Microbiology and Serology

Hi everyone,
Sasi here reporting from one of the private labs in Singapore. The first three weeks of my attachment I was attached to the Microbiology department. I basically had to run urine analysis via the URISYS 2400 urinalysis analyzer. The purpose of UAN (Urine Analysis) is for the semi-quantitative determination of pH, leukocytes, nitrites, protein, glucose, ketone bodies, urobilinogen, bilirubin, and erythrocytes in urine. Urine test strips are used to measure certain constituents in urine which signify renal, urinary, hepatic and metabolic disorders. In the mornings, I would start off by running the controls for the machine by loading the thawed controls. Then, I would record down both the positive and negative control results and make sure they tallied with the previous days' control results. In the afternoon, majority of the urine samples would begin to arrive. The urine specimen must be collected in a clean, dry container, either plastic or glass without preservatives. Once the specimens were received, they were labelled with the appropriate barcodes and loaded into the machine. When loading the samples, I had to make sure the sequence number on the monitor matched the first sample that was going to be loaded so that the results can be easily tracked down (via the sequence number) in the future in case of an emergency. This whole process of running the samples had to be done fast and accurately in order for the other follow up processes to take place. One of the follow up steps was to centrifuge the samples, print out the results of the Una, literally highlight the abnormal results and hand them over to the microscopy section of where urine FEME (Full examination microscopic examination)will be conducted.

The urine analysis results include:
- a description of color and appearance
- Specific Gravity- This detects ion concetration of the urine. Small amounts of protein or ketoacidosis tend to elevate results of the specific gravity.
- pH- pH of healthy individuals is usually between 5 and 6.
- glucose
- ketone bodies
- protein
- urobilinogen
- RBC number
- WBC number

The numbers and types of cells and/or debris present can bring about a great detail of information and may suggest a specific diagnosis.

Eosinophiluria - associated with allergic interstitial nephritis, atheroembolic disease
RBC casts - associated with glomerulonephritis, vasculitis, malignant hypertension
WBC casts - associated with acute interstitial nephritis, exudative glomerulonephritis, severe pyelonephritis
(heme) granular casts - associated with acute tubular necrosis
crystalluria -- associated with acute urate nephropathy (or "Acute uric acid nephropathy", AUAN)
calcium oxalate - associated with ethylene glycol toxicity

Also, the presence of urinary casts presence under microscopy evaluation hold significance as diagnostic and prognostic indicators of kidney disease. They are cylindrical and they are generated in the small tubules and collecting ducts of the kidney, and they generally maintain their shape and composition as they pass the lower parts of the urinary system.

Some common casts I spotted during my experience included:

Hyaline casts: Hyaline casts are solidified Tamm-Horsfall mucoprotein secreted from the tubular epithelial cells of individual nephrons. Low urine flow, concentrated urine, or an acidic environment can contribute to the formation of hyaline casts and thus, they may be seen in normal individuals in dehydration or vigorous exercise. They are cylindrical and clear.

Granular casts: They can result either from the breakdown of cellular casts, or the inclusion of aggregates of plasma proteins (eg, albumin) or immunoglobulin light chains. Depending on the size of inclusions, they can be classified as fine or coarse, though the distinction has no diagnostic significance. Their appearance is generally more cigar-shaped and of a higher refractive index than hyaline casts.

Epithelial cell casts: They are formed through inclusion or adhering of desquamated epithelial cells of the tubule lining. Cells can adhere in random order or in sheets, and are distinguished by large, round nuclei and a lower amount of cytoplasm. These can be seen in acute tubular necrosis and toxic ingestion, such as from mercury, diethylene glycol, or salicylate. Cytomegalovirus and viral hepatitis are organisms that can cause epithelial cell death as well.

In addition, I also did manual pregnancy testing(UPT- Urine Pregnancy testing) using a commercial kit called StanbioQuStick test kit. Its intended use was for the visual qualitative detection of the hormone hCG in urine. Human chorionic gonadotropin(hCG) is a glyco protein hormone secreted by the developing placenta after fertilization. When the test strip is placed into a vessel of urine, the urine migrates upward, transporting the coloured reagent onto the surface of the dye particles by immuno-reaction. Visual formation of one line is read as negative and two coloured lines represent a positive result.

I also observed how stool samples are tested using commercial test kits, for example to test for fecal occult blood. Also, for amoeba identification in the stool sample, the sample had to be stained with normal saline solution and iodine before microscopy examination. Besides, I also had the chance to observe how urine streaking was done on the culture plate. Basically, it was different from the streaking we normally do in school. In the micro lab, they streak it in the form of an inverted christmas tree.

Ok this marks the end of my micro lab experience. Feel free to ask me questions.

The following 3 weeks after microlab, I was attached to the serology department which I am in till now. Its the same lab Yeng Ting( from Med bankers group) was attached to in her first 3 weeks. Hence, the techniques she has posted in her blog are exactly the same as what I am required to do as well. So, please kindly refer to her blog. However, apart from what she has wrote about, I want to highlight another two particular tests I did.
One of them is is the WWF test. Not world wrestling federation or watsoever ah.. It is called the 'Widal Weil Felix ' test.
This test measures the level of warm or cold agglutinins in blood. Agglutinins are antibodies that cause the red blood cells to gather together. Cold agglutinins are active at cold temperatures. Warm agglutinins are active at normal body temperature.
These antibodies can cause a hemolytic anemia. This occurs when the body destroys its own red blood cells. Distinguishing between warm and cold agglutinins can help understand why the hemolytic anemia is occuring and directs therapy.
Basically, there will be 12 reagents present. These reagents help test for the different types of salmonella. In our lab, this is the main focus for WWF.
I will have to pipette out 40 microlitres of serum 12 times onto 12 test circles and also a drop of each reagent 12 times onto the same 12 test circles and let them mix on a rotator for 1 min. The timing is crucial as after 1 min, the results may be false positive.
If there is agglutination present, titre must be done.

Normal Values:
Warm agglutinins: no agglutination in titres at or below 1:80
Cold agglutinins: no agglutination in titres at or below 1:16

Abnormal Values mean:

Elevated levels of cold or warm agglutinins can cause hemolytic anemia. Some patients with moderately elevated levels will have no hemolysis and most likely won't require therapy.
The presence of warm agglutinins may occur with:
- Infections, including brucellosis, rickettsial disease, salmonella infection and tularemia Lymphoma
- Lymphoma
- Systemic lupus erythematosus
- Medications, including methyldopa, penicillin, and quinidine

The presence of cold agglutinins may occur with:
- Infections, especially Mycoplasma pneumonia. Also after viral, staphylococcal, and malaria infection.
- Cancer, including lymphoma and multiple myeloma
- Systemic lupus erythematosus

The other test I did was the mycoplasma pneumoniae test using the SERODIA-MYCO commercial test kit. It is designed for the sole purpose of detecting anti-Mycoplasma pneumoniae. Mycoplasma pneumonia is an infection of the lungs caused by Mycoplasma pneumoniae. Symptoms include fever, dry cough, sore throat, headache, chest pain etc.

Test method:
1. 100 microlitre of diluent in the first well.
2. 25 microlite of diluent in the 2nd-6th well.
3. 25 microlitre sample into the first well and mix and carry over 25 microlitre onto the subsequent wells (till the 6th well) and then discard the last 25 microlitre.
4. Add a drop of unsensitized cells to the 2nd well.
5. Add a drop of sensitized cells to the 3rd, 4th, 5th and 6th well.

Results:
If there is a distinct button formed in the middle of the well, it is negative. If there is no distinct button but a cloudy pinky suspension, its is positive. You have to note till which well this suspension forms and record the titre accordingly.


Alright, this again marks the end of my experience at serology department. Feel free to ask me questions. Thank you for reading!

Sasi
Tg02
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