Trepanema pallidum Haemagglutination Assay

Hi everyone..

For this week i was again attached to the Microbiology department.
I was introduced to a new test called TPHA (Trepanema pallidum Haemagglutination Assay) .This is a confirmatory test used to confirm the presence of Trepanema pallidum(syphillis) in patient's blood sample.


This test is usually done when VDRL result is positive. Since VDRL is not a confirmatory test, this test therefore is important in determining whether the patient really has syphillis or instead due to other contributing factors and conditions which gave a false positive VDRL result.

A false positive VDRL can be encountered in infectious mononucleosis, lupus, hepatitis A, leprosy, malaria and, occasionally, pregnancy.

Hence TPHA test is a very important test to ensure the condition/disease is diagnosed correctly.

This test uses the Serodia-TPHA test kit. It takes 2 hours incubation after which result can be obtained. This test makes use of the 96-well microtiter plate. However for every individual test, only 4 wells (in vertical order) will be used.



These are the reagents used,available in the kit:

1) Unsensitized particles
2) Sensitized particles
3) Sample diluent


The following are the procedures:

1) Pipette 100 ul of sample diluent into the 1st (top most) well
2) Pipette 25ul each into the 2nd, 3rd and 4th wells
3) Dispense 25 ul of serum into the 1st well and pipette up and down to mix the sample diluent with the serum
4) After mixing the contents of the 1st well, pipette out 25ul of the mixture and serially dilute it into the 2nd, 3rd and 4th wells consecutively.
5) The 4th well therefore should have the lowest concentration of serum
6) Using the droppers provided in the kit, add 1 drop of unsensitized particles (Control) into the 3rd well.
7) Add 1 drop of sensitized particles into the 4th well
8)Gently tap the sides of the plate to mix the contents together.
9) Incubate at room temperature for 2 hours,away from any vibrations.
10) After two hours, read the result from the 4th well and report it.

Only the result from the 4th well should be taken into account.





This is an example of how the result may appear.

This picture only shows the 4th well of 4 separate tests.
The 4th well of Test no. 1 and 2 show positive results. For both cases, samples are positive for presence of Trepanema pallidum.
However for the 4th wells of Test no. 3 and 4, results are negative. Hence this shows that no Trepanema pallidum is present and the previous VDRL result is a false positive.


Final result will be reported as REACTIVE (positive) or NON-REACTIVE (negative).


Since this kit takes up to 2 hours for the result to be out, it is thus used only for routine specimens.

For urgent specimens, since the turnaround time is 1 hour, the lab makes use of a different TPHA kit which only takes up 15 min and also has a much simpler procedure.



Alright, that is all for now. Feel free to comment if you have any questions!

Thanks =D

Siti Hawa
TG02
GRP 10

Clinical Chemistry: Laboratory Technique

Preparation of Haemolysate for Major Project Experiment

In my major project, i am tasked to evaluate a serum protein electrophoresis system.

Within the evaluation process,i intend to demonstrate the influence of haemolysed samples on the results of the tests. This portion of experiment is conducted for interference study of evaluation process.

As stated, my system separates serum proteins according to their charges along an agarose gel. It separates the proteins into 2 major fractions known as albumin and globulin. The fractions can be further catergorized into 5 minor distinct fractions, known as albumin, alpha-1, alpha-2, beta and gamma.

As you known, when a blood sample is lysed, haemoglobin is released into the serum. Haemoglobin is a protein and therefore may appear as an artefact on the gel presentation, increasing the concentration of alpha-2 and beta protein. Haemoglobin should not appear as a band, as only serum is used to be tested on the analyser.

Therefore, i prepared haemolysed samples to identify the extent of influence of haemolysis on the results. In this posting, i will explain the steps taken to prepare haemolysate. Haemolysate is the preparation of the resulting product of erythrocyte lysis.



HOW DO WE GET HAEMOLYSATE?

1. Obtain a normal blood sample collected in EDTA tube and spin down for 10mins, 3500rpm.
2. Remove plasma and add 10ml of saline to red cell pellet.
3. Resuspend red cells in saline by inverting.
4. Spin for 5mins,3500rpm and decant saline. Step 2-4 constitutes washing of red cells.
5. Conduct washing of red cells for 3 times.
6. Remove saline and add equal volume of deionised water. Mix well by inverting.
7. Freeze cells overnight in freezer.
8. Thaw cells til rtp.
9. Centrifuge mixture for 30mins.
10. Save supernatant. Supernatant = Stock Haemolysate

Stock Haemolysate will be used to spike normal serum to produce different levels of haemolysis status. We achieved 3 levels of serum status (mildly lysed, lysed and severely lysed). These samples will be used to identify the extent of haemolysis on the results of electrophoresis.


Here are some pictures to improve yr imagination of the above information.



These are the fractions along with some specific proteins found within the fractions. (above)










Concentrate on lanes 8-11. 8 is normal serum, 9 is mildly lysed sample, 10 is lysed sample, 11 is severely lysed sample. Look at the alpha-2 and beta region. You will notice increasing concentration.

Tng Wess Lee
0702570C

Sherman - Histopathology

Frozen sections

A procedure where a biopsy is taken, frozen and diagnosed by a pathologist within a short period of time

Frozen section is done on the account that the patient is currently undergoing an operation, and had a biopsy which requires immediate attention.
The entire procedures goes to such an extent that time is of an essence, a simple reason being that whiles all these are being done, the patient is still sedated.
A guideline for such procedures to be done is within 10mins per sample


The entire procedure in the laboratory goes as follows;

1. Retrieval of sample
The medical technologist will proceed to the operating theatre and collect the biopsy. The name, time of collection and sample type must be confirmed upon collection.
This entire procedure should take the most 10mins only. Be aware to have a hand free of gloves due to operation of the elevators

2. Transport of sample back to laboratory and processing
Clock in the time received and inform the pathologist which is involved with the case given. Ensure that the workbench is ready for the pathologist to trim the specimen

3. Scribing of information
As the pathologist trims, it is essential that all information that the pathologist disclose upon gross examination of the sample is recorded.

4. Preparation of the chuck for frozen section
Prepare the chuck with O.C.T compound (a compound which gives the freezing effect). The tissue is mounted into the compound and brought to the Tissue Tek (a microtome used for frozen section).

5. Sectioning of the tissue
The tissue is sectioned within the enclosed space of the Tissue Tek. The microtome operates similarly to a conventional microtome, only is done within the cold environment, otherwise the tissue will be damaged. All equipment (brushes etc) are to be kept in the cold environment too. A difference in temperature in the equipment can cause the tissue samples to stick onto it due to the thermal gradient, thus destroying the tissue. The tissue is mounted onto a slide and subjected to rapid stain




6. Rapid stain
- Formalin (after that rinse for 1min in running water)
- Hematoxylin for 1min (rinse the hematoxylin away in running water)
- Lithium carbonate
- Absolute alcohol
- Absolute alcohol
- Xylene
- Xylene

7. Mount in DPX
8. Present slide to pathologist for observation under microscope.
Clock in the time too when the pathologist has finished with the necessary diagnosis.

Histopathology Lab

Nadiah Sukma TG02


Helo0o..

for today's post, i'll be sharing with u what ive learnt in the main lab. basically it is a continuation of my first post.

Once the tissue are processed, they will have to be embedded, sectioned and stained. and all this takes place in the main lab.


Embedding

After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding.
Processed tissues are placed in molten paraffin (56 C melting point) such that after the paraffin cools, the tissue and paraffin wax will form a block of sufficient consistency to cut thin sections.
Embedding can done by:

1. Filling a mould of suitable size with molten wax
2. Orientating the specimen in the mould to ensure it is being cut in the right plane
3. Cooling the mass to promote solidification

Orientation of tissue specimen:

1. Tissue specimens must be embedded flat to ensure that a complete section is obtained.
2. Tissues must be placed such that the resistance to the knife flows from lower to greater amount. this is to achieve smooth sections as the knife cuts through the soft to the hard part of the specimen.
3. There should be adequate paraffin wax surrounding the tissue to provide maximum cutting support. (must ensure there is no air bubbles which will results to holes in the block)
4. Tubular structures such as veins and arteries and fallopian tubes must be embedded vertically in the mould such that the knife cuts across the lumen.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder and uterus must be positioned such that all tissue layers are cut across.
6. Multiple specimens should be placed side by side, close to each other so that all pieces can be sectioned.
7. Rectangular tissues should be placed parallel to each other with their long axis perpendicular to the plane of section.
8. Small bisected cyst (half of a sphere shape) must be embedded with the cut surface down and must ensure that no air bubbles are trapped in the praffin.
9. Muscle biopsies in 2 pieces should be embedded with one piece in a longitudinal and the other in vertical position.

Shaving

After solid blocks are formed and cooled, they are sent for shaving also known as rough cutting before they are ready for sectioning. Shaving at 20 microns is to removed the paraffin wax and to expose the entire surface of the tissue including the margins.

*If entire surface could not be exposed due to un-flat embedding, the blocks must be sent for re-blocks.

Once shaved, the blocks are then soaked in 10% fabric softener for 5 mins. This is to smoothen the tissue surface to ease sectioning to obtain very thin ribbons.But using a commercial softener is not essential. it is also possible to just soak them in cold water for 5 mins.

*Tiny biopsy tissues and fats should NOT be soaked.

Microtomy

After shaving and soaking, all blocks must be kept cool at all times , hence they are placed on a cooler before being sectioned.

Sectioning using rotatory microtome is to produce 4 microns (one cell thick) thick of section to be fixed on microscopic glass slide for easy examination under the microscope.

Things needed:

1. Rotatory microtome
2. Warm floatation water bath (48 +- 4C)
3. 1% alcohol cold floatation bath (95% alcohol 5ml and SRW 500ml)
4. Microscopic glass slides
5. Forceps, brush and pencil.

Sections are cut using an undamaged part of the knife. The motion must be continuous to produce even thickness sections and long ribbon of sections. Interrupted motion or change of speed during cutting will result in uneven thickness which can be seen after staining under the microscope.

The long ribbon of large tissue specimens produced are then transferred to the 1% alcohol foatation bath first before transferring to the warm bath for fishing. The ribbon is let to float in the alcohol solution to expand the tissue to prevent folds (or to unfold the tissue when the folds are difficult to get rid using forceps, brush). This is because alcohol having low vapour pressure will increase the surface tension of the tissue when transferring to the heated floatation bath. Once the section has spread sufficiently, they are then transfered on to the glass slides and sent for staining (H&E or Special stains).

OKIES thats all for now..

my next post will be on staining...

Thanks! have a happy happy day...