Clinical Biochemistry

Hello everyone, this is Joash here. The lab that i am posted to right now its just a very small routine diagnostic lab that only contains 3 main disciplines, which are Biochemistry, Hematology and Microbiology. i am going to talk about Biochemistry 1st.



The analyzer that we used in our lab is the Olympus AU400

This fully automated biochemistry analyzers contains 3 major components which includes robotic arms, called probes, controlled by pressure valves, next is the a photometer that measures the light from built-in cuvettes which are held at a constant temperature and lastly the microprocessor or software package which calculates the values from the signal and then displays the data in a user-defined format. The analyzer contains 2 probes, sample and reagent probe which has good accuracy and precision. The reagent chamber is refrigerated and accommodates 72 reagent bottles and can analyze up to 46 parameters at 1 time. The STAT table is also refridgerated and it is used for emergency analysis as it can hold other analysis 1st but run the samples on the STAT table. Routine parameters include liver function tests, renal function tests, glucose tolerance tests and special parameters such as free fatty acids, HbA1c, ketone bodies and much more. This analyzer can run samples such as serum, plasma and urine.

Daily, weekly and monthly maintenance is required for this analyzer and each involves different cleaning procedures such as cleaning and rinsing the sample and reagent probes. replacing the cleaning solotions and etc. QC have to be done daily before the analysis of samples can be done. This is to ensure that the results produced are accurate and reliable at that point of time. The QC menu of the software offers LJ plots, west guard rules, etc. for user review. The data can also be stored and printed. it allows us to review day to day control to monitor the QC for each components and it will flag if the QC results are out of the +/- 2SD off the mean.

After QC had passed, only that we can run our patient samples. The blood tube that we are using is the Red clot activator blood tube as we are analyzing the serum of the blood. We will 1st have to key in the required test into the LIS system for that particular patient and paste the barcode label along the blood tube that matches the patient's particulars. Then the blood tubes are centrifuged to spin down the clotted red and white cells with serum as the supernatant and after that, they are placed into the analyzer for processing. the analyzer will read the barcode and recognize what test to do for this patient. After the test is done, the results will be transmitted to the LIS system and the report is ready to be printed and summit to the vets for review.

Regards,

Joash(0702657H)

picture taken from:http://asia.olympus-global.com/lisg/diagnostic/au400e/index.cfm

Medical Microbiology(Cont')

Hi! This is Shameema. I was posted to Central Processing Area (CPA) in the Department of Diagnostic Bacteriology for the first 3 weeks. I worked on the respiratory, blood and stoot. On the forth week i was moved to parasite.

As Muna and Jennifer have done the posting about respiratory and urine, I will share my experience on stool in this blog.

Stool Culture

The stool culture test is used for the detection and identification of pathogenic bacteria in the stool. In the laboratory, the test is initiated by streaking small amount of a fresh fecal sample to a variety of media; details as follows:




The stool incubated in the selenite broth and alkaline peptone is cultured on to agar plates the following day:

Selenite --> MacConkey and SS agar plate

Alkaline peptone --> TCBS and blood plate

It is crucial that the subculture for alkaline peptone has to be done the following day. This is because the pH of the alkaline peptone which is set at pH 8 will decrease in the presences of vibro as acid is the end product of vibro. Decrease in the acidity increases the growth of other organism and decrease the concentration of vibro thus affecting the result. Selenite broth is the selective broth that will rule out the normal flora.


Vancomycin Resistance Enterococci Screening (VRE)

Enterococci are bacteria found in stool and some strains of the enterococci has become resistance to vancomycin antibiotic is referred to as VRE.


On the stool table I also had to do the widal weil- felix test. It is a diagnostic used for typhoid fever. It is an illness caused by the bacterium Salmonella enterica serovar typhi. The precense is detected by the aggultination of somatic (O) and flagellar (H) to Salmonella typhi in the patient's serum. Weil-Felix test is the detection of rickettsial antibodies thus indicating the presence of rickettsial species in the patient.

PROCEDURE

1. Blood sample is centrifuged at 2000rpm for 5 minutes to separate the serum from the blood.
2. Add 0.2ml of the patient serum to 7.8ml of normal saline in test tube to form 1/40 dilution.
3. Add 1ml of 1/40 dilution serum to 1 ml of normal saline to form 1/80 dilution.
4. Arrange Dryer’s tubes as demonstrated in table below and add one drop of commercial antigen in the Dryer’s tubes.
5. Add 0.8ml of the 1/40 dilution test serum to Dryer tube number 1-6 and 1/80 dilution serum to Dryer tube 7 and 8..
6. Shake the racks gently to remove trapped air.
7. Incubate overnight at 50 degree Celcius in water bath.

TO: Solmonella typhi Group D somatic antigen
TH: Salmonella typhi-H flagellar antigen d
AH: Salmonella parathyphi: A-H flagellar antigen a
BH: Salmonella parathypi B-H group B somatic antigen
OX19: Proteus OX19 antigen
OXK: Proteus OXK antigen

Will share my parasite experiance in my next blog.

Shameema :)

Medical Microbiology(Cont')

This is Jennifer from TG02. As what Muna had mentioned on the previous post, we were posted to CPA( Central Processing Area) in the Department of Bacteriology. I was rotated to the repiratory section during the third week of my SIP where i recieved specimens from patients from their upper or lower respiratory tract.

( Very sorry that due to some problems, i couldnt upload the pictures that i have downloaded. I will upload the pictures as soon as i have settled the problem.)


Upper respiratory tract infection

MRSA (Methicilin Resistant Staphylococcus aureus) Screening
MRSA can also be known as multidrug resistant Staphylococcus aureus as it is resistant to a huge group of antibiotics known as beta- lactams. This bacteria cause many infections which are difficult to treat since they are also resistant to cephalosporin and penicilins.

Usually, most of the samples that are taken to detect for the presence of MRSA are nasal swab. Screening for MRSA requires the use of BHIB (Brain Heart Infusion Broth) and MRSA agar which is a selective agar that only allows the growth and differentiate MRSA from other bacteria. BHIB contain a type of antibiotic called oxicilin and this antibiotic will kill most of the bacteria in the nasal swab except for MRSA as MRSA can withstand this antibiotic and is an enriched broth for staphylococcus species.

MRSA selective agar BHIB

Procedures
1. Incubate the cotton bud that contain the nasal swab into BHIB and incubate for 24 hours to give enough time for the antibiotic to kill off most of the other types of bacteria and allow MRSA to grow, in an oxygen incubator.
2. After 24 hours incubation, sub the cotton bud which was incubated in the BHIB on MRSA selective agar and do normal agar streak.
3. Incubate the agar in an incubator for 2days for the bacteria to grow.

Results
If MRSA is present and grow on the agar, the positive result is shown a purple colony growing on the agar which is shown on the diagram below.

Purple colonies growing on MRSA agar

Lower Respiratory Tract Infection

Sputum Culture
Usually, sputum samples are obtained from patients to detect for the presence of lower respiratory Tract Infection. The appearance of sputum can be watery, clear or mucoid. Before doing sputum culture, we have to do a gram stain for the sputum first as we need to identify whether the sputum sample is a good sample to do the culture or not.

After viewing the gram stain of sputum under the microscope, it can be determine whether the remaining sputum sample can be carried on to do the culture or should it be rejected.
A good sputum sample should have absence of epithelial cells and presence of PMN (Polymorph nuclear leukocyte) as presence of high number of epithelial cells shows that the sample obtained could be from saliva as saliva sometimes does contain epithelial cells from our cheeks. High number of PMN shows that the sample is as good sample as it shows that there is inflammation by the infections caused by pathogens.

Other types of samples such as CSF (Cerebrospinal Fluid) and ETTA (Endotracheal Tube Aspirate) also requires gram staining and cultures. The only difference is that these two types of samples do not require doing gram stain first before culture can be done. Both the culture and gram stain can be done at the same time.

Below is a tabulated table showing which agar or broth to use for each of the type of samples.

(Staphylococcus streak is done on all blood plates after normal streaking for any type of samples as staphylococcus provide a particular essential nutrient for the bacteria on the blood agar to grow.)

hm_med()

Procedures (For sputum)
1. The appearance of the sputum was checked. (Such as whether the sputum is mucoid, watery, clear, or purulent.)
2. Using a labeled slide, a small circle was drawn on the under side of the slide.
3. The surface of the slide was swab by alcohol swab.
4. Using a cotton bud, the sputum was stirred and a small amount of sputum was obtained.
5. A small amount of sputum was applied onto the slide between the circles that was drawn on the underside.
6. The sputum smear was fixed by putting the slide on the heater until smear was dried.
7. Gram stain was performed.
8. The slide was viewed under the microscope.

If the sample is a good sample, carry on with sputum culture. If the sample is not good sample, reject it.
For a good specimen of sputum, there should be no or minimal epithelial cells seen and PMN (Poly morphs nuclear). If too many epithelial cells are seen, it could mean that the specimen is the saliva of patient not the sputum as saliva may contain epithelial cells that are actually the cheek cells. Presence of PMNs show that the is inflammation of the patient’s lower respiratory tract as pathogens are present and cause the activation of PMNs.
Cont’
9. The remaining sputum was subbed onto Mackonkey and Blood agar.
10. Normal streaking was done.
11. Staphylococcus streak was done on blood agar.


References:
MRSA Screening: The Test. Retrieved from 12 July 2009 from Lab Test Online: http://www.labtestsonline.org/understanding/analytes/mrsa/test.html

Medical Microbiology

Hi, this is Muna. For the past 4 weeks, including this week, Shameema, Jennifer and I have been posted to the Central Processing Area(CPA) in the Department of Diagnostic Bacteriology. Each week, we rotate to a different testing bench. We receive patient samples from the wards and clinics for testing.

I shall start by posting what I did last week - Urine Culture.

The purpose of urine culture is to isolate and identify potential pathogenic organisms from urine. This helps in the diagnosis of renal or urinary tract infections (UTI). Since urine is mostly sterile, any presence of microorganisms is usually clinically significant, unless it is contaminated by normal flora.

The samples we receive are usually mid-stream urine (MSU) for aerobic culture and dipslides. Mid-stream urine, as its name implies is collected in the middle part of the urination flow. The initial part is not collected as it usually contains the flushed out normal flora and cellular debris from the urethra and urinary tract. This may cause a false positive reading.

The samples are inoculated onto a split plate containing CLED agar and blood agar (BAP). CLED stands for Cystine Lactose Electrolyte Deficient agar, which can differentiate between lactose fermenters and lactose non-fermenters. Lactose fermenters produce a lower pH in the media, thus causing the indicator (bromothymol blue) to change from the original greenish yellow to yellow. It also enhances the growth of dwarf-colony coliforms.

Blood agar plates (BAP) supports growth of fastidious bacteria. It is also able to detect the pattern of bacterial haemolytic activity, which can determine the pathogenicity.

CLED and BAP split plate.


The streaking of plates for urine culture is unique as it does not follow the ocnventional way of streaking plates. I was taught that this method is used so that the pattern of growth can be obseved and the calculation of colony forming units per ml(cfu/ml) of urine can be counted easily. Also, instead of a 10 ul inoculating loop, a 1ul loop is used since bacterial presence in urine should be minimal or none in normal persons.

Urine culture streaking technique.

Normally, the BAP is streaked first, followed by the CLED plate so that any residual inhibitory substances picked up from the cLED plate in streaking does not affect the growth on the BAP.


NOTE: I shall continue where I left off soon. Need to clarify some doubts before continuing.