Return to the Stamataki Lab

I’ve kicked off this summer with a trip to Birmingham to meet with Dr Zania Stamataki. During my prior work experience here, I improved my wet lab skills by learning essential techniques such as splitting cell lines, performing serial dilutions, and imaging plates with confocal microscopy. This year, I chose to hone my computational biology skills to best prepare me for a future in research; Dr Scott Davies introduced me to bioimage informatics through training me to work with the ZEISS Efficient Navigation (ZEN) microscopy software.

The ZEN software can be used to process the plethora of data that a single imaged experiment can yield. I worked with data from an experiment in which Huh-7 hepatocytes (from an immortalised liver carcinoma cell line) are co-cultured with live T-cells and imaged using confocal microscopy.

I personally found this sophisticated mode of imaging fascinating so I did a little more research into the bioengineering responsible for the high resolution and vibrancy of the cellular interactions captured by the confocal microscope. Unlike the humble optical microscopes of my school, which only need light and a pair of eyes to visualise cells, confocal microscopes require far more exotic elements: lasers and fluorescent dyes.

GFP

One of the most widely used fluorescent biomarkers is green fluorescent protein (GFP) which is used in this experiment to highlight the cell membranes. GFP is a very important tool in research as it enables us to look beyond the cell surface and visualise organelles and protein movements. GFP has a unique sequence of the amino acids serine, tyrosine, and glycine which can be triggered to undergo chemical transformations in the presence of UV light. This special amino acid sequence is buried deep within the molecule’s barrel structure of intertwined protein sheets.

When activated, a cyclisation reaction occurs in which glycine forms a chemical bond with serine. This new closed amino acid ring spontaneously dehydrates and oxygen surrounding the GFP molecule forms a new double bond with the tyrosine – creating the fluorescent chromophore. In this way, GFP automatically assembles its own chromophore which can be used by researchers as tracking dyes. For instacnce, through genetic engineering, the GFP protein can be incorporated into the genome of specific cells or be used to label proteins of interest. It is especially perfect for studying live cells since the alternative small fluorescent molecules (ex. fluorescein isothiocyanate) are highly phototoxic and inflict more damage upon live cells during imaging.

In the microscope images I worked with, GFP had been used to label the hepatocyte cell membranes. The T-cells were stained a vibrant blue whilst a cytoplasmic dye had rendered the hepatocyte cytoplasm red. The smaller dark regions enclosed within cytoplasm are the hepatocyte nuclei.

Over the course of the week, I became proficient in producing orthogonal videos, which showcase the full depth of the hepatocytes, from multiple micrographs in a time lapse. Whilst processing these videos, I witnessed countless interesting phenomena; with Dr Stamataki’s permission, I’d like to share a few of my favourites with you.

Binucleate Hepatocyte Division

When I first starting working with the experiment data, Zania pointed this event out to me and I was both riveted and shaken to my core; this cell defied everything textbooks had me believe about cell biology! In this orthogonal video, a binucleate cell successfully undergoes mitosis to produce not two but three daughter cells.

Binucleate cells can be caused by cytokinesis failure in which the cell membrane fails to form a cleavage furrow so two daughter nuclei are trapped in the same cell. Alternatively, during mitosis, sometimes multiple centrioles form making the cell multipolar- pulling the chromosomes in several directions and producing several nuclei in a single cell. Judging by how the binucleate cell proceeds to divide into an odd number of daughter cells, its original two nuclei probably arose from the latter mechanism.

Binucleation in vivo is pretty rare and, in a healthy human liver, such cells are incapable of dividing again, instead forced to remain in interphase indefinitely. Binucleate cancer cells, however, have a far different fate; more than 95% can undergo mitosis with a higher rate of chromosomal disjunction error – producing daughter cells with even more mutations. Despite binucleation rarely arising in healthy tissue, this phenomena is less of a rarity in cancer cells due to one of their unique quirks. A prominent biomarker of cancer cells is their multipolar spindles which increase the risk of binucleation and other mitosis abnormalities – leading to an accumulation of mutations in subsequent daughter cells in the cancer tissue.

Lamellipodia Network Formation

Inside the body, liver cells are always in contact with their neighbours with junctions between their lateral faces. In a lab culture, however, liver cells are seen tightly adhered to the plastic that they are grown on, practically clinging to the plate for dear life! The in vitro hepatocytes achieve this through multiple membrane projections known as lamellipodia.

Lamellipodia are made of the cytoskeletal protein actin and, in the video above, can be seen stretching out to not only tether carefully to the confluent monolayer but to also engulf some extracellular material. I’m mesmerised by the rapid yet tentative movement of these hepatocytes’ extensive threadlike network.

Another Kind of ‘Nuclear Fission’

The earlier orthogonal video of two nuclei becoming three was very surprising. In contrast, the phenomena below is absolutely incredible. Two neighbouring T cells apply a mechanical stress on the hepatocyte’s nuclear envelope great enough to break the nucleus into two ‘daughter’ nuclei without the hepatocyte undergoing mitosis! I find it truly fascinating that the nucleus is not as static an organelle as it is so often portrayed; instead, it is dynamic and capable of distorting and even bifurcating in response to changing cytoplasmic pressures.

As always, I’ve really enjoyed my time at the Centre for Liver Research this year and I’m so grateful to Zania for allowing me to learn so much about the cell biology and immunology of the liver and participate in her current work. Her team is currently in the midst of publishing some amazing cutting-edge research on a cellular mechanism similar to entosis; as soon as it is available to the public, I’ll share it here and, hopefully, write a review article about this interesting hepatocyte process!

WordPress does not support video uploads for this blog so the orthogonal videos in this post were compressed into gifs, hence losing resolution and quality. If you would like to see the original videos, I’ve also uploaded them to my YouTube channel:

Bibliography

Kim, Dong-Hwee et al. “Volume regulation and shape bifurcation in the cell nucleus.” Journal of cell science vol. 128,18 (2015): 3375-85. doi:10.1242/jcs.166330

Shi, Qinghua; Randall W. King (13 October 2005). “Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines” (PDF). Nature. 437 (7061): 1038–42. doi:10.1038/nature03958. PMID 16222248

Yang, F.; Moss, L.G.; Phillips Jr., G.N. “STRUCTURE OF GREEN FLUORESCENT PROTEIN.” Nat Biotechnol vol. 14 1246-1251 (1996). doi: 10.1038/nbt1096-1246. PMID 9631087

Give Me A Break: Bioengineering Solutions to Spinal Fracture

I’m writing this post in a lab at Queen Mary University in the heart of London. Through the EDT Headstart scheme, I have been given the brilliant opportunity to immerse myself in the field of bioengineering (from the physiological level right down to the molecular level) surrounded by leading academics and university lecturers as part of a 4 day residential course.

Absorbing fascinating new information about molecular bioengineering has been a major highlight of the course. On the first day, I attended Dr Karin Hing’s lecture ‘Engineering Biocompatibility for Bone Regeneration’ which inspired my independent group research project on the use of synthetic bone grafts, autografts, and growth factors in spinal fusion.

Biocompatibility lies at the intersection between biology and material science and it refers to the interactions between an artificial material and a living system. With its abilities to bear loads and control homeostasis as well as self-regulate and repair, bone is a particularly interesting natural material – but what is it actually made of? Bone primarily consists of inorganic materials, such as calcium hydroxyapatite, and organic materials, such as the protein collagen, which make up a rigid extracellular matrix. Within this matrix, resides a range of specialised bone cells: osteoblasts, osteoclasts, and osteocytes.

Like ourselves, bone has a life cycle. Our bones are constantly undergoing remodelling, cycling between phases of formation and resorption. In formation, mesenchymal stem cells in the bone marrow differentiate into osteoblasts (bone-forming cells). These osteoblasts create the extracellular matrix (known as the osteoid) and mineralise it with calcium deposits. Next, they find themselves trapped within the bone matrix and they mature into osteocytes which are no longer capable of bone formation, instead serving as a communication port- relaying signals between other bone cells (much like neurons).

Life is centred on balance, a yin and yang of sorts; as new bone is built up, old bone is destroyed through the process of resorption. Osteoclasts are a key player in resorption and prevent new bone formation from getting out of hand. Bone tissue contains rich deposits of minerals such as calcium and, when it is resorbed, the minerals leach out and enter the neighbouring blood stream. Through selective resorption of bone tissue, osteoclasts maintain homeostasis and regulate our calcium levels. Between formation and resorption is the reversal phase in which mononuclear cells inhibit osteoclasts and prepare the bone surface for osteoblasts to build the connective tissue up again.

With its constant repair and remodelling, bone can automatically fix virtually any hairline fracture or microdamage. But when it comes to breaks larger than 2.5 cm (known as critically large defects), the bone can no longer spontaneously heal and will require medical intervention. For instance, in cases of vertebrae fracture, spinal fusion is necessary to heal multiple vertebrae into a single, solid bone – restoring stability to the spine.

What do we use to bridge the gap between vertebrae? That’s where bone grafts come in. Traditionally, a bone graft involves removing a small portion of bone from the hip (where it will naturally heal back) and transplanting it between the vertebrae to promote bone growth at the fusion site. If the graft comes from the patient’s own body (which is currently the gold standard in spinal fusion) then it is referred to as an autograft. Allografts come from a cadaver or live donor and are used when the fracture is too large to bridge with the patient’s own bone.

harvesting an autograft from the iliac crest (hip)

The reason autografts are considered the gold standard is because they can integrate into the patient’s fracture more rapidly and completely and there is no risk of immune rejection or bio-incompatibility. However, autografts require the patient to undergo two surgeries – giving rise to an array of new complications including donor site morbidity and secondary infections. Additionally, multiple incision sites may be necessary to extract an ideal autograft which increases the risk of nerve injury and bone damage at the donor site. But allografts are not without their flaws either; they come with a risk of immune rejection and disease transmission and there are long waiting times to even obtain an allograft due to a shortage of eligible donors.

Recently, synthetic alternatives to bone grafts (such as calcium sulfate and collagen) have emerged. Initially, these synthetic bone grafts were bio-compatible but still did not promote bone healing. Let’s take a closer look into what is necessary for a graft to support successful bone formation. Firstly, we have osteoconduction which is the graft’s ability to support the attachment, migration, and growth of osteoblasts within the structure. Then, there is osteoinduction which refers to the graft’s ability to stimulate stem cell differentiation into bone cells (osteogenesis).

Ever tried gardening? The properties of an ideal bone graft are similar to those needed to grow a plant. Osteoconductivity (the scaffold properties of the graft) can be seen as the pot and soil, osteogenesis by the actual seeds, and osteoinductivity is best represented by the fertilisers and water that stimulate plant growth.

At the very least, a synthetic graft must be osteoconductive for a successful fusion. On the third day of the course, I had the chance to enhance my wet lab skills via testing protein adsorption in a range of synthetic grafts. The experiment involved incubating five types of graft material (both organic and inorganic) in an albumin solution and using a colorimeter and calibration curves to assess the efficiency of protein adsorption in each of the graft types.

The first synthetic grafts were bio-compatible but also bio-inert meaning that they had were osteoconductive but not osteoinductive. To overcome this issue, subsequent designs of synthetic grafts tend to be infused in growth factors. Growth factors stimulate differentiation of stem cells and growth of living tissue – accelerating bone formation.

The most widely studied growth factor is bone morphogenetic protein 2 (BMP) which acts as a chemotactic agent by recruiting stem cells to the fusion site and prooting their differentiation. BMP even stimulates the growth of blood vessels (angiogenesis) through the damaged bone which helps support new bone formation.

the structure of BMP-2

BMP is considered safe for use in spinal fusion and eliminates the need to take a large portion of the patient’s own bone cells in a graft. The only downside is that it is currently very expensive – increasing spinal fusion surgery costs by up to $15,000.

BMP works alongside, and can be incorporated into, both types of graft. Synthetic grafts can be made of bioabsorbable polymers (such as lab-grown collagen or calcium sulfate structures) which carry doses of growth factors. As the material dissolves in the body, the BMPs are gradually released, helping sustain bone growth. In autografts, the patient’s own cells can even be genetically engineered to express specific growth factors in greater quantities.

It has become increasingly clear that bone grafts cannot be developed solely from an engineering perspective. As Dr Alvaro Mata explained in his lecture on bioinspired materials, nature is a particularly important source of inspiration for designing any bioengineered system, particularly synthetic bone grafts. Structure at even the smallest levels, such as the arrangement of structural protein nanofibres, affects cell signalling and growth to a large degree.

Biomimetic design is developed through an appreciation of nature’s own design. Through dissecting a bovine metacarpal joint, I gained a far better understanding of the interplay between bone and cartilage that makes smooth movement effortless.

As the end of my week at Queen Mary’s bioengineering department crept closer, my team began to prepare a presentation showcasing our independent research in the fascinating field of spinal fusion. This presentation was the perfect culmination of the amazing molecular bioengineering techniques I had studied and carried out in the lab. To my absolute delight, we were awarded the 1st place prize for our level of detailed research and our debate style of presentation – a rewarding end to an incredible week!

Work Experience: Centre for Liver Research, University of Birmingham

Over the course of the two weeks of my work experience, I had the opportunity to try out new laboratory techniques and to explore various aspects of the research being done at the University of Birmingham, Centre for Liver Research. In this account I will focus solely on the major highlights (despite each day having been amazing and enriching in its own right).

On the first day, I was greeted warmly by Dr Zania Stamataki and given a tour of the Medical School building and the IBR building. We discussed about the research being done by her team concerning hepatocytes and their interaction with the immune system (mainly CD4+ T-cells). Having completed a FutureLearn course on the liver (Liver Disease: Looking after Your Liver – University of Birmingham) provided me with a decent background to understanding the fascinating concepts Zania introduced me to. I learnt the basics of cell-in-cell structures and the vital efferocytosis that hepatocytes perform and what these processes mean for inflammation and the development of cancer and metastasis.

In the afternoon, Janine Fear lead an induction in which I was exposed to the many fascinating laboratories and ‘behind-the-scene’ areas such as the waste disposal and incineration systems, the autoclave room, and the liquid nitrogen cooling systems. One of the many surprising things I was shown was the autoclave tape – I couldn’t help but marvel at the simple yet ingenious concept of tape that changes colour when exposed to the high temperatures of the sterilisation process. Janine Fear also informed me of the lab safety procedures and that the labs I would be working in are classed as Category 2 and that the majority of the labs are fitted with Class II biosafety cabinets. As part of the induction, Janine also taught me basic skills (such as how to use a micropipette and a pipette gun) which I later utilised when performing experiments.

After the induction, I was introduced to Adam McGuinness (a PhD student) who showed me several culture flasks (containing hepatocytes and T-cells) under a light microscope. He also explained that the nutrient medium provided to the cells contains an indicator which changes to yellow as the solution becomes acidic (because CO2 is produced as the cells metabolise) – indicating that the medium should be changed. I recalled that a similar process was used by the BactAlert machines in the serology lab in Mumbai (where I shadowed Dr Salunke last year); the machine detected low pH of blood cultures (which indicates sepsis: bacteria in the blood produce CO2 – making the blood acidic). The hepatocytes cultured in this lab were from a cancer cell line (Huh7) – I learnt that cell lines are ideal for research as they can survive and replicate better than primary cells in vitro.

On day two, Zania and I discussed the role of a researcher and the many facets of the job (including the process of writing and revising publications and some aspects of what makes for a high-impact publication worthy of a renowned journal such as Cell or Nature). Our discussion then drifted back to Zania’s current research and she explained to me that the host cells of cell-in-cell structures can fail to divide as the cell inside them becomes a physical obstruction to cytokinesis. Fascinated by this concept, I spent the rest of the morning reading publications* on cell-in-cell structures which Zania recommended to me (including one written by her former PhD student, Alex Wilkinson).

In the afternoon, I observed Adam performing an experiment to image a host cell hepatocyte failing to divide due to a T-cell inside it. He explained the various fluorescent cell tracker dyes and cytoplasm stains which would be used to image the experiment. We then visited Dr Omar Qureshi who introduced us to the impressive Celldiscoverer 7 (ZEISS) capable of fluorescence microscopy and performing time-lapse imaging.

The next day, imaging experts from ZEISS explained the process of analysing the images produced by the Celldiscoverer 7; the analysis software can be configured to automatically count the number of cell-in-cell structures and identify any host cells which failed to divide.

Afterwards, I followed Adam to a meeting with the rest of the students who are taking the same PhD course (Sci-Phy). One of the major advantages of shadowing both a student and a PhD supervisor was that I gained exposure to the many aspects of completing a PhD course (such as writing a thesis and preparing for a viva) from different perspectives. During this meeting, Adam presented his plans for a mini-project which involved using machine learning to identify and quantify cell-in-cell structures.

In the afternoon, I attended an interesting lecture (presented by Alejandra Tomas from Imperial College) with Zania regarding current research into improving pancreatic beta cell function.

On the fourth day, I had the very exciting chance to do some ‘hands-on’ work; under the direction of Zania, I ‘split’ a cell culture of hepatocytes. Since the hepatocytes are from a cancer cell line, they can be repeatedly ‘split’ to allow them to proliferate and be used for various experiments. It was delightful to have this opportunity which enabled me to sharpen my skills in handling pipettes and working with the hepatocytes. I also learnt how to care for the surfaces and machines in the lab; I cleaned all the worktops in the labs with 70% IMS (Industrial Methylated Spirit) and Trigene before commencing my work, which ensures that the surfaces are sterile.

The afternoon provided another opportunity to immerse myself further in the topic of cell-in-cell structures and I read two absorbing publications: ‘Cell-in-cell phenomena, cannibalism, and autophagy’ and ‘Entosis enables a population response to starvation’. I found the latter article intriguing as it introduced the idea of host cells obtaining nutrients and biomass through phagocytosis of neighbouring cells as part of a coordinated response to long-term glucose starvation.

On day five, I observed Adam perform another experiment which explored the relationship between the number of necrotic T-cells provided to hepatocytes and the number of phagocytosis events (in which cell-in-cell structures are formed). The experiment used serial dilutions of necrotic T-cells; hepatocytes can engulf dead T-cells easily (even in low quantities). Following this, we imaged the experiment using the smaller, yet equally impressive, CX5 fluorescence microscope.

In the afternoon, we analysed the images from this experiment to yield averages for the number of cell-in-cell structures observed with the different concentrations of T-cells. I performed some manual counts with the help of an image editing software (Image J) whilst Adam began to use Matlab (a programming language) to automate this process.

On the first day of my second week with the University of Birmingham, I attended a conference with Zania (who chaired the third session) on stromal microenvironments in health and disease. A variety of speakers from the University of Birmingham (and a plenary speaker from Barts Cancer Institute) presented their research on a vast range of topics connected to stromal cells (cells that make up the supporting tissue of organs) from potential cures for rheumatoid arthritis (presented by Andrew Filer) to identifying indicators of myeloma and hallmarks of cancer (presented by Dan Tenant). These presentations helped to further enhance my understanding of the incredible research undertaken at the University of Birmingham.

Two days later, I had the pleasure of meeting visiting Professor Lucie Peduto from the Pasteur Institute. Her research lab has a strong focus on stroma which relates to the liver stromal microenvironment researched here. In the afternoon, I attended her seminar on the relationship between stromal cells, fibrosis, and cancer; having attended the conference on stromal microenvironments earlier that week allowed me to gain a better understanding of her research.

The penultimate two days of my work experience were by far my favourite; I had the exciting opportunity to perform an entire experiment single-handedly. My detailed observation of the experiments performed by Adam proved very useful as I performed the same experiment he did on day five (which explored the relationship between the number of necrotic T-cells provided to hepatocytes and the number of phagocytosis events). It felt like a culmination of all my training and learning from the past two weeks and I utilised all the skills I had learnt by shadowing and observing Adam performing experiments. In the morning of my ninth day at the University of Birmingham, I observed the cells I had split and cultured the previous Thursday and was pleasantly surprised to find that they were thriving and ready to use for the experiment. Completing this experiment helped me to develop my organisational skills as I carefully followed the protocol Adam had created and balanced different tasks simultaneously.

Overall, my work experience at the University of Birmingham has been a fantastic experience and I am extremely grateful to Dr Zania Stamataki and Adam McGuinness for teaching and supporting me throughout my time here.

* I have provided the list of publications I had the pleasure of reading in case you would also like to immerse yourself into this fascinating field:

Gram Staining

Whilst observing Sunflower Laboratory, I got the exciting chance to Gram Stain several slides. I have created a little tutorial in which I carry out the procedure (Youtube link)

Serology in Mumbai (Part II)

I was incredibly fortunate to observe another serology lab whilst in Mumbai. Learning in the Sunflower Laboratory was a completely different to experience to my previous lab observation as the Sunflower Laboratory was much better equipped with over 30 fully automated diagnostic tools. Also, at this lab the staff actually spoke English- making it a lot easier to learn!

One of the most important automated diagnostic tools at the Sunflower Laboratory was the GeneXpert Machine as it was capable of, very accurately, diagnosing tuberculosis (TB) and producing results in under 2 hours.

To understand why this is so important, it is necessary to know about India’s history with TB and the nature of the bacterium itself.

TB is a horrific disease caused by Mycobacterium tuberculosis that severely affects the lungs and causes those with the illness to cough up blood as well as experience fevers, among other torturous symptoms. It also is a granulomatous inflammatory diseases- meaning that it causes many different types of white blood cells to clump together (aggregate) to form a giant multinucleated cell that can block blood vessels. Bacteria actually use these granulomas to essentially ‘hide’ behind- preventing themselves from being destroyed by the body’s immune system.

Mycobacterium TB as seen under an electron microscope (Credit: louisville.edu)

Granuloma as viewed under a microscope (Credit:http://pathhsw5m54.ucsf.edu/overview/text.html)

India is the country with the highest burden of TB- around 40% of the Indian population have TB. Whilst observing the lab, I had the chance to talk to the lab assistants as well as some of the local people and discovered the complex cultural factors that affect the spread of TB. TB used to be considered a “poor man’s disease” due to the fact that it spread remarkably quickly in slums. This is because slums are poorly ventilated and overcrowded. In addition to this, poor people are more susceptible to TB due to malnutrition. If a person in these conditions also has any prior diseases, such as HIV, they are even more likely to contract TB.

TB has only recently lost its reputation as exclusively a disease of poverty as the rich have begun to contract TB from their maids, drivers, etc. This is also, in part, because the rich and the poor have started to occupy the same areas. For instance, my native city, Mumbai, is the location of hundreds of stylish skyscrapers, four luxurious shopping centres and a grand airport- but also home to 10% of all slums in India (Source: Times of India).

A well-known industrialist from Pune was shocked when he was detected with TB. He said that he used AC car and always ensured to keep safe distance from crowd. But then when we enquired and checked people around him, we found that his driver who lives in a city slum had TB

– Times of India

Excessive coughing caused by the high levels of pollution in India, coupled with the fact that the country is very overcrowded, allows Mycobacterium tuberculosis to spread easily through the air. In addition to this, India is, for the most part, impoverished- meaning that the majority of TB sufferers cannot afford treatments. Leaving TB untreated means that not only will their condition worsen but also, they will likely spread the disease to others around them. According to tbalert.org, “just one person with untreated infectious TB can pass the illness on to 10-15 people annually”.

(This problem is currently being combated as the government is providing free drugs to any citizen who has TB.)

Prevention of diseases via vaccine was, originally, thoroughly researched; in fact, ever since the late 1970s, the BCG vaccine that was designed to give immunity against TB has been administered to almost all citizens of India. Unfortunately, exactly 20 years later, the final results of a 15-year-long BCG trial were released, confirming that the vaccine provided no protection against TB.

(I, myself, was given the BCG vaccine as a newborn- it left a small scar that, surprisingly, is still visible 15 years later!)

Even getting a diagnosis for TB is not usually very easy; diagnosing tuberculosis based only on signs and symptoms is difficult as the typical symptoms are also shared by other major diseases. The only definite way to diagnose the disease is to actually see the mycobacterium under the microscope. This can be impractical, however, as culturing this slow-growing bacteria takes anywhere from two to six weeks.

Moreover, in some areas of India, few people actually seek a diagnosis due to the overwhelming stigma surrounding TB. People could lose jobs and women may be divorced if they were found out to have TB.

So, to recap, TB is an airborne disease that affects just under half of the Indian population and is easily spread due to the high pollution levels and population density. Many of the people who contract TB are too poor to get it treated- allowing them to get worse in their ill state and to spread it to those around them. Even if the people who suspect that they have TB were to try to get a diagnosis, they would have to wait for around a month to get a formal diagnosis and start an effective treatment course. Essentially, contracting TB in India is a terrible, even life-threatening disease, that is shockingly easy to contract.

Enter the GeneXpert Machine.

This diagnostic tool detects the Mycobacteria TB complex DNA in sputum samples and Rifampicin (an antibiotic used to treat TB) resistance associated with mutations of the patient’s genes. This impressive machine automates sample processing, nucleic acid amplification, and detects target sequences in samples using real time polymerase chain reactions (PCR) and reverse transcriptase PCR.

The GeneXpert Machine at Sunflower Laboratory

The machine uses cartridges (which the sample is added to) that hold the PCR reagents and host the PCR process. This means that the process is entirely self-contained meaning there is no risk of cross-contamination.

A cartridge that is ready to be processed by the machine

The diagnostic tools were truly awesome to learn about, however I found the manual processes for detecting other pathogens equally exciting!

One of these processes was the culturing of bacteria for visual analysis or analysis by one of the other diagnostic tools.

In order for cultured pathogens to thrive, they must grow on a culture medium that is well suited to the pathogens’ needs.

The lab mainly used three different types of Agar (a gelatinous substance derived from algae).

Blood agar- an enriched medium consisting of a low concentration of blood that is used for growing fastidious organisms (those which have complex nutrient needs) and detecting haemolytic activity as the RBCs are digested.
Chocolate agar- a type of blood agar plate made up of lysed RBCs that is used for fastidious respiratory bacteria.
MacConkey agar- a selective medium specially designed for culturing gram negative bacteria

In order to transfer the pathogens that need to be cultured onto the growth medium, a streaking method that is well-suited to the process must be chosen. At the lab, I was introduced to quadrant streaking.

(Credit : Pinterest)

After this, the cultures are incubated for a certain amount of time depending on what bacteria is being cultured. After this incubation period, the cultures can be examined by eye or a colony from the culture can be processed by one of the automated diagnostic tools. I even got to handle some of the cultures!

Quadrant Streaking on Blood Agar

…And on Chocolate Agar (This patient’s sample shows profuse bacterial growth)

Quadrant Streaking on a split plate of Blood Agar and MacConkey Agar

(Interestingly enough, in the above photo, the pink appearance of the bacterial colonies on the MacConkey Agar indicates that the bacteria is lactose-fermenting. )

All in all, I had a wonderful experience observing the Sunflower Laboratory for a total of 3 days- learning about how TB and other bacteria are being identified at this clinic in both manual and automated procedures. Learning about TB in this depth enriched my understanding of the specific disease and of how diseases can be spread, prevented, and cured. Being introduced to quadrant streaking will certainly help in the required practicals I’ll complete this year in GCSE Biology!

I am extremely thankful to Dr Aditi Salunke and Reema for this fantastic opportunity.

Shukriya (thanks!)

(A quick selfie in the microbiology lab)

Serology in Mumbai (Part I)

Recently, I have been offered the fantastic opportunity to observe a couple of diagnostics labs in India. This past week has been an amazing, immersive learning experience in the field of diagnostics and serology, in particular.

mcl

I have been observing Marve Clinical Laboratory in Malad West, Mumbai in order to understand more about the practical aspect of diagnosing diseases. Initially, I was apprehensive about this undertaking as my knowledge of diagnostics beforehand was limited only to what has been covered in the GCSE course and the occasional article I have read in the NewScientist. Thankfully, my apprehension subsided quickly as the staff were keen to give me a tour of the lab and talk about the facilities at their disposal as well as briefly go over the basics of serology and diagnostics.

The main difficulty was communication; the majority of the lab technicians could only speak Hindi and, despite being of Indian origin, I barely know enough to get by. While they were very willing to explain procedures, I could only understand the gist of it (and that’s only because the more scientific words are in English). This meant that I frequently bombarded the doctor with questions (which he very kindly answered in spite of his busy schedule) and turned to the Internet for more information.

Despite this, I gained a lot from this experience and I am eager to share this newfound knowledge with you.

Most diagnostic labs use serology diagnostics. Serology is the study of serum (the blood plasma, not including fibrinogens) and other bodily fluids. Diagnostic labs identify antibodies formed due to infection in the serum and other bodily fluids in order to diagnose diseases. At Marve Clinical Laboratory, I learnt about various techniques used to diagnose disease using serum and urine.

On my first day, I was introduced to the diagnostic tool known as a Complete Blood Count. A Complete Blood Count has a range of uses from diagnosing deficiency diseases (by looking at the patient’s hemoglobin concentration) to assessing whether the patient’s immune system is fighting a disease (by checking their lymphocyte count). This process is fully automated through the use of a CBC Machine; the machine simply takes a sample of the blood and produces the result within a minute.

bcc

After this, I learnt about Erythrocyte Sedimentation; when blood is contained in a tall, thin tube, erythrocytes (red blood cells) gradually settle to bottom (sedimentation). When a patient is suffering inflammation, their erythrocytes clump together, becoming a denser mass of cells- meaning they settle to the bottom faster. In order to measure the progression of an inflammation, the erythrocyte sediment rate is recorded; the greater the amount of sedimentation, the greater the inflammation.

An ESR Tube

At Marve Clinical Laboratory, two methods for measuring the ESR were used: the Westergren Method and an automated method. The Westergren Method entails putting 2ml of blood into a test tube containing 0.5ml of sodium citrate and storing it at room temperature for 2 hours before transferring the blood to a Westergren-Katz tube (filling it to the 200mm mark). This tube is then placed upright for 1 hour at which point the distance ‘travelled’ by the erythrocyte sediment is measured. The ESR is expressed as mm/hour.

Another important role of the lab is to carry out ABO typing (blood typing)- in some cases, knowing your blood type can be vital to survival. Your blood type depends on which antigens (proteins), if any, you have on your red blood cells. In ABO typing, the person’s blood is mixed with a reagent containing antibodies against type A and B blood. If the blood cells clump together (agglutinate), it means that the blood has reacted with the antibodies. For instance, if a person’s blood type is B, agglutination will occur with the antibodies against type B blood. The same is true with type A blood also. If a person’s blood type is AB, agglutination will occur with both sets of antibodies. If their blood is type O, there will be no agglutination. Blood typing can also tell you whether you have a substance known as Rh Factor on your red blood cells. If agglutination occurs when your blood is mixed with a reagent containing antibodies against Rh Factor, your blood is Rh positi

Since there is agglutination when the blood is mixed with antibodies against type B blood and antibodies against the Rh Factor, the blood type is B positive

In this blood sample, no agglutination occurs with antibodies against both type A blood and type B blood – so the blood type is O

Can you tell which blood type this test shows?

By far the most fascinating machine in the lab was the Biochemistry Analyser. Capable of doing an impressive 400 tests an hour, this machine was able to process samples and produce results rapidly and with very little human involvement. It is used to test renal function (RFT) and liver function (LFT) as well as to carry out Lipid Profile Tests and Blood Sugar Tests.

The specific model was the Mindray BS-210

mindray

Urine analysis, necessary to diagnose renal diseases and endocrine disorders, is often done at Marve Clinical Laboratory as well. Important factors used in diagnosis are volume of urine as well as the presence of ketone bodies, occult blood, cells, casts, and crystals in the urine.

In 24 hours, a healthy person passes 1.5 to 1.8 litres of urine. However, if a person has kidney or blood sugar problems, they will pass 2.5 to 5.6 litres. This abnormal increase in urine is known as polyuria. The opposite extreme is known as oliguria and is seen in patients with acute nephritis (nerve damage). Anuria, the complete suppression of urine, is seen in patients with renal failure.

Ketone bodies are water-soluble molecules produced by the liver from fatty acids. Ketone bodies are only produced when the body is lacking glucose as a fuel. They are present in the urine of patients with diabetes mellitus- early detection of this is paramount.

Pus cells in the urine are essentially white blood cells (mainly neutrophils), dead skin cells, and bacteria. In order to confirm the diagnosis of a disease, the number of pus cells in a urine sample must be counted; at this laboratory, this task is done manually and so I was fortunate to have the chance to try this myself. The presence of high levels (more than 10 per microlitre of urine) of pus cells in the urine is known as pyuria and it indicates infection in the lower or upper urinary tract.

Stained pus cells as seen under a microscope

Urinary casts (microscopic cylindrical structures) are formed by a diseased kidney. There are several different types of urinary casts including hyaline casts (which suggest renal disease), RBC and WBC cells (which point to glomerular issues), granular casts, waxy casts, and fatty casts (which are caused by the degeneration of tubular epithelium).

(Credit: Medical-Labs.net )

In addition to casts, crystals can also be found in acidic urine. Calcium oxylate crystals (which is seen in patients with kidney stones) is the most commonly encountered at Marve Clinical Laboratory.

Calcium Oxalate Crystals viewed under a standard light microscope. This is very similar to the crystals I have observed in some of the urine samples at the lab. (Credit: Medical-labs.net)

Upon researching this crystal further, I found the surprisingly dramatic image of these crystals under an electron microscope.

(Credit: Wikipedia)

Fascinatingly enough, Calcium oxalate monohydrate crystals can also be found in the major organs of ethylene glycol (better known as a primary agent of Antifreeze) poisoning victims. When ethylene glycol is metabolised by the body these crystals are produced in spindle or ‘picket fence’ structures.

spindle

In total, I spent 4 days observing Marve Clinical Laboratory and gained an incredibly valuable insight into the field of diagnostics and serology in particular. I discovered the practical side of many processes (such as blood typing) that I had, up until now, only read about in textbooks. On the whole, the experience was amazing and I am incredibly grateful to Dr. Thakkar and the rest of the laboratory staff for giving me this opportunity.

Shukriya (thanks!)

Curiosity Killed The Cation

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