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

Biochemistry Lab
Tan Zhao Rong. Tgo1.

Blood gas analyses are performed to evaluate the respiratory function of patients namely the acid-base status (pH), the oxygenation (pO2) and the ventilation status (pCO2). This blood test is specifically performed on blood from an artery and thus is somewhat more uncomfortable and difficult to perform compared to venipuncture.

The reference range for blood pH should range between 7.35 and 7.45. The pH status would indicate if the patient is acidemic or alkalimic.

The reference range for pO2 ranges between 75 – 100mmHg. A low o2 indicates that the patient is not respiring properly (hypoxemic). At a pO2 level of less than 26mmHg, the patient is at risk of death and must be oxygenated immediately as insufficient oxygen is transferred to the vital organs such as brain and heart.

The reference range for pCO2 should range between 35.0 – 45.0 mmHg. Any abnormal pH status would indicate a respiratory problem as the pCO2 in arterial blood is determined entirely by ventilation. A high pCO2 indicates under-ventilation, where respiratory acidosis occurs and acidity of blood rises. A low pCO2 would indicate hyperventilation where there is increased alveolar respiration.

ABL 825 flex blood gas module is the instrument responsible for carrying out blood gas analyses. The specimen type required would be 1 – 3 ml of arterial blood in heparinized syringes.

Procedure:
1. It is necessary to mix a blood sample before introducing it to the system/instrument so as to ensure homogeneity. RBC is to be evenly distributed and mixed within the syringe so as to prevent machine from aspirating a plasma phase or packed RBCs from the specimen. This may result in inaccuracy in results produced.
2. Check for any clots by injecting the first few parts of the specimen into a waste container.
3. Ensure that the analyzer is in READY mode and scan the barcode. (Barcodes would be labeled on the patient’s request form and specimen once it arrive.)
4. The inlet is a section of the instrument in which the analyzer aspirates specimens. Therefore, the next step would be to place the syringe tip firmly in the inlet and press START.
5. Press Aspirate to start the measurement.
6. Remove the sample when prompted by the analyzer.

When specimen volume is too low, it would be difficult for the machine to aspirate fully from the syringe as some parts may remain at the tip of the syringe. So as to fully utilize the whole specimen, the specimen can be transferred to a capillary tube before inserting into the inlet. A capillary tube can hold up to 200ul of blood specimen and the minimal requirement for a blood gas analysis would be to fill up to three quarter of the capillary tube (150ul).


Factors that may cause result to be inaccurate:

- Frothy samples or visible air bubbles would affect the pO2 and pCO2 level in the syringe causing inaccuracy in results.

- Sample not properly anti-coagulated are rejected as clots can cause the instrument to break down. Therefore, blood samples in normal unheparinized syringes are not accepted.

- Sample should not be left in room temperature for more than 30 minutes.

- Sample should be delivered in ice so as to slow the metabolic processes which causes inaccuracy. Delays in analysis (without chilling) may result in inaccurately low

Cytopathology

Cytopathology

Hi guys!
How are you all doing? It’s already halfway through SIP!! :)

For the first 2 entries, they are all about Histology, but now, I’m going to talk about Cytology :)
For us, we have to take turns to be attached to Cytopathology Lab for a month. So I’m now in my third week in Cytology, and let me share about one component of it, ie. Erythrocyte lysis. And btw, Cytotechicians basically spend long hours (the whole day) screening slides, and they will take turns to be stationed in the processing room to prepare and process smears.

Cytology is different from Histology in that specimens sent to Cytology are fluid-based and are to be screen for pre-malignancy. Specimens sent to Histology are tissue specimens that consist of tumors, polyps (basically,already malignant).

The most common specimens received in Cytology are:
Non-gynaecological:
Sputum, Aspirate, CSF and urine

Gynaecological:
Cervical smear, vaginal smear

Some of these specimens may be blood-stained, and this blood contamination may obscure microscopic examination of cellular materials. (They only want to look at the specific cells for the tissue type and the inflammatory cells/WBCs)

So, we use hemolytic agent to help us.
1. Clarke’s solution, or
2. 0.9% Saline solution (equivalent to tap water)

Clarke’s solution
· Contains acetic acid and 95% alcohol
· Smears are placed in Clarke’s solution for 15-30 mins, until the RBCs are lysed (judging by the colour of the smear)
· Smears are then rinsed in water before fixing in 95% alcohol

0.9% Saline solution
· Smears are air-dried before placing in 0.9% Saline solution( or tap water)
· Smears are then fixed in 95% alcohol

Non blood-stained smears are fixed directly in 95% alcohol.



Ting Ying Chee
TG01

Urine Full Examination

Urine FEME

YAY WE SURVIVED HALF OF OUR SIP! Haha great job guys! :)

Anyway like what Lesile mentioned in his post, for the 3 of us in Raffles we are permanently stationed in a section each. I’m stationed at the urine section! Hence today I will share with you guys about Urine FEME.

Urine FEME stands for Urine Full Examination(FE) Microscopy Examination(ME).
As the name implies, there is 2 processes to perform upon receiving the urine samples.

I will share with guys what is FE this post and contiune with ME on my next post yeah:)

Full Examination
The 1st process to perform upon receiving a urine sample is urine full examination. This process uses urine dip stick to test for the content of the urine.

For my lab, we uses Combur-test strips and Miditron Junior II to obtain the results.

Test for:

S.G (Specific Gravity):
o Purpose: Check the amount of substances in urine, which shows us how well our kidneys balance the amount of water in urine.
o Range: 1.005-1.030

pH:
o Purpose: Measure how acidic or alkaline the urine is.
o Range: pH 4.5-8
o In our lab, we use the pH to determine the type of crystals observed in urine.
(Will be further evaluated under ME)

Pro (Protein):
o Purpose: Detect for kidney diseases; measures albumin level in urine
o Range: neg, trace, 1+, 2+, 3+
o If pro is > 1+, we need to look out for cast in ME
o Fever and pregnancy may cause protein in urine.

Leu (Leucocytes):
o Purpose: Detect for UTI
o Range: neg, trace, 1+, 2+, 3+
o If leu is > trace, we need to look out for WBCs in ME for confirmatory.

Nit (Nitrate):
o Purpose: Detect for bacteria infectioin
o Range: neg or pos
o Even if it is pos, report only if there is present of bacteria.

Glu (Glucose):
o Purpose: To detect for diabetes
o Range: neg, trace, 1+, 2+, 3+
o If glu is > trace, do a confirmatory test with Keto-Diabur-Test 5000 urine dip sticks.

Ket (Ketones):
o Purpose: To measure whether fat is metabolized properly in our body.
o Range: neg, trace, 1+, 2+, 3+
o If ket is > trace, do a confirmatory test with Keto-Diabur-Test 5000 urine dip sticks.

Ubg (Urobilinogen):
o Purpose: Detect for hepatic dysfunction
o Range: neg, trace, 1+, 2+, 3+
o If ubg is > 2+, perform confirmatory test by repeating Combur-test strips.

Bil (Bilirubin):
o Purpose: Detect for hepatic dysfunction
o Range: neg, trace, 1+, 2+, 3+
o If leu is > 1+, perform confirmatory test with bilirubin tablet (ICTOTEST® Reagent Tablet)
(If tablet turns absorbant purple, bil is pos)

Ery (Erthrocytes):
o Purpose: Pathologically significant for several diseases
o Range: neg, trace, 1+, 2+, 3+
o If leu is > 1+, we need to look out for RBCs in ME for confirmatory.

For more details, visit
http://www.webmd.com/a-to-z-guides/urine-test
http://www.anytestkits.com/utk-urobilinogen-in-urine.htm

Hui Min
TG01

Week 10

Hi everyone, it’s my turn again this week. For this week, I think I’m going to share about what I’ve learned from the microbiology section from my lab. I’m going to introduce this kit that can help to identify the different types of gram-negative bacteria. It is named the Microbact Gram-Negative Identification System.

Principle:
The Microbact Gram-negative system is a standardized micro-substrate system designed to stimulate conventional biochemical substrates used for identification of Enterobacteriaceae and common miscellaneous Gram-negative bacilli. Organism identification is based on pH change and substrate utilizations.

This product consists of two separate substrate strips, 12A and 12B. Each strip consists of twelve different biochemical substrate. The 12A strip can be used alone to identify oxidase-negative, nitrate positive glucose fermenters and can be used for screening pathogenic Enterobacteriaceae from enteric and urine specimens or identification of other common isolates. The 12B strip can be used together with the 12A strip for identification of oxidase positive, nitrate-negative, glucose non-fermenters and the Enterobacteriaceae.

Each kit contains one holding tray for the 12A and 12B strips and organism ID report forms including colour interpretation chart.

Examples of species of oxidase negative, Gram-negative bacilli that can be identified using 12A strip alone:
Acinetobacter spp., Shigella spp., Enterobacter spp., Escherichia spp., Salmonella spp., etc.

Examples of species of Gram-positive bacteria that can be identified by using combination of 12A and 12B strips:
Vibrio spp., Pseudomonas spp., Pasteurella., etc.

Set up procedure:
1.Isolation

To use the kit for identification of organism, a pure culture of that particular organism must be obtained. Appropriate agar media such as MacConkey, Blood or Chocolate may be used to grow the organism for approximately 18 to 24 hour. I believed you all would know how to do a pure culture plate by now so I won’t state the procedures here.

Before using the kit, an Oxidase test should be performed on that organism to be identified. As mentioned above, if it’s positive, 12A and 12B strips should be used and if it’s negative, 12A strips can be used alone.

2.Preparation of inoculum

Pick 1-3 isolated pure colonies from the 18-24hr culture and emulsify in 2.5 to 5 ml of sterile saline. Usually 1 colony is enough to emulsify in 2.5ml of saline if doing on the 12A strip alone. If it’s doing 12A and 12B strips, it may need 2 colonies to emulsify in 5ml of the saline. Then, mix thoroughly for a uniform suspension.

3.Inoculation

As the strips are sealed, the individual substrate sets can be exposed by cutting the sealing strip and slowly peel it back. Then, place the strip on the holding tray and using a sterile dropper, fill the bacterial suspension to half of each well in the set. After that, drop one drop of sterile mineral oil on the wells that have the substrates underlined (for 12A strip is well 1,2 and 3 while for 12B strip is well 8 and 12).


In case you all want to ask me what each well contains, here’s the answer:
For 12A strips-Well 1- Lysine
Well 2- Ornithine
Well 3- H2S
Well 4- Glucose
Well 5- Mannitol
Well 6- Xylose
Well 7- ONPG
Well 8- Indole
Well 9- Urease
Well 10- VP
Well 11- Citrate
Well 12- TDA

For 12B strips-Well 1- Gelatin
Well 2- Malonate
Well 3- Inositol
Well 4- Sorbitol
Well 5- Rhamnose
Well 6- Sucrose
Well 7- Lactose
Well 8- Arabinose
Well 9- Adonitol
Well 10- Raffinose
Well 11- Salicin
Well 12- Arginine

4.Incubation

Reseal the inoculated rows with adhesive seal and write the specimen identification no.(in my lab case is using barcode no.) on the seal with a marker. Then, incubate 33-37°C for 18 to 24hrs.

5.Reading the test strips

Remove the test strips from the incubator and unseal them for reading. Positive and negative reaction results are recorded down on the organism ID report forms by comparing the colour change with the colour chart. For some of the wells, there’s an additional reagent to add to before reading. For 12A strips, well 8 drop 2 drops of Indole (Kovacs) reagent and read within 2 minutes, well 10 add 1 drop of VPI and 1 drop of VPII and read within 15 to 30 minutes and well 12 add 1 drop of TDA reagent and read immediately. These additional reagents are required for the reaction to start as different bacteria can have different reaction to these reagents.

There’s also an additional test called the Nitrate Reduction Test. It is performed in well 7 (ONPG) after reading the ONPG reaction. 1 drop of Nitrate reagent A and 1 drop of Nitrate reagent B is added to the well and a change to colour red within few minutes indicates nitrate reduction to nitrite. The positive reaction indicates that it is an organism belonging to the family of Enterobacteriaceae.

6.Interpretation

For the interpretation is pretty easy as they adopted a coding system/program that interpret the type of bacteria simple by entering the recorded result. For every 3 reaction (every three wells), it is added up to a single digit of the code. So if only 12A strip is used, there will be 4 digits which make up the code. If using 12A and 12B strips together, there will be a total of 8 digits that make up the code. Simply just choose the correct program and enter the code and the system will tell you what kind of bacteria is it.


Alright, that's it for this week. Enjoy your life people, if you still can now. Lol.

Sharon
Tg01

Week 9

Hi all, here I am again..

For my MP, I have started on doing my 'control' assay on the 96-well microtitre plate. This experiment is to determine the concentration of cells to be use in future experiments.

Yellow section(Row A-D): Positive control
Red section(Row E & F): Negative control(blank)
Blue section(Row G): Negative control(with 100ug/ml of ampicillin)
Green section(Row H): Negative control(with 200ug/ml of ampicillin)

Methods:
1. After taking the OD reading(till the bacteria cells reached the log phase), dilute the cells to get
the concentration of cells needed.
2. Using a multichannel pipette and micropipette,
For Row A-F, add 10ul of autoclave water into each well.
For Row G, add 10 ul of 100ug/ml of ampicillin into each well.
For Row H, add 10ul of 200ug/ml of ampicillin into each well.
For Row A-D and Row G &H, column 1-3, 4-6, 7-9, 10-12, add different concentration of
bacteria cells into the well.
For Row E & F, add fresh TSB as this will be the blank control of the assay
3. After adding all the solution into the wells, incubate the microtitre plate at 37oC overnight.
4. Next day, prepare Resazurin which is a redox active dye.
5, Add 10ul of Resazurin dye into all the wells and incubate it.
6. At 1 hour interval, take the fluorescence reading.
7. After 5 hours, all datas collected are to be sort and plot graphs using those data.
8. From the graph, determine which concentration of cells can give a better sensitivity.
9. In future experiment, the specific concentration of cells will be use.

In the real experiment, the 10ul of autoclave water will be replace by tea extracts.

As for my SIP, other then data entry, I also help to prepare acidulants by measuring 0.6g of citric acid, lime juice, vinegar and tartaric acid, and add 2L of DI water into it, then mix them. Other then helping to prepare media, I also help to check some items in the lab making sure all items are in place. From doing this, I realised how tiring the work of the TSO can be, so make sure the next time you use the lab, put all items back to their original place if not TSO will have a hard time looking for them.

Justina
0605950E
TG01

Department: Clinical Biochemistry Laboratory

During the fourth week of my attachment, I was stationed at the DxI station where I observed and learnt about the Beckman DxI-600 and its day to day maintenance.

Every morning, the technician-in-charge of DxI would be responsible for its daily maintenance such as

Backing up the system and data – By doing so, the data (Eg. The type of test run on which specific specimen and its results) accumulated in the previous day would be saved into floppy disks and could be used for further reference.)

Checking Inventory – This is important as it ensures that all reagents required by the DxI to perform all its tests are topped up and sufficient to last the entire day.

Recording Test count – This simply requires the technician to record the total number of tests performed by the DxI the day before.

Shaking the solid waste container – The waste container is where DxI deposit solid waste such as pipette tips. Usually, the waste would accumulate at one side of the box (as the tips are dropped from the same spot) and shaking the box would balance out the level of the contents.

Running a daily special clean – This daily special clean is commercially prepared by the DxI manufacturer. The special clean solution is transferred to a reaction vessel (plastic holding cups) by a dropper and the reaction vessel is then placed on a specific rack that carries the solution into the DxI once the task is assigned for the rack to run. This daily special clean is used for cleaning probes in the DxI. Probes are in fact mechanical arms that the machine used to pipette and transfer specimens and reagents within the DxI. Although it is constantly washed by the machine, running a special clean very morning would ensure removal of possible specimen/reagent traces from the probe itself and its pipes.

Other than its daily maintenance, I have also researched on the one of the common tests carried out by the DxI – Insulin.

Brief overview of Insulin:
Insulin is a hormone secreted by the pancreas that facilitates the uptake of glucose from the blood and storing it as glycogen in the liver and muscle. Glycogen (fuel storage) would be broken down to glucose when energy is required by the body later on.

When insulin level is low or absent, glucose is not taken up from the blood and stored the liver or muscle cells. The glycogen storage would eventually deplete as the body utilizes energy, this would then results in the body utilizing body fats as energy source. At the same time, glucose in the blood remains high as insulin fails to function. This condition is known as diabetes mellitus.

Test Principle:
DxI is able to test the levels of insulin in patient’s serum by using the ultrasensitive insulin assay which is an immunoenzymatic sandwich assay.

1. A sample is added to a reaction vessel with monoclonal anti-insulin antibody and anti- insulin alkaline phosphatase conjugate.

2. Insulin in the patient’s serum will bind to the anti-insulin antibody on solid phase, while the conjugate reacts with different antigenic sites on the insulin molecule.

3. Unbound materials are washed away.

4. The chemiluminescent substrate LumiPhos 530 is added to the reaction vessel and light generated when this substrate react with the conjugate is measured with luminometer.

Additional notes:
Patient’s serum are used for insulin testing and as red blood cell contains insulin-degrading enzymes, badly hemolyzed samples are not suitable as insulin levels may be suppressed, causing pre-analytical variations and inaccurate results.

Tan Zhao Rong
Tg01

Special Stains

Subject: Histopathology

Hi guys!
This is my 7th week at Histopathology, and so we have gotten pretty familiar at how things are managed and operated there. My previous post mentioned a bit on Special Stains, so now I'm here to share about 2 of the more common ones.

*Note:

• Commerical reagents are usually used in the lab
  
• Timings in the staining procedure are for your reference only because I've learnt that different types and different sections of tissues absorb and expel dye molecules at different rate.
  So if you are staining 5 slides at the same time using the same timing, the results obtained for each slide will vary. Thus, it is important to perform microscopic examination for each slide after each staining.
  However, there are also some stains which can be left on the sections as long as you want as the duration does not affect the staining.
  
• All reagents have their specific "lifespan" so it is important to record the date when the bottle of reagent is opened and their expiry date. Reagents are changed as often as needed to ensure quality staining.
  
• All types of staining including H&E are to be done with a control (positive control) section. It can either be at the upper half of the same slide, or on another new slide. This is added for the pathologist as a reference during microscopic examination.

_______________________________________________________________________________________

Alcian Blue (ALBL)

Function:
· Stains acid mucins and acid mucosubstances

Where can acid mucins and acidic mucosubstances be found?
· Excessive amount of non-sulphated acidic mucosubstances can be seen in mesothelioma, with certain
amount occurring normally in blood vessel walls but increased amount in early lesion of atherosclerosis.

Principle:
· Alcian Blue is a group of polyvalent basic dyes that are water soluble
· Blue colouration is present due to presence of copper in the dye molecule
· Alcian Blue at pH 2.5 stains both sulphated and carboxylated sialomycins (gylcoproteins)
· Alcian Blue forms self-linkages with acidic groups of acid mucopolysaccharides

Control used:
· Small intestine or colon

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· 1% Alcian Blue
· 3% aqueous Glacial Acetic Acid
· Nuclear Fast Red

Reagent preparation:

1. Alcian Blue
· Alcian blue – 1g
· 3% aqueous Glacial Acetic Acid – 100ml

Allow the dye to dissolve and adjust the pH to 2.4 by using 1 M of sodium hydroxide. Store in a dark bottle to prevent reaction of dye with the light. This reagent is stable at room temperature for 6 months.

2. 3% Glacial Acetic Acid
· Glacial Acetic Acid – 6ml
· Type II water (DI Water) – 200ml

Store at room temperature.This reagent is stable for 1 year.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Rinse the section using 3% aqueous Glacial Acidic Acid solution
5. Stain in 1% Alcian Blue in 3% aqueous Glacial Acidic Acid for 20 minutes
6. Rinse in 3% aqueous Glacial Acidic Acid solution before rinsing in water
7. Counterstain with Nuclear Fast Red for 5 minutes
8. Rinse the section with water
9. Dehydrate the section using graded alcohol from 95% to absolute alcohol
10. Clear the section using 3 changes of xylene
11. Mount the slide with Depex

Results:
· Acid mucins – Blue
· Nuclei – Reddish pink

______________________________________________________________________________________

What is mesothelioma?

· Cancer of mesothelium
· Mesothelium is a thin membrane that surrounds most of our internal organs for protection purpose. The
membrane consists of 2 layers; one on the organ, another forms a sac around it. In the sac, there is a fluid
produced by the mesothelium to reduce friction as our organs glide against one another or against our
body structure such as the rib cage.

What is atherosclerosis?

· Hardening or arteries
· This is due to the buildup of deposits on the walls of the arteries. Common deposits are fatty substances,
cholesterol, calcium and cellular waste products.
· Buildup of deposits is known as plague and can rupture the arteries, causing the blood clots to obstruct
blood flow or travel to other parts of the body, resulting in heart attack, stroke etc.

______________________________________________________________________________________

Masson Trichome (MT)

Function:
· Stains muscle, fibrin and collagen

Principle:
· A section is first treated with Masson Ponceau Fuchsin (small molecule anionic dye) before treated with
phosphomolybdic acid or phosphotungistic acid solution
· Phosphomolybdic acid or phosphotungistic acid solution will compete woth the dye to gain acess to the
collagen, at the same time, expelling the dye in the process
· The collagen is then treated with a larger molecule green or blue dye

Control used:
· Liver tissue, skin or muscle

Fixative used:
· 10% Neutral Buffered Formalin

Chemicals required:
· Haemotoxylin
· 4% aqueous phosphomolybdic acid
· Masson’s Light Green
· Ponceau 2 R
· Acid Fuchsin
· Glacial Acetic Acid

Reagent preparation:

1. Masson Ponceau Fuchsin
· Ponceau 2 R – 3.5g
· Acid Fuchsin – 1.75g
· Glacial Acetic Acid – 5ml
· Type II water (DI Water) –500ml

This reagent is stable at room temperature for 6 months.

2. 2% Phosphomolybdic- tungstic Acid
· Phosphomolybdic Acid – 4g
· Phosphotungistic Acid – 4g
· Type II water (DI Water) – 200ml

Dissolve in water and heat it at 50-60oC overnight by putting it in the oven.
Filter and store at room temperature.

3. 1% Acetic Acid
· Glacial Acetic Acid – 2ml
· Type II water (DI Water) – 98ml
Mix before use. Stable at room temperature for 6 months.

Staining procedures:
1. Heat the slide containing the section on the hotplate for 3 minutes
2. Dewax the section using the auto-stainer (machine)
3. Bring section to water to prevent section from drying up
4. Post fix the section in Bouin’s solution for 30 minutes at 56oC
5. Stain nuclei with Weigert’s iron haemotoxylin for 10 minutes
6. Wash in tap water and blue for 5 minutes
7. Stain in Masson’d Ponceau-Fuchsin for 5 minutes
8. Wash in 1% Acetic Acid
9. Stain in Masson’s Light Green for 30 seconds to 2 minutes, until collagen is green. In order to
control intensity of the stain, it is better to stain for 1 minute first, then rinse in acid water before examining
under the microscope.
10. Rinse in 1% Acid Alcohol
11. Dehydrate the section using graded alcohol from 95% to absolute alcohol
12. Clear the section using 3 changes of xylene
13. Mount the slide with Depex

Results:
· Nuclei – Blue black
· Cytoplasm – Light red
· Muscle – Dark red
· Red cells – Bright red
· Hyaline & fibrin – Bright red
· Collagen & mucin – Green

________________________________________________________________________________________

Cheers!

Ting Ying Chee
TG01

Seminal Analysis

Section: Hematology
Task: Seminal Analysis


When is the test requested?
Seminal analysis is usually performed on 2 occasions:
1) To investigate whether a man might be infertile.
It is only a ‘screening test’, not a confirmatory test for fertility.
Studies shown that 30% of all patients with normal semen analysis have abnormal sperm function.
(It is a common request in couples planning for marriage!)

2) After a vasectomy to determine whether the procedure was successful.

How to collect?
A colleague joking told me that in olden days, hospitals have a special room playing RA-ted movies in order for males to collect the semen.

That I am not sure whether it is true, but now semen can be collected through:
1) Masturbation
2) If intercourse is the only way to collect sample, special nonreactive condoms are available. They are made up of silicone or polyurethane, instead of latex, which is non-harmful to sperm.

Semen specimen are collected into a sterile and transparent container and required to be analyzed within 1 hour of collection. Therefore, the patients should be strongly recommended to collect samples within clinic area.

Procedures
Upon receiving the semen, we will put it into the incubator for an hour before performing seminal analysis.

Why do we need to incubate for an hour?
- It is estimated that the sperms after being ejaculated, take an hour to pass through the vagina to the uterus to the fallopian tubes to reach the ovaries. Hence 1 hour is the standardized time to measure the amount of sperm that can reach the eggs of females.
- Putting in the incubator is to maintain the viability of the sperms.

Tests done:
1) Appearance
How? Observing the color of semen
Normal result: White or pale yellow
Comments: Reddish semen is abnormal

2)Viscosity
How? Observing the thickness of semen
Normal result: Liquid-like
Comments: Glue-like(very thick) is abnormal

3) pH
How? Use pH dip strip
Normal result: 7.2-7.8
Comments: Semen is alkaline by nature; Acidic ejaculate can be associated with blockage of seminal vesicles.

4) Volume
How? Using 1ml dropper
Normal result: 2-6 ml
Comments: Low volume may be due to absence of seminal vesicle component or decrease of retrograde ejaculation

5) Density
How? Count 10 squares of neuber chamber under microscope
Normal result: >20x106/ml
Comments: The result = (no. of sperm counted) ×10^6/ml

6) 1st hr and 2nd hr motility
How?
1) Drip a drop of semen onto the glass slide and place a cover slide on top
2) Count the motile and non-motile sperms under microscope
3) Motility can be calculated by dividing the motile sperms with total sperm count.
Normal result: >60% for 1st hr
Comments: 2nd count is usually lower as more sperms die.

7) Morphology
How? RBCs , WBCs , Abnormal sperm
Comments: Abnormal sperm includes those with short tail, either head that are too big or small, sperm with 2 heads etc

Factors leading to low sperm count:
Smoking:
Toxins in cigarettes affects the lifespan of sperm and it was also proven that smokers had far less sex drive than their non-smoking peers.

Alcohol:
Drinking in excess damages developing sperm and decreases healthy sperm.

Testicular overheating:
Putting laptop on your laps or wearing tight fitting underwear can raise scrotal temperatures, which slow down the rate of sperm production. Laptop heat insulators and loose fitting garments are recommended to allow air to circulate and keep the testicles cool

Vigorous exercises:
Exercises that cause repeated trauma or impounding damage to the testes and scrotum result in semen alterations. Examples of such exercises are cycling or mountain biking.

There are many more interesting factors! You can read more on www.spermtest.com/causes_of_abnormal_low_sperm_counts.php

Other sites visited:
www.umc.sunysb.edu/urology/male_infertility/SEMEN_ANALYSIS.html
www.labtestsonline.org/understanding/analytes/semen/test.html

Cheers,
Kum Hui Min =)
tg01

Hi people, hope you guys enjoy your SIP. I’ve been posted to the same lab as Maya, Lloyd, Azeimah and Ivan so what we learned are the same. But I realized even those who are not in our lab, they learned similar things as us too.

For the first 2 weeks I’ve been posted to Immunology with Maya and the 3rd and 4th week we are posted to Biochemistry. For these two sections they are closely link as they used the same automated track that helps them to automatically load samples into their machine. As mentioned in Maya’s and Ivan’s blog, for Immunology section they have this machine called Centaur and for Biochemistry, their machine is called Bayer Advia 1650 Chemistry System. Since both of them had already posted on the machines, I guess I’ll post on the manual test that biochemistry section does then.

Basically, the manual test procedures are not difficult or as many steps as we thought. The types of manual test that biochemistry does in out lab are OGTT, G6PD Qualitative test, and Drug Analysis (Drug Screen Test). I’ll share a little about the Drug screen test since no one has really talked about it.

Drug analysis (Drug Screen Test)

Samples to collect: Urine

The Drug Screen Test is a fast and easy test to determine drug abuse. The company purchases these cassettes (test device) that can be used to test for different drugs such as Amphetamine (AMP), Barbiturates (BAR), Benzodiazepines (BZO), Cocaine (COC), Marijuana (THC), Methadone (MTD), Methamphetamine (MET), Methylenedioxymethamphetamine (MDMA), Opiate (OPI), Phencyclidine (PCP) and Tricyclic Antidepressants (TCA). Simply just drop 3 drops of urine to the cassette well and the results will be out in a few seconds.

Signs and symptoms of patient with drug abuse:

Eg. Benzodiazepines— this drug is usually prescribed for treatment foe anxiety and sleep disorder. When this drug is taken more frequently, it will cause dependence and stopping abruptly can bring about symptoms such as trouble sleeping, gastrointestinal upset, feeling unwell, loss of appetite, sweating, trembling, weakness, anxiety and changes in perception.

Principle: Competitive binding.

During testing, the urine migrates upwards through capillary action. The drug present in the urine competes against their respective drug conjugate for binding sites on their specific Ab. If the presence of drug in the urine is below the cut-off concentration, it will not saturate the binding sites of its specific Ab. The Ab will then react with the drug-protein conjugate and a visible coloured line will appear at the test region. If the drug present in the urine is above the cut off concentration, it will saturate all the binding sites of the Ab, thus the coloured line will not appear due to drug competition.

Interpretation of result:

Coloured line appears—negative
No coloured line appears—positive—drug abuse


Note: The assay only provides a preliminary analytical test result. A more specific alternate method should be used to confirm this analytical result. The preferred confirmatory test is Gas chromatography/mass spectrometry (GC/MS). However these test are not done in our lab so I’m not sure how is it done. The lab would usually report positive cases to which ever clinic or hospital that requested for the test. When this preliminary test shows positive result, clinical consideration and judgment from professional are needed in order to make sure that the patient is really having drug abuse.


Sharon
TG01

Week 4 SIP...

Hi all, enjoying your SIP?? I hope you do enjoy it.. Left with 4 months!!!

Alright here to share with you my experiences during my attachment in school..

For my SIP, what I will be doing is mostly on data entry. Well seem very unrelated to what we doing right.. haha.. but it plays a major role in operating the whole lab smoothly. For examples what I dealing with is the purchasing or maintaining of equipments, chemicals products etc. So what I do is key in the amount and what is the amount use for then sending the invoices to the finance department. I will also be updating the actual prices for chemicals products for the next semester use. When lecturer wanted to buy chemical products, they will refer to the price stated to estimate the amount they going to spend on the items.

For my MP, I will be doing a project on 'Evaluation of anti-microbial activity in different tea extracts'. Tea is one of the most commonly consumed beverage(other than water) and reports have shown the benefits of tea to our health. So I will be setting up a whole cell-based assays which is(in my case) S.aureus to evaluate different tea extracts for anti-microbial activity. For now I will be focusing on what I have done in the past 4 weeks.

During the past 4 weeks I had done a series of experiments to determine the log phase of S.aureus. This is just the first part of my whole experiment.

Aims: To determine the log(exponential) phase of S.aureus.

Methods:

1. Preparing of 1L of Trypic-soy agar and 1L Trypic-soy broth.

2. Inoculate S.aureus into TSB and incubate at 37oC for 18 hours, shake with 150-200rpm, as

S.aureus is in beads form this is to dissolve the beads and allow the S.aureus to be release.

3. Streaking of S.aureus from TSB broth to TSA agar plate so as to obtain isolated colonies. The

streak plate is incubated at 37oC for 18 hours.

4. Gram stain of S.aureus, this is to check for the purity of the culture. For S.aureus, it will

appear purple and cocci in cluster.

5. Inoculation of one colony from the TSA agar plate to TSB broth and incubate at 37oC for

18 hours, shake with 150-200rpm.

6. Take OD reading at 600nm to find out the log phase of S.aureus at a specfic time(for me I

take the reading once every half an hour). The reading is to be continue until the culture

reached stationary phase.

7. Confirm OD reading and do a serial dilution. Serial dilution is necessary so that when doing

spread plate it will not be overcrowded.

8. After serial dilution do spread plate and incubate at 37oC for 18 hours. For a good plate/ good

dilution, the cells number in the plate should be in the range of 30-300 colonies.

9. Count plate to get the CFU/ml. This result will be use for reference for further experiments.

For the experiment above, I have repeated a few times so as to ensure the result is reproducible. Alright that is all I had done so far.

Cheers;)
Justina
0605950E
TG01

Biochemistry Lab

Hi All,

On the second and third week of my attachment in a Clinical Biochemistry lab, I was stationed at the TDX station to observe how blood levels of therapeutic drugs are tested.

For this week's post, I'm zooming in to one of the most commonly tested drug - Cyclosprine A.

Cyclosporine A is an immunosuppressive drug which is used to combat tissue rejection following organ transplantation. For example, after an organ (liver, kidney, skin etc) transplant, the immune system of the recipient may reject the introduction of a foreign tissue. T cells and other immune cells may be induced to destroy the foreign cells, leading to graft rejection. Cyclosporine A is thus administered to enable graft survival by reducing the activity of the recipient’s immune system.

Cyclosporine A has a narrow TI (Therapeutic Index) and is also associated with nephrontoxicity and hepatotoxcity, it is therefore recommended to have regular drug monitoring for the patients on this medication. (This explains why I notice a handful of repeated names everyday.)

Procedure

The specimens, which are whole blood specimens received in EDTA tubes, are labeled with a specific bar code that gives each specimen a code number.

1. Microcentrifuge tubes are labeled with patient’s code number. Control tubes are labeled with L (low), M (medium) and H (high).
   
2. Control reagents are provided and treated in the same way as a patient’s sample.
   
3. The samples are mixed by gentle inversion and 150ul of the sample are accurately pipetted into the labeled mircocentrifuge tubes accordingly. (During pipetting, it is important to make sure that blood do not stick to outside of the pipette tips as this would cause additional volume to be added to microcentrifuge tube)
   
4. 50ul of solubilization reagent and 300ul of whole blood precipitation reagent are pipetted into each mircocentrifuge tube.
   
5. After pipetting all the samples, each tube are capped securely and vortex for a full 10 seconds to ensure thorough mixing. After which, samples are centrifuged for 5 minutes at 10900rpm. A clear supernatant and a hard, compact pellet of denatured proteins would be obtained after centrifugation.
   
6. The supernatant of each specimen is decanted completely into the sample well of a sample cartridge. The cartridges are then positioned on a carousel and are ready to be placed into the TDX machine.


7. Other than the carousel, a reagent pack is also placed in the TDX machine. This reagent pack should be mixed by gentle inversion and bubbles should be removed prior to placing the pack on the reagent platform in the TDX.


8. When both carousel and reagent pack are placed stably on their respective platforms, the cover is closed and the analyzer would begin upon pressing RUN, ASSAY 50.


9. Allow time for the machine to operate and once the results are produced, the control values are matched to ensure it falls within the acceptable range. If it does, patient’s results are accepted. If it does not, a rerun of the test is required.

Tan Zhao Rong

TG01

Histopatology!

Fig1.1 Hotplate.
Added to explain in my reply to one of the comments.


Subject: Histology

I am posted to Histopathology/Cytology department, together with Ernest and Tira. ;) (Hope they can stand my craziness at times as the formalin is making us real high. LOl)
This week we are at the Histopathology department, and I heard that we will be in cytology for a month! We been there before, during our tour and I saw the Biosafety Hood (remember what Ms. Chew mentioned before? Yes. The expensive one. )
_____________________________________________________________________________________
The overview of the Histopathology was explained to us by one of our friendly colleagues.
Surgeon cuts specimen of interest-->sends to Histopathology department receiving room-->request forms are checked thoroughly and biopsy no is issued to each patient/case-->sends specimen to trimming room(2 steps away only. Haha)-->pathologists trim big/large specimen while medical technologists trims smaller ones-->cut specimen placed in cassettes--> send for tissue processing--> embedding-->microtomy-->heat slides before staining (H&E as the most common staining)-->sorting slides according to the different doctors and the request forms--> send out for pathologists to read the slides

_____________________________________________________________________________________
Assisting during trimming:
These few days, we were allowed to observe how our colleagues and help out what we could. I assisted the pathologist during trimmer by preparing and labeling cassettes (using a special tissue and cassette marker which cannot be dissolved by any solvent known to the histo-department) for them according to the specimen received and jot down the number of cassettes used for each case before putting them into formalin. The most common specimens received are breast, uterus, colon and kidney and the others are lungs, bile, prostate, fetus, and placenta. I even found out from our colleagues that they had received eyeball and an entire leg before! During trimming, the pathologist will also dictate what they observed, for instance, a cyst in found __ cm near the right margin. These dictations will be sent to the admin staff who will type it out as a report to be attached to the respective request form. Sometimes, the pathologists will also ‘colour’ the specimen using special dyes of different colours as to denote the different parts of the specimen.

Fishing:
Other than assisting during trimming, we also did fishing, heating of slides and sorting slides. After cutting the tissues using the microtomy, we will put the tissues into cold water first before putting them into the water bath. This is to increase the surface tension of the tissues, so that there will be no folds on the tissues which can hinder reading of slides. The water bath has an alarm system so that once the temperature is above 550C, the alarm will beep. The optimum temperature is between 500C to 540C. We will fish the tissues before labeling them according to what is stated on the cassettes. Everyday, we will fish from around 8am to 10 plus am, depending on the workload. Tissues are to be fished in the middle of the slide with the same orientation as the specimen embedded in the paraffin wax. If tissues are fished too high on the slide, it may be properly stained by the auto-stainer (machine for staining). If too low, tissue may be damaged during heating when not properly handled. The correct orientation facilitates checking of slides with cassettes before sending them out to the doctors and pathologists. Smaller tissues (app. 1cm by 1cm) are fished 6 in a slide, 2 by 2 by 2 slices, while normal size tissues are 1 per slide.

Heating:
After fishing, the slides will be tap dry before placing them slanted(partially) on the hot plate to remove all the water on the slide first. This is to prevent ‘smearing’ or moving of tissues out of their positions (on the slide) when they are heated on the hot plate for 3 mins at the highest temperature on the machine (the knob is pointing at 10, the maximum).

Staining:
After heating the slides, we will load the slides for H&E staining into this awesome machine called an auto-stainer. There is a total of 18 buckets of reagents, with 0.5% acid alcohol and lithium carbonate (not for H&E but for other types of staining). This machine also incorporates a part where it mounts the coverslip for you! So fast and with no air bubbles in it! Not like that time where our fingers trembled when placing coverslip. Some slides are requested for special staining like GMS and PAS where they will be placed in another machine. Some types of staining even need up to 1.5 hours!

Sorting:
After the slides are mounted, we will sort them according to their batch no and biopsy no before sorting them again according to respective request forms and the different doctors. These will be checked by our colleagues before sending them out.

The manpower in histopathology department is rotated throughout the different sections, for instance, receiving, trimming, embedding, microtomy, and staining. The hospital we are in receives the most no of specimens and requests everyday, which explains the large no of slides we do everyday. The staff there are also very experience, many of whom with at least 8 years of experience in histopathology department!

Posted by:
Ting Ying Chee =D
0606530D

My experience in bio/immuno lab :)

Subject: Chemistry and Immunology

Hi guys!

I'm posted to Biology and Immunology for this week:)

Basically in the bio/immuno section, we handle samples that requires chemistry and immunology testing. Upon receiving the samples, we are to sort them into different catergories and send them to respective machines for testing.

The categories are:
1) Chemistry
2) Immunology
3) Mixed

Chemistry
Types of tests:
Tchol, HDL, Tg, Chol, CO2, Glu, K, Na, Cl, TP, ALT, AST, GGT, alb, glob, ratio(A/G), T-Bil, Ca, Phos, CPR Quant/latex, HS CRP, Amylase, UA, APO-A, APO-B, D-Bil, Fe, D-Dimer, Mg

Those highligted in green are normal tests requested.
Here we test the sample under a machine called ModP. These samples are easy to handle as we only need to load them in the machine, press START and wait for the results that will be shown on the computer:)

Those highlighted in purple, they are samples we have to take note of.
ModP will not be able to detect them hence we use a more specialized machine Cobas501.

For Cobas501, it is able to detect all type of chemistry tests.
So why don't we just use Cobas for all chemistry tests?
It is because Cobas is a much smaller machine than ModP... Hence ModP is usually used as it is able to process more speicmens.

Immunology
Type of tests:
TSH, FTy, FT3, CA, CEA, PSA, Testro, HIV, FSH, LH, Prolactin, Progesterone, Ferretom, Abs, Ag, HAV, HBeAg, Afp, Trop T, CK-MB, B-hCG, anti-HBe, anti-HBC, anti-HBCIgM, anti-HCV, anti-EBV, anti-dsDNA, Rubella-IgG

Similarly to chemistry, there are samples that we run normally and others that we must take note of.
For normal tests requested, we use modEAO
For specialized tests, we use Eles2010. These samples we have to run maunally, where we have to key into the machine what specific tests to perform.

Mixed
The tests requested consist of both type! So samples are run in respective machines.

All samples are run in plain tubes except D-dimer where it is run in sodum citrate tube.


The procedures above are the general tests we perform. However we also handle other cases like:
-Oral Glucose Tolerance Test(OGTT)
test for: diabetes
sample: fasting glucose, 1st he glucose and 2nd hr glucose
machine used: modP
tube used: fluoride tube

-HbAIC (more specific test for diabetes)
machine used: D10
tube used: EDTA tube

-Micoalbumin(Quanti)
test for: urine(normal/abonormal)
uses: Clinitex and urine strips

-G6PD
test: manually perform on test strip and looked under long wave
results: If baby is normal, sample will fluoresce)

-Microsomal Ab
test: performed on microtitre plate


ok so heres a recap!
chemistry testing---- modP or Cobas
immunology testing-- modEAO or Eles2010
diabetes------------- OGTT or HbAIC in D10
urine(microalbumin)- Clinitex
G6PD---------------- Manual testing on test strip
Microsomal Ab------- Manual testing on microtitre plate

Additional info~
Test tubes:
Yellow- plain tube with gel
Red --- plain without gel
Blue--- sodium citrate (D-dimer)
Purple- EDTA
Grey--- fluoride (glucose)
Green-- lithium haprine

Posted by=)
Kum Hui Min
0605989C