Week 5: Histopathology Lab

Name: Nadiah Sukma
Admin number: 0705365E


hello people..

it is now my turn to share the experiences ive gained within this 5 week of very-tiring-yet-fun SIP... First, i have to explain the different parts of the working area. According to our schedule we'll be posted to 4 months of Histopathology lab and a month of Cytology.
In Histo lab, there are;

1. Outside area

• Reception/ Specimen sorting room
• Trimming room

2. Inside area- Main lab

• Embedding
• Microtomy
• Staining (H&E and Special Stains)
• Sorting slides + Verification of patient's form + Signed out to the respectives pathologists

For this month, im posted to the outside area (but most of the time im in the trimming room, assisting the pathologist).

All specimens that comes, will be received at the reception room, where the technologists have to first, verify the specimens against the checklist. If nature and the number of specimens (Specimens A, B anc C etc) correspond to the ones listed on the checklist, they are initialed, electronically dated and billed. Second, all the specimens are then given individual biopsy number (eg PB XXXXX) which will be its ID throughout the whole process.

The specimens will then be sorted out. The small and simple biopsies (eg; polyps, certain gallbladder and appendix) are given to technologists for them to pass (not necessarily trim, cos some are too small) and the large and complex specimens (eg; breast, liver, lung, kidney, nose, colon, ovarian cysts + fallopian tubes, enlarged gallbladder and prostate gland) are for pathologists to trim.

[[fyi: all specimens comes in a bag of formalin to be fixed. they have to be sufficiently fixed for atleast 6 hours before being trimmed]]

and..... all trimmings are done in the trimmning room... (haha!) thats where my main job is... which is to assist the pathologists. there are 3 stations in the room, so 3 pathologist are able to trim at a time. but sometimes more pathogists come in to do other stuff and thats just plain havoc for me...hehe

Before trimming, i have to prepare a set of cassettes (remember during HTech we use green colour cassette to embed?) for each specimens; labelling them with patient's ID, specimen ID and block ID. Then prepare the request/patient's form for pathologists. bla bla bla... n other necessary stuff.....that, in a way or another assist them in their trimming... hehehe

After trimming, the blocks are placed into an automated tissue processor. The process takes around 9.5 hours but is left over night (coz our lab, no night shift) and will be ready at the time that we set (eg; 7.30 am)

Principles of tissue processing:

3 stages of the tissue processing are designed to remove the extractable water from the tissue specimens and replace it with a medium that solidifies to allow sectioning. It must be firm enough to support the tissue and give it the rigidity and at the same time must be soft enough for the knife to cut through the tissue into thin sections with little or no damage.

1. Dehydration of tissue

Since the specimens are prior fixed in formalin, dehydration is to remove both water and the fixative (formalin) from the tissue using graded alcohol (dehydrating fluid). This step is essential because paraffin wax will not penetrate the tissue in presence of water and placing the specimens directly into 100% alcohol will distort the tissue.

2. Clearing the tissue

Since alcohol (dehydrating fluid) is completely not miscible with wax, it has to be removed from tissue and replacing it with fluid that is completely miscible with both dehydrating fluid and embedding medium (paraffin wax). common eg; xylene.

3. Infiltrating the tissue with paraffin wax.

Replacing the clearing agent with paraffin wax; infiltrating (impregnating) the tissue with embedding medium. Specimens are transfered from the clearing agent to molten wax at 60C and diffusion will occur. This step is different from the embedding step itself because at this stage the wax provides support internally and externally to the tissue.

Factors affecting tissue processing rate:

1. Agitation

Too slow => ineffective

Too vigorous => cause damage to soft and friable tissue.

2. Heat

Heat increases the rate of penetration.

3. Viscosity

Low viscosity => easier to diffuse into tissue

4. Vacuum

Removes air bubbles trapped within the tissue and able to bring the processing fluids into more intimate contact with parts of the tissue.

[i dont think stating the steps involved in the processing is appropriate, because there are many type of processing, each different in length time eg; 9.5 hr, 13 hr, 16 hr, and the steps involved are also different]

At the end of the processing, the blocks are ready to be embedded, sectioned and stained. (main lab)

and anw, unlike rachael, i didnt have the chance to witness a post-mortem.. =(

*First posted on THURSDAY 23 july, last editted on Monday.

=) =) =)

Week 4: Sherman

Hey guys! This is the 4th week, and it's my turn to post!


My topic will be on Biochemistry, Urinalysis.

The main aim of this test is to determine the perimeters that are present in the urine, for example, erythrocytes, proteins, bilirubin and leukocytes.

The material used is basically the dipstick.


Control:
2x Controls (1x Normal and 1x Abnormal)


The important perimeters that must be highlighted in the urine perimeter are the following;
1. Leukocytes
2. Nitrate
3. Protein
4. Erythrocyte
These 4 perimeters, under a normal condition, is tested negative of these component presence.
A deviation from JUST 1 of these perimeters from a normal condition, for e.g. a 4+ grade on Protein component, the urine sample is considered abnormal, and should be sent for microscopy examination.


Microscopy examination
Criteria: Urine is tested abnormal from dipstick test
Aim: To identify the different components present in the urine and count the number of cells present in the cells (e.g. White Blood Cells, Erythrocytes, Epithelial cells, Fungi)

* Counting of cells under microscopy examination
For control tests, all 81 grids of the counting chamber has to be counted, and records of the control test is to be taken down.
*The main perimeters to count are the RBC and WBC*
- If the WBC count exceeds >50 for a "Children Emergency" case, it's immediately sent for further testings.

Certain KEY perimeters to look out for in urine samples under the microscope:

1. Red Blood Cells
Variety of renal and systolic diseases, including trauma

2. White Blood Cells
Presence indicates an infection

3. Epithelial Cells
Active tubular degeneration
Squamous epithelil found in normal urine

4. Crystals
Urine is considered abnormal is the following are identified; cystine, leucine nd tyrosince
Varible to the pH of urine
E.g. Squarish crystals are calcium oxidate
Hexagonal crystals are uric acid

5. Bacteria
Evidence of infection (e.g. UTI)

6. Yeast
Urinary moniliasis

7. Casts


Further tests

Under certain circumstances, results reflected in the dipstick results will lead to further tests to confirm the reliability of the dipstick results (e.g. Bilirubin level)

If there is bilirubin detected in the urine, Fouchet is done

1. Mix well, 2 parts of urine with 1 part of Barium Chloride
2. Shake and observe for precipitation
3. If there is no precipitation, add 1 drop of saturated ammonium sulphate
4. Once the precipitate is obtained, filter the mixture and obtain the residue on the filter paper
5. Add 1 drop of Fouchet's reagent onto the residual area
5. Observe for a blue colour change
*A blue colour change is evident of bilirubin presence



Ok so that's all from me! =)
Any questions, feel free to comment and I'll answer it as soon as I can.

Till then, ciaos ~

Replies to Comments

Hello Wess,

sorry for not being very clear. The numbers are the values obtain when the absorbance is measured at 450nm.
And it when the control is 0.350, whether its the positive control or the negative control, the run has to be rejected and troubleshooting must be carried out.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Hello Siti,

for the negative results its less than 0.85 x COV, sorry for the typo.
as for false positive, yes it is possible as VZV IgG may crossreact with other antigens found in Herpes viridae species. And to confirm, VZV IgM immunofluorescence assay (IF) can be carried out to further confirm the diagnosis.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Hello YinLiang,

sorry for not stating, the absorbance used for reading is 450nm.
And DUHHHH, other viruses that can be detected using ELISA is dengue virus and rubella virus.


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

thanks,
kenneth

Medical Microbiolgy - Virology Serology Laboratory Techniques

Low Wei Qi Kenneth - TG02



Hello everyone,

SO sorry for a week delay of the blog sharing. For the few weeks of attachment, i'm attached to virology serology lab which performs tests on human serum mainly detecting Ag and Ab of specific virus. The test i'm going to share is ELISA ( Enzyme linked immunosorbent assay) or EIA (enzyme immunoassay).



Test - Human Varicella Zoster Virus (VZV) IgG Enzyme immunoassay ( EIA/ELISA)



The purpose of this test is to detect Immunoglobulin G antibodies to VZV in patients' serum where VZV is a virus that commonly causes chickenpox in humans.



In my lab, they use commercial test kits which provide all the reagents and pre-coated microtiter strip wells. The base of the wells serve as the solid phase are pre-coated with VZV antigens and by adding the patients serum and incubate it at room temperature, if the IgG antibodies are present it will bind to the antigens at the solid phase. After incubation, the wells are washed using the washing solution provided in the kit. Also, the washing step time is reduced using automated microtiter plate washer which is programmed to dispense a certain amount of wash solution into each wells and remove them. So after washing, conjugates are added. The conjugates are anti-human IgG antibodies with peroxidase conjugate. The anti-IgG is added and it will bind specifically to IgG antibodies that are bound to the solid phase. Again, the wells are washed to remove unbound substances. Later, substrate and stop solution are added the wells with VZV-IgG-Antibodies will produce yellow colour. The intensity of the colour is directly proportionalo to the amount of secondary antibody present in the well. And is determine by using automated ELISA microplate reader. After the absorbance of each well is being read, the result is printed out and is compared against cut-off values.

SOoooo heres the STEP BY STEP:

1. Reagents and samples are brought to room temperature.
2. 10 ul of Patients specimens are diluted in 1ml of dillution buffer. ( 100x dilution )
3. 100ul of Positive and Negative Controls and diluted specimens are added into each wells.
4. The plate is then incubated at room temperature for 30 minutes.
5. During incubation, the wash solution is being prepared by diluting the concentrate into distilled water. ( 20x dilution)
6. After incubation, the wells are washed using the microplate washer.
7. 100 ul of Conjugate is then added into each wells except the blank well which act as a control.
8. The plate is then incubated at room temperature for 30 minutes.
9. After incubation, the wells are washed using the microplate washer.
10. 100 ul of Substrate is added into each wells
11. The plate is then incubated at room temperature for 15 minutes.
12. 100 ul of stop solution is added to each wells to stop the reaction.
13. The microplate is then place into the microtiter plate reader for results.

Results interpretation :

Quality control criteria -

• Negative Control values should be less than 0.250
• Positive Control values should be more than 0.750
• +ve control / -ve control should be more than 5
• Blank ( empty well ) should be less than 0.150

If either of the values do not meet the criteria, the whole run is rejected and rerun has to be conducted.

Interpretation -

• Cutoff value (COV) = -ve control + 0.1 x +ve control
• Positive > 1.15 x COV
• Negative < 0.85 x COV
  

To interpret the results, calculations are carried out and these formulas are provided by the supplier of the kit.

Low positive will mean that the immunity is low and the patient is susceptible to re-infection

High/ mid positive will mean that the person is immune to the virus.

Negative will mean that is patient is not reactive or not immune to the virus.

So this is the end of my sharing, to summarise, mainly in virology lab, ELISA is one of the most common test that is conducted to detect viral infection as well as the immunity of the patient to specific virus.

****** THE END *******

Tng Wess Lee, 0702570C
TG02, Grp 10
Allocation: Clinical Biochemistry Laboratory

There are a total of 3 rooms in my working environment. We are allocated to 10 machines to learn 1 machine each week before being transferred to another lab. For the first 2 weeks, the machines which i have handled are basically non-analytical but an organiser.

The first week, i was allocated to Laboratory Automated System. The workstation requires me to sort sample according to their priority. (1. Urgent/Stat, 2. Ward, 3. Routine) For the entire duration i only received blood and urine specimens. The specimens are sent via a telelift system called a pneumatic tube system/carrier system. Upon arrival, the specimens are punched with time and date, to prevent dispute between the wards/clinics and the lab.

After sorting these samples, clerks will collect the sorted samples and enter the appropriate data in the LIS and print specific barcode labels, which are pasted onto the tubes. They will then pass the tubes back to me to place it at the inlet.

The samples then run along a line, where it is then sent to be centrifuged to separate the blood cells from the serum. After being centrifuged, the sample enters the Serum Level Detector to determine the volume of serum and if there is sufficient serum to be tested. After that, the sample is brought by the line into the decapper to remove the caps.

The sample is then sent to the labeller to label another tube if a separate test is required to be conducted. If there is an extra tube which exits from the labeller, both the original tube and the newly labelled tube will enter the aliquoter. The aliquoter aliquots the serum from the original tube to the new labelled tube. If no aliquots need to be done, the original tube will pass through the aliquoter.

The specimen then enters the analyser. The LAS is a preanalytical stage whereby it prepares the sample for analysis. It compromises of the Inlet, the Centrifuge, the serum level detector, the decapper, the labeller and the aliquoter.