Nikmat Perasaan

Jika kita terasa gembira kita harus bersyukur,

Jika Kita terasa sedih kita harus bersyukur,

Jika kita terasa marah kita harus bersyukur,

Jika Kita terasa cinta kita harus bersyukur,

Kerana Allah memberikan satu nikmat yang besar, iaitu mempuanyai perasaan

Second week - Histopathology and Cytology Lab

6 June- 10 June 2011 : The second week of industrial training. During this period, I have learned something new.     I learn what exactly histopathology and cytology is? Histopathology is the microscopic examination of tissue in order to study manifestation of disease. Cytology or more correct terminology using for this lab is cytopathology which is the study of cellular disease and use of the cellular change for diagnosis of diseases. Before I explain more about my experiences, I would to divide this article to several part which; general steps of histopatholgy and cytopatholgy, Histopatholgy( H and E staining), Cytopatholgy (Papanicalou staining and Wright staining), How to distinguish benign, and metastic tumour in breast cancer (histopatholgy).

General steps of Histopatholgy and Cytopatholgy

The figure below shows the general steps of histopathology and cytopathology. First step is the sample fixation. This step is crucial to get good microscopic slides. Second steps, is only for histopathology which is sample processing. The next step is sample staining. This steps basically to distinguish which part of cell are nucleus or cytoplasm. Last but not least is microscopic examination.

Histopathology

The histology samples from patients have several steps in order to diagnose their cells. It is involved several steps which; 1- sample fixation with formalin, 2- sample processing, 3- sample staining. and 4- Microscopic examination.

1: Sample Fixation

Sample fixation is a crucial step in histology studies. Fixation is a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated tissues. Fixation of tissue is done for several reasons. One reason is to kill the tissue so that postmortem decay (autolysis and putrefaction) is prevented. Fixation preserves a sample of biological material (tissue or cells) as close to its natural state as possible in the process of preparing tissue for examination. To achieve this, several conditions usually must be met:

First, a fixative usually acts to disable intrinsic biomolecules – particularly proteolytic enzymes—which otherwise digests or damages the sample.Second, a fixative typically protects a sample from extrinsic damage. Fixatives are toxic to most common microorganisms (bacteria in particular) that might exist in a tissue sample or which might otherwise colonise the fixed tissue. In addition, many fixatives chemically alter the fixed material to make it less palatable (either indigestible or toxic) to opportunistic microorganisms.

Finally, fixatives often alter the cells or tissues on a molecular level to increase their mechanical strength or stability. This increased strength and rigidity can help preserve the morphology (shape and structure) of the sample as it is processed for further analysis. In this cases, the sample from patient is immersed in 10% formalin.

2: Sample processing

To provide slides for microscopic examination, the sample must be process first. This step involved another several steps. First is sample grossing- Is the process referred to as grossing a specimen, where tissue specimens taken from routine surgical cases, autopsies, or other scientific investigations are examined, described and trimmed to proper size. The grossing method is vary from hospital to hospital. From this lab, the sample that fixed by 10% formalin is cut. The part of sample that being cut is depend on pathologist. Pathologist may choose the tumour part or healthy part as control.

Next step is sample dehydration by tissue processor which is automated or can be done manually.  The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Biological tissue must be supported in a hard matrix to allow sufficiently thin sections to be cut. For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. This is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene.The sample processing firstly immersed in formalin for 30 minutes twice, after that in Alcohol solution for 1 hour six times, next in xylene for 1 hour 3 times and in wax also 1hour  3 times.

Next is the embedding step. After the tissues have been dehydrated, cleared, and infiltrated with the embedding material, they are ready for external embedding. During this process the tissue samples are placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned.

Because Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, FFPE tissues are an important resource for historical studies in medicine. The last step for sample processing is the sample sectioning by microtome. This step can be divide by 2 which are trimming and sectioning. This step provide thin slide for microscopic examination.

3: Sample staining

Before we observed the morphology of cell/sample under microscope, first we must stained the sample. In histopathology studies, the routine stain is Haematoxylin and Eosin stain (H &E). The purpose of H & E staining is to distinguish the nucleus and cytoplasm. Haematoxylin which stains blue or purple on nuclei. Haematoxylin is oxidative agent that positively charged and bind to negatively charged phosphate on nuclear-DNA. Eosin which stains pink to red blood cell or connective tissue in cytoplasm. Eosin is acidic colourant that mainly bind to protein.Besides routine H & E staining, it is also has another type of staining which histochemistry staining and Immunohistochemistry staining. The need to do the staining is depend on the patient cases and diseases.

4: Microscopic examination

Under the light microscopes, the pathologist can observed the morphology of the cell to determine the type of tumour whether benign or malignant, the level of certain cancer and whether the tumour are metastatic or not.

Cytopathology

Cytology laboratory test can be divide by two which “Gynecological  Cytology” and “non-gynecological cytology”. This is the test that everyone known by as “Pap Smear”. It name comes from the Papanicolau test for cancer cells, developed in the 1940’s. Smears are made from cells collected from the cervix of the uterus, stained, and then examined microscopically for evidence of any abnormal cells. Non-gynecological cytology can be body fluid such as sputum,urine and spinal fluid and also fine needle aspiration.

Cytology samples are fixed in 95% alcohol at least for 30 minutes. After that, the samples are stained by Papanicalou stain. After staining, observed the morphology under  light microscope.

Principle of Papanicolou staining

       It is involves five dyes in three solutions, A nuclear stain, haematoxylin, is used to stain cell nuclei. The unmordanted haematein may be responsible for the yellow color imparted to glycogen.First OG-6 counterstain (-6 denotes the used concentration of phosphotungstic acid; other variants are OG-5 and OG-8). Orange G is used. It stains keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.

Second EA (Eosin Azure) counterstain, comprising three dyes; the number denotes the proportion of the dyes, eg. EA-36, EA-50, EA-65. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells. Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, and however it produces visually different results and is not considered satisfactory by some. Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.

How to distinguish the benign tumour and malignant tumour in the cases of breast cancer

From this lab, what interesting that I can learn is how to distinguish benign tumour and malignant tumour specific to breast cancer. During practical here, I try to compare between various slides of breast cancer patients and observed the doctor comment on the patients. By H & E staining, we know that Haematoxyline stain purple on nuclei, so that under microscopic observation I try to observed nuclei morphology on benign tumour and malignant tumour.

From microscopic observation, the nuclei in benign tumour cell are big, and clump together in one place but in malignant tumour, nuclei very small and disperse in all cytoplasm or tissues. The presence of nuclei in all tissues indicates that the tumour is invasive (malignant tumour).

Benign Breast Tumour

Malignant Breast Tumour

Diagnostic Microbiology Lab

30 May - 3 June 2011-  First week of industrial training!! My first rotation at diagnostic microbiology lab. Here the very interesting things that learned from here was how to translate the knowledge of microbiology for diagnostic purposes. In order to explain what exactly I learned from this lab, I divide to 2 part which ; The principle of Gram staining and Identification hierarchy.

Principle of Gram staining

Gram staining was introduced by Danish scientist called Hans Christian Gram (1853-1938) in 1884 to distinguish between Pneumococci and Klebsella pneumonia bacteria. Until now, no others staining can replaced the Gram staining as major tools to detection of bacteria. The purposes of gram staining is to distinguish between Gram positive and Gram negative bacteria. 

Gram staining has 4 basic steps; First the application of primary stain (crystal violet) to a heat-fixed smear of bacterial culture, followed by the addition of a mordant (iodine), after that rapid decolourisation of alcohol or acetone and lastly counterstain with safranin or basic fuchsin.

How gram staining can distinguish between gram positive and gram negative bacteria. Gram positive bacteria have a thick mesh cell wall made of peptidoglican and gram negative bacteria have thin layer of peptidoglican with additional outer membrane layer made of lipid. Crystal violet (CV) dissociate in aqueous solution to form CV+ ion and Cl- ion. These ion penetrate through the peptidoglican cell wall and cell membrane of both gram positive and gram negative bacteria. The CV+ ion interact with negatively charged components of bacterial cells and stain cells purple.

Iodine (I- or I3-) interact with CV+ and form large complex of crystal violet and iodine ( CV-I). When decolourization with alcohol  is added, it interact with the lipids of cell membrane. A gram negative bacteria will lose its outer membrane and the peptidoglican layer is left exposed. The CV-I complexes are washed from the membrane. In contrast, a Gram-positive cell becomes dehydrated from ethanol treatment. The large CV-I complexes become trapped within Gram-positive cell due to the multilayer nature its peptidoglican. After the decolourisation step, the Gram-positive bacteria remains purple and the Gram-negatives loss its purple colour. Counterstain, which is usually postively charged safranin is applied last to give decolorised Gram-negative bacteria a pink or red colour.

In clinical diagnostic purpose, it  is important to distinguish between Gram-positive and Gram negative bacteria is to screen the pathogen. Most Gram-negative bacteria are pathogenic but not in Gram-positive bacteria. Gram-positive bacteria depend on species of bacteria, type of samples, and also how they colonized  part of the body. If certain bacteria colonized to much in one area of the body, it may due to immunodeficiency that cause recurrent bacteria infection. After we identify whether it Gram-positive or Gram negative bacteria, to identify the specific species of bacteria we followed the identification hierarchy.

Bacteria Identification Hierarchy


After we stain bacteria via Gram staining and observe under the microscope, we can distinguish whether this is Gram positive bacteria or Gram negative bacteria. After that we followed the bacteria identification hierarchy to screen which specific bacteria species whether its pathogenic or not? Bacteria identification hierarchy can be divided by two which Gram positive  bacteria identification and gram negatives identification  hierarchy:

The figure above shows the identification hierarchy for gram-positive bacteria. By observing the gram-staining of bacteria culture under microscope, if the bacteria cultured stained purple, we observed its morphologwhether it shape cocci or bacilli, if cocci we can further down the hierarchy whether its Staphylococcus or Streptrococcus. So Catalase test must be done to both of them. The principle of  catalase test is all the Staphylococcus bacteria have catalase enzyme with act on hydrogen peroxide to release oxygen.  In this test, we can observed the immediate bubbles.

After we know the bacteria is Staphylococcus, we further down the hierarchy, at this point this is very important. Because we must do Coagulase test to distinguish whether the bacteria culture is the Staphylococcus aureus or not. S.aureus is well known dangerous pathogenic bacteria. Because the bacteria culture from patient sample, this is important to know whether the patient have S.aureus or not. The principle of coagulase tests is to know whether the bacteria have coagulase enzyme or not? S.aureus contain coagulase enzyme which catalyze the formation of fibrin clot in plasma. In this test we can observed the clotting/ clumping formation

The figure below is the gram negative bacteria identification hierarchy. As mentioned above, most of gram negatives bacteria are pathogenic. To identify the specific gram neagtive bacteria, just follow the identification hierarchy.

Both Gram-staining and Identification Hierarchy is the basic knowledge of diagnostic microbiology. Actually that has more diagnosis. For example, biochemical test and also sensitivity test. Right now, the more fast and accurate in bacteria identification that available in kit such as API test kit. As conclusion, what I learned from the industrial training in diagnostic microbiology laboratory is how I see the very simple Gram staining principle  is very useful in diagnosis of diseases. Furthermore, in analysis what exactly bacteria infect the patient, besides we analyse what the type of bacteria  in patient's samples, we must know the medical history of patient and also we must observed whether the bacteria is colonized enough in patient body to be considered its pathogenic.

Clinical Diagnostic

It time for Industrial training !! One of elective course in Genetic and Molecular Biology Bsc. program of  University of Malaya. I choose this course for  completing my degree because perhaps I learn something that can increase my knowledge and experience.

Its true!! This industrial training like an oasis of knowledge.From 30 May 2011- 22 July 2011, my industrial training begin at Advanced Medical And Dental Institutes (AMDI)  . Generally my industrial training all about clinical diagnostic. Within 8 weeks, my task is to do lab rotation per week which diagnostic microbiology lab, histopathology and cytology lab, haematology lab, chemical pathology lab and lastly at genetic lab. from 6-8 weeks, this duration for mini-project.

The exposure to the clinical diagnostic give me an excitement. From the beginning, my education style always about theory. In University of Malaya, it has different approach, they related knowledge with research. But the exposure to clinical diagnostic makes me see the science world bigger. It make me feel every second in my routine life is about science.Besides that clinical diagnostic makes me more appreciate about how important to keep our body in good health!!!