Haematology

Hi all… I was attached to sgh haematology department and will be in coagulation lab for the 1st four weeks. I was assigned to do the routine PT and APTT test using the Sysmex CA-1500.

Principle: Both PT and APTT tests are used for the investigation of haemostatic failure. The prothrombin time (PT) tests for factors I, II, V, VII, X of the extrinsic system whereas the activated partial thromboplastin time (APTT) tests for all factors in the intrinsic system (factors I, II, V, VIII, IX, X, XI, XII). For PT, the time between the addition of tissue thromboplastin to the presence of a detectable clot is the prothrombin time; ref range 9.2-11.2s.
For APTT, the citrated plasma is incubated with APTT reagent (for the activation of contact factors) after which CaCl2 is added and the clotting time is measured; ref range: 27.0-36.1s.

Procedure:
1) Check if the received requisition form tally with the patient sample. Make sure that the level of blood is above the marker found on the tubes.
2) Stamp and label form and test tube.
3) Centrifuge the tubes for 3000rpm for 180s.
4) Put the tubes in the rack and placed into analyzer. Order test through the computer system.
5) Record results. PT<8.0s, APTT<26.0s and delta check indicate that test have to be repeated (to make sure it is not a random error).
6) Results will then be keyed into the LIS.


QC: Level I and II controls (used in sysmex CA-1500) are performed at an interval for every batch of 40 samples. Results that are out-of control may indicate that the QC is expired, analyzer has problem, reagent has problem.

here is a very simplified coagulation cascade:



When: -PT ↑
-APTT normal
Most likely is F VII deficient.

-PT ↑
-APTT ↑
F X, V, II, I deficient.

-PT normal
-APTT ↑
Most likely F XII, XI, IX, VIII deficient.

Eunice
tg02
0503245C

Serology-Immunology

Hi all,

Really sry ppl. So pai sei leh. I made a big mistake in the TPHA test thing.

1. The test cells are sensitised cells that reacts specifically to the antibody produced in response to the causative agent. It is by default brown in color.

2. After everything is added in, the solution in the wells are mixed evenly by tapping gently.

3. How the particles settle after the incubation gives you the result. (so must take care not to agitate the plate during the incubation and even before you read and verify the results.)

4. Particles concentrated in the shape of a button in the centre of the well with a smooth round outer margin is read as non reactive.

5.Definite large ring with a rough multiform outer margin and peripheral agglutination is read as reactive.

6. Particles concentrated in the shape of a compact ring with a smooth round outer margin is read as a boderline case. This is reported as "possible biological false positive result". A repeat testing in 10 days along with a FTA-antibody assay would be suggested to clarify if the patient is positive or negative.



Hope that helps in clafying some doubts =)
Sincerely,
Yeng Ting

Serology-Immunology

Hi all,

I am posted to a private clinical lab for SIP. Of which, my first 3 weeks is spent at the serology department before moving on to other departments of the laboratories.

Serology is basically a branch of immunology that deals with testing of patient's serum.

During this 2 weeks, I was mainly assigned to do the VDRL testing and Human ASOT because the other tests would require the usage of the LIS, which attachment students are not allowed to access.

This is the summary of the VDRL testing.

Principle of the test

RPR measures IgG and IgM produced in response to lipoidal material released from damaged host cells and also the lipoprotein released from Treponema pallidum. Thus, the antibodies detected are not specific for T. pallidum, which is the causative agent for syphilis.

Qualitative test
1. Using a Pasteur straw, place 100 µl of test serum into a circle of the test card.
2. Use the flatten tip of the straw to spread the serum evenly over the circle area.
3. Shake a plastic dropper containing the carbon antigen provided in the test kit to evenly mix the carbon.
4. Invert the bottle, holding it vertically to dispense a drop of the antigen. Each drop is approximately 0.4 µl.
5. Place the test card on an automatic rotator and rotate at 100 rpm (rounds per minute) for 8 minutes.
6. Read and interpret the results.

Semi-quantitative test
This test is performed if the result for the qualitative test is positive. This is to determine the antibody titre to aid the doctor in the treatment of the disease because the antibodies tend to disappear after successful treatment.To confirm the diagnosis, TPHA (T. Pallidum Haemagglutination) is performed.

The steps are the same as the qualitative test but instead, dilutions of the patient's serum of up to 1:32 is made using saline solution.

This is the Summary of TPHA testing.

Principle of the test

Gelatin particles, sensitised by purified T. Pallidum, agglutinate in the presence of antibodies against T.pallidum in human serum. A purple colored button would be developed after 1 hour of incubation at room temperature. This is compared with the positive control for interpretation of the results.

1. Label the test plate with the last 3 digits of the patient's ID and "C" for the positive control.

2.Prepare serial dilution of the patient specimen as follows:

Well no Diluent Specimen Test cells

1 190 10 75 for all wells
2 100 100 (carried over)
3 100 100 (carried over)
4 100 100 (carried over)
5 100 100 (carried over)
6 100 100 (carried over)


Final dilution
well 1 1 : 20
well 2 1 : 40
well 3 1 : 80
well 4 1 : 160
well 5 1 : 320
well 6 1 : 640

3. Mix the content by gently tapping the tray and incubate for 1hour at room temperature.

This is the summary of the Human ASOT

This test is basically antigen- antibody reaction of latex particles coated with stabilised streptolysin O as antigen against anti-streptolysin O (ASO) antibodies of patient's serum. This antibody is produced in response to group A Streptococci bacteial infections such as rheumatic fever or glomerulonephritis.

The method is basically the same as RPR. Instead of using a white background card, a black or dark background card is used. Instead of carbon antigen in the RPR, latex particles are used (caracterised as a milky colored solution). Instead of rotating for 8 mins in the RPR, 2 mins of rotation is needed or else it would give a false positive result.

I have also learnt how to use the Bio-Rad coda for EBV, HSV-1, HSV-2 and chlamydia IgG/IgM testing and was allowed to attend the training for the usage of their newly purchased equipment, the Bio-rad Evolis, which has an extensive list of tests it is able to perform.

The senior I was attached to also taught me how use the Serodia-Myco II test kit to test for anti-mycoplasma pneumoniae antibodies although I have yet to perform the test.

Cheers,

Yeng Ting

Question and Answers: Microbiology

Q1) What does XLD plate stands for?
Ans: Xylose lysine sodium desoxycholate (XLD) plate

Q2) What makes the Salmonella colonies black?
Ans: The Salmonella(non-glucose fermenter) produces H2S when growing which causes the colonies to turn black
Q3) How Rotavirus Quick test works?
Ans: It works by detecting the Rotavirus antigen using monoclonal antibodies (Elisa mtd). The test kit has monoclonal antibody againts the Rotavirus antigen, if the stool contains the virus a coloured pdt will be formed.


Q4) What is a MUG test?
Ans: Is by using the principle of antibody binding to the specific antigen on the Ecoli and under UV light the Fluorence compound labelled on the antibody will emit light(Ecoli positive)

Q5) What do you mean by using 3 parts of the loop?
Ans: The picture shows a inoculation loop which i have colour coded and numbered it. When doing the primary streaking use the Black part of the loop, Green part when streaking the secondary, and red part when doing the tertiary streak.(seen above)


Q6) What is a Primary, Secondary, Tertiary streak?

Ans: Refer to diagram above.

Q7) Why must the stool samples be subcultured onto the 3 different types of media (XLD plate, Campylobacter Plate, Selenite F broth)?
Ans: We want to look out for Salmonella which will grow as black colonies on the XLD plates, Campylobacter plate to look out for Campylobater jejum species(tiny grey colonies on the campy plate), so we will be able know the specific cause of the diarrhoea. The selenite culture is just for subculturing the stool sample on to a XLD plate after incubation for 1day.

Q7) Growth and identifying campylobacter.
Ans: The Campylobacter jejum species looks like pin-point grey colonies on the black background of the campy plate. a test can be done to verify the bacteria(Hippurate test). By adding the colonies into the hippurate reagent and incubate for 10mins and then add 7drops of Ninhydrin reagent and incubate for 2hrs-positive will have a purple coloured ring on the upper part of the solution.

Q8) For the quick test for rotavirus antigen identification.. are there any negative or positive controls to be done when doing it?
Ans: actually the blue band is already a control band so we do not have to run a seprate control. If the blue band does not appear, the test is not considered and the test must be redone.

Q9) For the vp3 test, how useful is it in diagnosis of microorganisms infection in the female reproductive system?
Ans:According to the manufacturer, the test kit's sensitivity is 100% and the specificity is 99%.

Q10)for UTI diagnosis,what would be done if its negative for the test, is there any further investigations that would be performed if the results turned out to be negative (not E.coli)?
Ans: It depends if the FEME count is it high if it is high we must issue a prelim report to the doctors to indicate no significant/no bacteria growth but also reflect the WBC count, so the doctor can monitor the patient's progress

Q11) What is a zig-zag line on the agar?

Ans: Refer to above diagram. the sequence of streaking is written in the picture. for the top part the streak is very close together and then when it comes close to the bottom the space is wider(but remember to maximize the space avalible at the sides. Also no note that the streaking must be done fast to prevent the urine from drying up and the streak must be consistent.

Q12)What are the main differences between MacConkey and CLED?

Ans: for CLED it is a more specialized media used for isolation, presumptive identification, for my lab we use the CLED for those overnight samples that is sent to us after working hours as if urine is not directly plated within 2hours it must be kept at 5degrees C. By using a dip slide which contains 3 types of agar: Mac, CLED, colourless base medium which is for identification of Ecoli. Mac agar inhibits gram pos bacteria which are unlikely causes of UTI thus we plate it on mac to rule out the gram pos bacteria.

Cheng Hong: Mircobiology

Subject title: Microbiology

Urine culture for diagnosis of Urinary Tract Infection(UTI)

• To culture patient’s urine sample to look for bacteria growth(mainly E.coli) which is the main cause of UTI
• Using a 1µl inoculating loop and dip just below the surface of the urine sample and draw a straight streak down the plate(both MacConkey and blood plate), then steak zig-zag lines across the agar(this is to obtain isolated colonies for identification and testing
• Incubate the plates overnight aerobically
• Obtain the plate the next day for identification of bacteria colonies
• If there is growth(pure/mix bacteria growth), look out for E.coli which morphology is white, slightly bigger than other bacteria colonies and has a very “typical smell”
• Do a indole test on the suspected E.coli colonies: if test result is green(positive), if its pink(negative)
• If the bacteria is a fermentative Ecoli the MacConkey plate should be reddish(ferment lactose) if it is not(yellow plate), we have to do a MUG test to confirm if it is a E.coli using Bacticard-Ecoli test kit which is bought commercially
• First, 1drop of rehydrating solution is added, then pick up some of the colonies using a inoculating loop and add to the test card and incubate at RT for 15mins
• Then add 1 drop of substrate and look under fluorescence light, if there is fluorescence (positive for Ecoli) vice versa
• At the same time for each patient a Urine FEME(WBC,RBC,Epithelial cell count) is also used to help in the diagnosis
• If the patient’s WBC count is high(50,000cfu/ml onwards there is a high chance of the patient being diagnosed with UTI if there is Ecoli growth on the agar
• After reporting the results to the doctors through LIS the culture is sent for verification and sensitivity testing(Antibiotic susceptibility testing)
  
  
  Stool culture for diagnosis of cause of diarrhea
• For diagnosis of diarrhea there are 3 main types of bacteria we are looking for: Shegella, Campylobacter, Salmonella
• For stool culture there are 3 types of media which is needed to be inoculated on(XLD plate, Campylobacter Plate, Selenite F broth)
• (Working behind a fume hood) Use a wooden stick to obtain some stool sample and inoculate on the XLD and Campy plates) each plate can have 2 different patient sample)-do streaking
• Use the remaining stool sample on the stick and put it into the Selenite broth
• Incubate the plates overnight(XLD, Selenite in 5% CO2 incubator and Campy plates in anaerobic jar with gas pack at 42oC-2days)
• Obtain the plates for interpretation the next day(XLD plates)
• If there is growth of black colonies(colonies are not individual, merge with neighboring colonies) we can interpret as growth of salmonella(cause of diarrhea due to ingestion of uncooked/ partially cooked meat)
• Send the plates for verification and sensitivity testing
  
  
  Rotavirus Antigen Identification
• Pick up patient’s stool sample and inoculate in to 1ml of buffer(comes with the commercial test kit)
• Gently mix the sample with the buffer
• Add 4drops of the solution from step 1 into the Rotavirus Quick test kit
• If only a blue line is seen the patient is negative for rotavirus antigen
• If a blue and red line is seen, the patient is positive for rotavirus antigen
  
  
  VP3 test
• Vp3 test is done using a vaginal swab to test for Trichomonas, Gardnerella, Candida
• The swab is placed into a tube and the excess is cut off using a scissors(remember to disinfect with isopropyl alcohol)
• 12 drops of lysis solution is added and the tube is capped and incubate it in a heating block for 10mins at 85oC
• Then add 12 drops of buffer and gently flick the tube and cap it with a filter nozzle
• Add 4 drops of substrate solution to the 7th well of the test kit and dispense the solution from the tube into the first well
• Then the PAC(test card) into the first well and place the whole kit into the Affirm VP3 machine and run the machine
• The machine will automatically move the PAC from the first well to the last well
  *those who want the contents of the individual wells please let me know
• At the end of the run if there is a blue colour on any of the respective organism it shows that the patient have the infection

*When streaking the plate always use 3different sides of the inoculation loop to make the primary, secondary and tertiary streak to be able to get isolated colonies.

*When doing biochem test or other test(eg:indole/oxidase test) use colonies grown on natural media(eg:blood agar)

If there is any question regarding the postings please comment, thanks

*Cheng Hong*

General: Order of Posting

Hi all,

We need to decide on the order on who is posting for the weeks during our SIP. So far as I know, we have 1 in research (post every 2 mths), and 5 in clinical labs (post every 6 weeks).

I was thinking of having the order like this (due to the timing):
[EDIT]
1. Clinical lab - Cheng Hong
2. Clinical lab - Yeng Ting
3. Clinical lab - Eunice
4. Clinical lab - Pei Shan
5.Clinical lab - Elaine
6. Research - Debra

Please do add comments if you have better suggestions. If this is ok, we need to sort out who takes what slot for clinical labs. Also for these future posting, do remember to sign off your own name so the lecturer can see it!

All the best for tommorrow's SIP!

Cheers,
Debra

LMQA

Hi all,

Our LMQA presentation is on CAP accreditation. This stands for College of American Pathologist accreditation program.
I have found the website to the college of american pathologist website, which I feel would have all that we may need for the presentation.

http://www.cap.org/apps/docs/laboratory_accreditation/standards/standards.html

There are 4 standards in the CAP accreditation.

Here are the references that are recomended by the site.

1. College of American Pathologists. Criteria for the Clinical Laboratory Director. Appendix O in: Policies and Guidelines Manual. Northfield, IL: February 1993.

2. US Department of Health and Human Services. Clinical Laboratories Improvement Amendments of 1988. Code of the Federal Register, Title 42, Part 493. Washington, DC: 1988.

3. Webster's Third New International Dictionary. Springfield, MA: G & C Merriam Co; 1976.

4. Joint Commission on Accreditation of Health Care Organizations Glossary. In: Accreditation Manual for Pathology and Clinical Laboratory Services. Chicago, IL; 1996.

5. American Board of Pathology.

6. College of American Pathologists. Appendix EE in: Policies and Guidelines Manual. Northfield, IL: August 1992.

7. College of American Pathologists. Glossary. Appendix J in: Policies and Guidelines Manual. Northfield, IL: February 1993.

8. Bartlett R, et al. Quality Control in Clinical Microbiology. Chicago, IL: American Society of Clinical Pathologists (CCE); 1968.

Related Links
Standards for Forensic Urine Drug Testing Accreditation (FUDT)



Cheers,
yt