Lab 1: liver transplantation

Liver transplantation is a surgery to remove a diseased liver and replace it with a healthy liver.

This kind of surgery has been done for more than 38 years.

Many people have had liver transplants and now lead normal lives.

When a patient receives liver transplant, his or her entire liver is removed during surgery. It is then
replaced, either a new complete or a portion of healthy liver.

- Ability to regenerate; higher success rate.

Reasons for needing a liver transplant:

Liver transplantation is needed for patients who are likely to die due to liver failure. Many diseases can
cause liver failure, the most common on is cirrhosis which is the scarring of the liver tissues. Most
commonly cirrhosis is caused by hepatitis C virus in adults.

Other Conditions which require transplants include diseases of bile duct; gall stones; biliary cholic, biliary
atresia (it is a blocking in tubes that carry bile from the liver to gall bladder. This blockage may occur due
to developmental error. This disease is present in infants and most commonly causes liver failure. Biliary
atresia appear after 8 weeks of birth (jaundice, clotting factor lack leading to bleeding, controls hunger));
primary biliary cirrhosis/primary biliary cholangitis: autoimmune disorder which relates to progressive
destruction of bile ducts which leads to buildup of bile and toxins in the liver and this condition is known
as cholestasis.

These all conditions are the major cause of liver failure.

Inherited disorders:

Wilson's disease: it is associated with dangerous levels of copper buildup in the body which, if not
treated, these copper levels damage liver, brain and cornea.

Hemochromatosis: it is associated with abnormal buildup of iron in the body especially in the liver.

- Many techniques, injections to mobilize the iron to stool.

Primary liver cancer or cancers that affect only liver.

Another reason is alcoholism which can cause cirrhosis also leads to liver failure.

Liver transplant in adults and children:

The donor operation is done through an incision in the upper abdominal layer. If the LDLT recipient is a
child, only a portion of the left part (smaller lobe) of the liver is removed from the donor and about 25%
of the donor liver is required for use.

If the LDLT recipient is an adult, a larger portion of the liver needs to be removed from the donor.
Usually the right portion of needs to be removed from the donor. Usually the right portion which is
about 60% of the donor’s total liver is required.

The blood vessels supplying the portion of the liver to be removed are separated out. The liver itself is
divided and the portion to be transplanted is removed. This portion is separated out in different
operating rooms.

Transplant is then performed. The donor operation takes 6-8 hours.

Liver transplant symptoms:

People who have liver diseases may feel many problems including:

1. Jaundice – yellowing of the skin and eyes

2. Itching – toxins; skin problems
3. Dark tea colored urine
4. Gray or clay colored blood movements
5. Ascites – swellings
6. Abnormal buildup of (interstitial) fluid in abdomen
7. Vomiting
8. Blood vomiting – lack of clotting factor synthesis in liver leads to internal bleeding
9. Mental confusion and forgetfulness – lack of glucose to brain; disturbed brain function

Types of transplant:

There are two types of liver transplants:

1. LDLT (living donor liver transplantation

2. Cadaveric liver transplantation

1. LDLT:

Living donor liver transplants are an option for some patients with end stage of liver from a healthy
living donor and implanting into a recipient.

Both, the donor and recipient liver segment will grow to the normal size in a few weeks.
The donor in LDLT may be a relative or friend. Blood type and body size are critical factors in
determining appropriate donor.

The LDLT donor must be active so the chances of transplantation are with excellent success rate.

2. Cadaveric liver transplant:

In this type of transplantation, the donor may be a victim of an accident or head injury but the donor’s
heart is still beating but the brain has stopped functioning. Such a person is considered legally dead
because his/her brain permanently and irreversibly stopped working.

The heart continues to beat because the donor is attached to a respirator. It delivers an adequate supply
of oxygen to all vital organs. In some hospitals, the death is kept confidential.

Test required before getting liver transplants:

1. X-ray
2. Liver biopsy slides
3. Previous doctor records
4. Record of all medication
5. Update all previous tests
6. Computed tomography – this test uses x-rays and computer to generate picture of liver which
shows size and shape of liver
7. Doppler ultrasound – which helps to determine blood vessels of the person.
8. Echocardiogram – to find out the proper function of heart
9. Pulmonary function test – determines lung’s ability to exchange of CO 2 and O2
10. Blood type
11. Clotting ability
12. Biochemical testing
13. AIDS and hepatitis must be tested before.

Surgery of recipient:

Liver transplant surgery takes 6-12 hours of recipient. During it, doctor removes diseased liver and
replaces it with donated liver.

Most patients stay in hospital for up to 3 weeks after surgery.

The surgeon will disconnect diseased liver from bile ducts and blood vessels before removing liver.

The blood flow in the liver will be blocked or sent through the machine to return to the rest of the body.

The surgeon will put the healthy liver in place and reconnect it to the bile duct and blood vessels.
After surgery:

The stay in hospital is 1-3 weeks according to the liver working.

In this duration, medicines will be continued to prevent rejection of new liver and to prevent infections.

- Opportunistic – so they are kept in ICU – sterilized environment

Doctor will check the internal bleeding.

Return to food: liquid  solid  semi-solid

Complications associated with liver transplants:

Two common are rejection and infection.

1. Rejection:

Immune system works to destroy foreign substance that invades the body.

The immune system cannot distinguish between transplanted liver and unwanted invaders such as virus
and bacteria.

Therefore, immune system may attempt to attack or destroy new liver, this is called rejection episode
after transplantation.

About 70% of liver transplant patients have some degree of organ rejection prior to discharge.

Anti-rejection medications (immunosuppressants) are given to these patients to avoid immune attack.

2. Infection:

Because of the anti-rejection drugs that suppress the immune system, there is increased risk of
infection. This problem diminishes as time passes and most infections can be treated successfully as
they occur.

Side effects of liver transplant:

1. Fluid retention
2. Raised BP
3. Headache
4. Diarrhea
5. Nausea
Lab 2: Options in diagnostics of blood-borne infections (yesterday, today and tomorrow)

In the first 50 years in the 20th century, many diagnostically useful tests were developed. Primarily, they
were focused on making diagnosis and therefore, applied after a physician had narrowed the differential
diagnosis to shortlist the possibilities. The main goal was diagnosis and treatment, if possible.

In the same period of time, serology, as a laboratory discipline, was established. Serology is the scientific
study or diagnostic examination of blood especially with reference to the response of immune system to
pathogens or introducing substances in the body.

Antibody-antigen detection developed different tests like:

 Gel precipitation
o Immunodiffusion
o Counter immunoelectrophoresis
 Agglutination
o Direct
o Passive
o Inhibitory
 Radioimmunoassay
o ELISA
 Flow cytometry – minute sample ag-ab

The sensitivity of various immunoassays differs and with each new method, increases.

The specificity of the diagnosis will also increase with new methods.

 Monoclonal antibodies
 Recombinant antigens

In the 21st century, with automation and molecular probe techniques and bioinformatics, clinical
laboratory has shifted from diagnosis to prognosis and:

 Risk stratification
 Treatment selection
 Patient monitoring

Lab 3: Epidemiology

Epidemiology is the study of diseases in population of humans or other animals.

Epidemiological evidence only shows the risk factors associated with the higher incidence of disease in
the population exposed to the risk factor.

- Europe  AIDS
- Atomic bombing  mutations
- Different risk factors according to region

Epidemiology is the study of how often disease occurs in different groups of people and why.

Epidemiological information is used to plan strategies to prevent illnesses and as a guide to

management of patients in disease has already developed.

Events in which epidemiological study is used:

1. Environmental exposures (lead and heavy metals)

2. Air pollutants and other asthma triggers (CO, etc)
3. Infectious diseases (food-borne illness – typhoid fever; food poisoning – botulism; pneumonia,
malaria, influenza)
4. Injuries (e.g. traffic accidents; domestic violence)
5. Non-infectious diseases (localized or widespread rise in particular type of cancer in one area)
6. Increase in major birth defects (e.g. in Japan, Hiroshima, Nagasaki  radioactive waves; iodine
deficiencies – lack of thyroid hormone  developmental error; congenital disease)
7. Natural disasters (e.g. earthquakes – Japan, china; volcano eruption)
8. Terrorism (why it is increasing, are they not getting rights so they rebel and turn to crime; lack of
food)

Objectives of epidemiological research:

1. To identify the causes of diseases – etiology

2. To determine the extent of diseases – mortality rate
3. To study the progression of diseases
4. To find out the preventive and therapeutic measures for a disease or condition
5. To develop the public health policies – polio vaccine till 5 yrs of age

Types of epidemiological research:

 Types of epidemiological
research

Non-experimental Experimental research

(observational studies) (interventional studies)

Population- Individual- Non-randomized

based based Randomized control trialquasi-
trials or clinical trial experimental field
trial

Descriptive Analytical Descriptive Analytical

Cross- Case-
Cohort
sectional control
study
study study

Experimental research is also called interventional studies  patients’ or participants receive some
intervention, usually medicines. This study is used in drug development

- Randomized control trial or clinical trials  control group and drug given group
- Non randomized  community –based quasi experimental
- Non-experimental studies  observable changes
o Population-based
 Descriptive – health surveys
 Analytical – ecological studies
o Individual based
 Descriptive – case studies
 Analytical – 3 types
1. Cross sectional study is the observational-based study and a type of descriptive research in
which we see the prevalence of specific disease in a specific area
2. Case control study  this study requires case reference which link with the specific outcome
e.g. population has smoking rate increase, so outcome → lung cancer; thus smoking is linked
with lung cancer. This study is designed to help determine if an exposure is increased with
smoking can associate with lung cancer or cannot associate with lung cancer.
3. Cohort study, also known as follow up study. In this study we use the similarity of different
groups
  Healthcare workers → exposed
 Females → oral contraceptives
 Iron deficiency
 Managers → sedentary lifestyle

Lab 4: ethical issues in epidemiological studies:

1. Planning an epidemiological research:

1. Formulating a research topic should focus on the health and welfare of the population and
should not be based on feasibility alone.
2. Selection of research should benefit the population
3. Ethics demand that area where trust is needed
4. This area may exist in literature on that aspect and priority importance to the public should be
emphasized than the research

2. Study design:

The study design should be proper for the research. It should be addressed specifically in the research
protocol

3. Conduct of study:

Conduction of study with research question that is needed to be answered, the study must have sample
size and selection of standard protocol.

4. Informed consent:

Informed consent is another important aspect in epidemiological research in which it is important to

explain the procedure carefully and preferably to locals, keeping in mind local cultures and traditions.

The risk and hazards in any procedure if involved in the study should be properly explained and every
effort should be made so that the expected participants clearly understand.

5. Confidentiality:

Confidentiality of any data obtained should be guaranteed.

6. Analysis:

Plan for analysis of the study should be developed ahead of time, before the study is conducted.

7. Publication:
The study should be published so that it is easy to use or access for the normal people.

Lab 5: Molecular diagnostics of viral blood-transmissible infections:

Nucleic acid amplification technique (NAT) for viral testing have experienced a rapid development
during the last 30 years and have been used in the diagnosis of infectious diseases particularly in viral
diseases.

NAT has reduced the use of viral culture based method in serological assays because of faster
turnaround, time and significant shortening of window period.

 Window period is till the antibody is produced after the incubation period.

The introduction of fully automated platform has allowed molecular diagnostic laboratory to report
sensitive and accurate results.

NAT provides faster, highly sensitive, specific and accurate results which improves patient care.

Do use of molecular techniques such as real time PCR (polymerase chain reaction) in which sequence-
specific oligonucleotide probe (PCR-SSOP) for virus detection, genotyping and quantification have high
sensitivity and reproducibility.

A great number of qualitative and quantitative molecular virus assays mostly based on RT PCR
technology. Automation of these methods provide analysis of smaller volume of sample, ease in
performance and speed, low contamination risk, as well as better standardization.

NAT testing is implemented in many developed countries in blood screening for detection of HIV, HCV,
and HBV that is a priority for blood transfusion all around the world.

Emerging and re-emerging agents also can be detected by this technique including chikungunya,
dengue, West Nile virus, malaria; these infections are also transmissible via blood.

Lab 6: Flow Cytometry:

In biochemistry, flow cytometry is a laser-based biophysical technology employed in cell counting, cell
sorting, biomarker detection (C reactive protein, cancer cells) and protein engineering by suspending
cells in a stream of fluid and passing them by an electronic detection apparatus.

It allows multi-parametric analysis of physical and chemical characteristics of thousands of particles per
second.
Flow cytometry is routinely used in diagnosis of health disorders, especially blood cancers in basic
research.

This Technique is also used to purify population of interest.

Components of flow cytometer:

Flow cytometer has five basic components:

1. A flow cell: flow cell is a cell from which liquid sample can flow. This cell aligns the cells so that
they pass single file of cell through the light beam for sensing.
2. A measuring system: it is commonly used for measurement of conductivity. This system consists
of:
i. Optical system – consists of lamp (mercury, xenon)
ii. High power water cool laser – argon, krypton, dye laser
iii. Low power air cooler laser – made up of argon
iv. Diode layer – these are lamps providing light signals when interacted to the specific cell
population in the sample.
3. Detector and analogue to digital converter (ADC): this system basically converts analogue
measurements of ‘forward scattered light’ (FSL) and ‘side-scattered light’ (SSL), as well as the
specific fluorescent signals into digital signals that can be processed by binary computer.
4. An amplification system: amplification system Produce actual signal proportional to input
signals.
5. A computer: to analyze the report.

Application:

Flow cytometry is successfully used in study of:

 Apoptosis
 Quantification and measuring of DNA degradation
 Mitochondrial membrane potential
 Permeability changes
 Cell pigments – chlorophyll
 Study of cell surface antigens
 Total DNA content of cell
 Total RNA content of cell
 Enzyme activity in different cells
 Used in the field of molecular biology, pathology, immunology, plant biology, marine biology
 Have a vast application in the field of medicine, especially in transplantation, haematology,
tumor immunology, chemotherapy, genetic disease detection, etc.
Lab 7: Serological diagnostic of blood-borne infections:

Reported blood borne infections are due to virus, bacteria, protozoa, and nematode agents.

Obligatory serology tests in different countries is very important and blood donor testing, including:

 HBsAg – hepatitis B surface antigen

 Anti-HCV – antibody of HCV testing
 HIV ag/ab
 Anti-TP (treponema pallidum)
 Anti-CMV
 Anti-EBV (Epstein-Barr virus)
 Anti-Toxo (T. gondii) → gonorrhea
 HTLV – I/II – human T lymphocytic virus type I and II
 Hepatitis A, D, and E.

Serological testing or diagnostic used from 1970s to present and now progress is very good. By
development of new methods, new markers and highly sensitive test. This testing also decreases the risk
of post-transfusion infection as well as morbidity.

The sensitive serological testing includes enzyme assays: ELISA (enzyme-linked immunosorbent assay),
CMIA (chemiluminescent microplate immunoassay), ELFA (enzyme-linked fluorescent assay) – latest
version of enzyme assay.

ELFA and ELISA work in a similar manner but ELFA decreases the window period of diagnosis. It may
diagnose in about within 5 days of infection, while ELISA requires more days.

These all tests of serology are approved internationally by ISO (international organization of
standardization).

Immunoblot test called Western blot, used for protein estimation, protein markers of cancers, antigens,
antibodies as well.

Lab 8: Analysis and research of serological testing of solid organ donor

The success of transplantation and graft survival are closely related to the absence of infection of
recipient by infected graft, reactivated recipient, latent infections, nosocomial infections, and
community acquired infections.

- Nosocomial infections → occurs by microbes resistant to antibiotics → difficult to treat

- ACs have streptococcus in them
The exact risk of infection associated with organ transplantation is unknown but correlates with multiple
factors including epidemiology of specific infection, exposure, tissue tropism (it is capacity or capability
of cell or tissue of host to help or support growth of particular virus or bacteria like HIV – CD4 cell
decreases), and the transmissibility of potential pathogens through transplantation and from the
recipient’s side, it depends on the type of immunosuppression.

In USA, the highest number of transplanted organs approx. 30,000 per year and the infection is
estimated about 1%.

During transplantation, transmitted infections include viral infections (HBV and HCV, HTLV I & II, West
Nile virus, rabies, HIV, virus B19), bacterial infections (mycobacteria, meningococcus, treponema
pallidum), parasitic infections (plasmodium, toxoplasma → gonorrhea, trypanosoma → trypanosoma
cruzi).