Medical Microbiology - Virology Serology Laboratory Techniques

Low Wei Qi Kenneth TG02


Hello everyone,

this time im going to share another serology technique in detecting viruses total antibody for Influenza A, Influenza B, Herpes Simplex and many others. The test is known as Complement Fixation Test ( CFT). This test uses the properties of complement to lyse cells in the presence of antigen-antibody complexes.

CFT is used to detect the presence of specific antibody in the patient's serum. this test utilizes sheep red blood cells (sRBCS), anti-sRBCS antibody, complement and specific Ag. If Ab is present in the serum, the complement will be completely utilized and sRBC lysis will be minimal. If the Ab is not present in the serum, then complement will bind to anti-sRBC antibody and cause sRBCs to hemolyse.

Sooo here is the STEP BY STEP:

1. 50ul of serum specimen is diluted with 350ul of veronal buffer (VB)
2. diluted specimen is then heat inactivated at 56 degree celsius for 30 minutes
3. after inactivation, 25 ul of VB is added to the microtiter wells except the first row
4. 50 ul of specimen is added to the first well
5. titrate 25 ul of specimen from the first well downwards
6. 25 ul of viral antigen is added to each well
7. 25 ul of complement is added to each well
8. the microtiter plate is then incubate at 2-8 degree celsius overnight

Next day,

1. sRBC is washed using VB for three times
2. 24 ul of sRBC is mixed with 24 ul of hemolysin
3. 50 ul oof sRBS mixture is added to the wells
4. the plate is incubated for 30 minutes at 37 degree celsius
5. after incubation, the plate is centrifuged at 2000rpm/ 4 degree celsius for 10 mins
6. reading of results

Controls involved

Antigen control - using positive and negative antigen

serum control - using serum only

complement control - complement at different concentrations and insensitized cells

Reading of Results

Positive - no hemolysis/ button observed - meaning patient serum contains Ab to virus

Negative - hemolysis/ no button observed - meaning patient serum does not contain AB to virus

for more understanding you can visit

http://www.cehs.siu.edu/fix/medmicro/cfix.htm

thanks,

kenneth

Sherman - Histopathology

This time I'm gonna post something which I saw and witness. Doesn't really have much principles in it, but it's just a good experience to see it.

If you find it disgusting or anything in whatever I'm gonna state, you may close and try to read again soon ~ but it's really interesting!



In the Histopathology department, post mortem refers to the medical examination done to a dead body. In this case, it’s a baby.
It's kinda scary when you first see it...cos it's like a formed baby, then it's gonna get cut open and have its organs removed and all, like a nice body being physiologically ruined.
These incisions and cuts have to be done very carefully, and very gently. Stillborn babies can range from those barely formed to those that are already fully formed, near the 5th month of pregnancy etc.
The state at which the pathologist has to adopt to retrieve the organs have to be dependent on the stillborn's age. For example, a not yet fully developed foetus is most likely to have a liquified brain after the skull has been cut open, so the pathologist has to be careful so as to not "spill" the brain contents.


The necessary sharps are needed like surgical scissors, scapel, metal ruler and forceps.


The steps are crucial and the body has to be treated with absolute care.

The body is examined for physical deforms. Actions done are as follows;
- Probing the nose with a probe till the end of the skull (check clearing of nose)
- Checking of the eyes
- Opening of the mouth
- Checking of the ears, with reference to the distance from the eyes
- Measuring the limbs, chest width, abdomen width, spine
- Ensure fingers and toes are separated
- Check the genitals and anus
- Check for a straight spine

Preparation:
Formalin
- To fix the organs that was removed.

Organs (removed in their order from superior to inferior sections of the body)
- The brain is removed first
The scalp is sliced open, followed by an incision of the skull with a surgical needle along the line of the skull.
The skull will then be pried open (like a banana skin) to expose the brain
The linings of the brain is cut, and the brain is removed and then fixed into formalin

- The pituitary gland is removed next
It is located just below the brain and is cut out using a scalpel blade. The gland resembles a rectangle shape. It is located in the middle, behind the posterior cartilage. You can picture the inside of the skull from a bird's eye view...the pituitary gland is right in the "bull's eye" of the cavity.

- The torso is then sliced open. A T-shape cut will be done, as per normal for an adult autopsy. The straight line will be cut across the chest, from shoulder to shoulder, and a vertical line cut down from the neck to the pelvic area. The skin and subcutaneous tissues are to be cut through, as there is no need to remove muscle by muscle.
The attachment under the rib-cages is to be cut, along with soft tissues.

- Remove the thoracic cavity

- Remove the thymus (located below the neck)

- Check the morphology of the organs
E.g. check pulmonary veins, branches from the aorta, isomerism, and pathological findings
* All pathological findings are to be photographed *


- Removal of the intestine
The mesentery is cut between the intestines. It suspends the intestine in the abdominal cavity and stops it being disturbed when a person is physically active.
The intestine is removed as a long strand. Keep in mind to take note to make sure that the intestine has the bile stain
For the small intestine, remove it until it reaches the duodenum, cut it off
For the large intestine, remove it until it reaches the rectum, cut it off.
Measure both intestines and fix formalin into it via needle (the mucosa will not fix easily under external exposure conditions to formalin)

- Removal of stomach, kidney, pancreas, adrenal, and spleen.
All linings of the internal organs are to be cut from the esophagus to the rectum, and the entire digestive system complex is removed by cutting through the urinary tract.

- Removal of salivary glands

- Removal of skeletal muscles (to look out for myopathy etc)

- Removal of ribs for sampling (3 ribs maximum)

- Removal of portion of spinal cord

- Breaking down of digestive system complex
From the posterior
a. Removal of kidneys (for pediatrics, do not remove kidneys from capsules). Keep in mind the removal of renal tubules along with the kidneys
b. Removal of adrenal
c. Removal of pancreas
d. Removal of heart and lungs as a complex. Formalin is injected into the lungs and heart to inflate the organs.
e. Removal of stomach (the stomach is cut open)
f. Liver: Removal of anterior (more effective fixation)


Finishing up

- Disposal of all sharps
- Excess parts are to be returned to the body (the incisions will be patched up by glue, returning the baby's physiological shape to the way it is before the autopsy)
- Fix all organs that were retrieved

Processing in cassettes
- All parts that are taken from the body are to be placed into cassettes for processing in the tissue processor before embedding
- All organs that are to be put into cassettes are to maintain a spare portion in case more samples are needed.



It may be disgusting, especially to imagine it, but after a few looks, it'll seem fine. Hahahaha!
Amazingly too, if you're not very confident about your Human Anatomy, somehow just by looking at an autopsy, where all the organs are removed piece by piece, somehow just by looking you can just memorised the entire basic anatomy of the human body...it's easier somehow just by looking at it in real life.



Well there isn't much to ask also~ it's just steps and procedures. But I'll try to answer questions if you have them. I'll try my best! =D


Happy SIP!

Week 7: Administrative Department =D

Hello everyone..=)
This week is my 3rd week in the administrative section of the lab, as every month i will be rotated between the Microbiology and Administrative section with another student.

Although the admin department do not carry out diagnostic tests and experiments,
it is still a very important section of the lab as every sample that reaches the lab will be registered by the admin staff first before any tests can be done.

The admin department is responsible for ensuring the lab conform to the turnaround time for every specimen. They are also responsible for ensuring all urgent specimens are prioritized.

In the administrative department, i was introduced to all
aspects of the Laboratory Information System (LIS),
where registration of samples and tests that are ordered for each sample are done.
Apart from that LIS is also linked to HIS (Hospital Information System) and CMS(Central Management System), from which details and particulars of patients, tests previously ordered, as well as approved test results can be derived from.

Occasionally, we would receive calls from staffs ranging from nurses, doctors, patients themselves and even the dispatchers.

I was trained for the first week on how to attend to the wide range of queries the callers will make.

Doctors and nurses from other clinic branches under the hospital will at times
call to query on the availability and test codes of a particular test that
they are interested in doing.
Dispatchers may also call to ask if there are any samples to be dispatched
to a certain area in Singapore they are in charge of handling.

However most of the time the administrative department would receive calls from the different centres in the hospital (eg. Aesthetic centre,Women's Centre, Operating Theatre, different wards,etc) requesting for the time test results will be out in the system.

We would then have to dial the laboratory med tech in charge of the test
( eg. dial the Hematology section to query on Full Blood Count result; etc) and transfer back to the caller to provide them with necessary details and progress of the test they previously queried on.

Registration of specimens are the most common task in the admin department.

Peak hours for registration of samples is usually from 4.30pm-5.30pm as dispatchers will then arrive in the lab with bags containing specimens coming from different clinic and branches under the hospital all over Singapore.
Every specimen from a patient will come with a request form where tests ordered for the patient are indicated.

Different codes are assigned to different types of specimens ( eg. 01 for EDTA blood, 02 for PLAIN blood, 13 for FLUORIDE, 23 for URINE, etc).
Every lab test has its own 6-digit test code; and 6-digit profile codes on the other hand are assigned to different test packages (lab tests ordered in bulk).

Every admin personnel is given an ID and password to access the LIS.
Hences, specimen registration as well as ammendment of tests
and patient particulars can be traced back to the personnel
who made the changes to the data. This way the management is able to trace who is responsible for any changes and hence maintain a high level of security to the data in the LIS.

The admin section can be refered to as the central unit of the lab
as it is connected to all sections of the lab (Haematology, Immunology,etc ) as well as the rest of the hospital and its clinics.

They are responsible for communicating with patients, nurses,
doctors and laboratory medical technologists themselves.
Hence, they have a huge role in maintaing the overall workflow of the lab.

In all, i had a great experience in the administrative department =D
Good communication is key when working in the admin department. Even though the tasks assigned is different with that in Microbiology department, there are definitely more unexpected challenges in the administrative department.

I hope this gave you an idea how administrative work in the lab is like.
Feel free to comment me if you have any questions!

Siti Hawa Bte Azali
0704087F
TG02

Analysis of RBC Folate using DXI

Tng Wess Lee, 0702570C
Clinical Biochemistry Lab (CCHEM)

Test: Analysing RBC Folate using DXI.

The test for RBC folate analysis falls under the anaemia panel category. It is used to diagnose the specific cause of anaemia. As we know, folate is used in the synthesis for RBC. Folate deficiency would cause megaloblastic anaemia.

Clinicians request for folate test to identify the specific cause of anaemia. As Vit B12 or iron deficiency might lead to anaemia too. The reason to call for RBC folate is because RBC folate is the best indicator of long-term folate stores. A low value would indicate a prolonged folate deficiency.

Before manually loading the sample (which requires RBC Folate analysis), the blood tube is treated first. Blood is received in an EDTA tube to prevent the clotting of the red cells, as the test requires analysis through the red blood cells and not the serum. This tube cannot be loaded into the LAS system to the prepared for analysis as the EDTA tube is of a smaller size compared to plain tubes or fluoride tubes.

Prior to treatment of the blood sample. Prepared (frozen) controls are thawed. Controls come in 3 levels. There are 3 levels of controls. Level 1 (low), Level 2 (medium) and Level 3 (high). Each level contains a standard and stated value, which falls within the reference range. Controls are use to determine the accuracy of the calibration.

After controls are thawed. Treatment of patient’s sample and controls proceed.

Treatment of blood sample

1) Lyse 50uL of sample and all 3 levels of control with 1000uL lysing agent.
2) Vortex all tubes
3) Wrap lysates with aluminium foil and store them in the cupboard for 1.5hrs
4) Remove wrappings, and manually load them into DXI analyser
5) After controls are verified and passed, load patient sample to be analysed

Reference interval of RBC folate: 776-1784nmol/L