LAST WEEK

Finally it’s the end of SIP and I guest everyone would be busy with their MP now. This would be the last entry and we are done with it! Alright here goes..

This week I would introduce a test done in serology called SERODIA-ATG .

Intended use

SERODIA-ATG is a semi-quantitative microtiter particle agglutination test use for detection and titration of thyroglobulin Abs in human serum.

Introduction

Autoimmune disease such as chronic thyroiditis (Hashimoto’s disease) may commonly produce Abs to thyroglobulin or microsomal Ag of the thyroid. Other than being found in thyroiditis, these Abs may be found in other thyroid disorders such as primary myxedema, hyperthyroidism, goiter and thyroid tumors.

Thyroglobulin Abs can be demonstrated by several procedures, such as passive agglutination. SERODIA-ATG is prepared using gelatin particles sensitized with purified thyroglobulin. As thyroid autoimmune disease may demonstrate an immunological response to Ags other than throglobulin, SERODIA-ATG would be recommended to use in conjunction with clinical findings or other immunological thyroid test.

Principle of the test

This test is based on gelatin particle carriers sensitized with thyroglobulin, extracted and purified from human thyroid tissue. Serum containing specific Abs will react with the thyroglobulin-sensitized coloured gelatin particlesto form a smooth mat of agglutinated particles in the microtitration plate. Negative reactions are characterized by a compact button formed by the settling of the nonagglutinated particles. The test is designed to be used with microtitration techniques.

Materials

- Reconstituting solution: for reconstituting the sensitized and unsensitized particles
- Sample diluents: for diluting human serum in assay
- Sensitized particles
- Unsenitized particles
- Positive control
- Dropper: to dispense approximately 25µL per well

Test procedure

1. Use a ‘U’ shaped microplate sideways. One row consisting 12 wells is necessary to test one
patient sample.
2. Drop 2 drops of sample diluents into the first 2 wells and 3 drops into the third to twelfth
well.
3. Using a pipette, add 10µL of positive control or patient’s serum into the first well. Mix well
by pipetting u and down several times. Then transfer 25µL of the diluted serum or control
from the 1st well to the 2nd well. Mix well again and transfer 25µL to the 3rd well. Repeat
mixing and transferring till the twelfth well.
4. Drop one drop of Unsensitized Particles into the 2nd well and drop one drop of Sensitized
Particles into the 3rd to 12th well.
5. Repeat the above step for every patient sample and positive control.
6. Mix the contents in the well by tapping the plate with finger. Then cover the plate and
place on a level surface. Allow it to stand for 3 hrs at room temperature.

Final dilution of each well:
Well 1- 1:6
Well 2- 1:27
Well 3- 1:100 (10)2
Well 4- 1:400 (20)2
Well 5- 1:1600 (40)2
Well 6- 1:6400 (80)2
.
.
.
Well 12- 1: 26214 (5120)2

Interpretation of results

Settling patterns of particles:
1. Non-reactive (-): particles are concentrated in the shape of a button at the center of well.
There is a smooth round outer margin.
2. Indeterminate (+): particles are concentrated to form a compact-ring shape with smooth
outer margin.
3. Reactive (+): particles form large ring with a rough multiform outer margin. Peripheral
agglutination occurs.
4. Reactive (++): firmly agglutinated particles spread out covering the bottom of the well
uniformly.

Expected results

Thyroid Abs are seldom found in serum of normal patient. However, 2 to 17% of the normal population may show signs of low titers of these Abs with no symptoms of disease. This happen more on women and increases with age. The occurrence of these Abs may also indicate that there’s previous autoimmune disorders. Patients with low titer of Ab should be tested periodically as the presence of the Ab may be an early sign of autoimmune disease.

In active cases of thyroid autoimmune disease and in some cases of thyrotoxicosis, moderate (1:1600) to very high (1:25600) Ab titer may be observed. The observation of very hugh Ab titers in an individual with a firm, hard, fast-growing, symmetrical goiter strongly suggest Hashimoto’s goiter.

Note: in any case of reactive result at any dilution should be interpreted in accordance to the clinical findings. Diagnosis of thyroid autoimmune disease should not be based on the test alone but in conjunction with other immunological tests, physical examination, familiar studies and if necessary, biopsy.

Sharon
Tg01

Last second week..

YOzzz..... finally this will be the last posting from me.. i bet most of you guys cant wait for it to end..

I will post on my SIP work for this post cause you all might be interested in what i am doing.

Most of the time during my SIP, i will be doing on paper work. First to photocopy all the purchasing orders, the invoices, delivery orders/service reports. Then i will need to update the purchasing orders into a folder so that everything can be easily located and to keep track. Purchasing orders are actually forms filled in by TSOs or lecturer to buy certain stuffs from a specific company and this purchase will be needed to be approve by a person with higher authority. After which the purchase orders can be process. After purchases are made, the company will give invoices and delivery orders/service reports whereby the invoices will be updated by me and send to finance department to make the payment. All documents are to be file according to the purchase made into different files. Other than purchasing, i will also update ont the estimated price and the actual price for chemicals and acessories items(use every semester for purchasing). Estimated price is a reference to buy items and to prevent over badget. After getting the quotation from different companies, we will choose the company that fulfill our criteria, we can then update on the actual price given by the company. Usually for chemicals, we will try to get from the same company from the previous purchase to ensure that the chemicals content will not vary too much that they will affect the results of experiments.

Other than those paper work, i will also help in doing some preparation in the lab. As my TSO is in-charge of food preparation labs, thus i will have to help out in the food lab. I need to prepare media before classes which is quite simple.

The tiring part is when it come to checking the items in the lab. In food lab, there will be around 12 sets of items, each set meaning 4 drawers containing all the cutlery and 2 cupboards containing the pots, pans, plates, bowls etc. So i will go around checking if all items are in their place. This may sound easy but it is not as a lot of the items will be misplace by the students and i have to search for them one by one. Just thinking of it make me feel tired.

Other than that, i will also check if the ovens are in working condition, clean the water bath, check the first aid box to see if there is anything to replenish. Oh and i also help to validate the micropipettes. Some of the equipments in labs are validate by TSO over a period to check if the equipments are working normally. If there is any abnormality, they will then send them to external to calibrate them.

Alright that's about all that i did for my SIP. Enjoy yourself for the last week(but remember to continue working at the same time)!!!

Justina
0605950E
(opps i really forget bout my name)

Tan Zhao Rong
Tg01
Pathology/Biochemistry Lab

For the past two weeks in the pathology lab, I've been trained to carried out RBC cholinesterase testing. Here goes..

What is Cholinesterase?
Cholinesterase is an enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid. They are important in the proper functioning of the nervous system in human, other vertebrates and insect.

There are mainly two types of cholinesterase in human are Acetylcholinesterase and Pseudocholinesterase. Acetylcholinesterase are mainly found in nervous system, RBCs, lungs, spleen and grey matter of the brain while Pseudocholinesterase are mainly found in heart, liver, pancreas, serum and white matter of the brain.

Why is Cholinesterase (ChE) measured?

1. Monitor pesticide poisoning

Pesticides can be ChE inhibiting and may enter the human body through skin absorption, inhalation and ingestion. When individual is overexposed to ChE inhibiting pesticides, these pesticides will combine with acetylcholinesterase at the nerve endings in the brain and nervous system, resulting in cholinesterase inhibition. Acetylcholine will build up causing symptoms of pesticide poisoning to show. Therefore, ChE measurement is a useful indicator of pesticide poisoning.

2. Test Sensitivity to Succinylcholine

Succinylcholine is a short acting muscle relaxant administered during surgery. It is hydrolyzed and eliminated by ChE. Therefore, individuals without sufficient ChE or with certain genetic enzyme variants may be unable to metabolize the drug quickly, resulting in prolonged apnea.

3. Alzheimer Disease

In Alzheimer’s disease (AD), the production of acetylcholine is decreased. Cholinesterase inhibitors (ChEI) are drugs prescribed to treat symptoms resulting from the early and middle stages of AD. ChEI block the activity of ChE, thereby making more acetylcholine available to nerve cells in the brain. Monitoring cholinesterase levels is often used for therapeutic drug monitoring purposes.

How is RBC cholinesterase measured?

a. Wash EDTA blood 1 time with 0.9% saline.
b. Centrifuge to pack the cells at 3000rpm for 10 minutes.
c. Remove the saline and buffer coat completely
d. Pipette 200ul washed packed cells into 200ul normal saline to obtain 1:1 RBC suspension.
e. Mix the suspension.
f. Pipette 50ul of 1:1 suspension into 500uL of 0.5% saponin for lysis.
g. Mix well and stand at 2-8 degree C for 10 minutes.
h. The haemolysates would then be ready to send for testing for ChE at LX20.
i. The packed cell volume (PCV) is determined by filling a hematocrit tube with the 1:1 suspension from step d.
j. Centrifuge at 12000 rpm for 5 minutes.
k. Read off PVC value from a calibration rule.
l. Divide ChE value by PVC and multiply by 100% to determine the actual ChE value.

Histopathology

SHAVING

Hi guys!
This is the last month of our internship. Wonder how you guys feel. I think time really flies. 20 weeks of attachment and 16 (or rather 17) weeks of it has already gone! :)
This month, I was assigned to.. Shaving of tissue blocks!
The pro is- this does not really require much brain cells but you will discover the trivial things that affect shaving.
The con is- it is pretty tedious having to shave 500 blocks every morning. I’m going to develop muscles soon.

Shaving
Shaving is the common term for rough cutting which is to remove the excess wax on the paraffin block to expose the tissue. This is very important for obtaining full-face sections during Microtomy. Shaving is quite similar to Microtomy just that shaving is done at 20um while Microtomy is done at 4um. After shaving, the paraffin blocks will be soaked in fabric softener for 5 min to soften the tissues while those containing bones or stones will be soaked in RDO (acid) and be the last batch of blocks to be shaved. So the blocks in RDO will be decalcified for quite some time since they will be the last to be shaved. The paraffin blocks are washed with tap water after soaking and are then cooled on the cryoplate (coldplate) before they can be sectioned.

Things to look out for:
- Warm blocks: Blocks that have yet to cool down after embedding are not ideal for shaving. They are too soft and the tissues can drop out easily due to the lack of support. Also, it is difficult to fit the block onto the block holder without ‘poking’ your finger into the warm and soft wax. This will cause a depression on the surface of the block and one may need to shave deeper to get a fully-exposed surface.

Solution: Cool the blocks in ice water (ice scraped from the inside of the fridge) for immediate shaving

- Wax on the sides of the tissue cassette: Excessive wax used during embedding will overflow and form an extra width around the cassette so the block cannot fit into the block holder.

Solution: Melt the surrounding wax on the heating block

- Tiny tissue: There are tissues the size of a ‘full-stop’ in this entry and sometimes they are not dyed (so they are white/colorless). Excessive shaving can result in block exhaustion which means the entire tissue is gone (irreversible effect). So one has to be extra careful with these blocks.

Solution: Instead of shaving at 20um, shave at 10um. And also, once the block is almost fully-exposed, stop shaving. The medical technicians sectioning can trim it at 4um.

- Thin tissue: The same as ‘tiny tissue’. If the tissue is a trucut (very small and thin), do not shave the block and just pass it to the medical technicians who will shave at 4um.

- Hard tissue: Hard tissues such as fibroids and bones can cause a loud sound during shaving. Cutting these calcified tissues will damage the blade, causing kinks which will produce score lines on the tissue sections.

Solution: Try to use one side of the blade (the more blunt side –all shaving blades are used blades from routine Microtomy) specially for hard tissue and change the blade once it is blade or with kinks. Always soak these shaved blocks in RDO for decalcification.

- Uneven surface of tissue: This can be due to a few reasons. Improper embedding of tissues (not flat on the mould), nature of tissue (eg. Bone), and improper cutting of tissue during trimming by pathologists. All these are inevitable so it is common to have blocks with uneven surface. These blocks will require deeper shaving done to expose a full surface. However, this runs the risk of exhausting one side of the tissue even before a fully-exposed surface can be obtained.

Solution: Re-block the block. However, if it is not caused by improper embedding, even after re-blocking, it will be uneven and the medical technicians would have to shave and section the block by adjusting the position of the block.

- Presence of sutures or staples: Similar to presence of bones and stones, shaving cannot be done smoothly. These sutures or staples are used in operation to hold the organ together or to be in the correct orientation. However, they are missed out during trimming and embedding because they are too small. So they are usually spotted during shaving and they can cause score lines.

Solution: One can only remove the sutures or staples immediately with pilers or forceps once they are detected.

Shaving of blocks is not that difficult but speed is quite important. Sometimes (rather most of the time), when there are more technicians sectioning, it is not easy to catch up with their speed as there is only one person shaving the blocks. It takes the cooperation of others to make work easier. Eg, when the blocks are embedded flatly, a few turns on the handle will do the job, but if it is not properly done, it will take quite a while to obtain a fully-exposed surface.

Ting Ying Chee
TG01

Immunological Fecal Occult BLood

Hey guys!

Actually I promised you guys to continue the part II of my urine FEME, but I realised Azeimah had already done a very good job! Haha yup so think today I will share with you guys what I'm doing for my MP instead :)

I believe many of you guys had read on quite a few posts on testing for fecal occult blood(OB) using the guaic-based stool card. This is a screening test to detect for bleeding from anywhere of the GIT, particularly for colorectal cancer.

For my MP, i'm researching on the incidence OB. But instead of using the chemical method of stool card, my lab uses a machine called OC-Sensor DIANA.

OC-Sensor DIANA


OB casette

Pic 1: The whole OB casette
Pic 2: The body of OB casette and the 'cap' of OB casette
Pic3: The tip of the 'cap' of the OB casette where we smear stool around it and cap it back to the casette body

This is an immunological method that uses the principle of latex agglutination reaction and an optical measurement method. The method is quite simple actually... the stool usually arrives in an OB casette. After that we just need to place the casette into rack and run the machine to get the results!

How it works?
If a person suffers from OB, there will be present HbAo hemoglobin in the stool.
OC-Sensor DIANA uses a latex reagent, which is prepared by sensitizing anti-human HbAo antibodies to polystyrene latex particles, to detect for the hemoglobin in stool. Upon addition of stool sample to the latex reagent, a latex agglutination will result due to the reaction of sensitized HbAo antibodies to HbAo hemoglobin in the stool sample.

Sensitized HbAo antibodies(in latex reagent) + HbAo hemoglobin(in stool) = latex agglutination

This reaction is then analyzed accordingly to the change in optical density. The higher the concentration of HbAo in the sample, the greater the change in the optical density.

Why is immunological method better?
-Unlike the stool card that indicates positivity through colour changes, OC-Sensor DIANA is able to provide an exact value of the amount of OB in the stool. If the optical density > 100 ng/mL, it indicates that the person is suffering from OB. Hence with the exact value known, we can tell whether the patient is suffering from mild or serious fecal occult bleeding.

-If using the stool card, a patient need to avoid a variety of food 24-72hrs before the test to prevent false results. This is because the hydrogen peroxide used to detect for OB can be interfered by many food. For OC-Sensor DIANA, no dietary restriction is required.

-For OC-Sensor DIANA, the sample can be kept for 21 days if refrigerated. But for stool card, if the stool is harderned, the colour change will not be obvious.

Disadvantages of immunological method....
The only disadvantage of using OC-Sensor DIANA is that the test must be performed in laboratory. For stool card, if you have the necessary materials you can simply perform the test yourself at home. So the stool card is a more conveneint method.

Week 15

Finally, it’s my third entry. For this entry, I decided to post about Occult Blood Test.

As many of you would have already know that Occult Blood Test is to test for occult blood in the fecal and I believe it is a very common test to be carried out in many labs. However, different labs may be using different kit or materials and methods so I decided to share this method that I learned from my lab.

My lab uses this slide call Hema-Screen Slides which is a guaiac slide test for the qualitative detection of occult blood in fecal. It can be used to diagnose some gastrointestinal disorders and is usually used in routine physical examinations, routine hospital test, and mass screening for colorectal cancer. The occult blood detection is very important in many gastrointestinal diseases. The existence of occult blood can mean that there’s gastrointestinal pathology like hemorrhoids, diverticulitis, fissures, colitis or even colorectal cancer. Hema-Screen provides an easy, cheap and visual test designed for use in the collection and preparation of stool samples.

Principle of the test:

Hema-Sreen is made up of guaiac impregnated paper together in a cardboard frame which allows a maximum of two samples to be applied on one side of the paper and develop the result on the reverse side of the paper. The guaiac paper tests for occult blood through the oxidation of phenolic compounds (guauaconic acids) present in the guaiac to quinines causing the production of blue colour. Due to its similarity to the prosthetic group of peroxidase, the hematin portion of the hemoglobin molecule catalyzes the oxidation of guaiac by acting in a pseudoenzymatic way.

When the stool containing the occult blood is being applied to the test paper, the contact made between the hemoglobin and the guaiac will cause a pseudoperoxidase reaction to take place when the developer solution is added. A blue chromagen will form proportionally to the concentration of hemoglobins. The reaction usually takes about thirty seconds.

Specimen collection and handling:

It is suggested to have patient to go on a high residue diet starting two days before the test and continue throughout the test.

After stool samples are taken from the patient, use the applicator provided to spread a very thin smear of stool to the HemaScreen slides. Allow the smears to dry. The slide smears may be prepared and developed immediately or stored up to 12 days prior to development. Care should be taken to prevent any contact with blood to the specimen. Patient specimens and all materials in contact with them should be handled as potentially infectious materials and should be disposed in proper precautions.

Procedure
Materials provided:

-Hema-Screen slide with On-slide monitors (quality control)
-Hema-Screen developer
-Specimen applicators

Methods:

Write down the information to the empty lines of the front flap of Hema-Screen slide
Open the front flap
Use the applicator stick provided to collect a small amount of stool samples from the container of the patient stool and apply a very thin smear to the box.
Allow samples to dry and close the cover
Open perforated window on the back of the slide
Drop 2 drops of developer to the back of the area where the samples are being applied
Read results after 30 seconds
Any blue colour traced from the stool is considered positive for occult blood.

Limitations:

Results aabtained with the Hema-Sccreen cannot be considered conclusive evidence for presence or absence of gastrointestinal bleeding or pathology. It is used only for preliminary testing which cannot replace any other diagnostic procedures.it will only detect hemoglobin released upon hemolysis of the red cell. If whole blood is applied, it is required to hemolyse the red cells by addition of a drop of water before adding the developer. Positive result can be due to a couple of reasons such as red meat in the diet, diverticulitis, hemorrhoids, colitis to colorectal cancer.

Sharon
Tg01

Week 14..

Hellooooo.. this is already the 14th weeks think everyone should already get use to working life.. another 6 more weeks till the end everyone jia you! anyway this week is my turn to blog again have a nice day reading..

I have been doing the control assay since the last post and I had gotten the result. You might be wondering why I took so long to get just one result. Due to some mistakes make and repeating of the same experiment a few times in order to confirm the results. Anyway some of you asked me about showing the graph during my last post. Now i shall show you some of the graphs that i had plotted.

Graph A Graph B

Graph C Graph D

Different gain are shown in the graphs. The higher the gain value, the higher the reading due to the higher sensitivity of the machine(Tecan plate reader). From the raw data graph, the curves for positive control show increasing reading over time, while the negative controls are quite constant over time. After getting the raw data, i will plot them into ratio(which mean positive control/negative control). From there, i can decide which gain value and which concentration of cells will give me better stability and better reading.

From graph B and D, gain 40 will give a stability for 2 hours and gain 50 and 60 will give stability for 4 hours.

My next experiment will be on the standard inhibitor.. This is to find out how long it will take for the bacteria cells to be inhibited.

Brief steps:

1. Inoculate and take OD reading of S.aureus.

2. Serial dilute and dilute S.aureus culture.

3. Prepare 96-well plate, incubate overnight.

4. Prepare ampicillin of different concentration.

5 Add ampicillin into the 96-well plate.

6. Prepare Resazurin.

7. Add Resazurin into 96-well plate.

8. Take fluorescence reading at 1 hour interval.

Have a nice day reading!!

Justina

0605950E

TG01