Validation of Dried Tube Specimen (DTS) as Internal Quality Control for HIV testing in Blood Banks
A dissertation submitted in partial fulfilment of
M.D. Immunohaematology and Blood Transfusion (Branch XXI) Examination of the Tamil Nadu Dr M.G.R. UNIVERSITY,
CHENNAI to be held in May 2018
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PLAGIARISM CERTIFICATE
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Table of Contents
PLAGIARISM CERTIFICATE ... 5
LIST OF ABBREVIATIONS USED ... 9
LIST OF TABLES ... 11
LIST OF FIGURES ... 12
ACKNOWLEDGEMENTS ... 13
ABSTRACT ... 14
AIM ... 16
OBJECTIVES ... 17
INTRODUCTION ... 18
REVIEW OF LITERATURE ... 20
METHODS AND MATERIALS ... 48
Detailed diagrammatic Algorithm of the study ... 53
RESULTS ... 66
DISCUSSION ... 78
LIMITATIONS ... 84
CONCLUSION ... 85
REFERENCES ... 87
ANNEXURES ... 94
I - INSTITUTIONAL REVIEW BOARD (IRB) APPROVAL LETTER ... 94
II - INSTITUTIONAL REVIEW BOARD (IRB) FUND LETTER ... 98
III- CONSENT FORM ... 99
IV- STANDARD OPERATING PROCEDURES (SOP)... 101
SOP for MICROLISA-HIV™ (J. Mitra & Company Private Limited™, New Delhi, India) ... 101
SOP for Trueline™ ( Alere Medical Private Limited™, Gurgaon, Haryana, India) ... 106
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SOP for HIV TRI-DOT™ (J.Mitra & Company Private Limited™, New Delhi, India)... 108
SOP for CombAids-RS Advantage™ (ARKRAY Healthcare Private Limited™, Surat, Gujrat, India) .. 111
SOP for Preparation of Phosphate Buffered Saline (PBS) with Tween 20 ... 114
SOP for making and use of HIV dried tube specimen (DTS) ... 116
V- CLINICAL RESEARCH FORM ... 118
VI -THESIS DATA – EXCEL SPREAD SHEET ... 119
VII – Data of Homogeneity testing ... 120
VIII - Costing of a DTS ... 121
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LIST OF ABBREVIATIONS USED
DTS – Dried Tube Specimen
HIV – Human Immunodeficiency Virus
AIDS – Acquired Immunodeficiency Syndrome ELISA - Enzyme Linked Immunosorbant Assay
CMIA – Chemiluminescence Microparticle based Immuno Assay NACO -National AIDS Control Organization
WHO – World health organization HTLV - Human T Lymphotropic Virus FFP – Fresh Frozen Plasma
IQC - Internal Quality Control EQC – External Quality Control
SIV - Simian Immunodeficiency Virus
PPTCT: Prevention of Parent to Child Transmission MSM: Men having Sex with Men
IDU: Injectable Drug Users HBV: Hepatitis B Virus HCV: Hepatitis C Virus
NBTC: National Blood Transfusion Council NACP: National AIDS Control Programme ART – Anti Retroviral Therapy
RNA - Ribo Nucleic Acid DNA – Deoxyribonucleic Acid
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QA – Quality Assurance NAT – Nucleic Acid Testing DBS - Dried Blood Spot DPS - Dried Plasma Spot
PBS - Phosphate Buffered Saline SOP – Standard Operating Procedure SD - Standard Deviation
OD – Optical Density
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LIST OF TABLES
Table 1: Prevalence and Incidence of HIV (41) ... 28
Table 2: HIV - Therapy and Death: An Inverse Relationship (41) ... 28
Table 3: The Various Proteins and Glycoproteins that differentiate HIV - 1 and HIV – 2 in the bands observed (53) ... 38
Table 4: Break-up of the number of aliquots used... 59
Table 5: Break-up of the volume (200 µl) of DTS shared by Microlisa and Rapid platforms ... 60
Table 6: Number of positive and negative tests each per platform for 26 weeks ... 60
Table 7: Evaluation of DTS on CombAids™ in comparison to reference tests on baseline plasma ... 67
Table 8: Evaluation of DTS on Tri Dot™ in comparison to reference tests on baseline plasma ... 67
Table 9: Evaluation of DTS on Trueline™ in comparison to reference tests on baseline plasma ... 67
Table 10 : Linear Regression of DTS in Microlisa Reactivity Ratios with Time ... 69
Table 11: Linear Regression of DTS in Microlisa™ Optical Density Strength with Time ... 71
Table 12: Linear Regression of DTS in Architect™ with Time ... 73
Table 13: Linear Regression of DTS in EVOLIS™ Reactivity Ratios with Time ... 74
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LIST OF FIGURES
Figure 1 : Taxonomy of HIV ... 23
Figure 2 : Distribution of the transmission modes of HIV in India(44) ... 26
Figure 3 : Estimated Prevalence of HIV in Indian Adult Population in the last One and a half Decades(48).... 27
Figure 4 : Inversely Proportional Relationship between AIDS Related Deaths and People Living with HIV on Anti Retroviral Therapy (ART) in India(48) ... 29
Figure 5 : Estimated HIV Incidence in India (1998-2015)(48) ... 29
Figure 6 : HIV Prevalence among the High-Risk Groups in India(48) ... 31
Figure 7 : Inverse relationship between voluntary blood donations and HIV prevalence in blood donors in Africa (56) ... 33
Figure 8 : Schematic figure of an HIV-1 virion (64) ... 35
Figure 9 : The Various Stages of HIV Replication (65) ... 37
Figure 10: An Artistic Diagram of HIV - 1 Versus HIV – 2 (46) ... 38
Figure 11 : Window Period Reduction by various Platforms (68) ... 39
Figure 12 : Changes in Serology after HIV Infection ... 39
Figure 13 : HIV RNA Concentration Variations with Temperature and Time in DTS (84) ... 45
Figure 14 : CombAids™ – Both Positive and Negative Reactions while Positive Ones of Trueline™ ... 68
Figure 15 : Tri Dot™ – Both Positive and Negative Reactions while Negative Ones of Trueline™ ... 68
Figure 16 : Pictorial representation of Microlisa™ Strips with Positive and Negative tests ... 70
Figure 17 : Trend of reactivity ratios of DTS and controls on Microlisa™ platform ... 70
Figure 18 : Trend of DTS Results on Microlisa™ platform in Optical Density (OD) Strength ... 72
Figure 19 : Trend of Negative Control DTS on Microlisa™ platform in Optical Density (OD) Strength ... 72
Figure 20 : Trend of Cut-off Values of Microlisa™ in Optical Density (OD) Strength ... 72
Figure 21 : Trend of DTS and controls on Architect™ platform ... 73
Figure 22 : Trend of DTS and negative controls on EVOLIS™ platform ... 75
Figure 23 : Levey Jennings chart of Positive IQC used for Microlisa ... 75
Figure 24 : Levey Jennings chart of Negative IQC used for Microlisa ... 76
Figure 25 : Estimated Prevalence of HIV in Indian Adult Population in the last One and a half Decades(48) .. 78
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ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to my mentor, Dr Joy Mammen, Professor and Head, Department of Transfusion Medicine and
Immunohaematology, for giving me the opportunity to complete my post-graduate dissertation under his able guidance. His expert advice, refined knowledge,
constant enthusiasm and dedication were of immense encouragement to me.
I am thankful to Dr Rajesh Kannangai, Professor and Head, Department of Virology. His deep knowledge and attention to details were inspirational and helped me in building up concepts and executing the project.
I would like to thank my teacher Dr Dolly Daniel, Professor, Department of Transfusion Medicine and Immunohaematology, for her constant support.
I am grateful to Mr Prasanna Kumar, Department of Virology, for explaining the concepts of ELISA with clarity. I extend my thanks to all the technicians in the department for their support with logistics and other assistance in the study.
I am thankful to Mrs Gnanamani, Mr Guru, Mr Louis and Mr Amal Raj, Blood bank supervisor, for helping me with sample collection.
I thank Dr. Visalakshi, Department of the Biostatistics for the analysis of the data and interpretation of results.
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ABSTRACT
Internal quality control for HIV testing of Blood Donors - Dried Tube Specimen as a cost effective alternative
Background: Serological testing for HIV is mandatory in every blood bank to
prevent its transmission by transfusion. The practice of third party internal controls is however uncommon. Most often cited reason for this is the cost, lack of easy access to reliable material and the need for freezers for storage of plasma. We explore an alternative cost-effective method for production and maintenance of in-house controls to overcome these issues.
Aim: Validation of Dried tube specimen (DTS) from HIV positive blood donor plasma as a low cost, stable material for use as Internal Quality Control material in blood banks.
Methods and Materials: Fresh frozen plasma prepared from consenting blood donors, confirmed HIV positive by serological and molecular methods was retained over a period of six months and pooled (4 bags). Equal numbers of seronegative FFP bags were pooled together. Aliquots of 20µl were made in plastic micro-centrifuge tubes and air dried overnight in a biosafety cabinet at room temperature. These samples were stored at 2-6⁰ C refrigerators and tested once every week on a variety of platforms that included three rapid tests, a second generation ELISA and a fourth
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generation Chemiluminescence immunoassay. A fourth generation ELISA was included on a monthly basis. This protocol was followed over a 6 month period to study extended stability of the control material.
Results: Epidata™, MS Excel ™and SPSS™ were used to tabulate and analyse the data. A total of 109 positive samples (DTS aliquots) were tested with 100% samples showing consistent positive results on all the platforms over the test period of 6 months. Similarly the negative samples (n=109) showed consistent negative results on all assay platforms over the six month period. It was observed that the procedure for reconstitution of DTS if not followed rigorously resulted in change of expected results.
Conclusion: The plasma in the form of DTS maintained stability when stored at 2- 8°C, over the study period of 6 months. The results were consistent and samples did not show any deterioration. Issues to be highlighted include the need to follow stringent safety measures when handling the plasma during preparation and the reconstitution of the DTS prior to use. This provides evidence that the DTS can be a modality for production of cost effective stable in-house control material for resource restricted countries.
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AIM
To validate a low cost and stable quality control material for HIV testing in blood banks
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OBJECTIVES
1) To characterize pooled Human Immunodeficiency Virus (HIV) positive donors plasma and prepare aliquots of Dried Tube Specimen (DTS) from it.
2) Perform tests on DTS once a week on three Rapid and one conventional Enzyme Linked Immunosorbant Assay (ELISA) platforms approved by CDSCO for the purpose of HIV screening in blood banks.
3) Perform tests on DTS once a month on a CDSCO approved 4th generation ELISA on automated platform and once a week on highly sensitive 4th generation CMIA.
4) To evaluate the stability of the DTS over a period of 6 months when stored at 2- 8°C.
5) To design a protocol for the use of DTS as a low cost internal quality control material for the above mentioned tests in blood banks.
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INTRODUCTION
Safety is a concern for all of us in our daily lives. When it comes to the ill who are weak and dependent on various drugs for their health, safety becomes more important. Blood transfusion has been used as a life-saving intervention in various clinical conditions. With the advancing technologies, the availability of blood components has increased the use of blood in medical sciences. However, the ever present risk of blood borne pathogens like HIV and Hepatitis virus mandates adequate screening of the transfused blood as it has direct implications for their transmission. An infected person remains asymptomatic on an average for 10 years during which he will still be spreading HIV inadvertently.(1)
With time, various cost effective and easy to use platforms for testing of HIV have mushroomed, especially when the number of blood banks is growing day by day. Access to blood banks has improved for people in rural areas and small towns with many blood banks being present in suburban areas. As per the last Census of India - 2011(2), 68.84% of Indian population resides in rural area.
Of the 2774 towns born in the decade prior to the census (2011), about 90%
belong to the category of census towns or small towns (3). Regular quality control of the infectious diseases screening tests is necessary to ensure that testing systems are performing to the expected level and not missing donors who may be
potentially infected. As the currently available materials for this are most often imported commercially available liquid controls that are very expensive, it becomes a significant barrier for blood banks to perform regular internal quality
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control. Increased false negatives compromise the safety of the recipients while false positives cause wastage of this precious resource and added cost to follow up and confirm truly infected donors. Traditionally frozen (-800C) aliquots of
characterized donor plasma has been suggested for this purpose. This is expensive in terms of equipment and power requirements and not a viable alternative. Thus there is a pressing need for an alternative material for quality control.
Dried tube specimen (DTS) can be easily prepared and reconstituted without the requirements of high levels of expertise and does not require deep freezers and hence are less expensive to store. If their stability can be validated over extended periods. it can be considered a a strong low-cost contender for internal quality control material. This type of specimen has already been assessed as an external quality assessment material and has been found to be quite suitable for it, especially in African countries with very hot climate. Prior studies have assessed stability at periods ranging from 4 – 8 (4,5) weeks. These studies focused on storing the specimen at ambient temperatures for the sake of transport of the specimens without cold chain requirements for external quality assurance purposes (6–8). Since we are considering its use in blood banks for the purpose of Internal Quality Control (IQC), we are restricting this study to storage only at 2-8 deg C as such refrigerators are available for reagent storage in all blood banks. The
volume of production and frequency will then be a function of frequency of usage and available space.
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REVIEW OF LITERATURE The Tale of HIV
Acquired Immune Deficiency Syndrome (AIDS) is a disease caused by HIV. HIV is thought to have evolved from close genetically related Simian Immunodeficiency Virus (SIV) and got transmitted from primates to humans in the recent past (as zoonosis) in Central and West Africa (9). The global pandemic may have had its origin from HIV-1 subgroup M.(1,10) A rough estimate of the viral mutation rates and molecular phylogenetics suggest that this spring from primates to humans probably took place during late 19th or early 20th century, which was the era of colonisation and urbanisation in the continent of Africa.
Of the two types of HIV-1 and HIV-2, the former is more infectious and has caused the most of the HIV infections around the world(11).The strains of HIV-1 are related to a virus found in either chimpanzees Pan troglodytes
troglodytes (SIVcpz) or gorillas (Gorilla gorilla gorilla) (SIVgor), which live in forests of Central Africa.(12–16) The infections caused by HIV-2 are largely confined to West Africa. Interestingly, its closest genetically matched relative SIVsmm, a virus of a type of wild ape known as sooty mangabey Cercocebus atys atys is also geographically confined in West Africa (17).
Pathogenicity of SIV in non-human primates
It is said that most primate species do not develop any fatal illness due to an SIV
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infection.(18) However, a long-term study of chimpanzees naturally infected with SIVcpz in Tanzania found that chimpanzees with SIVcpz infection do have an increased mortality and suffer from a Human AIDS-like illness.(19) The study stated that SIV pathogenicity could exist in other chimpanzee subspecies and other primates as well but has stayed unrecognized due to the lack of long term studies.
How did this transfer occur?
The most popular theory is the natural transfer theory, also known as
"hunter theory" or "bushmeat theory"(15). The SIV or HIV (post mutation from SIV) was transmitted from some primate species to a human who was a bushmeat hunter or butcher. The resulting exposure to blood or other bodily fluids of the animal resulted in SIV infection(20). By natural selection, the SIV virus might have transformed into HIV after infection. A survey for SIV infections in humans in Africa showed an infection rate of 7.8% in villages where the meat of primates is used and 17.1% in the people who were directly involved with them as a part of occupation in these villages (21).
The factors that led to rapid spread among people of Africa were
colonisation and urbanisation leading to the start of railways, booming commercial sex trade due to a mobilisation of more males than females in search of work, rapid population growth and unsterilised needles used in health clinics and vaccinations. The bushmeat hunting increased to compensate for the increased need to supply colonial labourers and because firearms became more available (22,23).
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Identification of the virus
Before the discovery of HIV, AIDS was described in US in 1981 (24).
Dr. Luc Montagnier et al at Pasteur Institute in France isolated a retrovirus from lymphoid ganglions and called it Lymphadenopathy-Associated Virus in 1983. They described it as the causative pathogen of AIDS. (25)
Studies by Robert Gallo et al, US confirmed his discovery in 1984 and they called it Human T Lymphotropic Virus type III (HTLV-III).(26)
Dr. Jay Levy et al, in 1984, named it as the AIDS-associated Retrovirus (ARV).(27)
The first description of acute HIV infection was of a "mononucleosis-like"
illness.(28)
In 1986, the International Committee on Taxonomy of Viruses named it as HIV (Human Immunodeficiency Virus).(9,29)
In 1987, US banned visitors with HIV and that was lifted by President Obama in 2010.
Nobel Prize
Montagnier and his colleague Francoise Barre-Sinoussi were awarded one-half of the 2008 Nobel Prize in Physiology / Medicine for the discovery of HIV.(30)
Subtypes of HIV
The virus is divided into two major types, HIV type 1 and HIV type 2 (on the basis of the presence of proteins and glycoproteins specific to them). These are
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then subdivided into groups (Figure 1) (24).
Each group represents an independent transmission of Simian Immunodeficiency Virus into mankind.(25) This large variability in the genetic makeup of HIV is the result of its evolution and thus making the therapy of its infection more
difficult.(31)
HIV-1: It is subdivided into 4 groups - Group M and Groups N, O and P (the latter three are minor groups).
Group M
The 'M' stands for "major", and as its name indicates, it is the most common type of HIV. 90% of HIV and AIDS cases belong to this group. This group is further subdivided into recombinant and non recombinant subtypes. There are 9 non recombinant subtypes [ A-K except for E and I] or clades. The subtypes may be further split into sub-subtypes (Eg A1, A2 and F1, F2).
Apart from these, there are ―circulating recombinant forms" or CRFs which have evolved from the recombination of different virus subtypes. Eg, CRF12_BF derived from a recombination of the subtype B and subtype F, both belonging to
Figure 1 : Taxonomy of HIV
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the M group of HIV-type 1. In India, subtype C is the dominant one.(32) Overall in Southeast Asia, CRF01_AE is predominant.(1)
Group N
The 'N' stands for "new". Till 2015, there has been less than 20 Group N infection reports worldwide.(33)
Group O
The O or the "Outlier" group is seen only in West-central Africa (1,34).
Group P
In 2009, a new HIV sequence was described and it had been isolated from a woman from Cameroon living in France. The virus has been placed in a new proposed Group P "pending the identification of further human cases".(12)
HIV-2
The first case of HIV-2 was described in the United States of America in 1987.
There are 8 known HIV-2 groups (named after alphabets A till H). Of all these, groups A and B have spread over large geographical areas. Though both are concentrated in West Africa, unlike Group B, Group A has dispersed to many other parts of the world and India too is not untouched.(17,35) The other six HIV- 2 groups (C to H) have been found in just one person each. They have probably descended from independent transmissions from sooty mangabeys to human
beings. Man is probably the dead-end host in these infections.(17,35) These HIV-2 strains are probably dead-end infections(17,35,36).
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History of HIV in India
Before April 1986, the rare HIV positive cases detected in India were among Indians who had acquired the infection abroad or among foreign tourists or foreigners residing temporarily in India. It was believed that AIDS was not going to be a major problem in India.(37) In 1986, the first six cases of HIV in India were diagnosed amongst female sex workers in Chennai, Tamil Nadu.(38,39) The samples were tested on ELISA platform, a rarely available test at that time in India, in the laboratory of Christian Medical College in Vellore(40,41). The samples were later confirmed on Western Blot technique. It is thought that the disease came to India along with the foreigners who were quite frequent at that time.(42)
Later, Lele et al reported another case in 1986 from Mumbai. These led to the realization that HIV infection may become a big problem in India. Blood products were shown to be infected with HIV virus by Tripathy and others in 1991 (43). In 1991, Naik and group also reported about an explosive outbreak of HIV-l infection among Injectable Drug Users (IDU) in Manipur.(37)
Routes of Transmission
In India, unprotected sex (87 % heterosexual) is the predominant route of HIV transmission, followed by vertical spread (5%) and non-verified causes (3.0%).
Injectable drug abuse and homosexuality among men contribute about 2% each.
Transfusion transmission attributes to 1% of HIV spread in India (44) (Figure 2).
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Figure 2 : Distribution of the transmission modes of HIV in India(44)
PPTCT: Prevention of Parent to Child Transmission, MSM: Men having Sex with Men, IDU: Injectable Drug Users
UNAIDS (45) mentions that the chance of acquiring HIV is 10 times more in a sex worker while it is 24 times more in people who inject drugs and in people practising homosexuality.
Current HIV Scenario in India
After South Africa and Nigeria, India leads with having the third largest HIV affected population in the world (46). The prevalence of HIV in India in 2015 was 0.26% (0.22% – 0.32%) in the adult population (15–49 years) with an
estimated gender wise breakup of about 0.30% among males and 0.22% among the females. This 0.26% figure is small compared to most other developing countries but due to India's huge population of 1.2 billion, it implies that 2.1 million Indians potentially have HIV infection. India today stands at a rank of 80 among all the countries in the world when the adult HIV prevalence rate is considered (2016) (46,47).
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The north-eastern and the southern regions of the nation have the highest HIV prevalence. These include the states of Manipur, Mizoram, Nagaland, Andhra Pradesh and Karnataka. Manipur has the highest adult HIV prevalence (1.15%) followed by 0.80% in Mizoram. The highest number of people living with HIV is in undivided Andhra Pradesh and Telangana, 3.95 lakhs, closely followed by Maharashtra having 3.01 lakhs patients.(47)
The Indian adult HIV prevalence has seen a gradual decline (Table 1). It has gradually slid from about 0.38% in 2001-2003 to 0.26% in 2015 (Figure 3). While children (less than 15 years of age) account for 6.54%, females form two-fifth (40.5%) of the total HIV infections.(47)
Figure 3 : Estimated Prevalence of HIV in Indian Adult Population in the last One and a half Decades(48)
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Table 1: Prevalence and Incidence of HIV (41)
People living with HIV (all ages) New HIV infections (all ages)
2010 2016 2010 2016
Global 33.3 million 36.7 million 2.2 million 1.8 million Asia and
Pacific
4.7 million 5.1 million 3,10,000 2,70,000
India 2.2 million 2.1 million 1,00,000 80,000
Table 2: HIV - Therapy and Death: An Inverse Relationship (41) People living with HIV on
antiretroviral treatment (all ages)
AIDS-related deaths (all ages)
2010 2016 2010 2016
Global 75,01,100 1,70,25,900 1.5 million
1 million
Asia and Pacific
9,07,600 20,71,900 2,40,000 1,70,000
India 7,26,824 10,00,000 1,20,000 62,000
There was a continuous rise in the number of AIDS related deaths occurring in India till 2007. The access to Anti Retroviral Therapy has lead to a downward trend and the annual AIDS related deaths have reduced by 54% between 2007 and 2015. (Figure 4) About 62,000 people died of AIDS-related causes in 2016 (Table
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2). The decline in incidence rate of HIV over the same period is 32% (86,000 in 2015).(47) (Figure 5)
Figure 4 : Inversely Proportional Relationship between AIDS Related Deaths and People Living with HIV on Anti Retroviral Therapy (ART) in India(48)
Figure 5 : Estimated HIV Incidence in India (1998-2015)(48)
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The high-risk populations include (Figure 6):
1) Sex workers
2) Men having sex with men 3) Injectable drugs
4) Transgender people (emerging group).
Truck drivers and migrant workers form the ‗bridging population‘.
The first two groups have experienced a recent reduction in HIV prevalence while the third group has seen a rise in India.
The estimated number of female sex workers in India is 6, 57,829 and about 2.2% of them are infected with HIV. As per UNAIDS, 90% or greater of the
commercial sex workers have adopted condom use in Asia Pacific‘s five countries, which includes the two larger ones, China and India(49).
Of the estimated 2, 38,175 men who have sex with men in India, 4.3% are living with HIV. Injectable drug abusers in India have an estimated head count of 1, 27,532 and about 9.9% of them carry HIV in their blood stream. IDU has been the major route of the spread of HIV in north-east India. The population of transgenders in India is about 25,984 with 7.2% prevalence of HIV among them.
There are about 1, 85,182 prisoners in India. The prevalence of HIV in them is unknown.(47,50)
There are an estimated 72 lakhs migrant workers in India. The prevalence of HIV in this population is 0.99% and that is quite high in comparison to the national prevalence rate of 0.26%.(51) The migrants from the ‗bridge population‘
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linking urban and rural areas and also linking high and low-risk groups. As per UNAIDS, studies in the year 2014 showed that of all the women who tested positive in our country, about 75% women‘s husband was a migrant labourer.(45) Apart from that, the prevalence of HIV in the population who moved from the countryside to cities was 0.9%. (45) HIV prevalence among the estimated 20,00,000 truckers in India is 2.59%.
Figure 6 : HIV Prevalence among the High-Risk Groups in India(48) ANC: Antenatal Checkup, FSW: Female Sex Workers, MSM: Men having Sex with Men, TG: Trans Genders, IDU: Injectable Drug Users
BLOOD TRANSFUSION SERVICES
In July 1982, three patients with haemophilia who had received factor VIII concentrates developed infections commonly seen in severe immune-compromise.
(CDC 1982) (52). The index case of transfusion related AIDS was an 18-month- old baby. The baby had severe combined immunodeficiency syndrome and had been transfused multiple times. A follow up of one of the donors showed his
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subsequent development of AIDS. During the 1985 – 2015 period, gays could not donate blood in the US (53).
The safest blood donor is the one who is donating voluntarily, is non- remunerated and is from low-risk group. The World Health Assembly passed a resolution in 1975 which became the World Health Organization‘s goal for all nations to have all of their blood supplies via voluntary and non-remunerated donors.(54) A voluntary non remunerated donor is one who donates blood on his/her own free will without distinction of caste, creed, religion, color and status of recipient and does not expect any monitory benefit from the collecting facilities or other sources at the time of donation or in future (55).
As per World Health Organization (WHO), about 13 million units globally went untested for transfusion transmittable infections in 1998-1999 leading to 1,60,000 HIV/HBV/HCV (Hepatitis B Virus, Hepatitis C Virus) infections
annually(56). The figures for untested units came down to 6 million units in 2001- 2001.(56) After its survey in 152 countries, the Global Database on Blood Safety reported a low quality of HIV tests performance and infrequent and unpredictable supply problems are leading to HIV spread via transfusions.(57) Jayaraman et al in 2009 showed the residual risk of getting an HIV, HBV or HCV infection post a blood transfusion in sub-Saharan Africa was 1, 4.3, and 2.5 infections per 1000 transfusions respectively. The study estimated that transfusions alone would be causing about 28,595 HBV, 16,625, and 6650 HIV infections annually if WHO estimated transfusion demands would be satisfied in sub-Sahara.(58)
Of all the people with HIV, about 2-5% acquired the infection post a
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transfusion and a big chunk of these occurred before the screening of HIV had started.(59) HIV is a quite infective virus and 90% of the patients who got transfused with an HIV positive unit developed the infection.(60)
Figure 7 : Inverse relationship between voluntary blood donations and HIV prevalence in blood donors in Africa (56)
An African study showed a good correlation between voluntary blood donations and HIV prevalence in the blood donors in 16 countries in the Africa (Figure 7).
How India fought back its TTI woes
HIV screening was made mandatory in blood banks in 1988 in India(61).
As there was a lack of trained technicians to do the tests, Zonal Blood Testing Centres carried out the tests for them. Due to the HIV epidemic, the Government of India launched a 7 year National AIDS Control Programme (NACP I) in 1992 (48) with the objective of curbing the spread of HIV infections. In 1996, Supreme Court ordered (48) and paved the path for the creation of National Blood
Transfusion Council (NBTC) as apex policy-making body for blood transfusion
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services. NBTC became a part of NACO for the effective administration of
NACP. The Supreme Court then banned remunerated or professional donors in the year 1998(61,62). In 2002, the Government of India adopted the National Blood Policy ―An action plan for blood safety‖ to ensure safe blood supply. As a part of the National Blood Policy, the concerned blood bank must notify a donor his status if he is found reactive so that he can get himself confirmed and start therapy (51).
The current programme, NACP-IV (2012-2017) (48)has been working to reduce HIV incidence by 50% providing comprehensive treatment, greater
awareness, care and support for the high risk as well as the general population. Its targets also include making the second line ART available free to the patients.
The incidence of HIV in India is going down and has fallen by more than half since 2001, yet the number of new HIV infections in India in 2015 was a striking 86,000 (48). HIV testing and counselling centres in India have increased from a meagre 67 centres in 1997 to 20,000 centres in 2016. This has prolonged the lives and improved the quality of life of the patients. India currently spends about 5% of its health budget on HIV/AIDS. Currently, the blood transfusion services are supported by 2760 licensed blood banks spread across all the states, trying to meet the requirement of blood in India which is about 12 million units annually.(47)
UNAIDS has launched a 90-90-90 target which aims to achieve that by 2020, 90% of the population suffering with HIV should know about their diagnosis, 90%
of them should receive sustained ART and 90% of the ones being treated should have achieved viral suppression (63). This ambitious strategy will go a long way in
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bringing the AIDS epidemic down. As per UNAIDS (2017 report) (50), in 2016, 77%
of seropositive Indians know about their being diseased and 49% are on ART. The viral suppression status is unavailable. It lacks in the availability of adequate resources in its treatment centres and most of the diagnostic centres for the
assessment of viral counts. Hence, in India, the complete achievement of the 90-90- 90 target by 2020 appears difficult at this stage.
THE STRUCTURE OF HIV
Figure 8 : Schematic figure of an HIV-1 virion (64)
Retroviruses are membrane coated RNA viruses (Figure 8). There are mainly four viruses in the retrovirus family that can cause disease in man - HIV-1 and HIV-2 of the lentivirus group and human T-cell lymphotropic viruses (HTLV-I
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and HTLV-II) of the corona group. Like the HIV, the HTLV-I and HTLV-II are also thought to have evolved from simian retroviruses.
The 100-120 nm, spherical virus has its double stranded RNA (Ribo Nucleic Acid) packed in a cylindrical core along with two structural proteins and RNA dependent DNA (Deoxyribonucleic Acid) polymerase enzyme, the reverse transcriptase. The nucleoprotein core is enclosed in a cone-shaped shell of p24 protein. This 24,000 Da molecular weight major core protein is similar in HIV-1 and HIV-2. The whole unit is called viral capsid. The capsid is covered by two layers. One is a shell made up of pl7 matrix protein to which the small proteins that stick out from the virus particle‘s surface are bound to. This is then embraced by a lipid bilayer. The transmembrane proteins poke out through the lipid layer.
The gp41 transmembrane proteins are attached on the inner side to the p17 matrix and externally to the gp l20 envelope proteins. They look like small projections on the viral outer surface. These projecting and their anchoring proteins are the major difference between HIV-1 and 2. The equivalents of these proteins in HIV-2 are gp l10/130 and gp 36. Antibodies formed against these 2 proteins do not cross- react.(55)
HIV uses CXCR4 and CCR5 co-receptors to enter CD4 cells (Figure 9).
Being an RNA virus, it first uses reverse transcriptase to create a copy of its DNA, which later like any other virus integrates with the host DNA to control the host cell for producing its own progeny.
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Figure 9 : The Various Stages of HIV Replication (65)
HIV has 9 genes of which 3 are structural genes and their encoded proteins are antigenic:
1. Gag gene - codes for the core proteins, 2. Pol – codes for polymerase enzyme,
3. Env gene - coding for envelope glycoproteins.
In addition, genes that encode proteins with regulatory and accessory functions and help in the expression of the above genesinclude:
Tat, Rev, Nef, Vif, Vpr and Vpu170.(24)
Though HIV 1 and 2 share core and pol protein epitopes, they share only 40- 50% of the nucleotides with each other. They mainly differ in their envelope nucleotides and proteins. (Figure 10) (Table 3)The RNA of HIV-2 is more similar to the SIV (66)
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Table 3: The Various Proteins and Glycoproteins that differentiate HIV - 1 and HIV – 2 in the bands observed (53)
Gene HIV - 1 HIV - 2 Protein
Gag p18, p24, p15 p16, p26, p55 Core
Pol p31 Endonuclease
p51, p65 p68 Reverse transcriptase
Env gp41 gp36 Transmembrane protein
gp120, gp160 gp140, gp125 Envelope unit
gp = glycoprotein (number indicates molecular weight); p = protein
Figure 10: An Artistic Diagram of HIV - 1 Versus HIV – 2 (46)
HIV Testing
Screening all blood donors for antibodies to HIV-1 with EIA first started in 1985 in US. Testing for HIV-2 got a go-ahead in 1992.(67) The platforms available today for screening HIV -1 and 2 include the quantitative ELISA (available in various generations based on its increasing sensitivity and reducing window period), qualitative Nucleic Acid amplification Tests (NATs) and various
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qualitative immunochromatography based rapid diagnostic tests. Other diagnostic platforms for HIV include quantitative CMIA and the ‗gold standard‘ Western Blot. (55) These all have their own window periods. (Figure 11)
Figure 11 : Window Period Reduction by various Platforms (68)
Figure 12 : Changes in Serology after HIV Infection
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The incubation period of HIV spans from 2 to 10 weeks (mostly asymptomatic or mild flu like features), HIV RNA is detectable by 6–9 days and the viral antigens appear in the blood stream by next 5–10 days. Antibodies against HIV become detectable at about 22 days after infection. During the window period when no antibody is detected, viral antigen (p24, gp41) could be detected. The time period during which the antigens may be detected is quite small, often about 1-2 weeks.
Viraemia is maximum during the window period and after Aids Related Complex and AIDS develop. (Figure 12)
HIV has very high genetic variability, more so in their env gene which codes for the major outer glycoproteins which bear the epitopes reacting with anti-HIV antibodies. Extreme genetic diversity has negative effects on the viral detection and its treatment.(66)
Residual risk of infection
Highly sensitive serologic testing for HIV screening in the blood banks has significantly reduced the risks of HIV transmission via transfusion. But there still exists a residual risk of infection in a transfused unit.
It can be due to:
1. Donation in window period
2. Inadequate quality of TTI screening 3. Variants of pathogens
HIV-NAT-negative blood has also been reported to cause transfusion-
transmission of HIV. The HIV reverse transcriptase lacks a proof reading function.
So, any error in transcription will not be registered and hence not corrected. This
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causes HIV RNA to be undetectable in such cases.(69) Hence, even with the use of most sensitive of the assays, zero risk is not yet achievable.(1)
Even in developed countries like the UK, the residual risk of HIV spread via blood transfusion was 0.14 per million blood donations during 1996 to 2003.(70) There has been an increase in the residual risk to 0.22 per million (UK data) due to an increase of the disease prevalence in the general population, even though many more sensitive tests have come up now. The number of donors donating in the window period is about 1 in 4 million in US.(71)
Most of the deaths due to blood transfusion globally are not due to its immuno-haematological causes but due to its capability to spread the various infectious agents: viruses, bacteria and protozoa. As human civilization infringes its limits into the forests, the germinating suburban wilderness and the ever increasing global travel assure the emergence and dispersal of ‗new‘
pathogens.(66)
The nuisance of false-positives
Though the tests used nowadays in blood banks for screening infective diseases are highly specific, they being a screening tool are designed to keep the sensitivity in more focus than the specificity. And in a low prevalence scenario as like in apparently healthy blood donors, the specificity is negatively affected (noise in screening assays)(72), causing blood banks to permanently defer and lose their scarce resource of suitable blood donors. This creates a mental stress on the donor and reduces the confidence of the public in blood banks. It also leads to financial loss to the blood banks. Long term follow-up of indeterminate HIV status of the
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blood donors on ELISA and Western Blot platforms did not show seroconversion in them.(73,74) Enhancement of procedures designed to reduce mistakes will go a long way in preventing such scenarios.
The trouble of false-negatives
The four sources of false negatives leading to residual risk of virus transmission to a blood recipient include: (i) Window period donation (ii) Mutants and variants of the pathogen (iii) donors who mount an inadequate immune response to infection by the donors (iv) procedural errors in performance of tests.(75)
Quality Assessment and Controls
Quality assurance (QA) is a means of systematically verifying the results of a laboratory with the help of a sample with known parameters and is tested for ensuring the trueness of the tests performed. By detecting procedural errors and manufacturing defects, QA ensures good quality of the service provided or the product manufactured. It monitors all facets of a testing process, namely pre- analytic, analytic and post-analytic phases. Analytic QA is performed via:
1) Internal Quality Control (IQC) 2) External Quality Assessment (EQA).
All laboratories (including the infectious disease screening laboratory in a blood bank) should have quality control measures for ensuring the validity of tests it performs. The ensuing data is recorded, producing trends and statistics for the reviewing of daily results. Regular usage of internal quality control is a good way
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of doing it. Whenever the quality control data is detected outside pre-defined standards, a planned action is taken for troubleshooting and its correction and finally, measures can be taken to prevent such errors in the future.(76)
The sheer volume of the HIV screening required in our blood banks does not leave much elbow space for testing errors. A bare 0.5% error rate in 12 million tests may result in 60,000 donated blood units wrongly labelled, either dangerously as false negative or wastefully as false positive. (77)
The Global Database on Blood Safety (56) stated that the coverage for HIV testing was 100% in 152 countries in 2002. But low quality in the execution of the tests still plague many blood banks worldwide increasing the chances of HIV transmission by transfusion.(78) The lack of proper validation and quality
management programmes lead to persistence and non-correction of errors. There may be inconsistent refrigeration, clerical errors, equipment malfunction, pipetting errors, low quality reagents, failure to follow kit manufacturer‘s instructions, batch to batch variation in commercial assays (79) which can be detected by the regular use internal quality controls. The type of testing kits used, the operating
procedures followed and the technical skills of the staff affect the screening outcomes of a blood bank.
A major constraint in underdeveloped and developing countries is the use of quality controls during screening of transfusion transmissible infections because of the high costs of the commercially available IQC materials and the lack of infrastructure for freezing and long term storage of seropositive positive samples
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in many blood banks.(67, 68) A study in by Dogbe and Arthur et al in Ghana (2015) looked at infectious disease screening facilities of 5 blood banks in using rapid immunochromatographic tests found high false positives and negatives in their results. Overall, the facilities were graded poorly.(82) Hence, there is a pressing need for an effective yet cost-effective control system for screening of infective diseases in donated blood in under-developed and developing countries.
A study by Parekh et al at CDC, USA validated the use of Dried Tube Specimen (DTS) for the use as an external quality control or proficiency testing material for HIV screening in blood banks on rapid immunochromatographic and ELISA platforms. They showed the stability of DTS samples at 40C and 250C for 4 weeks while it had marginal deterioration at 370C and 450C over the same time period.(4) The DTS were tested at higher temperatures as their objective was to design sample specimens for transport to other centres at ambient temperature.
Chopra et al also showed that HIV antibodies in DTS are stable at 37⁰ C for up to 30 days.(83) Ramos et al evaluated DTS as a proficiency testing tool for HIV viral load determination on molecular platforms. Samples with viral RNA concentrations ranging from 10² to 10⁶ ·⁵ copies/ml examined up to 8 weeks showed no viral load reduction at 37 °C while a little deterioration in DTS stored at 45 °C. (5) DTS for HIV has been used in many countries for EQA purposes.(3–
5, 71–74) A study by L Cabuang et al (84) showed that HIV RNA was quantifiable in DTS stored at 4⁰ C and -70⁰ C for 24 weeks and 52 weeks respectively, though there was about 50% reduction from the baseline in the
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number of copies at 4⁰ C at its final reading (Figure 13). A study by National Institute of Biologicals, India showed that the DTS with HBsAg gave good results on ELISA and rapid tests till one month period at ambient temperature and
below.(85) Studies have been done on DTS for syphilis as well showing stability for 3 weeks period.(86,87)
Figure 13 : HIV RNA Concentration Variations with Temperature and Time in DTS (84)
Dried Blood Spot (DBS) on filter paper has been studied as an alternate sample material for unrefrigerated transport from rural and remote areas to a laboratory in low and medium income countries with hot ambient conditions. The goals for the DBS material have been to increase the population access to
diagnostic tests that are not available to them in their vicinity and as a good tool for various surveillance studies. It can also be used for sample collection in paediatric populations where venepuncture may be difficult.
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DBS has been studied for use in the diagnosis of viral infections like HIV, hepatitis B, measles and protozoa like Plasmodium by serological tests (88–90).
Solomon et al from South India tested DBS of HIV positive and negative samples for anti-HIV antibodies by ELISA and Western Blot after subjecting the DBS to ambient temperatures and high humidity for 6 days got a sensitivity of 100% and 92% on the two platforms respectively with no false positives or negatives.(91)
DBS specimens have also been proven to be usable for HIV-1 virus load measurement (92) and testing for development of resistance to first-line
antiretroviral drugs.(93) HIV-1 RNA concentrations remained stable in DBS for 2 weeks and longer at ambient temperature with no apparent degradation.(94–96) A study comparing DBS with Dried Plasma Spots (DPS) for HIV-1 nucleic acids showed that both DBS and DPS started showing deterioration after 1 month of storage at 37⁰ C while they were good till 3 months on storage at 20⁰ C. DBS fared better in comparison to DPS.(97) However, Schmitz et al noted and cautioned that the ability to do molecular testing on DBS differed on similar platforms by different manufacturers. They gave a minimal viral load threshold of
>1000 copies/ml for field evaluations without a long term storage.(98) For long term storage, a significantly higher viral load is a must to prevent false negatives.
Behets et al demonstrated that DBS made from high and low titres HIV-1 positive blood on storage in tropical ambience remained positive for HIV-1 antibodies for 6-7 weeks and then the hot and humid conditions led to gradual deterioration of the samples. The negative controls though did not show any false
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positive reactions over 20 weeks of storage. The tests had been performed on ELISA and Western Blotting and rapid platforms.(99,100) DBS samples can be stored at ambient temperature for up to 1 month for quality control of HIV testing with serum on ELISA platform.(101)
External quality control plays an important role in ensuring the proficiency of a laboratory(102) but the samples are not adequate to be run daily. There is a need for internal quality control which is easily available at less cost for day to day testing. Internal quality controls provide a continuous and critical evaluation of the daily working of any laboratory. Precision of the laboratory for the particular test is verified through IQC, allowing for detection of random errors and alerting to onset of systematic errors. EQA verifies accuracy (103). The control samples are to be run in parallel, without any discrimination, along with the routine samples on a regular basis.
Note: Zotero Version 5.0.21 reference manager software has been used in this literature review.
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METHODS AND MATERIALS Setting of the study
This study was carried out in Christian Medical College, Vellore, Tamil Nadu. It is a 2300 bedded tertiary care hospital and referral centre. It caters to the needs of people of Vellore and surrounding districts of Tamil Nadu. It also receives patients from all the other states of India in varying proportions. Apart from these, patients from Bangladesh, Nepal, Bhutan, Sri Lanka, Maldives and UAE also visit the centre.
This prospective observational study was conducted in the Departments of Transfusion Medicine and Immunohaematology and the Department of Clinical Virology.
The study was approved by the Research and Ethics committee of the Institutional Review Board, Christian Medical College, Vellore. (IRB minutes number: 9826 dated 7th of January 2016) (Annexure-I)
This study was conducted in the year 2016-2017.
Sample Size
In our blood bank, we get about 30,000 donations per year of which only about 20% are voluntary and the remaining are replacement donors. The donors are screened for HIV and other infectious diseases as mandated by regulatory authorities (done here by 4th generation ELISA - EVOLIS™ platform by
BIORAD™, France). These blood samples also undergo individual NAT testing in our blood bank (done on Procleix Panther System™ by Grifols™, San Diego,
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USA). All the HIV screen positive samples are sent to the department of Virology in our Institute for confirmation on three platforms, including a 4th generation chemiluminescence microparticle based assay, a 4th generation ELISA and Western Blot. All HIV positive samples (either by serology or NAT) are
discarded after autoclaving as per national regulatory requirements. The national prevalence of HIV is 0.26% (104).We collected all the HIV positive Fresh Frozen Plasma (FFP) bags donated prospectively over the first six months of our study period based on our inclusion and exclusion criteria.
The sample collection began in 12/07/2016 and continued till 11/12/2016.
Of the total of 25 donors who were reactive for HIV on initial screening 4 donors were confirmed as true positive based on confirmatory testing including viral load and fulfilled all our inclusion criteria. These 4 FFP bags were quarantined for long term storage. Equal numbers of HIV negative bags were also collected. The index reference paper has also used 4 HIV positive plasma samples for pooling
together.(4)
Source of plasma:
Donors who donated blood in our blood bank after adequate screening by Public Relationship Officer, a technician and a blood bank physician but turned out to be HIV positive in blood investigations and fulfilled the following criteria were recruited for this study. Consent was taken (Annexure-II) prior to their blood donation in the blood bank. The identities of the donors were delinked from the study to maintain their anonymity. There was no follow up of the participants
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other than the mandated regulatory notification and linkage to ICTC.
Inclusion Criteria:
HIV Positive samples
1. The age of the participants was to be more than 18 years.
2. The donor samples had to be strong positive for HIV in our department‘s Evolis platform and Nucleic Acid Testing platform.
3. They had to be confirmed HIV positive by Western Blot platform in the department of Virology.
4. They must have signed the consent forms (Annexure III).
Exclusion Criteria:
1. Donors who were detected to be reactive for any other disease screened in blood- bank were to be excluded from the study.
2. Plasma that were found to be lipaemic or icteric.
Controls:
Plasma units from four donors that were negative for all the diseases screened in blood-bank were chosen as negative controls.
Variables:
Duration of storage was the independent variable while deterioration of samples over time was the dependent variable.
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Storage temperature could act as an effect modifier. So, pooled and aliquoted specimens were stored in a cooler at 2-8⁰ C and were taken out only when they were to be reconstituted for use.
Volumes were measured accurately using micropipettes to reduce any variation due to incorrect dilution.
All the coolers, freezers and micropipettes that were used had been calibrated and were within the valid period of calibration.
The tests were considered satisfactory if all the controls produced expected results.
Expected Outcomes:
Outcomes Time-points
Primary Outcome The stability and
significant deterioration of the Dried Tube Specimen (DTS) was looked into.
1) Tests were started on 21/12/2016 and done every week till 21/06/2017 (Week 26 or 6 Calendar months). Every week, the integrity of the samples was checked.
2) Statistical analysis was done after the end of 6 months of the study (21/6/2017).
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Secondary Outcome 1) Whether should we continue testing of DTS further after the initial 6 months of the study are over (if the DTS does not show significant
deterioration)
2) Should this idea be extrapolated for other infectious diseases
screened in blood banks - Hepatitis C and Hepatitis B (if the study gives good results for HIV).
After the end of the study - 21/6/2017.
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Detailed diagrammatic Algorithm of the study
Over 6 months period, plasma of donors detected as HIV positive in blood- bank, confirmed by virology and having high titres of antibodies were pooled together into 1 pool. A total of 4 FFP bags were collected over the period.
Equal number of FFP bags (4 bags) that were negative for all infectious disease screened in our blood bank were chosen as negative controls and pooled together.
20µl aliquots of plasma were dried in Eppendorf tubes overnight at room temperature on day 0 and stored at 2-8°C. A total of 300 aliquots of HIV positive and negative DTS were prepared.
Aliquots were reconstituted with 200µl of Phosphate Buffered Saline once every week for the tests.
Tests were done on the following platforms: Microlisa (2nd generation manual ELISA), Immunochromatography based rapid tests – Trueline, Tridot and CombAids and ARCHITECT (4th generation automated CMIA) every week and EVOLIS (4th generation automated ELISA) once every month.
Data collected every week was analyzed statistically after the end of 6 calendar months period (26 weeks).
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Pooling of samples
All of the 4 HIV positive and negative FFP bags each were quarantined and stored in -80⁰ C freezer until the time they were to be used to prevent any
deterioration in them. They were thawed at room temperature without the use of any thawing device as they would not be available at smaller centres nor was any water-bath used to avoid any contamination of the plasma. The plasma bags were brought to room temperature before use to prevent any water of condensation diluting them.
Using sterile 10 ml syringes and needles, 10 ml of plasma was collected from each of the four seropositive and mixed together in a 50 ml sterile plastic container. The same process was performed with the four negative FFP bags. The positive and negative samples were pooled for storage on different working days to prevent any possible contamination. The procedure was done with adequate personal protective care in laminar flow hood cabinet. No distinction was made between positive samples and controls in terms of safety and care.
The reference article (4) from which the study derived its protocol had used Kroger brand apple green food grade dye for providing a green hue to the faint straw coloured plasma to make it better visible after drying. This would be of high importance considering the conventional low illumination in the walk-in- coolers and reagent fridges. A trial was performed with the locally available green food grade dye but it was noted that the dye caused inhibition of the strength of reactions in Microlisa. Hence, the idea of staining the plasma was abandoned and the DTS was prepared in native state without the use of any dye.
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Preparation of the DTS
Colourless micro-centrifuge tubes of 0.5 ml capacity were sterilized by ethylene oxide gas sterilisation in the Central Supplies and Sterility Department of our institute. This volume of micro-centrifuge tubes was chosen as bigger tubes would result in higher dead volume. Sterilization was done to prevent any microbial growth in the protein rich plasma, which was to be stored for a long time and otherwise could lead to their early degradation and false results. The tubes were labelled as positive or negative with a water-proof marker before samples were added to them and were kept segregated. 20µl of seropositive and seronegative plasma were added to the respective labelled micro-centrifuge tubes with the help of micropipettes and disposable tips. A total of 300 positive and negative tubes each were loaded in a laminar flow hood. They were kept open in separate boxes in the laminar flow hood for 14-16 hours at room temperature allowing them to dry completely. The volume of 20µl was adopted from the same reference article (4) where they stated that with greater volume, the plasma was difficult to dry and lesser volume would reduce the available anti-HIV antibodies in the plasma. All the samples were loaded consecutively without any break. The next day, after the plasma had dried completely, there was a visible shrinkage of volume and loss of mobility of the fluid. All the tubes were carefully capped to prevent any sort of leakage or contamination. Separate boxes that were clearly labelled were used to store the positive and negative samples. They were stored immediately in 2-8°C walk-in cooler in a dry location. A total of 300 positive and negative test aliquots each were prepared so as to ensure adequate samples are available for initial
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practice, weekly tests, homogeneity tests and any further tests if required.
Reconstitution Steps
Each week, 4 positive and negative samples were reconstituted for the rapid tests, Microlisa-HIV™ (J. Mitra & Company Private Limited™, New Delhi, India) and for the Chemiluminescence tests. Every month, one additional set of samples were used for testing on Evolis™ (BioRad™, France). Phosphate Buffered Saline (PBS) with Tween 20™ was used to reconstitute the samples. (Tween 20™ is a
commercially available scientific grade liquid detergent). 200 µl of PBS(4) were added with micropipettes for the reconstitution of each DTS. This provided a 10 times dilution of the plasma after compensating for the dried up plasma volume but was treated as undiluted for all testing platforms. The PBS was added on the evenings prior to the day of experiments into the required number of samples to ensure that the dried plasma sample was completely dissolved. The samples were tapped gently 10 times at the tip end to dislodge the dried plasma. Care was taken that the micro-centrifuge‘s cap did not open and spill during the taps. They were stored overnight in 2-8°C storage walk-in cooler prior to testing. The methodology to prepare PBS with Tween 20™ is attached as its Standard Operating Procedure (SOP) (Annexure-IV).
Preparations before doing the tests each week
All the rapid and Microlisa™ test kits along with the DTS samples were kept out at room temperature for at least half an hour before doing the tests. One positive
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and negative quality control tubes would be taken from the -20°C freezer and thawed at room temperature before use each week. The DTS were inverted for 10 complete cycles and mixed with the help of micropipettes again for 10 times to enhance their dissolution. Care was taken to change the disposable tips wherever applicable to avoid any sort of inter-tube contamination. These procedures for dissolving the DTS were used as vortexes may not be available in most blood banks. In order to compare this with the mixing done by vortexes, on some occasions, vortexes were tried and they gave similar results as with the manual mixes with respect to the semi-quantitative Microlisa™ results. Thus these mixes were equally efficient to vortexes.
Adding PBS overnight helped in the adequate dissolution of the dried plasma. Plasma is made up of multiple proteins each having different solubility.
The dried plasma is not readily dissolvable and macro particles would be visible to naked eyes even post dissolution. The particles would be larger in size if the PBS were added just prior to their usage.
To resolve this issue, a homogeneity test was done in which 10 samples taken from the 4 corners and centres of 2 boxes each of HIV DTS were tested in duplicate on Microlisa-HIV™ (J. Mitra & Company Private Limited™, New Delhi, India) platform. The negative control DTS was tested similarly. (n=40)
Platforms tested
The tests were done each week for 6 calendar months, making it a total of 26 weeks. Each week, the samples were tested on 5 different platforms including 3 immunochromatography based rapid tests, namely:
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1. CombAids-RS Advantage™ (ARKRAY Healthcare Private Limited™, Surat, Gujrat, India),
2. HIV TRI-DOT™ (J.Mitra & Company Private Limited™, New Delhi, India), 3. TRUELINE™ ( Alere Medical Private Limited™, Gurgaon, Haryana, India)
A 2nd generation ELISA, Microlisa-HIV™ (J. Mitra & Company Private
Limited™, New Delhi, India) was used as a manual ELISA. All these platforms were chosen as they were the notified kits by Drugs Controller General of India (DCGI) to be used for HIV screening in blood banks.
The rapid tests have a short turnaround time (about 20 minutes), are easy to use, more affordable than other available screening tests and require no additional infrastructure. These make them a very attractive option in small setting blood banks. A 4th generation Chemiluminiscence microparticle based Immunosorbant Assay (CMIA), Architect™ (Abbott™, Pennington, New Jersey, USA) was included to test these DTS too as it being a highly sensitive platform used in the department of Virology could pick up any deterioration of DTS at an early stage.
A 6th platform to test the DTS was the EVOLIS™ (Automated ELISA used in our blood bank, Manufactured by BioRad™, France), a 4th generation ELISA. The samples were tested once every month on this platform to validate DTS on automated platforms used in bigger blood banks.
All the positive samples and negative control samples for rapid tests and Microlisa-HIV™ (J. Mitra & Company Private Limited™, New Delhi, India) were tested in triplicates to resolve any discrepancy that may have arisen had a single sample been tested. So every week, 4 positive and negative samples each
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were reconstituted (1 for Architect and 3 for the other tests for doing in triplicate) while once a month, 1 extra pair of samples were reconstituted for EVOLISTM
(BioRad™, France).
All the tests were performed as per the SOP for the respective platforms (Annexures IV). It was planned that the tests will be repeated the next day in case of any discrepancy. Such a situation never occurred during the period of the whole study.
The results of the rapid tests were imaged every time in a camera for
maintaining a visual record. The results were entered into secure form developed using Epidata™ software.
Number of sample aliquots tested Positive aliquots: 109
Negative aliquots: 109
The break-up of number of sample aliquots used shown in Table 4 is for positive and negative samples each. Table 5 mentions how each DTS was used.
These were apart from the DTS tubes used for Evolis™ and Architect™ which were tested with individual DTS samples. Table 6 shows the number of tests (positives and equal number of negatives) done on each platform.
Table 4: Break-up of the number of aliquots used PLATFORM Architect™ Microlisa™ and Rapid
platforms
Evolis™ TOTAL ALIQUOTS NO. OF
ALIQUOTS
26 26 x 3 = 78 05 109
FREQUENCY OF TESTS
Singlet per week
Triplets every week Singlet, once a month*
