PHOTO-SYNTHETICALLY ACTIVE RADIATION (PAR) EFFECTS ON BEAN PLANTS THAT DEVELOPED DUE TO RATION OF LIGHT : DARK (L:D) PERIODS (PHOTOPERIODS)
12th-December 2007

Abstract

Plants are bombarded by a myriad of signals, not just from their physical environment, but from friend and foe alike. As a consequence, they have evolved a remarkably sophisticated system of receptors and signal transduction pathways that generate appropriate responses, light plays a major signaling role in plant development. The plant’s ability to maximize its photosynthetic productivity depends on its capacity to sense, evaluate, and respond to light quality, quantity, and direction. Photo-synthetically active radiation (PAR) effects the general morphological development of bean plants with responses in leaf area, plant elongation (shoots and roots), and biomass accumulation measured as dry weight. The variables were assessed using Light : Dark (L : D) reception period ration, and the plants adjusted favorably to altered environmental illumination, in order to coincide or synchronize their growth to these conditions. The results show that the plant-growth tallied with the already existing hypothesis, ‘Plants synchronize to PAR with due changes in their morphological characters that is to say with changes in their photo-morphogenesis. The plants synchronized to altered PAR conditions and showed an reliably good pattern at growth conditions L : D = 60 out of 108 hours of PAR radiation per week. Although not statistically significant, the growth differences for continued growth variation of third and fourth week were not assessed due to time limitation.

1.0 Back ground
Research into photo-synthetic active radiation effect began more than 60 years ago when the existence of short-day and long-day plants was discovered (Borthwick., et al 1945). Its analytical stage started after botanical institutes had been equipped with climatic chambers where plants could be cultivated under controlled light and temperature programs. Plants have evolved to respond to light illumination differently using different receptors. A red, far-red reversible chromoprotein, phytochrome, were the first photoreceptor to having been identified. It is now known that multiple phytochromes (A, B, C, D, and E) exist and sometimes act independently of one another, sometimes redundantly, sometimes antagonistically, sometimes at the same time in development, and sometimes at different times. The first blue-light receptors to be identified were the two cryptochromes, and chromoproteins that mediate several responses, and more recently, another blue-light-absorbing chromoprotein accumulation (Briggs and Margaret. 2001) Photoperiodism was seen to involve synchronization of the photo-synthetic active radiation (PAR) and the rhythmical events in lifecycle of organisms. The major development activities of the plants were thus conclusively known as being affected by seasonal PAR changes, activities including among others diapauses, hibernation, cryptobiosis, sleep, migration, mating, dormancy, flowering, and germination. It was also discovered that it is not the day length (light period) that is decisive for stimulation of plant activities but the dark period, and hence a minimal light period was required for the production of enough assimilates. But the dark period alone can not exert both a stimulating and an inhibiting influence on general plant functions. In this study the effect of photoperiodism as the effect of more dark periods and light periods, was undertaken to define its effect on general bean plant growth relationships including leaf area, shoot and root elongation and biomass.

2.0 Introduction

Plants’ photo-responses to light include; photo-taxis, photo-morphogenesis, and photo-periodism. Photo-taxis (or phototropism) is a light-induced movement of organisms consisting of a single or just a few cells towards the light source. Plants show a positive photo-tactic growth towards a light, and this is very typical for multicellular plants though many chlorophyll-free, non-plant egg cell, plus the sporangiophore of the fungi Phycomyces also perform a similar response (Halliwell, 1981). Photo-morphogenesis is PAR light-induced activity of plant growth and differentiation. Certain wave lengths of visible light function as a signal causing the generation of information within the cell that is used for the selective activation of certain genes. Photoperiodism on the other hand is the ability of plants to measure the length of periods of light. Certain species (short-day plants) stop certain growth activities such as flowering as soon as the day length has attained a critical value, whereas other species (long-day plants) begin to flower only after such a value has been passed. With the onset of a light-period, plants begin a physiological activity called the photophilic (Light-loving) stage. After 9 -to-12 hours of light regime, development of plant being exposed is thought to be inhibited by all further exposure to light. After this stimulation the plant enters its skotophile, that’s to say its darkness-loving phase. The next photophilic phase begins just a few hours later, long before dawn and thus quite independent of the actual conditions of alternation of day and night. A reduced light program where the plant receives just a few hours of light every 168 hours of the total week duration, were hypothetically thought to be varying before the start of this experiment and the variations in bean study groups were to be synchronized to the alternation of longer skotophile and shorter photophilic phase all week round. But in natural sense growth and differentiation are dependent on light so do physiological activities of plant need a dark period as speculated in the hypothesis? Yes, during this period its thought that a pool of Phytochrome red (PR) is replenished. This pool does not grow unlimited but is broken down again into far red (PFR), during long periods of darkness so that the skotophile phase returns periodically. Phytochrome is a homodimer, (or two identical protein molecules) each conjugated to a light-absorbing molecule. Plants make 5 phytochromes: Phy-A, Phy-B, as well as C, D, and E, and there is some redundancy in function of the different phytochromes but there also seems to be functions that are unique to one or another. The phytochromes also differ in their absorption spectrum; that is to say, which wavelengths (e.g., red vs. far-red) they absorb best. phytochromes exist in two interconvertible forms; PR because it absorbs red (R; 660 nm) light, and PFR because it absorbs far red (FR; 730 nm) light. These have relationships in that, absorption of red light by PR converts it into PFR, and absorption of far red light by PFR converts it into PR, and in the dark, PFR spontaneously converts back to PR. Thus in description long-day plants (such as Nicotiana sylvestris), are long day + adequate PFR hence growth is accelerated in short skotophile phase, while short-day plants (such as Kalanchoe blossfeldiana)are, short day + enough PFR so growth is limited due to short the skotophile phase.
Beans just like any other flowering plant use the pigment phytochrome to sense seasonal changes in day length, but beans behave are known to be neutral plants because they were found to flower irrespective of photoperiodicity, they rather use PAR-induced temperature conditions (vernalization) to control the different growth activities, such as flowering, transpiration, and the generally plant growth. The behavior of phytochrome described above provides the study model — called the hourglass model — of the mechanism of photoperiodism in plants, but this model fails to account for the fact that night-time exposure even two hours extra of darkness when all the PFR has already been converted to PR affects plant function. Light : Dark ratio responses were studied by varying bean plants’ groups to 1, 2, 3, 4, 5, 6, 7, and 8, (or A-to-H), for 0 –to- 7 days of the week illumination ration respectively, after which treatment four (4) samples in each group were harvested and various growth variable changes assessed and these included among others; the number of leaves, the leaf area of lower leaves, the wet weight, and the dry weight and elongation of the shoot and roots in bean plants for two (2) consecutive weeks.

3.0 Objectives

3.1 Major objective
Study photo-synthetically active radiation (PAR) effects in bean plants developed on basis of rationed of light : dark periods (photoperiods) of the two weeks’ growth period.

3.2 Specific objectives
 Grow model bean crops in eight different buckets that were subjected to different Light : Dark (L:D) ratio through the week.
 Weekly harvest of four plants from each bucket and then measuring the growth variables that were initially set to be number of leaves, shoot and root length, leaf area of the lowest leaves, wet weight of the plant, and the dry weight of the plant.
 Collect results in two (2) consecutive weeks, take average of each group treatment and then analyze them.

4.0 Justification

The research project findings were aimed in disclosing the influence of Photo-synthetically active radiation (PAR) responses of a well renowned photo-period neutral plant (Phaseolus vulgaris, Beans). These results will help to describe the cause and find a solution to early maturity, declining in productivity and food-quality yields, increased susceptibility to pests and infections, and poor adaptability of some breeds of beans in some areas, witnessed in tropical zone of the world today. These changes can be thought at as being determined by the entrained photo-period rhythms, which is due to a reputation global climatic change in temperature (global-warming). Global temperature increases are estimated at 0.6-0.7oC rise, this has caused a lockstep within the PAR cycle of day and night. The tropics have balanced exactly 12 hours for light and dark regimes of illumination, but with altered cycles of PAR-induced temperature, variations in plant modules of growth and general environmental impact on life is thought by many scientists to result into ‘environmental disease’, there fore much efforts like one sited in this work still need a hand resolve before the environmental problem worsen.

5.0 Methods and materials

The reliability of this study was mostly dependent on the basis of good, health, and homogeneous-ness of the bean planting material. The conditions of growth were standardized, such growth factors included; fertile loam soils, similar watering regimes, well screened sowing seeds, and equally aerated light and dark gardens. Neither fertilizer application, nor pesticides were required in any trial experiment. In the optimizations of the study, a few bean seeds were planted out in an open garden and the period they took to sprout was recorded. In the main study this period of sprouting was deducted from the experimental due life of the first week’s trial, meaning it was not accounted for as part of the light response in terms of duration of bean seedling growth. The main study plants were grown in well labeled buckets signifying the ration study conditions in weekly hours for Light : Dark = 0:168, 12:156, 24:144, 36:132, 48:120, 60:108, 72:96, and 84:84, named as conditions A, B, C, D, E, F, G, and H (or 1-to-8) respectively. In each study condition, the bean garden was arranged as shown in the diagram below.

A diagram showing the lay-out of the sample gardens in each of the eight labeled buckets (A-to-H)
Garden (bucket)
Plant

As shown in the diagram bean plants were grown out in two (2) rows thus each garden held to a minimum of eight plants, therefore a total population of 56 bean plants were assessed. Equal spacing was utilized to avoid any competitions for light and nutrients. For two weeks, the plants were treated to varying ratios of both light and dark, this was done by standing the gardens outside of the laboratory window for light regimes (during day), and for night regimes plants were transferred to a dark room for artificially manipulated darkness which were used to supplement normal nights for the dark regimes. A weekly harvest of the four plants in one row was made, washed of any soil on the roots, and the measured for the variables which were; number of leaves, shoot and root length, leaf area of the lowest leaves, wet weight of the plant, and the dry weight of each sample plant.

6.0 Results
Garden

Condition A
(1) B
(2) C
(3) D
(4) E
(5) F
(6) G
(7) H
(8)
Exposures
d=day, n= night 0d
14n 1d
13n 2d
12n 3d
11n 4d
10n 5d
9n 6d
8n 7d
7n
Weekly exposure rational (Hours) L=light, D=dark 0L
168D 12L
156D 24L
144D 36L
132D 48L
120D 60L
108D 72L
96D 84L
84D
Week 1
Number of leaves 2

(small open) 2

(small open) 2

(small open) 2

(big open) 2

(big open) 2

(large open) 2

(broad
open) 2

(broad
open)
Average length Roots 16.8 11.3 13.8 13.3 12.5 13.5 12.5 9.3
Shoot 35.5 26.5 22.0 24.5 24.3 20.3 22.8 19.5
Total 52.3 37.8 35.8 37.8 36.8 33.8 35.3 28.8
Lower leaf area (cm2) 7.7 14.1 24.1 31.6 30.3 38.1 47.5 50.2
Wet weight (g) 2.7 2.9 2.7 2.5 3.4 4.0 3.8 3.4
Dry weight (g) 0.15 0.20 0.22 0.28 0.30 0.45 0.40 0.35
Week 2
Number of leaves Wilted 2
(chlorosis)

2 + bud 5 5 + bud 5 + 3 young ones 8 + 2 buds 8 + 2 buds
Average length Roots 5.0 5.5 12.0 15.0 11.0 17.0 18.0 9.0
Shoot 39.0 24.5 21.0 26.5 28.5 33.0 25.0 23.0
Total 44.0 30.0 33.0 41.5 39.5 50.0 43.0 32.0
Lower leaf area (cm2) - 21.5 22.5 31.2 36.0 52.5 50.0 50.2
Wet weight (g) 2.3 2.6 3.3 4.2 3.6 4.3 4.0 3.8
Dry weight (g) 0.4 0.7 0.8 1.0 1.1 1.5 1.1 1.2

7.0 Discussion
7.1 Plant-elongation variable and PAR growth response in the examined bean plants.

Plants in groups 1 showed abnormal maximum elongation in the length because of elongation in the body cells, and also because these plants are positively photo-tactic they try to reach out in areas of maximal illumination. Such a characteristic is also shown by the forest twinning climbers and there implies climber plants actually act abnormally due to increased dark stimulation, but they have elongated roots which enable them collect water from below sub-surface level, as shown in figure 1.

Fig 1: Total plant (shoot + root) elongation within the two weeks

Other plant groups such as group 3, 4, and 5 show a positive correlation with the increase in illumination regimes signifying that plant elongation factor is directly proportional to duration of light reception and but plant length is affected negatively by increased period of dark stimulation there fore breeds of short stem beans should be planted in open gardens which must also be well watered due to short root length which can not collect water from very deep the soil. However there is a remarkable fall in plant length in groups 7 and 8, this can be explained as a direct effect of increased temperature on plant growth there fore this extreme of PAR stimulation lessens growth and thus will affect normal activities of the plants. In addition to increase in temperature, decay of roots, root excretion and consumption of roots by parasites and symbionts can have led to shortening of roots, and this directly translated in low net primary productivity (NPP).
7.2 Plant biomass-accumulation variable and PAR growth response by the bean plants.

Plant bio-mass accumulation (or NPP) was measured as the plant dry weight. And it was found to be positively proportional to the light illumination in the treatment condition. Therefore plants in groups 1-to-6 show a positive coloration with increase in light than with dark stimulation as shown in the figure 2.

Fig 2: Plant dry weight growth in the two weeks

But plant groups 7 and 8 show an abnormal negative response to increase in light treatment; this can due to increase in temperature conditions caused by the increase in PAR stimulation.

7.3 Plant lower-leaf area variable and PAR growth response by the bean plants.

Lower-leaf area growth is proportional to light stimulation, and there fore its affected by dark stimulation as shown in figure 3. Plants in lower illumination conditions 1, 2, and 3, the plants lost chlorophyll (or they developed chlorosis) and many leaves dropped off the plants. Low leaf area growth in 1-to-5 is an affecting factor in net primary productivity (NPP). Other factors that affect NPP are shading of leaves, and decay of some parts of the leaf.

Fig 3: Lower leaf area growth in the two weeks

8.0 Conclusion

Bean plants show maximum elongation and accumulation of biomass at L : D = 60:108 hours in a week, this condition was optimum for growth, but since they showed shorter root system, they there fore require a sub-optimal water supply. These conditions can help revive normal growth in plants especially those in a tropical countries such as Uganda. But these plants at the end of week 2 they had less development of root nodules than in their counterparts of groups 7 and 8. There fore such conditions if they are to be integrated national agriculture, efforts have to be taken to improve on their nitrogen fixing ability. Groups 6 and 7 showed maximum leaf area growth, there fore such plants can be grown for forage (or leaf harvest) so normal conditions of balanced L : D = 84:84 hours can be utilized. But since such conditions occur with minimized root length, it implies that adequate water supply should be availed since such plant will not fetch water from deep crust of earth.

9.0 Appendix

Fig 4: Root elongation growth within the two weeks

Fig 5: Shoot elongation growth within the two weeks

Shoot length growth influences total plant length than does the root length growth but still bean plants in conditions 6 and 7 show the normal patterns of elongation.

ENVIRONMENTAL DISCIPLINARITY
12th-December 2007

BACK GROUND

Environmental discipline scientific research works have formed basis for attempts to solve environmental problems, but despite some positive results, such as reduction of air and water point source pollution, major environmental issues (e.g. climate change, biodiversity loss, air pollution, eutrophication, chemicalisation) need more research and practical applications derived from research results. Typically, modern environmental problems have multiple and cascading effects. Study of such multidimensional environmental problems requires cooperation between different research fields and traditions. However, understanding the complex effects of various human actions on the environment is just the first step in the process to resolve them.
After a breakthrough of environmental social science (Policy making), nature has been understood as an integral and essential part of the society. This means that questions concerning environment can be also seen as problems of the multi-discipline sciences of the environment including: political decision-making, economical development, societal planning, social conflicts, environment economics and jurisdiction. Thus, to succeed in resolving an environmental problem after the identification and basic studies of the issue, environmental social research has to integrate the results with the society in a way that they serve as a basis for political decisions and steer further actions. Encouraging high-quality and multidisciplinary environmental research with possibilities for applications in the society

1.0 INTRODUCTION

The environmental discipline encompasses a number of fields and is harmony with other disciplines such as environmental geology, hydrogeology, mining and remediation, toxicology, risk assessment, microbiology and bioremediation, and environmental-metallurgical engineering. This discipline also is involved in corrosion studies and related failure analysis, and in health and safety-related issues. The environmental field is multi-disciplinary by nature and, for maximum effectiveness we shall divide environmental discipline as follows;
1. Environmental policy
2. Environmental sustainable development

Environmental policy is any (course of) action deliberately taken (or not taken) to manage human activities with a view to prevent, reduce or mitigate harmful effects on nature and natural resources, and ensuring that man-made changes to the environment do not have harmful effects on humans.

Environmental policy can be sub-grouped into the following environmental themes:
Air, biotechnology, chemicals, civil protection and environmental accidents, climate change, environmental economics, enlargement and neighboring countries, health, industry and technology, international issues, land use, nature and biodiversity, noise, soil, sustainable development, waste, and water. These can be generally categorized under a broader environmental discipline, or they can be studied each separately, in this reported we tried the first option.
Successful environmental policy depends on the ability of its makers to bring together scientific information, analytical thinking and an awareness of the legal, social and political realities of environmental regulation. Its objective is to develop in its students the capacity to identify environmental problems, assess their nature and scale and provide solutions. For example, de-gazetting forests for commercial plantation like sugar cane or palm oil trees is a problem due to our political leaders’ corrupt activities like undermining the forestry-environmental policy. There fore we need some legal intervention from policy makers and scholars of environmental science.
Practitioners of environmental policy need to master suitable analytical and quantitative skills which are sensitive to the social and political context of environmental regulation. The discipline usually includes; studying rural environment, urban environment, and public environmental policy, plus environmental planning in institutions of development.
The discipline studies the social values, institutions and processes through which society arrives at choices about environmental policy. A core question is how environmental ethics, environmental politics and alternative approaches to environmental economics can help inform environmental valuation and policy decisions.

Environmental sustainable development
The objectives of environmental sustainable development can be separated into three objectives – economic, social and ecological - these are different disciplines that relate to very wide field of environment disciplinarity, each with its own theoretical and methodological perspective: Almost all disciplines have something to contribute to the study of environment disciplinarity and there is need to seek comment on all of them.
 Economics (biotechnology, chemicals, industry and technology)
Is concerned with human beings interacting with each other as decision-makers, with the emphasis on the individual entity; nature is typically treated as a material resource/constraint.
 Anthropology or sociology (civil protection, neighboring countries, health, international issues, nature and biodiversity, and noise)
Sociology is thus concerned with human beings interacting with each other not only as decision-makers but also as meaning-makers, with the emphasis on the collective requirement. Nature is regarded both as a resource/constraint and as a locus of meaning.
 Ecology (Air, climate change, soil, waste, and water)
These are concerned with human beings as a species, interacting as biological beings, both with their own and other species and with the inorganic environment, the emphasis is on the whole as an eco-system.

2.0 DISCIPLINES INTERACTION (inter-disciplinarity)

Environmental economics and ecological economics.
The environmental economics is a well-established and reputed sub-discipline of economics and ecological economics is a hybrid rather than a sub-discipline, not well-established, and viewed with some suspicion by both economists and ecologists.

Environmental anthropology (or sociology) and socio-biology.

The environmental anthropology is a well-established sub-discipline of anthropology, while socio-biology is either a hybrid or a sub-discipline of biology. It (socio-biology) is viewed with suspicion by most anthropologists, and mixed views by biologists. One in particular, geography, has, with some justification, claimed that it alone brings together the key disciplines within a single sphere. Other hybrids may also be found within some of the cells of the matrix. It now increasingly appears to be a variant of psychology.

Comparing the three disciplines: economics has been particularly powerful (some say imperialistic). Thus the perspective of economics has been applied to different fields of environmental study - notably, here, the ecology and society have been the least powerful. As a result, the environment has been the object of suffering from politics and poverty, but the associated perspective (ecology) has been undermined.
The significance of economics that becomes apparent from this brief analysis for example of the large populations of historian Member of Parliament (environmental policy-makers) relates also to the power of different disciplines to influence policy.

4.0 ENVIRONMENTAL ECONOMICS

This includes many activities of man in his environment and some of these are;
1. Mining & Mineral Resources

The mining and mineral resources disciplines involve a number of activities ranging from developing or reviewing mineral exploration programs for potential financial investors through developing mining plans to environmental permitting. For example the oil mining project in the mid-west (Lake Albert) region of Uganda.
Other discipline activities may range from economic analysis of the ore reserves, the projected product-flow, mine dewatering, water-power supply development or protection, and mine environmental impact assessments and environmental geology.
Causes for litigation often focus on the highly subjective or potentially ambiguous aspects of the projects, such as ore-reserve assessments, cash-flow realizations, and cost estimates to meet environmental regulations. Disagreements in methods, interpretation of drilling data, chemical analyses, and geophysical surveys often result in litigation. Any of the above activities, analyses, evaluations, or assessments may have be conducted in a biased manner, by inadequate methods, or by personnel without appropriate training and experience and the associated professional geological certifications and/or state licenses.

2. Mineral exploration and environmental investigations

These two have many common characteristics, both require a familiarity with the geologic literature and both involve drilling, sampling, and analyzing for anomalous compounds present at or near the surface of the earth. Mineral exploration involves the search and evaluation of concentrations of economic metals and other elements found in naturally-occurring deposits at or near the surface of the earth (For example the Kilembe copper deposits). Value is created by mining a mineral or commodity for use by society in making a product of value to society. Successful mining projects consist of multi-disciplinary activities, such as in heap-leach, precious metal projects for example, and require a careful blend and balance of geological, chemical, geotechnical, engineering, financial, environmental and managerial expertise.
4.1 Impact of environmental economics to the environment

In the process of making a product, waste and by-products are produced which have historically been improperly handled and disposed off at locations that often threaten the health and well being of humans and the environment. For example the green house gases that cause ozone layer depletion and thus leads to global warming. There fore environmental investigations involving the search and evaluation of concentrations of anthropogenic waste or by-products such as metals, hydrocarbons, solvents, pesticides, herbicides, and other industrial compounds found in and around industrial centers, should report concentrations considered, in many cases, to be potentially dangerous to human health and the environment, i.e., to other fauna, flora and other natural resources and not to be corrupt.

4.2 Use economics in environmental policy
The main reason of using environmental economics is that in our society the environment has become a scarce resource. Since economics is about how to tackle scarce resources, it can often be useful when dealing with environmental problems. One way of using economics is to ensure that the costs and the benefits of environmental measures are well balanced. Although it is difficult to estimate costs and benefits, there is an increasing demand that this is done before new environmental policy is decided on Ugandan level. With the use of market-based instruments, environmental goals can sometimes be reached more efficiently than with traditional command and control regulations. For example, mining and mineral resources are directly linked to the environmental field, Thus through mining as the first stage of supplying society with its building blocks and the mineral resources as the last stage of cleaning up after society's needs have been met, society will learn to produce the products it needs in more environmentally-friendly ways by reducing waste and improving handling techniques.

5.0 ANTHROPOLOGY (or SOCIOLOGY)

Environmental sociology is essentially the science of identifying and measuring the amount of species interactions in the environment, natural or man-influenced. It also includes the study of the fate and effects of these interactions in the environment. It includes such tasks as defining the intended use of analytical data, preparing sampling plans to satisfy the intended use, selecting appropriate analytical methods, advising on the collection of samples in the field, interpreting laboratory analytical results, and assuring the validity and legal defensibility of analytical results. As indicated, any of the above activities can be involved in litigation, inappropriate or misrepresented data or methods are, in many instances, the basis of litigation involving activities conducted within the discipline involved, practiced in some cases by unqualified individuals not having appropriate training and experience and the associated professional sociological certifications and/or state licenses. For example suppose some one wants to harvest honey combs, will it be professional to kill all worker bees plus the mother bees? Or if some one wanted to make an industrial park will it be appropriate to site it in Queen Elizabeth Park to interrupt wild life conservation?

5.1 Impact of environmental anthropology (or sociology) to the environment

Over populated social groups for example of wild beasts in the Tsavo national game park in Kenya, as been reported to be stressing the environment through over grazing on plants, making open patches due to trampling.
Over population of people in slum areas of urban environments has led to ethic and moral degeneration. Contamination of water sources due to poor disposal of human sewage and other solid wastes.
Health-wise; today, there is evidence that factors such as particulate matter in the air, noise and ground-level ozone damage the health of thousands of people every year. Environmental pollutants, including pesticides, endocrine disruptors, dioxins and PCBs persist in the environment, accumulating over time and we do not know enough about their long-term effect on our health. A range of specific policy actions are being taken to address the issues, and many acute environment and health related problems have been solved. However, there are areas which require more investigation, in particular with respect to the health implication of chronic exposures, as reported by organisations such as the World Health Organization (WHO) and a number of Uganda national organisations (such as NEMA). The effect of the environment on health is a major concern of the Ugandan public.

5.2 Purpose of the environmental anthropology (or sociology)

This increased security to members living in groups, for example mother elephants are able to guard their calves from predation by lions, and protect their feeding territories with the help from alpha-males.
Togetherness of living species is linked to development of strong and distinct cultures.

6.0 ENVIRONMENTAL ECOLOGY

This also includes many activities of man in his environment including interactions to biotic and abiotic spheres, some of these are;
1. Climate change

Climate change is one of the greatest environmental, social and economic threats facing the planet. The warming of the climate system is unequivocal, as is now evident from observations of increases in global average air and ocean temperatures, widespread melting of snow and ice, and rising global mean sea level. The Earth's average surface temperature has risen by 0.76° C since 1850. Most of the warming that has occurred over the last 50 years is very likely to have been caused by human activities. In its Fourth Assessment Report (AR4), published on 2 February 2007, the Intergovernmental Panel on Climate Change (IPCC) projects that, without further action to reduce greenhouse gas emissions, the global average surface temperature is likely to rise by a further 1.8-4.0°C this century.
Impacts of climate change that have already been observed

Glaciers are melting in many places across the world. Regional climate change is already affecting many natural systems. For instance, it is increasingly being observed that Rwenzori (mountains of the moon) ice are melting and frozen ground is thawing, hydrological and biological systems are changing and in some cases being disrupted, migrations are starting earlier, and species' geographic ranges are shifting towards the poles.

2. Environmental Geology

The environmental geology discipline, often a part of engineering geology, it involves studies, investigations, and reviews that often include:
a) Environmental site assessments (ESAs) for real-estate transfers, mergers, or of the impact of long-term production of oil and gas or injection well operations.
b) Environmental hazard investigations of growth faults in urban areas, water-well failures, hillside slumping, landslides, subsidence, etc.
c) Soil contamination investigations resulting from leaks from sewage and oil at service stations, and other industrial sources of leaks, spills, and accidents.
d) Remediation studies (including bioremediation) of ground-water contamination and,
e) Other subsurface investigations requiring sampling, interpretation and assessment of hydro-geologic data and hydro-chemical and soil analyses.

3. Hydrogeology

The hydrogeology discipline involves field investigations and reviews of the following typical activities:
a) Analysis and assessment of subsurface conditions by geotechnical drilling, geophysical surveys or other subsurface sampling methods,
b) Evaluation and determination of the direction and rate of ground-water flow,
c) Modeling of contaminant fate and transport in shallow, subsurface sediments and ground-water systems,
d) Assessment of contaminant impact on ground- water supplies,
e) Evaluation and assessment of human exposure and risk assessment relating to contaminated soil and ground water for example the out break of Cholera infections in Uganda’s populations today
f) Development and assessment of ground-water remediation system design, operation and effectiveness,
g) Forensic investigations, such as in evaluating likely sources causing illness or death to humans and animals for example Ebola, and
h) A number of other aspects in the field of hydrogeology.

 Water:
Water is life! It is a precondition for human, animal and plant life as well as an indispensable resource for the economy. Water also plays a fundamental role in the climate regulation cycle. Protection of water resources, of fresh and salt water ecosystems and of the water we drink and bathe in is therefore one of the cornerstones of environmental protection in Africa. The stakes are high and the issues transcend national boundaries and concerted action at the level of the UN (WHO) is necessary to ensure an effective protection.
Impact of water that has already been observed

Not always that water is life, especially in Uganda which as just recovered from a series of flooded Teso region and more recently Zana on Entebbe road sparing to talk about Bwaise a tradition flooded plain and the likely places. It is important for Uganda to prevent the advent of floods and to protect areas which are likely to be affected by such events. It is also vital to prepare Ugandan citizens to cope with the potential occurrence of floods. This will be important if new legislation obligating the nation to assess flood risks, to inform citizens in potentially affected areas and to involve them in the planning process in lobbied. There fore although floods can play a natural role in revitalizing the functioning of ecosystems, they can also cause widespread environmental damage. Pollution transported via flood waters can spread to areas where drinking water is extracted and extreme floods can wreak havoc with delicate ecosystems.

7.0 CONCLUSION
By assessing innovation induced discipline sciences on environment policy, Eco-innovation was found to be a key element in the quest for sustainable development. Environmental technologies (like use of cleaner-production technology) have the potential to sidestep the classic dilemma between economic growth and environmental improvement. In the past, numerous new technologies have been introduced in the Uganda that have contributed to pollution prevention, environmental clean-up, and the conservation of energy and resources (for example Bio-filtration by the Nakivubo effluent channel). But this and many others have not led to reductions in costs and/or reinforced the competitive strength of Uganda industry, as ‘clean’ technologies that were developed in Europe and are successful export products on the world market.
Uganda government also needs to put in structure environmental policy instruments. These are tools used by governments to implement their environmental policies; they may use a number of different types of instruments. For example, economic incentives and market-based instruments such as taxes and tax exemptions, tradable permits, and fees can be very effective to encourage compliance with environmental policy.

UGANDA VIRUS RESEARCH INSTITUTE (ENTEBBE)
AN INDUSTRIAL TRAINING REPORT IN THE
IMMUNOLOGY DEPARTMENT /
ENTEBBE MOTHER AND BABY STUDY (EMABS)

KAGGWA JOHN BASABOSE
A STUDENT OF NATURAL SCIENCES (BIOCHEMISTRY AND BOTANY) MAKERERE UNIVERSITY, KAMPALA
(05/U/3229)

AUGUST 2007

ACKNOWLEDGEMENT
I extend my regard thanks to my Programme Supervisors;
§ Ms. Namujju Prossicovia
§ Mr. Dennison Kizito
§ Mr. Patrice Mawa
§ Mr. Jimmy Lutangira
§ Mr. Dr Robert Tweyongyere
And to all ENTEBBE MOTHER AND BABY STUDY staff.
To my fellow colleagues;
§ Nabukko Sarah
§ Bukama Collins
§ Nabbumba Catherine
All of who are my Course mates at Makerere University Kampala in the Biochemistry Department.
§ Oren Gersten (Student of Wesleyan University, USA)
§ Kibuuka Hamza (Student of Kyambogo University Kampala)
§ Wasswa Solomon (Student of Uganda Christian University Mukono)
§ Heyle Evans (Master Student London School of Tropical Medicine)
§ Lauren Hall (Master Student LSTM)
And to Uganda Virus Research Institute, Medical Research Council Uganda (Wellcome Trust), Entebbe Mother and Baby Project Managements at large who have supported my study and special Thanks go out to Dr. Allison Elliott (E-MABS-Project Principal Investigator)
.

DECLARATION

I declare that this report has been compiled by the sole author (Kaggwa John Basabose), it has never been previously published by any person else, and anywhere. The report has been compiled with outmost good faith and I proclaim that all this information is true and complete unless otherwise cited all content is original and my own.
Student’s approval
Name; Kaggwa John Basabose
Signature …………………………………………………………….
Date …………………………………………………………….

Supervisors’ approval
Name; Dennison Kizito (Training Coordinator)
Signature; …………………………………………………………....
Date; …………………………………………………………….

Name; Patrice Mawa (Laboratory Manager)
Signature; ………………………………………………..……………
Date; …………………………………………………………….

DEDICATION

I dedicate this work to all those who have been my supervisors (tutors and mentors) during the course of my Industrial training, to my fellow colleagues and all the people who will find this report useful in their studying, and to all those who have made it possible for me to complete this study especially my mother Mrs. Nalongo Maxencia Nalubega who provided almost all the financial support and courage throughout the course of study.

TABLE OF CONTENTS

Acknowledgement…………………………………..……………………………………. 2
Table of contents…………………………………………………………………………. 6
Lists of tables…………………………………….………………………………………
List of figures……………………………………….……………………………………. 7
List of acronyms and abbreviations……………….……………………………………... 7
Objectives (justification and objectives of the training)…………. …………………….. 8
Summary / Abstract………………………………….………………..………….. ………9

INTRODUCTION
Sub theme;
The Role of Clinical Laboratories and Research work, and their Integration into Our
Nation’s Health Service.

I. MANAGEMENT
II. LAB INSTRUMENTATION AND DESIGNS SYSTEMS
III. LAB SAFETY
IV. SAMPLE COLLECTION AND TRANSPORTATION
V. SAMPLE TREATMENT AND DISPATION OF RESULTS
VI. STORAGE SYSTEMS, ARCHIVING AND LAB INFORMATICS
VII. QUALITY CONTROL

Chapter One
MANAGEMENT
1.1 Introduction / back ground of UVRI institute, Organization structures (Departments), and Mandate (Work) of UVRI institute ………………………......………....………10

1.2 Immunology Section (Department)………..………………………………....……....14

1.3 Back Ground and purpose of EMABS/MRC/WellcomeTrust Project…...………......14
-Organizational structure (Organogram of E-MABS Research Project)
-Mandate of the project
-Dissemination and marketing of services
1.4 Lab management…………………………………………………………….………..23

Chapter Two
LAB INSTRUMENTATION AND DESIGNS SYSTEMS
2.1 Lab instrumentation systems use and function ……………..……………….….……26
2.2 Lab design………………………………...…..............................................................30
2.3 Safety and Laboratory Designs……..…………....……………………………….…..30

Chapter Three
ETHICS AND SAFETY IN THE LABORATORY
3.1 Laboratory ethics
Ethical Duties of the laboratory personnel...................................................................32
3.2 Safety in the laboratory
Safety Equipment.........................................................................................................32
3.2.1 Packaging Biological Waste........................................................................................33
3.2.2 Safety Labelling...........................................................................................................33
3.2.3 Transport of biohazard material..................................................…...............................34
.
Chapter Four
COLLECTION, DOCUMENTATION, TRANSPORTATION AND SHIPMENT OF BIOLOGICAL SAMPLES, PROCESSING (OR TESTING), DISPATION OF RESULTS
4.1 Sample collection at the field safety and Research laboratory (E-MABS project Specimen / Result flow chart).....................................................................................35
4.2 Collection of samples...................................................................................................36
4.3 Documentation.............................................................................................................36
4.4 Transportation and Shipment of Biological Samples.............................................…..37
4.5 Sample Processing (or Testing)....................................................................................38
4.6 Dissemination and marketing of services
4.6.1 For E-MABS (field and Immunology research laboratories)........................................39
4.6.2 For VCT / PMTCT (Entebbe hospital Grade B Antenatal clinic)
(Flow chart for CD4 tests done as part of VCT at Entebbe hospitals).........................40

Chapter Five
IMMUNOLOGICAL ANALYTICAL LABORATORY TECHNIQUES
5.1 Rapid Immunological Tests..........................................................................................41
5.2 ELISA................................................................................…........................................43
5.3 ELISPOT.......................................................................................................................44
5.4 Whole Blood Assay (WBA).........................................................................................46
5.5 CELL SEPARATION or PBMC processing...............................................................47
5.6 Whole Blood Killing Assay.........................................................................................48
Chapter Six
STORAGE SYSTEMS, ARCHIVING AND LAB INFORMATICS
6.1 Sample storage systems, Archiving ..................................................................……50
6.2 Protocol for freezing cells (PBMCs)..........................................................................52

Chapter Seven
Quality Assurance and Quality Control in Clinical Research Laboratories
7.1 Quality Assurance………………………………………………...………………...55
7.2 Quality control………………………………………………………..…………….56
7.2.1 Quality control in laboratory designing.......………….......................................…...56
7.2.2 Quality control in Specimen Transport...........................................................….......57
7.2.3 Quality control of Tissue Cultures and Media...............................................…........58
7.2.4 Quality control of Reagents and Kits..............................................................….......59
7.2.5 Quality control of Instruments........................................................................…............59
7.2.6 Summary of Quality Control Measures...............................….......................................60
7.3 Basic Elements of QA Policies and Procedures…………………..……………………62

Chapter Eight
Conclusion and Recommendations
8.1 Conclusion and recommendations…………………………………………..
8.2 Results and Discussion ………………………………………………………

REFERENCES………………………………………………………………… ….

APPENDICES
Microbiological Safety Cabinets ………………………………………

LISTS OF FIGURES
Figure 1 Organization Structures (Departments) At UVRI
Organogram of UVRI……………………………………………….…….………..13
Figure 2 Organizational structure of E-MABS
Organogram of E-MABS…………………..………………………………..….....18
Figure 3 E-MABS work-flow organisation structure…………………………………...…..19
Figure 4 Life cycle of malaria in man…………………………………………….....…..….22
Figure 5 Standard hirachechial laboratory management set up………………..……...……25
Figure 6 Laboratory Instrumentation………………………….……………………………28
Figure 7 Safety Biohazard signs…………….…………………..……………….…………38
Figure 8 E-MABS project Specimen / Result flow chart………...…………………………39
Figure 9 Profile of the assays performed on samples in the Immunology Laboratory…..…42
Figure 10 Flow chart for Blood and Body fluid (urine) sample tests done as part of VCT / PMTCT at Entebbe hospitals……………………….………..…………………..46
Figure 11 ELISA techniques Assay Principle……………………………..…………………50
Figure 12 ELISPOT Assay Principle………………………..………..……….……………..51
Figure 13 Sample processing for Whole Blood Assays………………...……………………52
Figure 14 WBA Plate Step (with SWA and SEA included)…………….………. ………….53
Figure 15 PBMC processing Procedure………………………………………….…………..54 Figure16 Classes of Biological Safety Cabinets …………………………...…..……………68

LIST OF ACRONYMS AND ABBREVIATIONS

UVRI Uganda Virus Research Institute

E-MABS Entebbe Mother and Baby Study

AIDS Acquired Immune Defiency Syndrome

HIV Human Immunodefiency Virus

EDTA Ethylenediamine Tetra Acetic Acid

WBA Whole Blood Assay

ELISPOT Enzyme Liked Sorbent Spot Assay

LAB Laboratory

PBS Phosphate Buffered Saline

HEPES N-2-Hydroxyethyl Piperazine-N¹-2-Ethane Sulfonic Acid.

PHA Phyto Haemagglutinin Antigen

PBMC Peripheral Blood Mononuclear Cells

RPR

VCT Voluntary Counselling and Testing

PMTCT Prevention Of Mother To Child Transmittion

DMSO (freezing medium)

CLS Clinical Laboratory Services

OBJECTIVES OF THE TRAINING (EXPECTATIONS BEFORE THE STUDY)
1. Receive training and motivation in the role of laboratory and its integration into our Nation’s health service especially ELISPOT, WBA assays, Archiving, Storage systems, Quality control, Testing for HIV/AIDS and Examination of Malaria parasite infection.
2. Learn a professional code of conduct in all atmospheres of life including laboratory work, social life education and employment for my future life.
3. Meet or make and coordinate with new personal relationships with other people outside my current education status.
4. Experience employment satisfaction and have opportunities for Continuing Education and Career development through improving on my curriculum vitae (C.V) and experience all of which are main requirements for the current employment market in our Nation.
5. Utilize my Recess term or intenership course properly and satisfactorily more so by joining my hands in slowing the pace of Africa’s deadly AIDS epidemic in my community.

SUMMARY
My Eight weeks Industrial training at UVRI / E-MABS (Wellcome Trust) began with the orientation week 25th to 29th June 2007.
Ø My orientation was;
25th –Welcoming / introductory remarks, laboratory and general safety code at Rabbit House (Immunology Section /UVRI).
26th – Introduction to Hospital Research Laboratory (Antenatal Clinic, Entebbe Hospital Grade B)
27th –Introduction to immunology research laboratory (Rabbit house)
28th –Introduction to Clinical Research Laboratory (E-MABS Private Clinic Entebbe hospital Grade A)
29th –Introduction to Field section Research work (Field Trip in E-MABS study community)
Ø For the rest of the Seven Weeks at UVRI, I was introduced to various Pre-Analytical, Analytical, and post-Analytical Techniques in Research Work And these comprise of but not limited to Sample collection, Sample Transportation, Sample Reception in the Lab, Sample Documentation and Processing, Immunological-analytical testing of samples Sample storage and Archive systems, Laboratory Safety measures, Management of a Research, Build Relationships (Capacity Building) and the last week analysing work done at ANC (PMTCT / VCT) Hospital Research section of E-MABS.

E-MABS is a Current research project based on an on-going cohort study of worm infection in mothers and their infants on lake Victoria shores Entebbe Sub-community.
This study comprises two randomised, double blind placebo-controlled trials: of anthelminthics in pregnancy, (all mothers received anthelminthics after delivery) and of regular three-monthly deworming in young children (all children receive anthelminthics if indicated by results of annual stool samples).
During the course of my study with E-MABS project, i choose to research on the topic Management and Quality Control in Research work (At UVRI, E-MABS project). Of which I presented a ten minutes Power point show on 16th August 2007 to the audience at E-MABS Private Clinic (15:00 Hours).

CHAPTER ONE
INTRODUCTION

1.1 Background of Uganda Virus Research Institute.
The Uganda Virus Research Institute (UVRI), located in Entebbe, Uganda, was established in 1936 as the Yellow Fever Research Institute by the Rockefeller Foundation. In 1950, after gaining regional recognition it was renamed the East African Virus Research institute (EAVRI). After the collapse of the East African Community in 1977, it became a Uganda Government Public health research institution and was renamed the Uganda Virus Research Institute. The Government of Uganda now administers and funds the institute through the Uganda National Health Research Organization (UNHRO) which is an umbrella research body with in the Uganda Ministry of Health.
1.2 Role of Laboratories in Research and Health services
Laboratories are divided into three (3) main categories these are;
1. Chemistry laboratories
2. Physical sciences laboratories and
3. Biosciences or Life science laboratories.
Biosciences (or Life science or Biomedical) Laboratories are divided into;
A) Hospital Laboratories; these are attached to a hospital and perform tests on patients.
B) Private (Or Community) Laboratories; these receive sample from General Practitioners, Insurance companies and other Health clinics for analysis.
C) For extremely specialized tests, samples may go to Environmental science or RESEARCH laboratories for example Uganda Virus Research Institute (UVRI).

1.3 Organization Structures (Departments) At UVRI
Figure 1 Organogram of UVRI

There several Departments at the UVRI and these include;
· Departments of Immunology
· Departments of General Virology
· Departments of Arbovirology
· Departments of Epidemiology
· Departments of Entomology
· Departments of Zoology
· Departments of Medical Research Council Programme on AIDS (MRPA)
These are sub-stationed under respective collaborating programmes, which meet some of projects’ research fundings. Several programmes at the institute include;
Collaborating Programmes.
Ø MRC/UVRI- RESEARCH UNIT ON AIDS (MRC=Medical Research Council)
Ø MRC/Wellcome trust Projects
Ø IAVI (international AIDS Vaccine Initiative)
Ø RHSP (Rakai Health Science Programme)
Ø CDC (Centres for Disease Control and Prevention))
Ø EPI/WHO (Expanded Programme)
Ø Entebbe Hospitals (ministry of health)

Field Stations
Arua (UVRI), Kalisizo (Rakai), Kyamulibwa (MRPA), Masaka (MRPA), Zika (UVRI) and Nyapea Hospital Plaque Lab (UVRI).
1.3.1 Departments of Laboratories and Clinical Laboratory Services at UVRI.
Several laboratories are divided into a number of disciplines and these include;
I. Microbiology
This receives Swabs, Faeces, Urine, Blood, Sputum, Medical Equipment as well as possible infected tissue. They culture this (samples) for any Pathogenic microbes.
II. Haematology
This receives whole blood (Heparin or Green top tubes) and Citrated plasma; they do full blood counts, blood films and Coagulation investigations.
III. Biochemistry
This usually receives serum (Yellow top tubes); they test the serum for different components including DNA.
IV. Immunology
This test for antibodies in serum and also do the whole blood assays.
V. Serology
This receives samples to look for evidence of diseases such as hepatitis or HIV (through CD4-counts).
VI. Histology
This process Solid tissue samples taken from the body to make slides and examine cellular detail.
VII. Cytology
This examines smears of cells (such as of the cervix) for evidence of cancer and other conditions.
VIII. Cytogenetics
This section is involved in using blood and other cells to get Karyotype. This is useful in prenatal diagnosis (for example of Down’s syndrome), Cancers as well as developing vaccines.
IX. Virology and DNA Analysis
These are involved in developing primers for DNA synthesis and recombinant DNA manufacture a process useful for synthesing vaccines.
Others include Entomology department, this provides services such as an Insectary for raring possible Vectors such as mosquitoes, and an Epidemiology department.
Most of the sections and Field stations at UVRI however have at least one or more of the Departments in one Laboratory facility but not all at once. There fore the Institute as a joint support programme of Clinical Laboratory Services, which provides most of Departmental roles, named above on request from the respective Laboratories concerned.
In addition Clinical lab services provide;
a) A Biosafety level 3 (P-3) lab
In this the work of isolating Mycobacterium tuberculosis varities a T.B causative microbe is done.
b) A Media section
This prepares tissue and cell culture media for example Chocolate agar, Blood agar, Candida chromogenic agar and many others most of which are essentially needed by the microbiological and virological culturing work.
c) Parasitological Department
This processes samples for possible haematological parasites such as protozoa that cause Malaria, schistosomiasis, Filariasis, Ascariasis and Hookworm infections.
There fore different Sections of laboratories and shared services at UVRI define clearly the definition Biosciences laboratories.

1.4 Mandate (Work) of UVRI institute
The mission of the institute is to carry out Scientific Research concerning Communicable diseases, especially viral diseases of public health importance and to advise the government on strategies for control and prevention.
1.4.1 Medical Research Council (MRC), Uganda research unit on AIDS.
It is based at UVRI, was established following a request in 1988 from Uganda government to British government for assistance regarding the research and control of HIV infection and AIDS. MRC is committed to supporting sustainable, relevant research and research training in Africa.
In Uganda MRC program works with the Ministry of Health, TASO and with the Uganda Virus Research Institution.

1.5 Immunology Department (Section)
What is immunology?
Immunology is a Medical and Biological science study that deals with the immune system and the Cell-mediated and Humoral (Humoral = relating to or being the part of immunity or the immune response that involves antibodies secreted by T-helper cells that are circulating in bodily fluids) aspects of immunity and immune responses.
The Immunology Department at UVRI is under an Independent Laboratory facility (Rabbit House). The Laboratory is a Biosafety Level 2 laboratory and is shared among two projects the IAEA (International Atomic Energy Agency) HIV / AIDS vaccine development and Entebbe Mother and Baby study. (Definition of Biosafety Level 2, follow the text Chapter two)

1.6 Entebbe Mother and Baby study.
Back Ground and purpose of EMABS / MRC-WellcomeTrust Project
What is E-MABS and how is the Study Conducted?
E-MABS is a Current research project based on an on-going cohort study of worm infection in mothers and their infants on lake Victoria shores Entebbe Sub-community.
This study comprises two randomised, double blind placebo-controlled trials: of anthelminthics in pregnancy, (all mothers receive anthelminthics after delivery) and of regular three-monthly deworming in young children (all children receive anthelminthics if indicated by results of annual stool samples).
The principal outcomes are responses to immunisation and illness events in infants, the study also provides many exciting opportunities to explore other controversial areas, including the effects of helminths on allergic disease and on growth and development.

Two Randomised, Double-Blind Placebo-Controlled Trials
What do the words mean?
A “randomised” study is one in which usually two possible drugs are given to the participants – one is the active drug which is believed to hold some promise as an effective treatment, the other is a “placebo” – a drug which has no expected treatment value and which looks, tastes, feels as close as possible to identical to the active drug.
The randomisation is a code (usually generated by computer), which allocates each individual by chance to receive either the active drug or the placebo. This aspect of the trial designed tends to make up for any variation in the nature of the illness affecting the patients enrolled (such as differences in severity or duration of symptoms), and therefore allows successful statistical evaluation of any difference response rate between the two groups.
The “double-blind” is the procedure in the study design which makes every attempt to ensure that neither the patient nor the researchers are aware which drug (active or placebo) each subject is receiving.
The “blind” is very important in this type of research as it ensures that neither the patients nor the researcher can have any direct effect on the outcome of the treatment due to factors such as the effect of believing that one is receiving the active drug which in itself may produce some benefit.
Similarly, the researchers may inadvertently or otherwise affect the outcome it he is aware of which treatment a patient is receiving.

1.7 Organisation of E-MABS Research Project
Figure 2 Organogram of E-MABS

Figure 3
1.7.1 E-MABS work-flow organisation structure

PARTICIPATANTSEnrolled pregnant mothers and their borne infants provide specimen (In terms of Blood and stool samples)

FIELD PRACTIONERSCollect specimen from the rural set participants.
RESULTS DISPATION Immediate and long term results of interest to participants health

FIELD LABS AND CLINICSCollect specimen and treat sickness cases in the participants
RESULTS

RESEARCH LABS (IMMUNOLOGY and CLS)Process Research interest samples, carry out assays on them and store them for future possible purpose under refrigeration in +4, –20, -80oC and –196oC respective of the sample storage life and proposed assaying time.

MRC STATISTICSEntry and Analysis of research results.
RESEARCH CORDINATORInterpret the Results of the successful statistical evaluation of any difference response rate between the two groups of the “randomised, double blind, placebo-controlled trials” And publishes the results to the community.

1.8 Mandate of the E-MABS project
Hypotheses of the E-MABS study;
Amended in response to preliminary findings, are that ‘Maternal and Childhood helminthes infections reduce the effectiveness of childhood immunisations and increase susceptibility to viral and bacterial infectious diseases, while reducing the incidence of diseases mediated by poorly-regulated inflammatory responses; treatment of maternal and childhood helminthes infection improves the effectiveness of childhood immunisations and modulates disease incidence in childhood, with both beneficial and detrimental effects’.
Specific aims:
· To continue to examine the impact of helminthes infections on the response to immunisation and on infection and disease incidence in early childhood through follow up of the established cohort of 2500 mothers and their children.
· To examine the impact of the trial interventions on additional outcomes of importance for public health, through the opportunity provided by the cohort.
· To inform Public health policy on de-worming in pregnancy and early childhood ages.
1.8.1 Why did E-MABS Research aim at worm infections?
Human worm parasitic infection and their prevalence
There are over 100 types of parasite worms living in human bodies; some are microscopic in size while others can be seen quite easily.
These common organisms are found everywhere in our environment, in the air we breathe, in the water we drink, in the food we eat and through transmitting agents (like mosquitoes) and through sexual conduct.
In Uganda most of the worm parasitic prevalence is common along the shores of Lake Victoria that’s why E-MABS cohort is carried out in this section of community.
Symptoms of parasitic infections
People with (intestinal) parasite worm infections are usually under-nourished and weak, infected with viral, fungal, bacteria and have various types of chemical, food and metal poisoning (allergies) this is due to parasitic secretion of toxins (Auto intoxication) and stealing on vital nutrients from our bodies.
There are Different varities of Worm parasites.
Worm Parasites irritate or exaggerate health problems but the body can as well develop immunity due to early primary exposure, which helps to build immunity against secondary infections.
There are 3200 varieties of parasites in the four main categories: -
Protozoa, Trematoda, Cestoda, and Nematoda.
1. Nematoda
Common forms are Round worms (Ascaris lumbricoides), Hook worms, Whip worms, Pin worms, Heart worms, Strongyloides, Stercoralis, Ancylostoma, Caninum, Toxocara worm and Trichinosis.
Most frequent symptoms
Upper abdominal discomfort, asthma, eye pain, insomnia and rashes due to Auto-intoxication. Large numbers can cause blockages in the intestinal tract, haemorrhage when penetrating the intestinal wall, appendicitis, peritonitis, loss of appetite, and insufficient absorption of digested foods.
In Hook worm infection, symptoms include iron deficiency, abdominal pain, craving to eat soil, protein deficiency, dry skin and hair, skin irritations, edema, distended abdomen, stunted growth, delayed puberty, mental dullness, cardiac failure and death.
In Pinworm infection, symptoms include itching and irritation of the anus or vagina and irritability or nervousness since female adult worms crawl out of the anus to lay eggs (about 15000 per day) in our living environment.
In Whipworm infection, worms cause bloody stools, pain in the lower abdomen, weight loss, rectal prolapse, nausea and anaemia. They also cause haemorrhage when they penetrate the intestinal wall, which is usually followed by bacterial infection.
2. Protozoa
These are single cell parasites; - Amoebae, Protozoa, Neosporin, Toxoplasmosis, Cryptosporidium, Giardia, Sarcocystis and Trichomonas vaginalis.
Amoebae infection; amoebiasis is the most common infection caused by Amoebae (An irregular shaped microorganism) from the species Entamoeba histolytica that causes ulcers or abscesses where they can enter the blood stream.
Protozoa infection; Malaria is the most prevalent and debilitating disease among the protozoan types of infection, it is caused by Plasmodium.
In Uganda malaria parasites are protozoan belonging to class sporozoa, sub class coccidian, family plasmodiidae and these are: -
Family Plasmodium
Sub genus Laverania Sub genus Plasmodium
P. falciparum (This causes falciparum malaria the commonest in developing countries) P. vivaxP. ovaleP. malariae

Female Anopheles mosquitoes transmit malaria parasites, while other intestinal coccidia are transmitted by the ingestion of oocysts.
1.8.2 Life cycle of malaria.
Figure 4 Life cycle of malaria in man.

Understanding the malaria cycle:
Malaria is a potentially deadly disease and the cycle of infection can take place very quickly. The malaria life cycle is as follows:
1. Infected mosquito bites human
2. Parasite rapidly goes to the liver within 30 minutes
3. The parasite starts reproducing rapidly in the liver, some parasites (from the ovale and vivax species of malaria) lie dormant in the liver, to reactivate and cause diseases often long after the initial infection.
4. This gets into the blood stream, attaches and enters red blood cells. Further reproduction occurs.
5. Infected red blood cells burst, infecting other blood cells
6. This repetitive cycle causes fever and depletes the body of oxygen, caring red blood cells. Additionally, infected red blood cells clog up the circulation in vital organs such as the brain and kidney.
As infection progresses, sexual forms of the parasite (gametocycles) are released into the blood stream. When a mosquito bites, it takes up these gametocycles and the cycle of infection is perpetual placing others at risk.
3. Trematodes (FLUKES)
These include Flat worms, Blood, Liver, Lung, Kidney, and Intestinal flukes. Human infections with flukes (Schistosomes) are known as schistomiasis. They cause severe disease of the gastro intestinal tract, bladder, and liver they as well destroy blood cells.
These worm infections are the primary priority of the E-MABS project in its follow up of the Participants.
4. Spirochetes
These are very tiny organisms that are spiral-shaped and multiply in the blood and lymphatic system. Spirochetes (largest), Saprospira, Cristispira, Treponema (smallest), and many more. The primary hosts or carriers are usually lice, ticks, fleas, mites, and flying insects, which then transmit infection to humans through bite.
Spirochetes are responsible for relapsing fever, infectious jaundice, Lymes disease, sores, ulcers, and Vincent angina and Wyles disease.
1.9 Dissemination and marketing of services in E-MABS
Why is it a Randomised, Double-Blind Placebo-Controlled Trials?
This type if trial design is the only accepted way of establishing a new treatment into Medical practice. It is highly demanding for both Participants and Researchers, and carries with it inevitable difficulties such as the Stress of the Uncertainty of which treatment was received by a given Participant.
Every Participant in the study is monitored in an identical fashion (in the study trials, with report forms of symptoms and ability to participate in daily activities as well as Immunological testing).
At the completion of the trial, that is when every Participant has completed the treatment course and completed the follow-up, a double blind placebo code will be broken which details which Participant received the active and which Participant received the placebo treatment. An analysis will then be made of the data, which has already been collected for each Participant, as to whether a larger number of Participants who received the active drug than the Placebo showed a response to the treatment.
Participants who enrolled at the beginning of a large trial (Pioneers) may have to wait a long period until all the following Participants have completed the trial and the code is broken to find out which treatment they were given.
It is a usual practise to offer the active drug to all Placebo Recipients after the trial if the statistical analysis of the data shows the drug is effective
Testing for malaria parasites at E-MABS Private Clinic (Entebbe Grade A Hospital)
Laboratory confirmation of infection of Participants with malaria parasites is by finding the parasites in their stained blood films/slides observed under a light microscope. However in malaria surveys and control work in the cohort, immudiagnosis is used.
For other different E-MABS project results reached In-House Research work (For example; A Research for Developing an Immunological based method for detecting Tuberculosis infection in Latent stages tried by Mr. Lutangira Jim), Results are discussed through;
§ Presentations
§ Seminars
§ Conferences
§ Tutorials

1.10 Lab Management
Figure 4 Standard hirachechial laboratory management set up

1.10.1 Role and purpose of each office in laboratory work set up
A) The Laboratory Director
1. Plans, organizes, coordinates, directs and evaluates the work of staff through supervision of the performance of assigned personnel, selecting and training employees recommending appointments, transfers, reassignment, termination and displinary actions.
2. Analyses and prepares program goals in accordance with department’s needs, reviews and approves laboratory testing reports, researches, formulates and applies testing procedures to meet current public health needs.
3. Prepares scientific, technical, administrative reports and correspondence to address appropriate application of laboratory data to public health communicable disease prevention activities.
4. Prepares and analyses the financial and statistical data.

B) The Laboratory Manager
1. Establishes and maintains cooperative relationships within the lab or amongst labs, community groups, the general public, health officials and medical staff.
2. Interprets, apply and explain laboratory rules and regulations governing the operation of a healthy laboratory.
3. Recommends the techniques, equipment, and terminology used in the laboratory diagnosis of disease and abnormal conditions in the examination of body fluids and tissues.
4. Another responsibility of the lab manager is planning the laboratory physical facilities that are commensurate with the function of the laboratory that is to say to set up appropriate laboratory designs which is the primary tool in response to laboratory accidents (safety)
C) Operation Manager
Operation managers in the laboratory play a role of assigning each of the laboratory users a role at a specified period of time. That is to say he/she draws out the timetable of using and work in the laboratory.
D) Medical or Clinical Laboratory Technologists and Technicians
They examine blood and other body fluids and also perform complex chemical, biological, haematological, Immunological and bacteriological tests.
They do these through;
· Making cultures of body fluids and tissue samples, to determine the presence of bacteria, fungi, parasites, or other microorganisms.
· Analysing samples for chemical content or a chemical reaction and determine concentrations of compounds such as Antibodies, glucose and cholesterol levels.
· Evaluations of test results, develop and modify procedures, establish and monitor programs, to ensure the accuracy of tests.
E-MABS immunology research laboratory comprises of;
Immunology Technologist
They examine elements of Human immune system and its response to foreign bodies.
For example in some assays like whole blood assay the mimic the blood sample to body conditions to study the T-Helper cells Responses to introduced Antigens {for example No-Antigen (NA-Negative Control), CFP-Antigen, Antigen 85(Ag85), Tetanus Toxiod Antigen (TT), Schito Worm Antigen (SWA), Schito Egg Antigen (SEA), and PHA (Positive Control) Antigen.
Clinical laboratory technicians perform less complex tests and laboratory procedures than technologists perform.
Laboratory technicians
Prepare specimens (Collect, Document, Transport, Receive, Process, and Store) and they Operate automated analysers and other duties in the ordinally laboratory schedule.
Examples of technicians include;
Phlebotomist
Collects blood and other body fluid samples from patients or research study participants.
E) Laboratory Safety Supervisor
Enforces health and safety precautions and procedures in bacteriological, serological, mycological, parasitological, virological, and environmental and other microbiological safety principles.
F) Data capture clerks
Clerks feed the laboratory informatics with results data and do other purposes concerning capturing laboratory progress and purchase data.
G) Quality Assurance (QA) supervisor
Quality Assurance supervisor monitors the Quality of all laboratory systems from Policies and Procedures to Process control, Documents and Records, Facility Safety, Equipment, Reagents and Supplies.
Quality in Research work includes;
Quality Assurance, Quality Control, Quality Improvement, Quality Indicators, Quality Systems, Quality Management.
F) Receiving office supervisor
Receiving office supervisor monitors the collect reception of laboratory sample and also signs for those specimens being shipped out of the laboratory and other Laboratory supplies.
G) Courier
Monitors or even Transports the purchased laboratory Supplies and ensures proper Transportation mechanisms of laboratory sample from centres of their collection.

CHAPTER TWO
LABORATORY INSTRUMENTATION AND DESIGN SYSTEMS
2.1 Figure 6 Laboratory Instrumentation
Instrument or Equipment Brand name Type Use or function.
Sample Collection, Carrier and Shipment containers
1. Blood collection tubes BD Vacutainer® SST®- (Yellow stopper) tubesHeparin (Green stopper) tubesK2E- (Purple stopper) tubes -Contain a coagulant Gel for separating serum from whole blood after spinning.-Contain heparin for collecting whole blood.-Contain anti-coagulant K2E for separating pellet and plasmas after spinning.
2. Stool and urine collection tubes Nalgene® Stool and Urine collection Containers Collecting stool and urine samples respectively
3.Cotton end swabs Wenqi-Industry Mucillage (mucus) sample Collection tools These are medical-liquid-filled plastics sticks. With frictional edge on both ends used for collecting mucus-fluid samplesAnd to clean Laser Lens without scratches.
Sample transport containers. Plastic or rubber carriesContainerMetallic carrier containerShipment containersShipment and Cryogenic storage containers Transportation of non-infectious samples like urine and stool in their sample containers.Transportation of containers containing infectious samples like Blood and Body fluids.Transportation of sample under –196oC like isolated cellsTransportation and Cryostorage of cells and samples between on country and another.
Laboratory Plastic ware and Plastic Laboratory ware
Laboratory Utilities Beakers, Measuring Cylinders, Tip Boxes, Elisa Assay Plates, Slide Boxes/Mailer , Funnels, Bottles , Pipette Pump, Easen the laboratory work for example storage of reagents, holding distilled water, supporting test tubes and many other purposes.
Reagent Reservoirs Corning incorporated Costar reservoirs. Sterile polystyrene 50ml capacity reagent reservoirs. Holding sample mixtures with culture media (RPMI) and also plate coating and washing buffers
Laboratory Heavy Instruments
Safety cabinets Other diagrams in appendix. Class 1Class 2Class 3 -Provide safety to the user but not sterile for samples being handled.-This provides safety for both user and the sample.-This is used in very sensitive and sterile work
Centrifuges Rotanta 46R (maximum RPM 120,000)Labofuge 200 (maximum RPM 40,000) -Swing type-Fixed angle type-Freezer or non-freezer type.-Ultra centrifuge These types are better used for separating serum, pellet, and plasmas.Centrifuge separation of cell organelles.
Centrifuge accessories -Micro Centrifuge Tube-Centrifuge Tube Conical Bottom -Holding sample in separation of cell organelles.-Holding sample in separating tissues.
Microscopes ZEISS-CORNING -Binocular Research Microscopes-Trinocular Stereo Zoom Microscopes Both types and other types are used in microscopy study of cells and microscopic pathogens
Microscope accessories -Slides-Slide box and mailers.-Gram's Stain Kit (Leishman reagent and trypan blue solution)-Immersion oil (Zeiss immersol® 518N) -Coating thin films for microscopy viewing.-Storage of coated slides in a rack order.-Staining cells for easy identification.-Improving the resolution at 100x magnification.
ELISPOT Readers Advanced Imaging Device ELISPOT Plate Reading to count number of spots formed and measures their diameters.
ELISA Plate Washer Well wash®ElX405 Auto Plate Washer They are all used for washing ELISA coated Plates.
ELISA Plate Reader DYNEXMRX Revelations ELISA Plate Spectrophotometeric Reader
Incubators REVCO HABITAT CO2incubator For culturing Whole Blood Assay Coated plates
Fridges and Freezers +4oC fridges,-20oC Freezers,-80oC Freezers All these are for Storage of Processed or Un processed Samples Under Refrigeration. Serums are kept in +4oC while Plasmas and Pellets are kept Strictly in –80oC, reagents and Pre-coated Culture plate are Kept in –20oC.
Diagnostic test Kits.
HIV Test Kits 1. Abbott DetermineTM2. Stat-pak (ChemBio)3. Uni-gold® All for invitro visually read qualitative immunoassay for detection of antibodies to HIV-1 and –2 in human serum, plasma and whole blood.
Syphilis test kits RPR test kit TPHA test kit ‘Human’ test kit Rapid Plasma Reagin charcoal and enhanced flocculation test. Hemagglutination test for antibodies against Treponema pallidum
Human urine test strips Combina 10MCombina 3ACON® Combina 50 test stripsUrinalysis Reagent Strips All strips are designed for Rapid determination of pH, Proteins, Glucose, Ketone, Urobilinogen, Bilirubin, Leucocytes and Blot in urine.
ELISA Sets BD OPTIATM1.Human IFN-γ ELISA set2.Human IL-5 ELISA set3.Human IL-13 ELISA set4.Human IFN-γ ELISpot kit LIQUID solutions setLIQUID solutions setLIQUID solution set.LIQUID solution set ELISA assays for detection of human IFN-γ antigens.ELISA assays for detection of human IL-5 antigens.ELISA assays for detection of human IL-13 antigens.ELISpot assays for detection of human IFN-γ antigens.
Whole Blood Assay Culture Plates NunclonTM Delta surface cell culture Plates WBA cell Culturing.
Laboratory Mediums and Reagents.
Medium Name Content Use
Complete tissue culture medium Gibco-BRL (RPMI-1640) 10% FBS,1% penicillin-streptomycin, L-Glutamine, Hepes Buffer Culturing human, non-human, primate and rodent cells.
Coating and plate washing buffers. 1. Phosphate buffered saline.2. PBS-Tween 8g Nacl, 0.2g KCl, 1.44g Na2HPO4.7 H2O, 0.24g KH2PO4 in 1L sterile dH2O.0.5ml Tween®-20 per 1L PBS. A coating non-used plate well, which prevents evaporation of the samples in sample wells. Washing WBA, ELISA and ELISpot culture plates.
Disinfectant solutions VIRKON® ETHANOL 2% Virkon solution.70% Ethanol solution Surface and apparatus disinfectant.Surface disinfectant.
Albumin Bovine serum, Hanks balanced salt solution, penicillin-streptomycin solution, Hepes Buffer, Histopaque Lab guard hand soap, Reagent Grade water All these and other laboratory consumables are used in day-to-day laboratory work.

2.2 Laboratory designs
Lab facility design
The design of the facility is critical for providing protection to persons outside the laboratory and in the community in the event that an infectious agent is accidentally released in the laboratory.
There three facility designs
1. Basic laboratory
This lab provides general space appropriate for work with defined viable agents which are not associated with disease processes in healthy adults or which do not colonize in humans. All activities are regularly conducted on the open bench using standard laboratory practices. This includes most of the work in Biosafety Level 1 laboratories.
2. Containment laboratory
This laboratory provides general space appropriate for work with infectious agents or potentially infectious materials when the hazard levels are low and laboratory personnel can be adequately protected by standard laboratory practices. Work is commonly conducted on the open bench with certain operations confined to BSCs.
Public areas and general offices to which non-laboratory staffs require frequent access should be separated from spaces which primarily support laboratory function. This includes most of the work in Biosafety Level 2 laboratories

3. High Containment Laboratory.
This laboratory has special engineering features, which make it possible for laboratory workers to handle hazardous materials without endangering themselves, the community or the environment. The unique features which distinguish this laboratory from the basic and containment laboratories are the provisions for access control, a specialized ventilation system and vacuum line isolation. This includes most of the work in Biosafety Level 3 and 4 laboratories.

How many Biosafety Levels are there?
There are four Biosafety Levels in Laboratory Designs.
But the E-MAB project Runs a Biosafety level / class Two laboratory although the Uganda Virus Research institute Runs a class or P four T.B (Mycobacterium bovis or tuberculosis) isolating Laboratory as well. In Uganda there are no established facilities running BSL4 laboratories (and only two of there kind in the all of Africa, S.Africa and Gabon)
Biosafety Level 1 (BSL1)
· Involves minimal or no known hazard to lab personnel or environment.
· Wear lab coat, gloves, closed-toe shoes and safety glasses with side shields if there is splash potential.
· No special hood or cabinet requirements.
· Examples of agents involved include: Bacillus cereus, Canine distemper, Newcastle virus, influenza virus or Lactobacillus acidophilus.

Biosafety Level 2 (BL2)
· Involves agents of moderate potential hazard to personnel or environment.
· Wear long sleeved lab coat, gloves, safety glasses w/ side shields, closed-toe shoes.
· Use Class I or II Biological Safety Cabinet for procedures with high aerosol potential.
· Examples of agents include: Bacillus anthracis, Salmonella, Streptococcus pyogenes, Penicillium marneffei, Toxascaris or Hepatitis.

Biosafety Level 3 (BL3)
· Involves indigenous or exotic agents, which may cause serious or potentially lethal disease as a result of the inhalation route.
· Wear front or wrap around gowns, coveralls or scrub suits, gloves, safety glasses with side shields, covered shoes and appropriate respirator.
· Use Class I or II Biological Safety Cabinets for all procedures.
· Examples of agents include: Mycobacterium bovis or tuberculosis, sheep poxvirus, Arboviruses, Hanta or Rabies street virus

Biosafety Level 4 (BSL4)
· The use of these agents, which are extremely infectious to human beings and are restricted to be moved from on area to another.
· Involves agents extremely hazardous or have potential to cause serious epidemic
· Use Class III Biological Safety Cabinets exclusively.
· Examples of agents include: Ebola fever, tick borne encephalitis or Herpesvirus simiae.

CHAPTER THREE
ETHICS AND SAFETY IN THE LABORATORY

3.1 Laboratory ethics
Ethical Duties of the laboratory personnel
1) Clinical laboratory professionals are accountable for the Quality and Integrity of the laboratory services they provide. This obligation includes maintaining individual competence in judgement and performance and striving to safeguard the patient from incompetent or illegal practice by others.
2) Clinical laboratory professionals maintain High standards of practice. They exercise sound judgment in establishing, performing and evaluating laboratory testing.
3) Clinical laboratory professionals maintain strict Confidentiality of patient information and test results. They safeguard the Dignity and Privacy of patients and only provide accurate information to other health care professionals about the services they provide and which are of interest to the patient’s health.
4) Clinical laboratory professionals actively strive to establish Cooperative and Respectful working Relationships with other health care professionals with the primary objective of ensuring a high standard of care for the patients they serve that is to say they always over see Team work in their normal practices.
5) Clinical laboratory professionals Comply with relevant laws and regulations pertaining to the practice of clinical laboratory science and actively seek, within the dictates of their consciences, to change those which do not meet the high standards of care and practice to which the profession is committed.

3.2 Safety in the laboratory
Safety Equipment
Safety equipments include biological safety cabinets and a variety of enclosed containers. The biological safety cabinet (BSC) is the principal device used to provide containment of infectious aerosols generated by many microbiological procedures. Three types of BSC’s (Class Ι, ΙΙ, ΙΙΙ) used in microbiological labs are described in Appendix, in the E-MABS project Immunology Lab there two types of BSC and these are classes I and II.
Open-fronted Class Ι and ClassΙΙ BSC’s are partial containment cabinets which offer significant levels of protection to laboratory personnel and to the environment when used in conjunction with good microbiological techniques.
The gas-tight Class ΙΙΙ provides the highest attainable level of protection to personnel and the environment.
Safety equipment also includes items for personal protection (PPE-personal protection Equipment) such as gloves, coats, gowns, shoe covers, boots, respirators, face shields and safety glasses. These personal protective devices / equipment (PPE) are often used in combination with biological safety cabinets and other devices which contain the agents, animals and material being worked with. In some situations in which it is impractical to work in biological safety cabinets, for example in archiving. PPE may form the primary barrier between personnel and the infectious materials.

3.2.1 Packaging Biological Waste
1) Biohazard bags – used for the initial collection of certain biological wastes.
All biohazard bags must meet impact resistance (165 grams), tearing resistance (480 grams), and heavy metal concentration (<100 PPM total of lead, mercury, chromium and cadmium) requirements, they are Auto clavable and used in shipment of laboratory-generated soft waste to disposal sites.
2) Sharps
Place needles, scalpels, razor blades, pipette tips, and Pasteur pipettes in red plastic or metallic sharp’s containers. These containers are puncture-resistant or are placed in puncture-resistant cardboard boxes and then into a biomedical/biological waste box before disposal. It is also recommended that pipette tips be placed in puncture-resistant cardboard boxes prior to placement into the biomedical/biological waste box.
3) Corrugated biomedical/biohazardous cardboard boxes
Place all biological waste in rigid, specially labeled, puncture resistant boxes as the terminal receptacle.

3.2.2 Safety Labeling
Biohazard bags and sharps containers must be labeled even though they will be placed inside a secondary container for final disposal. All packages containing biological waste should be labeled as follows:
1) Date
Biohazard bags should be labeled with the date they were put into use. Sharps containers should be labeled with the date the container is full.
Corrugated boxes (biomedical/biological waste boxes) should be labeled with the date the biohazardous waste was treated. Boxes used for non-biohazardous waste collection should be dated when the box is sealed.
2) Biohazard sign
Use only manufacturer containers with the preprinted universal biohazard symbol and the words "biomedical," "biohazardous," or "infectious in order to notify other user the infection potentiality of the constituents.
Figure 7
Examples of some biohazardous signs

3.2.3 Transport of biohazard material.
Transport biohazardous waste outside of the laboratory (i.e., to an autoclave or incinerator) in a closed leak-proof container labeled "biohazard". Only trained personnel may transport biological waste. Labeling may be accomplished by use of a red biohazard bag or a biomedical/biological waste box with the universal biohazard symbol.

CHAPTER FOUR

COLLECTION, DOCUMENTATION, TRANSPORTATION, SHIPMENT, PROCESSING (OR TESTING) OF BIOLOGICAL SAM PLES, AND DISSIPATION OF RESULTS

4.1 E-MABS project
Figure 8 Specimen / Result flow chart

Hospital or field ClinicSample collection and simple blood testing
↨
ANC LAB1.Urine sample tests; (Glucose, Protein and pH)2.Blood rapid testing; (Abbott or Stat-pak or UniGold tests and RPR Test or TPHA).3. Documentation of results

Shipment of samples

IMMUNOLOGY LAB 1. EDTA Whole blood plasma and pellet processing.2. SST-Serum processing.3. Heparin Whole blood culturing.4. Heparin PBMC processing.5. ELISA, ELISPOT, Whole blood killing assays performed on specific samples.6. Documentation of sample reception, processing, assay results and storage.

CLINICAL LABS1.Selogical tests2.Microbiological tests3. Whole blood cell counting4. HIV EIA and western blot confirmation.5. Stool processing

RESULTSSent to appropriate hospital and field clinics and to MRC statistics department.

4.2 Collection of Samples
Collection of Blood samples or other Body fluids are collected by trained personnel.
Phlebotomists are trained to draw a blood sample of venous blood from a Patient by doing a venipuncture or a skin puncture or arterial collection. They use blood collection sets and blood draw needles to collect blood in collect vacutainer tubes.
The main qualifications in doing this are to understand the infection control and sterilization practices.
Fingerstick samples are collected by puncturing either the index, middle or ring finger wiping away the first blood drop and holding an EDTA capillary sample collection tube mouth in the second drop and gently squeeze the finger.
Other samples such as Stool and Urine are collected into their proper containers using a container enclosed spoon (for stool sample) or a small jar (for urine sample) while Virginal mucus and other Mucilage samples like Pus are collected using cotton-end swabs.
Although other samples may require more experienced practices, the first step in collecting the sample is to appropriately label the collection containers to avoid contaminations and mix-ups.
4.3 Documentation of Samples
A given sample should bear the full identification of the Patient and the sample type that is to say,
A. Names of the patient / participant in the study
B. Mother sample identification
1. Antenatal Clinic Number (ANC No)
2. Worm study number (WSN)
3. Age and a given History record.
C. Baby sample identification
a. Baby Identification number (BID)
b. Baby’s Age
c. Sex
D. Purpose of sample collection for example;-
· CD4-count or storage
· Measles sample
· Quality control sample or
· Normal study sample
E. Physician or Person who collected the sample
F. Time of sample expiry.

4.4 Transportation and Shipment of Biological Samples
Samples are transported almost immediately after their collection to the prescribed laboratories.
The transportation personnel examines the sample for complete documentation and appropriate attached requisition forms, testing expiry period and the transportation conditions such as temperature. (Some specimens need to be refrigerated during transportation)
The samples are examined while wearing appropriate personal protection equipment (PPE) for example gloves to avoid any body contacts with samples.
The courier container is disinfected and the appropriate container for specific sample is chosen for example for blood and other body fluids, a metallic container in chosen because in case of any accident they really expose off the infectious sample carried. While for stool and other sample in the same category can be carried in plastic containers. Also to note container for carriage of sample containing tubes must have containment racks to prevent tube collisions, which can lead to breakage and spillage of samples.
When the samples have been delivered they must be handle over to the laboratory personnel in-charge or handed over to the reception personnel who must sign for the delivery.
For transportation of sample within the research institute facility, samples must be well packaged in Biohazard bags.
For shipment of biological sample from on region to another or one country to another, speculated United Nations shipment rules and international Air transport association (IATA) must be meant.

4.5 Sample Processing (or Testing)
A. Sample processing at the Immunology research Laboratory (E-MABS project)
Sample processing will usually start with a set of samples and a request form. Typically a set of vacutainer tubes containing blood, or any other specimen arrives in the laboratory in a small plastic bag within a metallic sample carrier container, along with the form.
4.5.1 Figure 9 Profile of the assays performed on samples in the Immunology Laboratory

Samples from the field/clinic

Core immunology laboratory UVRI

EDTA tube (Purple) Heparin (Green) SST tube (Yellow)

Plasma for anti body studies /virology.
Whole blood culture, cell separation.
Serum for antibody studies.

Measles HIV immunology TB diagnosis BCG study Hook worm
Immunology study

Main study immunology

Whole blood cytokine assay

4.5.2 Reception and Documentation of the Samples
The Sample request form and the specimens are given a laboratory number. Specimens of the same individual (Heparin, SST, and EDTA tubes) usually all receive the same number, often inform of a sticker that can be placed on the tubes and forms.
Entry of requests onto a laboratory management system that involves recording of laboratory numbers with respect to entered patient’s identification as well as any test requested in done. This allow the laboratory staff to know what tests are pending and also gives a place for results to be clearly followed.
For clinical laboratory services samples for example Microbiological request samples, 1ml aliquots are sent to respective Labs for processing. The remainder,
SST and EDTA vacutainer tubes are centrifuged (Spinned) at 3000RPM for 5 minutes.
In SST tubes with coagulant gel, Serum separates as a top supernant over the gel and coagulated pellet settled below it after spinning, aliquots in doubles into White top cryogenic vials are made.
In the EDTA without coagulant, the Pellet separates below the Plasma and both are aliquoted in separate cryogenic vials Blue topped and Yellow topped respectively.
This allows proper storage systems and archiving of samples the samples are aliquoted in doubles to provide a back up in case one of the tube is used.

B. Sample Processing From ANC Laboratory (PMTCT / VCT services)
1. Collection of Blood and Urine samples as requested on the ANC request form.
· The mother is bled for Blood sample is collected in a green top vacutainer by a phlebotomist while the mother collect her own Urine sample into the container provided by the Lab technician.
· The Blood sample is left to stand for some time before both rapid tests for HIV and Syphilis are carried out on the Plasma or Serum that separates out of the whole blood.
· A test for haemoglobin concentration is also done by adding 0.2μl of the well mixed Blood sample with 2ml of 0.04% ammonium solution and then the whole solution read for absorbance using a potable Haemoglobin analyser.
· Urine sample are tested with Urinalysis test kits for the detection of Glucose, Proteins and measuring the urine pH and the rank score of a give sample is taken by comparison of the strips formed colour with that standards on the kit container label.
2. Aliquoting samples to send to Research Labs (both UVRI and E-MABS).
Some Samples which test HIV antibody Reactive are sent to UVRI for mostly two purposes;
a. CD4 and CD8 count
This is aimed at find the number of T-Helper cells in the blood sample of the patient. Its done using a counter machine (Microbiology Department). And the all process helps is determining whether the Patient should start taking Anti-retro Viral therapies (ARVs) or not.
b. Quality control samples
These are also categorised into two forms
Monthly Quality control samples and Referred Quality control samples.
i. Monthly Quality control samples
An extra aliquot of
-Every first 10 samples tested for HIV and Syphilis at the beginning of each month are sent to the immunology Lab and these include.
-All HIV indeterminate samples and Syphilis RPR reactive only
-All samples testing HIV 1 / 2 on the same strip.
ii. Discordant sample results
These are samples that test positive on some test strips but also test negative on others using a serial testing Arigolithm. For example discordance on Abbott and STAT-PAK or UniGold test strips. (In the serial Arigolithm testing the sample is treated first to the most selective kit test and if it shows reactivity its successfully treated to the second and third specific kit tests. That is to say from most selective to the most specific)
3. Records of sample results are noted both on forms and in the Lab desk book
Results are filled in the respective forms carefully and on the patient Antenatal cards.
All details are copied from the laboratory work sheets into the book.
And the book is updated every morning.

4.6 Dissemination and marketing of services
4.6.1 For E-MABS (field and Immunology research laboratories)
Since E-MABS is a randomised “blind” trial, this type of research ensures that neither the patients nor the researcher can have any direct effect on the outcome of the treatment due to factors such as the effect of believing that one is receiving the active drug which in itself may produce some benefit.
Similarly, the researchers may inadvertently or otherwise affect the outcome it he is aware of which treatment a patient is receiving.
There fore every patient is monitored in an identical fashion (in test trials, with report forms of symptoms and ability to participate in daily activities as well as Immunological testing). Results of the trial testing important for the participant’s health are forwarded to the respective clinics, which take action of treating the infection.
At the completion of the trial, that is when every will patient have completed the treatment course and completed the follow-up, a code will be broken which details which patients received the active and which patients the placebo treatment. An analysis will then be made of the data, which has been already partly collected for each patient, as to whether a larger number of patients receiving the active drug than the placebo showed a response to the treatment.
For different project results reached In-House, Results are discussed in;
· Seminars
· Conferences
· Presentations, and
· Tutorials

4.6.2 For VCT / PMTCT (Entebbe hospital Grade B Antenatal clinic)
Figure 10 Flow chart for Blood and Body fluid (urine) sample tests done as part of VCT / PMTCT at Entebbe hospitals

Entebbe hospital sends sample and requisition/ result form

MRC /UVRI Reception (new building)

MRC central laboratory service (perform test on samples)-fills results in requisition / result forms

Requisition / result form sent to reception (form photo copies-2copies)

1 copy sent to E-MABS laboratory
Original sent to MRC statistics

Form results sent to the appropriate hospital clinic.

1 copy to project coordinator

The respective hospital clinics carry out the rapid tests on the collected sample but for research work and more quality control of the results obtained sample are sent to the research institute as shown in the flow chart above.
CHAPTER FIVE
IMMUNOLOGICAL ANALYTICAL LABORATORY TECHNIQUES
There are several laboratory techniques employed in laboratory work and these include;
1. Rapid Immunological Tests
2. ELISA
3. ELISPOT
4. Whole Blood Assay (WBA)
5. CELL SEPARATION or PBMC processing
6. Whole Blood Killing Assay

5.1 Rapid Immunological Tests
Assay Formats
Most rapid assays are in kit form that include all necessary reagents and require no other specialized laboratory equipment.
Rapid tests include; HIV rapid tests, syphilis tests and pregnancy tests.
HIV rapid tests
How rapid HIV tests are used
Rapid HIV tests are used as screening tests to detect antibodies to HIV as part of multi-test algorithms to aid in the diagnosis of infection with HIV. Positive (reactive) rapid HIV test results are preliminary and must be followed up with an approved confirmatory test.

The three most common assay formats that can be used with whole blood are;
I. Particle agglutination
II. Immunoconcentration and
III. Immunochromatography.

a. Particle agglutination assays
These assays typically require 10 to 60 minutes or more when a patient specimen containing HIV antibodies is mixed with latex particles coated with HIV antigen, cross-linking occurs and results in agglutination, Results are interpreted visually. Most of these assays are used with serum or plasma, while some have been developed for use with whole blood.
b. Immunoconcentration assays
This assay employs solid phase capture technology that involves the immobilization of HIV antigens on a porous membrane. The specimen flows through the membrane and is absorbed into an absorbent pad. A dot or a line visibly forms on the membrane when developed with a signal reagent (usually a colloidal gold or selenium conjugate). The flow- through tests are designed for use only with serum or plasmas and usually require several steps for the addition of specimen, wash buffer, and signal reagent so they can usually be performed in 5 to 15 minutes.
c. Immunochromatography (lateral flow) strips.
These are the most recently developed and are incorporated with both antigens and signal reagent into nitrocellulose strip. Many lateral flow tests require only a single step. The specimen (usually followed by a buffer) is applied to an absorbent pad. Alternatively, the specimen is diluted in a vail of buffer into which the test strip is inserted longitudinally. The specimen migrates through the strip and combines with the signal reagent. A positive reaction results in a visual line on the membrane where HIV antigen has been applied. A procedural control line is usually applied to the strip beyond the HIV antigen line. A visual line at both the test and control sites indicates a positive test result, a line only at the control location indicates a negative test result and the absence of a line at the control site means the test result is invalid.
The test results can be obtained in 20 minutes or less many can be used with whole blood, serum, or plasma and some can be used with finger stick blood specimen, saliva or oral fluids. In most lateral-flow devices the test strip is encased in a plastic cartridge for example UniGold test kits.
Test Kit Usage in E-MABS (ANC- Laboratory facility)
At the ANC-Laboratory they use a serial HIV/AIDS testing Arigolithm Instead of the parallel one. In this they test the sample with the most selective test kit first and if it shows reactivity it is then treated to the successful specific test kits to confirm the result.
Test 1 Test 2 Test 3 Comment
Abbott (Determine Kit) STAT-PAK Kit Uni-Gold Kit
Negative Leave out Leave out Results Given as HIV Negative
Positive Positive Leave out Results Given as HIV Positive
Positive Negative Negative or Positive Results not Given, And the SampleReferred for Quality control / Testing
But when the results show discordance with in the two test kits then the results of the test are not issued to the Patient but rather the sample is referred for Quality control testing
Using the Enzyme immune assays or Western blots.
Serial HIV / AIDS Testing Arigolithm as applied at the ANC- Laboratory.
Manufacturer Product Principle Performance
Sensitivity % Specificity %
Abbott laboratoriesAbbott park, Illinois USAChemBioChembio, Inc.Medford New York USATrinity Biotech plcBray, Ireland Determine HIV-1/2/OHIV 1/2 STAT-PAK DIP STICK1.UniGold recombinant HIV-1/2 LFLFLF 10099.6100 99.499.8100
2.Serocard HIV-1/23. Capillus HIV-1/2 FTPA 100100 97.9100
Notes to table: FT=flow-through; LF=lateral flow, PA=Particle AgglutinationSensitivity and Specificity represent published reports against multiple HIV-1/2 sub types from independent (WHO) evaluations.
E-MABS/UVRI Antenatal Clinic Services (VCT / PMTCT) Frequently Used HIV-Test Kits and their Evaluation.
As copied from R.Junker J Labmed 2003; 27 (7/8) Online journal page 291.
How ever Serocard and Capillus test kits are not very commonly used at the ANC they are included to show the selectivity and specificity of the few among the many HIV/AIDS test kits on Market.

5.2 Enzyme-linked immunosorbent assay (ELISA)
ELISA and Solid-Phase Immuno assay have similar techniques.
An antibody specific for a protein of interest is attached to a polymeric support such as a sheet of polyvinyl chloride.
A drop of cell extract or a sample of serum or urine is laid on the sheet, which is washed after formation of the antibody-antigen complex.
Antibody specific for a different site on the antigen is then added and the sheet is again washed. This second antibody carries a radioactive or fluorescent label so that it can be detected with high sensitivity. The amount of second antibody bound to the protein is proportional to the quantity of antigen in the sample.

Figure 11 ELISA Technique
Assay Principle

ELISA
The sensitivity of the assay is enhanced even further if the second antibody is attached to an enzyme such as alkaline phosphatase. This enzyme rapidly converts an added colourless substrate into a coloured product, or a non-fluorescent substrate into an intensely fluorescent product.
ELISAs are rapid, convenient and less than a nanogram (10-9g) of a protein can readily be measured.
For example immunoassaying of the presence of Human Chorionic Gonadotropin (hCG) a 37-kd protein hormone produced by the placenta, in female cord blood sample and a baby’s blood sample is used to determine the possibility of mother’s blood having mixed with the baby’s in labour.
or in female urine sample is used to determine Pregnancy within a few days after conception. But for very small quantities of a protein of interest in a sample or in body fluid, it can be detected by an immunoassay technique called Western Blotting.

5.3 Enzyme-linked immunosorbent spot assay (ELISPOT)
The enzyme-linked immunosorbent spot (ELISPOT) is a common method in immunology research work for monitoring immune responses in humans and animals (several immunology researches include; Cancer, Transplantation, Infectious diseases and Vaccine development).
These assays are based on or were developed from a modified version of the ELISA immunoassays, they were originally developed to enumerate T-cells secreting antigen-specific antibodies, and they have subsequently been adapted for various tasks especially the identification and enumeration of cytokine-producing cells at the single cell level. Each spot that develops in the assay plate well represents a single reactive cell. Thus the ELISPOT assay provides both Qualitative (type of immune protein produced for example IFN-γ, IL-3, 4, 5, 10 and 13) and Quantitative (number of responding cells) information. Modern ELISPOT analysis is typically performed using ELISPOT readers these employ computer vision techniques to enumerate the actively producing cells. This also allows much of the analysis process to be automated, and permits a greater level of accuracy than if manual inspection is done.
Figure 12 ELISPOT Technique
Assay Principle

Procedure
The ELISPOT assays employ a technique very similar to the sandwich Enzyme-linked immunosorbent assay (ELISA) technique in which either a monoclonal
or polyclonal capture antibody is coated a aseptically (in a sterile safety cabinet) onto a Polyvinylidene fluoride (PVDF) backed 96-micro well plate. These antibodies are chosen for their specificity for the analyte in question. The plate is washed with a washing buffer and the blocked usually with a solution of Human serum albumin (HSA) that is non-reactive with any of the antibodies in the assay. After this, cells of interest are plated out at varying densities along with the reactive antigen or mitogen (this antigen is aimed at stimulating the cells to produce Cytokines) the plate is incubated at 37oC in humidified CO2 incubator for a specified period of time.
Cytokines (or other cell products of interest) secreted by activated cells are captured locally by the coated antibody on the high surface PVDF membrane. After washing the wells to remove cells, debris and media components, and a Biotinylated Polyclonal antibody specific fro the chosen analyte is added to the wells. This antibody is reactive with a distinct epitope of the target cytokine and thus is employed to detect the captured cytokine. Following the wash to remove any unbound Biotinylated antibody, the detected cytokine is the visualised using an Avidin-HRP enzyme, and a precipitating substrate (for example AEC, or BCIP/NBT). The coloured end product (a spot, usually blackish blue) is formed and it typically represents an individual cytokine-producing cell. The spots can be counted manually (for example with a dissecting microscope) or using an Automated reader to capture the micro well images and to analyse spot number and size.

5.4 Whole Blood Assay (WBA)
Processing Samples for Whole Blood Assays
A blood sample in the Green Stopper (Heparin) vacutainer is treated as shown in the figure below, Equal amount of a blood sample for example 2.0ml is dilute with equal amounts of the Complete Culture medium RPMI.
Figure 13 Sample processing for Whole Blood Assays

Plate processing.
Plates already prepared with required antigen and the PBS in other non-specific well are thawed as well as the reagents (RPMI for culturing the sample) are removed from their respective refrigerated storages. Plates are labelled with the Sample Lab number (that was given to it at reception) at the head and the date of culturing at the tail.
For schito positive sample checked in records of after delivery and cord samples that were priory studied in expecting mothers, the schito ant