ for too long after incubation

period causes exposure to high levels of proteolytic enzymes.

• Kits that are of poor quality or analytically less sensitive.

• Kkit does not detect IgM-- a main component of early antibodies, both in vitro and vivo.

• When looking for the stimulation for additional antibodies (higher O.D. readings) in the

SMARTube™ supernatant, the dilution (1:4 i.e.X5) of the plasma was not taken into

account.


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15. Extracts of Clinical Trials in different parts of the world Clinical studies of the HIV&HCV SMARTube™ for HIV/HCV have been performed in

• China

• USA.

• South Africa,

• Mexico,

• Israel,

• Kenya

China:

Clinical Trials in China were conducted, executed and reported by the Department of Cell

Biology, National Institute for Control of Pharmaceutical and Biological Products. The trials

were done in 5 different regions in China (Total samples tested: approximately 6,000).

1. Trial in high risk population (IVD) in Sichuan District:

HIV

• 653 individuals tested.

• 149 Seropositive.

• 151 Seropositive after pre-treatment in the SMARTube™.

HCV




2. Trials in blood banks:

HIV

• Beijing Blood Bank: 2000 low risk samples, no positives.

• Clearance of false positives by the SMARTube™.

U.S.A Studies were performed in monkeys. naïve monkeys were infected with a very low dose of

SIV virus (the equivalent to HIV in monkeys).

• 4 monkeys tested.

• 4 seronegative (one week from infection).

• 4 Seropositive after pre-treatment in the SMARTube™ (one week from infection).

All monkeys seroconverted between 1-5 months from infection.


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South Africa Clinical trials were carried out in South Africa among high risk population (blood donors):

HIV


• 3 Seropositive.


Mexico

Clinical trials were carried out in Mexico, by an approved government agency.

HIV

• 200 Individuals tested, very high risk, multiple, current exposures.



Israel Several high risk populations were screened using the SMARTube™ as a blood pre-treatment

device in a number of trials (total: over 2,000 individuals).

1. Immigrants from High risk areas:

HIV




HCV


• 1 Seropositive


2. Low risk populations were screened using the SMARTube™:

HIV

• Over 1,500 individuals tested – no positives.

HCV

• Over 600 individuals tested – no positives.


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Kenya Clinical trials were carried out in Kenya.

1. Screening of high risk population:

HIV




2. Additional trials conducted in the blood bank in Kenya for complete detection of HIV

infected blood units:

HIV

Adults:




Youth:




HCV

• Over 300 individuals tested.



3. A study was conducted on pregnant women:

HIV

• 40 Seronegative women tested.

• 8 out of the 40 Seronegative women, were positive after pre-treatment in the

SMARTube™.


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16. Case Histories – Applications & Benefits

A baby saved:

Mayama was 22 when she came to the antenatal clinic. This was her third pregnancy, yet her fist

visit to that clinic. She has come because her friend told her that she could save her baby if would

go there. She was five months pregnant, and the nurse explained the risks of transmitting HIV to

the un-born baby, and that there was medicine that could save the baby form getting AIDS.

Mayama was tested for HIV, using a rapid test, which was negative. The nurse explained that such

a result does not mean that she is not infected for sure, as she could have been infected in the last

few months, and then the test will not detect it yet. Mayama was worried. She was sure one of her

regular clients on the truck route was sick with AIDS and seeing she got pregnant… When she

shared her fears with the nurse she was told that she could come back in 3 months or so and re-

test. Mayama wanted to know. She was worried, and she really wanted to give that baby the best

chance possible. “I cannot wait for 3 months; if I am infected I want to take the medicine now. In

three months I will give birth, it will be too late. Plus, I cannot come back here heavy with

pregnancy – everyone will talk! The nurse shrugged her shoulders. “There is nothing we can do for

you now. We cannot see the infection during the window period, when the virus is hiding and the

tests are negative. Mayama started crying. The head nurse took her into her office. “There is a new

way we can use to see if you are infected, even if it happened recently. But for that we need to

draw blood and send it to the laboratory in town. The results will come back next week. You will

need to come back then, and if you're positive we will give you the ART.” Mayama agreed to come

back. A test tube with her blood was sent to the laboratory. There they treated the blood with the

SMARTube™, an innovative blood pre-treatment which closes the window period and thus

eliminates the false negative results in the early stages of the infection. On the fifth day, the lab

sent the results back to the clinic. While negative on the tests using the regular methods,

Mayama was clearly positive after the SMARTube™ step that was added in the laboratory to the

testing. When Mayama came back, she got the results with tears of fear and a smile of relief.

She was going to save her baby; she was going to get the drugs to protect him from the virus

that has invaded her. As she was walking out of the clinic, holding on to the medicine for both her

and for the baby, when it will be born, she turned around and ask the head nurse “How do they do

it, there in the laboratory? How can see what is still hidden”. “Well” answered the nurse, “ it is as

if they go behind the stage and peak into the dressing rooms, this way they know about the actors

even before they get on stage”. Mayama gave birth to healthy baby girl.


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If we only knew!

Katub had her fifth child less than a year after she immigrated to Israel. Upon arrival her whole

family had their blood tested for different things, including HIV. Her husband was the only one

that tested positive for HIV. He figured he must have gotten it in the camp on route to Israel.

Katub was upset, but relieved for herself and the unborn baby. The baby was born slightly

underweight, but pink and beautiful. When he was six months old he had a bad cold that would

not go away. Then, the doctor said it was probably some infection in the lungs. The antibiotics did

not help. The baby was hospitalized, but could not be saved. In the blood tests, he was found to

be HIV positive, but it was too late, he died of lung infection typical to AIDS patients. The doctors

were upset. “ If only you would have told your doctor that you are HIV positive, you could have

saved that baby. We know how to treat these type of infections, we just do not suspect it in a baby

without an HIV record.” Katub was very bewildered. How could she transmit HIV to her baby if she

is not infected? Did they not tell her in the immigration center that she tested negative? She told

the doctors it must be a mistake. “No”, said the young doctor, “it is not a mistake. Unfortunately

we cannot detect the HIV infection in the first few months. You must have gotten infected shortly

before the pregnancy, this is why the results were negative, but it was not a true negative result.

A year later, in a scientific-medical conference, An immunologist presented some interesting

results with a new method which enables the detection of those infected even when still missed

by regular testing in the first months of infection. She called the method “Stimmunology”, as it

stimulates the immune system in the blood sample to “tell” us about the infection “right away”. “I

would like to share with you some alarming results we got when studying some families with one

seropositive HIV carrier. We used the Stimmunology process for stimulating antibody production

even in blood samples form infected individuals during the window period. This was we can detect

them using the regular diagnostic antibody tests.” On the screen appeared results showing

seronegative wives who were actually infected, and their infected babies. The doctors in the

audience sighed “If only we would have known”.

Building a new relationship.

Seth and Diane decided to move in together and formalize their relationship. They both went

together for HIV testing, and, to their relief, both tested negative. Because of their lifestyle, Seth’s

doctor recommended to do an additional blood test using an experimental pre-treatment of the

blood in the university laboratory. They agreed. The following week the doctor called them in for

consultation and told then that using the experimental new technology; Seth was found to be

infected with HIV. “It must be a recent encounter, in the last half a year or so” said the doctor.


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“But it is still experimental, right” said Seth hopefully… Diane was silent all the way home. They

have been together for some time now, she wanted to believe Seth that “it could not be”, yet she

insisted that for their future they should use precaution “Just for the next few months. The

window period is not forever, right?” Three months later, Seth tested positive in a routine

testing.

Organ donor

Sheila has been waiting for a kidney transplant for 2 years. The phone finally rang with the news –

we have a donor. The young motorcyclist was brain dead and his family agreed to donate his

organs. A battery of tests was run, including HIV and HCV antibody tests. All came negative.

Additional testing was using very sensitive molecular biology techniques to detect the virus even

before the antibody tests detect the infection. They were negative for both HIV and HCV. Sheila

got the kidney, and stayed on immunosuppressive drugs to reduce the risk of rejection of the

transplanted kidney. Less than a year later, Sheila was diagnosed with HIV and HCV infection.

The source of the infection was the transplanted kidney. All the recipients from that donor were

now positive for HIV and HCV. When Sheila sued the hospital the doctors testified that they have

used all known measures for testing the donor for these infectious and deadly viruses. “But even

with the most sensitive tests, there is a window period in which we cannot detect the infection,

and this window period can take three to six months and sometimes even longer” testified the

laboratory expert. “So there is nothing that could have been done?” asked Sheila’s lawyer. “Well,

responded the expert “there is a way to eliminate that window period. It is a simple system of pre-

treating the blood in a way that expresses the antibodies prior to their appearance in the body. It

works like magic; it exposes those early infections we currently miss.” “So if you would have used

that method, you would have been able to prevent all those terrible infections! Why did you not

use it?!”. “We do, but only experimentally, and unlinked, as it has not yet been approved for use in

our country...” responded the expert.


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Follow Up Questions / Clarifications:

Finally, please do not hesitate to contact us with whatever technical or practical

questions or comments you might have. When you send us the data from these

evaluations, we can assist with the data analysis, sharing thoughts and ideas as to

the implications of the data which come up during the evaluation period.

For More Details Contact:

Eternal Health & Wellness Foundation (USA)

India Branch Office

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Health & Medicine

Feasibility study outline (final)