Feasibility study outline (final)
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Feasibility study outline (final) Feasibility study outline (final) Document Transcript

  • CORP OFFICE: 10160 MEDLOCK BRIDGE ROAD, DULUTH, GA 30097 (USA)PH: 770-495-0011, FAX: 770-495-0012INDIA OFFICE: C-33A SHASTRI NAGAR, GHAZIABAD-201002 (UP) INDIAMOB: 9999989066, 9818666863
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 IndexS.No Description Page No. TM1 Introduction to SMARTube 32 Back Ground 43 Purpose and Scope of the Study 44 Study Design 55 Expected results 76 Outcome of studies 77 Benefits to the participating organization 88 Research Procedures 99 Methodology 1010 Comments & Notes 12 Administrative and Financial Issues11 Economic/ Financial Implications 1412 Equipment and Material Required 1513 Defining of Responsibilities 15 Additional Inputs for Clinical Trial14 Instructions for Use 1815 Guidelines for Running a laboratory evaluation 1916 Notes & Tips 2017 Trouble Shooting 2518 Extracts of Clinical Trials in different parts of the word 2719 Case Histories – Applications & Benefits 30 2 Page20 Follow up questions and clarifications 33
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Introduction to SMARTubeTMSMARTubeTM being introduced for the first time in India – is a first of its kind, innovative medicaldiagnostic product that will revolutionize HIV & HCV detection. Stimulating Maximal AntibodyResponse Tube – SMARTube™ enables earlier, better and complete detection of HIV/HCV just aweek after exposure. SMARTube™ not only enables the detection of all the patients who arediagnosed in the conventional testing - but also enables detection in additional patients that areinfected, but otherwise would have gone undetected at that testing time. As a cost effectivemethod that increases the SENSITIVITY and SPECIFICITY of other known HIV & HCV detectiondevises—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 thehighest 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 iscertified 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, ResourcesSMARTube™ has been tested in controlled clinical trials on over 10,000 patients/individuals inseveral countries like China, Israel, Kenya, Mexico, Romania and South Africa. Most of theseclinical trials and tests were done by reputed government agencies, blood banks, reference 3 Pagelaboratories, academic and professional bodies.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Background:The window period of HIV and HCV infection is a major concern for the governments, healthauthorities and professionals, blood banks, vaccine and drug developers all over the world, asmany 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 theblood sample, prior to testing it for HIV (and/or HCV) antibodies, has been developed, to solve thewindow period. This has been implemented in the SMARTube™ HIV&HCV, a blood pre-treatmentdevice, which enables the detection of HIV and HCV infections (during the window period) byovercoming, in vitro, the specific immune suppression exerted by the virus, and which is the causefor the window period.This process involves placing 1ml of blood sample inside the SMARTube™ for a 3-5 day incubationperiod, leading to the formation of HIV and/or HCV antibodies in detectable levels in all thoseinfected, including those in the window period. The detection of the antibodies is done by thecurrent serological assays and antibody detection kits (ELISA, WB), following the same proceduresand 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 4 evaluate the feasibility of using the SMARTube™ technology in the Indian settings, Page become opinion leaders on this issue for curtailing the HIV and HCV epidemics in India.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668632. 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 5 for 1-3 months, testing 500-2000 samples. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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. 6 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668633. 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 kitsused 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 followthe same guidelines as those for plasma results for HIV and/or HCV antibody screening – leading toa healthier society and safer blood supply.The studies will be the initiation of an implementation plan, initially in the participating institutesand eventually in others in the city, district and eventually the country, with the first users offering 7leadership and guidance to others. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668635. 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. 8 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668636. 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 36.5-37.5C, 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”. 9 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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 -80C (or > 20C), 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 10 plates. Diluent should be adjusted to obtain the same final volume, as mentioned in the instructions for use. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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 10ul plasma + 90ul diluent = 50ul ST-Plasma + 50ul diluent Partial compensation 20ul plasma + 80ul diluent = 100ul ST-Plasma + 10ul diluent 50ul plasma + 50ul diluent = 100ul ST-Plasma + 10ul diluent No compensation 100ul plasma = 100ul ST-PlasmaStimmunology has been tried using different ELISA kits and different plasma volumes required. Inall 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 Wednesday6.4 Interpretation of results:Any sample, with or without the SMARTube™ pre-treatment, which tests positive for HIV or HCVantibodies according the site’s algorithm, is positive for HIV or HCV infection respectively. 11Sensitivity and specificity of the results are dependent on the kit itself and its intrinsic qualities. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668637. 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. 12 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Administrative and Financial Issues 13 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668638. 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) 14 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668639. 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 SMARTubeTMs FREE • Medium Level Study (Sample Size 201-500) o Eternal Health & Wellness Foundation will provide 125 SMARTubeTMs FREE • High Level Study (Sample Size 501 – Upwards) o Eternal Health & Wellness Foundation will provide 200 SMARTubeTMs FREE 15 • Additional number of SMARTubeTM s required for the trails/ efficacy studies will be provided Page at the rate of 3 Euro per SMARTubeTM
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686310.2 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. 16 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Additional Inputs for Clinical Trial 17 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686311. Instructions for Use:For using SMARTube™ HIV&HCV as a blood pre-treatment prior to HIV or HCV antibody testingof a blood sample.1. Limit exposure of SMART media to fluorescent lights (refrigerators for SMARTubeTM storeshould not be with glass doors).2. Blood should be collected into heparin and transferred to the laboratory within 20 hours atroom temperature (RT) (15-25oC).3. Transfer 1ml of blood (mixed well) into a properly marked SMARTube™ at RT underaseptic/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% CO2incubator, at 37 oC (CO2 gas phase in the incubator can be anywhere between 5% and 7%, bestset at 5%). Write the date on the tray. (You might want to already mark the planned “out” datealso 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 theincubator, 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. Forlater repeat testing, transfer the supernatant fluid to another, properly marked sterile test tube toprevent 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, wasput into 2ml of SMART medium), it is best, when possible, to Compensate for this dilution (x5) inthe sample volume used on the ELISA (See “Notes and Tips” #8). 18 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686312. Guidelines for running a laboratory evaluation:For using SMARTube™ HIV&HCV as a blood pre-treatment prior to HIV or HCV antibody testingof 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: 19 • Every infection missed, means tens of lives risked. • Every infection diagnosed means saving the life of just as many, if not more. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686313. Notes and Tips:For using SMARTube™ HIV&HCV as a blood pre-treatment prior to HIV or HCV antibody testingof 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 20 environment for the transfer of the blood into the SMARTube™. Work close to the Page flame.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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: 10ul plasma + 100ul diluent = 50ul ST-Plasma + 60ul diluent 10ul plasma + 90ul diluent = 50ul ST-Plasma + 50ul diluent Partial compensation 20ul plasma + 80ul diluent = 100ul ST-Plasma + 10ul diluent 50ul plasma + 50ul diluent = 100ul ST-Plasma + 10ul diluent No compensation 100ul plasma = 100ul ST-Plasma 21 The SMARTube™ has been tested using different ELISA and different plasma volumes Page required. In all settings, plasma positives were also positive using the SMARTube™.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 98186668639. 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). 22 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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 ELISAwith full compensation for the dilution by using a larger volume of sample on the ELISA. The cutoffwas 0.140Sample # (for Plasma O.D. (10ul+ SMARTube™ O.D. Diagnosis & [comment]ref. only) 90ul diluents) (50ul+50ul diluents)001 0.012 0.016 Negative002 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]006 0.508 0.783 Positive [increase in antibody levels -> early seroconversion]007 1.882 2.701 Positive [increase in antibody levels -> early seroconversion]008 2.850 3.000 Positive [increase in antibody 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]012 0.026 0.250 Positive [window period]013 0.043 0.600 Positive [window period] 23014 0.077 0.822 Positive [window period, probably close to seroconversion] Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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 withno complete compensation for the dilution by using a larger volume of sample on the ELISA. Thecutoff was 0.142 Sample # Plasma O.D. (100ul SMARTube™ O.D. Diagnosis & [comment] (for ref. only) + no diluent) (100ul+no diluent) 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] 008 2.850 2.822 Positive [increase in antibody 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] 011 0.087 0.172 Positive [window period] 012 0.026 0.202 Positive [window period] 013 0.043 0.300 Positive [window period] 24 014 0.077 0.377 Positive [window period, maybe close to seroconversion] Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686314. Trouble-shooting:For using SMARTube™ HIV&HCV as a blood pre-treatment prior to HIV or HCV antibody testingof 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.) 25 9. When comparing plasma and SMARTube™ results, antibody levels in the SMARTube™ are Page the same or lower(and not higher) than in the plasma. An increase in antibody levels
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863 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. 26 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686315. Extracts of Clinical Trials in different parts of the worldClinical studies of the HIV&HCV SMARTube™ for HIV/HCV have been performed in • China • USA. • South Africa, • Mexico, • Israel, • KenyaChina: 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 • 653 individuals tested. • 389 Seropositive. • 391 Seropositive after pre-treatment in the SMARTube™. 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). 27 All monkeys seroconverted between 1-5 months from infection. Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863South Africa Clinical trials were carried out in South Africa among high risk population (blood donors): HIV • 90 individuals tested. • 3 Seropositive. • 4 Seropositive after pre-treatment in the SMARTube™.Mexico Clinical trials were carried out in Mexico, by an approved government agency. HIV • 200 Individuals tested, very high risk, multiple, current exposures. • 20 Seropositive. • 25 Seropositive after pre-treatment in the SMARTube™.IsraelSeveral high risk populations were screened using the SMARTube™ as a blood pre-treatmentdevice in a number of trials (total: over 2,000 individuals). 1. Immigrants from High risk areas: HIV • 537 individuals tested. • 26 Seropositive. • 28 Seropositive after pre-treatment in the SMARTube™. HCV • 67 individuals tested. • 1 Seropositive • 4 Seropositive after pre-treatment in the SMARTube™. 2. Low risk populations were screened using the SMARTube™: HIV • Over 1,500 individuals tested – no positives. 28 HCV Page • Over 600 individuals tested – no positives.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Kenya Clinical trials were carried out in Kenya. 1. Screening of high risk population: HIV • 555 individuals tested. • 28 Seropositive. • 42 Seropositive after pre-treatment in the SMARTube™. 2. Additional trials conducted in the blood bank in Kenya for complete detection of HIV infected blood units: HIV Adults: • 513 individuals tested. • 45 Seropositive. • 66 Seropositive after pre-treatment in the SMARTube™. Youth: • 332 individuals tested. • 12 Seropositive. • 22 Seropositive after pre-treatment in the SMARTube™. HCV • Over 300 individuals tested. • 13 Seropositive. • 14 Seropositive after pre-treatment in the SMARTube™. 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™. 29 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 981866686316. Case Histories – Applications & BenefitsA baby saved:Mayama was 22 when she came to the antenatal clinic. This was her third pregnancy, yet her fistvisit to that clinic. She has come because her friend told her that she could save her baby if wouldgo there. She was five months pregnant, and the nurse explained the risks of transmitting HIV tothe 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 sucha result does not mean that she is not infected for sure, as she could have been infected in the lastfew months, and then the test will not detect it yet. Mayama was worried. She was sure one of herregular clients on the truck route was sick with AIDS and seeing she got pregnant… When sheshared 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 bestchance possible. “I cannot wait for 3 months; if I am infected I want to take the medicine now. Inthree months I will give birth, it will be too late. Plus, I cannot come back here heavy withpregnancy – everyone will talk! The nurse shrugged her shoulders. “There is nothing we can do foryou now. We cannot see the infection during the window period, when the virus is hiding and thetests are negative. Mayama started crying. The head nurse took her into her office. “There is a newway we can use to see if you are infected, even if it happened recently. But for that we need todraw blood and send it to the laboratory in town. The results will come back next week. You willneed to come back then, and if youre positive we will give you the ART.” Mayama agreed to comeback. A test tube with her blood was sent to the laboratory. There they treated the blood with theSMARTube™, an innovative blood pre-treatment which closes the window period and thuseliminates the false negative results in the early stages of the infection. On the fifth day, the labsent 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 thetesting. 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 virusthat has invaded her. As she was walking out of the clinic, holding on to the medicine for both herand for the baby, when it will be born, she turned around and ask the head nurse “How do they doit, there in the laboratory? How can see what is still hidden”. “Well” answered the nurse, “ it is asif they go behind the stage and peak into the dressing rooms, this way they know about the actorseven before they get on stage”. Mayama gave birth to healthy baby girl. 30 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863If we only knew!Katub had her fifth child less than a year after she immigrated to Israel. Upon arrival her wholefamily had their blood tested for different things, including HIV. Her husband was the only onethat 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 slightlyunderweight, but pink and beautiful. When he was six months old he had a bad cold that wouldnot go away. Then, the doctor said it was probably some infection in the lungs. The antibiotics didnot help. The baby was hospitalized, but could not be saved. In the blood tests, he was found tobe HIV positive, but it was too late, he died of lung infection typical to AIDS patients. The doctorswere upset. “ If only you would have told your doctor that you are HIV positive, you could havesaved that baby. We know how to treat these type of infections, we just do not suspect it in a babywithout an HIV record.” Katub was very bewildered. How could she transmit HIV to her baby if sheis not infected? Did they not tell her in the immigration center that she tested negative? She toldthe doctors it must be a mistake. “No”, said the young doctor, “it is not a mistake. Unfortunatelywe cannot detect the HIV infection in the first few months. You must have gotten infected shortlybefore 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 interestingresults with a new method which enables the detection of those infected even when still missedby regular testing in the first months of infection. She called the method “Stimmunology”, as itstimulates the immune system in the blood sample to “tell” us about the infection “right away”. “Iwould like to share with you some alarming results we got when studying some families with oneseropositive HIV carrier. We used the Stimmunology process for stimulating antibody productioneven in blood samples form infected individuals during the window period. This was we can detectthem using the regular diagnostic antibody tests.” On the screen appeared results showingseronegative wives who were actually infected, and their infected babies. The doctors in theaudience 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 wenttogether for HIV testing, and, to their relief, both tested negative. Because of their lifestyle, Seth’sdoctor recommended to do an additional blood test using an experimental pre-treatment of the 31blood in the university laboratory. They agreed. The following week the doctor called them in forconsultation and told then that using the experimental new technology; Seth was found to be Pageinfected with HIV. “It must be a recent encounter, in the last half a year or so” said the doctor.
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863“But it is still experimental, right” said Seth hopefully… Diane was silent all the way home. Theyhave been together for some time now, she wanted to believe Seth that “it could not be”, yet sheinsisted that for their future they should use precaution “Just for the next few months. Thewindow period is not forever, right?” Three months later, Seth tested positive in a routinetesting.Organ donorSheila 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 hisorgans. 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 evenbefore the antibody tests detect the infection. They were negative for both HIV and HCV. Sheilagot the kidney, and stayed on immunosuppressive drugs to reduce the risk of rejection of thetransplanted 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 werenow positive for HIV and HCV. When Sheila sued the hospital the doctors testified that they haveused all known measures for testing the donor for these infectious and deadly viruses. “But evenwith 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 thelaboratory 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. Itworks like magic; it exposes those early infections we currently miss.” “So if you would have usedthat method, you would have been able to prevent all those terrible infections! Why did you notuse it?!”. “We do, but only experimentally, and unlinked, as it has not yet been approved for use inour country...” responded the expert. 32 Page
  • Corp Office: 10160 Medlock Bridge Road, Duluth, GA 30097 (USA) Ph: 770-495-0011, Fax: 770-495-0012 India Office: C-33A Shastri Nagar, Ghaziabad-201002 (UP) INDIA Mob: 9999989066, 9818666863Follow Up Questions / Clarifications:Finally, please do not hesitate to contact us with whatever technical or practicalquestions or comments you might have. When you send us the data from theseevaluations, we can assist with the data analysis, sharing thoughts and ideas as tothe implications of the data which come up during the evaluation period. For More Details Contact: Eternal Health & Wellness Foundation (USA) India Branch Office Mob: 9999989066, 9818666863 33 Page