PCT

Hello, for my SIP I have already started on my Major project which is on the evaluation of a machine that analyses on PCT. I will describe the PCT in detail in this post.

The prohormone precursor for CT is a 116-amino acid polypeptide which has three component peptides, a 57-amino acid peptide at the amino-terminus, named aminoprocalcitonin (aminopro-CT), a centrally placed 33-amino acid immature CT and a 21-amino acid CT carboxyl-terminus peptide-I. its expression is induced several fold in the systemic bacterial infection and sepsis to elevated levels found in blood.
PCT has been demonstrated to be the best marker for differentiating patients with sepsis from those with systemic inflammatory reaction not related to infectious cause. Procalcitonin provides support for early diagnosis and allows critical decision making which could direct an effective treatment at the proper timing and also to save unnecessary costs in the use of appropriate antibiotics for critically ill patients. Produced in numerous neuroendocrine cells such as the thyroid glands and present in very low concentrations in blood, it has been shown that systemic bacterial infection causes a marked increase in PCT expression (encoded in the Calc-1 gene), hence its clinical utility as a indicator of sepsis of bacterial origin. Very significant concentrations of procalcitonin (up to 1000 ng/ml) can be detected in the patient sample with severe bacterial infection/sepsis

The inflammatory and sepsis markers present now are IF-2, IF-6, IF-8, PCT, CRP and TNF-α. PCT has currently show in recent studies that it has better specificity and sensitivity over the classical sepsis markers in systemic sepsis, moreover, it has a higher and earlier increase during sepsis, and the level of PCT present usually correlates with the severity of the disease.

PCT compared to CRP

In this diagram PCT is shown to have a higher pooled sensitivity and specificity over C reactive protein, which is a classic inflammatory marker.

References: http://www.procalitonin.com/
http://www.jle.com/

yuxuan

Week 9 - Virology

Hello people! For the 4th to 8th week, I was at the virus isolation section of virology.
For this area, it is mainly responsible for specimen processing, virus inoculation, maintenance of culture tubes and repassaging (Repassaging is to subculture or split cells into new culture tubes to prevent overgrowth and degeneration.)

Basically, after the specimen is processed, it is inoculated into respective culture tubes and incubated. The tubes are examined for cytopathic effect (changes in appearance of caused by the virus that is growing) everyday and results are recorded. If any cytopathic effect is observed, then the specimen will be sent for confirmatory tests – immunofluorescence, acid test and neutralization test [This will be elaborated in the next post :)]

The culture tubes are maintained by changing their media on every alternate day. This is done to maintain the viability of the cells - as nutrients will deplete and waste products produced by the cells are detrimental to growth. However, this will still depend on the condition of the cells. If the cells are going to be repassage the next day or they are still healthy, then there is no need to change media on that day itself. As for repassaging, it is usually done on the 7th and 14th day after inoculation when degeneration of cells is observed.

Changing media for culture tubes

1. Remove media in culture tubes by using a 1ml disposable plastic pipette.
2. Discard media in disinfectant, sodium hypochlorite.
3. Add in 1ml of new media using a new pipette.
4. Cap tightly and arrange it back into roller drum.
5. Incubate in respective incubator, depending on the type of virus.

ROLLER DRUM (Picture taken from http://www.nbsc.com/rollerdrums.aspx )

Repassaging for culture tubes

1. Select the culture tubes according to the original cell type (Eg, Hela tube needs to be repassage, the new tube chosen must also be hela.)
2. Check the selected tubes under microscope to ensure cells are present and confluent.
3. Label the newly selected tubes with its original lab number and the date of repassage.
4. Loosen the caps of the tubes [Done in BSC-II].
5. Scrape the cells in the original tube by using a bent pipette.
6. Flush the cells up and down to ensure that it well mixed.
7. Inoculate 0.2ml of the cell suspension into the newly selected tubes.
8. Cap tightly and arrange it back into roller drums.
9. Incubate in respective incubator, depending on the type of virus.

In this section, we will receive many different specimens. Each of them will come with a request form, indicating what virus is suspected and needs to be isolated. Hence, we will only perform that particular test.

Here are some of the commonly received specimens and the virus that is suspected of…

ETDA Blood -- Enterovirus/ Neurotropic/ Cytomegalovirus

Lung tissue -- Respiratory virus

Heart/intestine/brain tissue -- Enterovirus/ Neurotropic virus

Urine -- Cytomegalovirus /Mumps /Adenovirus

Stool -- Enterovirus/ Neurotropic virus

Vesicles/ ulcer / genital swab -- Herpes virus



Now, let me share with you guys on specimen processing.

Urine Processing

1. Centrifuge the urine sample for 10minutes at 200rpm.
2. Add 1.2ml of media to a clean sterile container.
3. Pipette 1.2ml of supernatant and mix it with the cell deposit, thereby forming a cell suspension.
4. Transfer the cell suspension to the container with media.
5. Inoculate 0.3ml of cell mixture into respective shell vials and tubes.
6. Preadsorb for 1 hour before incubation (preadsorb will help in virus penetration)

Stool Processing

1. Add 10 glass beads into a sterile plastic tube.
2. Add 10ml of Dulbecco PBS, 1ml of antibiotic and 1ml of chloroform to the tube above.
3. Take a pea-sized amount of stool and add it in.
4. Seal the tube with parafilm to prevent leakage.
5. Vortex the tube and place it on the horizontal shaker at 200rpm for 20 minutes.
6. Centrifuge at 2600rpm for 20 minutes.
7. Transfer 5ml of the supernatant into a clean sterile 5ml tube.
8. Select the tubes and label them with date of inoculation and lab number.
9. Inoculate 0.2ml of the supernatant into each tube.
10.Preadsorb tubes before incubation (preadsorb will help in virus penetration)

Glass beads- serve as stirring purpose
Dulbecco PBS- contains calcium ions that will help in stabilizing the virus
Antibiotic- prevent bacteria contamination
Chloroform- helps to disperse virus aggregate and kill any bacteria or fungus present

Tissue processing

Lung tissue (to test for respiratory virus)
1. Transfer the lung tissue into a mortar and cut it into small pieces using a scissor.
2. Add in alumdum powder and grind it with a pestle.
3. Transfer the contents into a sterile centrifuge tube and centrifuge at 3500 at 20 minutes.
4. Separate the supernatant from the deposit ( supernatant into a sterile 5ml tube while deposit back to the original container)
5. Select the tubes and shell vials before labeling them with date of inoculation and lab number.
6. Inoculate 0.3ml of supernatant into the tubes and shell vials.
7. Centrifuge the shell vials at 1800rpm for 30minutes.
8. Preabsorb the tubes at 33±0.5̊̊ C for 1 hour.
9. Incubate both tubes and shellvials overnight and observe for cytopathic effect the next day.

Alumdum- To provide sharp edges for grinding the tissue


That’s all for now. Hope that you guys can understand.

Shihui
0607135A

Week 8 Microbiology

This week I will be sharing with you about ‘Motility’. In the Investigation lab, motility test is one of the biochemical tests carried out. It is a characteristic to classify and identify the type of bacteria. Examples of motile bacteria: Proteus vulgaris and Pseudomonas aeruginosa. Examples of non motile bacteria: Staphylococcus aureus and Streptococcus pyogenes.
There are two ways to determine motility.
1. Through the use of a medium.
2. Microscopic examination.

1. Using a medium
The medium used is OF (Oxidative-Fermentative) medium.
[Oxidative-Fermentative is another biochemical test.]
OF medium has an agar that is in semisolid form which can also be used to determine if the bacteria are motile or non motile. Semisolid agar (soft agar) allows the bacteria to be diffused throughout the medium by the presence of flagella. Since one tube of OF medium can be used for 2 biochemical tests (Oxidative-Fermentative test and motility test), there is no need to use the motility medium which can only be used for motility test.

Methods:
a. Pick a few colonies and mix with a tube of saline to obtain a McFarland turbidity standard of 0.5.
b. Use a disposable inoculating needle to obtain a drop of the suspension and stab into the medium.
c. Incubate at 35°C in O2 conditions for 18 – 24 hours.
d. Examine the medium.

Results:

Motile bacteria: The inoculation line cannot be clearly seen as the bacteria are able to grow away from the inoculation line and diffuse into the surrounding medium.

Non motile bacteria: The inoculation line [stab] is clearly seen whereas the surrounding medium is clear. (The bacteria remain localized at the inoculation line).












(non motile bacteria)

2. Microscopic examination
This method is known as the hanging drop. It is used when the results of the previous method is not significant (Not sure whether it is motile or non motile).

Methods:
a. Prepare a glass slide with a ring of plasticine.






b. Use a wooden stick to obtain a drop of suspension from the tryptophan broth containing the bacteria and place it on the cover slip. [Tryptophan broth is used for another biochemical test. The OF medium used in the previous method cannot be used as it is not in liquid form.]

c. Invert the glass slide and press it against the cover slip. The drop of suspension should be hanging inside the ring of plasticine.

d. Observe under the microscope at 40X objective.

Results:
Non-motile: The bacteria appear to be stationary.

Motile: The bacteria are moving in forward or in spinning direction.


LeeJin
TG02

Week 7 - Special Coagulation (Haematology)

For this week, I was attached to the Special Cogaulation Lab where there are many SPECIAL tests carried out. These tests require reagents that are expensive thus I do not really have any hands-on. All I can do is just observed. Some tests done in the lab are automated while some are done manually. For those tests done manually, timing is very important as reactions take place constantly.

For this entry, I shall share with all of you about the soluble fibrin monomer complexes test which is done manually in the lab.

Soluble fibrin monomer complexes test

USE:
It is a rapid qualitative test for the presence of the monomer in the plasma by the hemagglutination technique.


PRINCIPLE:
Fibrin monomers are intermediate products between fibrinogen and fibrin. They are produced during the proteolysis of fibrinogen by thrombin. [Proteolysis: digestion of proteins by enzymes called proteases] When the thrombin concentration in plasma is low, fibrin monomers are in insufficient quantities to aggregate to form a fibrin clot. Thus, fibrin monomers associate themselves with fibrinogen/ with fibrinogen degradation products to form soluble complexes. The presence of small amounts of soluble fibrin monomer complexes in the plasma can quickly be visualized with the F.S test by the agglutination reaction of human erythrocytes coated with purified fibrin monomer which do not agglutinate with normal plasma.

TYPE OF SAMPLE: Blood in sodium citrate tube.

PROCEDURE:
1) 100mL of patient’s plasma, 100mL of positive control and 100mL of negative control are added to 3 separate tubes.
2) 50mL of F.S test reagent is added to each tube and mixed well.
3) The tubes are incubated for 10 minutes in the water bath.
4) The tubes are removed from the water bath and dried. The contents in the 3 tubes are transferred using a pipette to a circle on the hemagglutination plate.
5) The plate is rocked gently to make the liquid swirl around the circle for 6 minutes.
6) The hemagglutination patterns of the patient’s plasma are then compared with those of the negative and positive controls.


REFERENCE RANGE:
Normal plasma does not produce agglutination of the F.S test.


INTERPRETATION:
Positive F.S test may be found in the following clinical conditions:
-Liver disease/cirrhosis
-Angioma
-Leukemic cancer
-Infection/Septicemia
-Polytraumatic condition
-Obsteric complications
-State of shock
-Thrombotic disease
-DIC of other organs


DONE BY:
Malerie Goh
TG02

Hello i hope everyone is having a great time at SIP now. its been so long now. For today i'll be blogging on osmolality. and my lab uses a different equipment from our school's. it seems to be more advanced. well on to it =)

Osmolality is an expression of total concentration of dissolved particles in a solute without regard to the particle size, density or electrical charge. The measure of total solute concentration can only be made indirectly by comparing one of the solution colligative properties with corresponding cardinal property of pure solvent.
Vapro osmometer is based upon a measurement of vapor pressure depression made possible by thermocouple hygrometry. This sensitive temperature sensor operates on the basis of a thermal energy balancing principle to measure the dew point temperature within the chamber.
10 micro liter of specimen is aspirated into a micropipette tip and then inoculated into a solute free paper disc. The paper disc is then pushed into the instrument and sample chamber is locked. The sensing element is a fine wire thermocouple hygrometer. This is suspended in a unique, all metal mount, which forms a small chamber enclosing the specimen when joined with the sample holderAs vapor pressure equilibrates in the chamber airspace, the thermocouple then seeks the dew point temperature within the enclosed space, giving an output proportional to the differential temperature. The difference between ambient temperature and dew point temperature is the dew point depressionSample used- serum or urineStepwise.push sample chamber level upwards and pull the sample slide out from the instrument.
use forceps supplied to place a single sample disc in the central depression of the sample holder. If more than two discs are used, results will slightly elevated

Using a micropipette, pipette 10ul of sample

Rest pipette tip on the notch of pipettor guide. It should be about 5mm away from center of sample discsmoothly depress micropipette plunger to release sample onto sample discwith plunger still held down, lightly touch the pipette tip to sample disc, then lift it away. Tip must briefly contact sample disc to press the disc flat against the holder

Sample disc should appear fully saturated, with a slight liquid meniscus on surface.

Note: when cleaning the sample chamber, use only lint free tissue. Facial or soft tissue will produce excessive lint residue that will contaminate the thermocouple sensor

Yuxuan