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