week 14

Subject title: Clinical chem/ Haem
Name of test: G6PD screening test

Principle:

• G6PD test carried out in our lab is only a QUALITATIVE and SCREENING test
  
• G6PD is an enzyme which catalyzes the reduction of NADP+ to NADPH
  
• NADPH keeps glutathione in its reduced form


• Reduced glutathione or GSH protects red blood cells from oxidant damage and hence prevents the lysis of RBCs


• The reagent used in this test contains NADP


• Test principle is as follows:

G6PD (this is supposed to be on top of the arrow in the equation..but i juz can't move it)

Glucose-6-P + NADP+ ----->Gluconate-6-P + NADPH+H+

• The NADP in the reagent will be converted to NADPH if G6PD is present
  
• NADPH produced in the reaction will fluoresce under long-wave UV-light
  
• If there is G6PD deficiency or total absence of it, no fluorescence will be observed
  

Materials:

1. Filter paper

2. Hitachi cup

3. G6PD reagent

4. UV light

5. Incubator

6. Patient's sample in EDTA tube

Method:

1. Pipette 100uL of reagent solution into the hitachi cup which has been labelled with the patient's lab no

2.Add about 2.5uL of patient's sample into the same cup

3. Mix well and pipette 5uL onto the filter paper(1st spot)

4. After 5 mins, pipette another 5uL onto the filter paper(2nd spot) and wait for another 5 mins to pipette again 5uL of patient's sample(3rd spot)

5. Then, put the filter paper into the incubator

6. After 5 mins, observe the filter paper under long-wave UV light

ok the filter paper will look something like this, u would have three spots of blood which are made in 5 mins interval by pipetting 5ul of the patient's sample

Results:

1. Fluorescence indicates presence of G6PD

• usually the 1st spot of blood would not fluoresce well, the 2nd and 3rd ones will

2. Absence of fluorescence indicates the absence of G6PD

Note:

• Deficient cases will be followed up with G6PD quantitative test which is a send-out test


• Negative control is done before our first sample of the day

Nur Farhana Binte Ramlan

0604834B

13 th week

hello guys...

for this entry, i will blog on one of the test that is carried out in microbiogy section.

the test is STOOL CULTURE

Principle Analysis
Acute infectious diarrhoea is caused by a number of different agents: bacteria, viruses and protozoa. Each bacterial agent of infectious diarrhoea has unique pathogenic mechanism that cause a specific symptoms. These symptoms and an adequate patient history are clues that will help the physician catergorise the patient's disease. Antimicorbial therapy can alter the host's normal protective microbiota and thus predispose the patient to bacterail overgrowth that result in diarrhoeal disease caused by organism such as Clostridium difficile, staphylococcus aureus, Candida spp. and Pseudomonas aeruginosa.

For our lab, we routinely cultures stools only for the presence of Salmonella, Shingela and Vibro spp. because of their role as causative agents of bacterial diarrhoea and other enteric diseases.

Organism such as Aeromonas spp., Plesiomonas shigelloides and Yersinia spp. are identified only wen they are present in pure or heavy growth or upon request.

Specimen
stool

• collect the specimen in a sterile, dry, leak-proof container
• select portion containing pus, blood or mucus for culture
• 1 to 2 g of sample is sufficient

Reagents
media

• Alkaline Peptone Broth, AP
• MacConkey Agar, MAC
• Selenite-F Broth, SF
• Thiosulphate-Citrate-Bile salts-Sucrose Agar, TCBS
• TSA with 5% Sheep Blood, BA
• XLD

supplies

• Sterile cotton swabs
• Applicator sticks
• Inoculating loop/straight wire

storage requirements

• store all media (agar plates and broth) at 2 to 8 degree celcius
• leave the media at room temperature overnight before use

Procedures
primary inoculation

• note the appearance of the stools i.e watery, loose, bloody, semi-formed and form
• inoculate stools according to Table 1
• subculture selective broth after 18 to 24 hours of incubation as in Table 2

Culture examination
After incubation, examine all primary and subculture plates for the growth of enteric pathogens.

For Salmonella/Shigella/Vibrio cholerae screenig, examine all the primary and subculture plates for the growth of Salmonella, Shigella and V. cholerae.

For the Vibrio cholerae screening, examine all the primary and subculture plates for the growth of V. cholerae.


Reporting of results


dats all...
i hope you guys learnt something from my posting.
thanks for reading..

sutiana
TG01
0604651J

12th week

Ever since i got back from cytology, i have been at the shaving station in histo. Shaving is the process of removing excess wax and exposing the tissues in the embedded blocks. This will provide a smooth-sailing sectioning of the tissues. Shaving is done using the microtome machine. The difference between sectioning and shaving is, sectioning is done at 4-5 microns while shaving is done at 20 microns.

In addition, the person at the shaving station will be responsible for placing the tissue blocks which require decalcification into RDO. RDO is actually an acid which will decalcify the tissues. I have actually come across tissues which i cannot shave at all because it is too hard. For tissues of that nature, it will have to be placed in RDO even before it is shaved. For other tissues which do not require decalcification, they are placed in a detergent after shaving. This detergent will soften the tissue surface to the perfect level to which that it will be easier to section it. These blocks are placed in the detergent for five minutes and then washed under running water. If the blocks are placed in the detergent longer than five minutes, the staining of the sections on the slides will be compromised. Once, it was left in there for more than 5 minutes, blobs of stains were seen on the slides. This is perhaps due to chemical interactions. The detergent which has penetrated into the tissues will interact with the chemicals from the H&E stains.

There are many things to look out for during shaving. Firstly, score lines. Score lines are lines that appear on the surface of the tissue blocks. These lines are caused by the blunt blades. When score lines are observed, the blades must be removed immediately. Then, the blocks will be shaved again until the lines have disappeared. It is very important to ensure that after shaving a block which requires decalcification, the blade is changed to shave the next block. This is because, obviously, the blade will be blunt after shaving the hard tissue from the block which requires decalcification. If the blunt blade is used to shave a tissue block containing small tissues, it is hard to remove the score lines without taking the risk of losing the tissue after continuous shaving. Thirdly, removal of sutures and staples. Sutures are what seemed to be black threads used during an operation. Amazingly, sutures and staples are used by the surgeons during the operation for the orientation of the organs. Orientation of the organs aid in the trimming of the organs by the pathologists. Sometimes, i believe, unknowingly, when the pathologists trim the tissues into the cassettes, sutures or staples are hidden in tissues. The presence of the sutures and staples will apparently remain unknown throughout tissue processing as it does not distrupt the processing. It will usually be found during embedding and most of the time, during shaving. Which is pretty unpleasant. Not only will the staple or sutures cause the blade on the microtome to be instantly blunt, it will also require much digging and pulling out the staples and sutures from the hardened wax block. Fourthly, tissue blocks containing certain breast tissues are not required to be shaved. This is because they are exceptionally thin. Finally, i have to ensure that the wax in the tissue blocks are completely hardened before it can be shaved. This is to prevent the risk of the tissues dropping off from the block. When that happens, the tissue will have to be sent for re-blocking.

I realize that embedding affects shaving a lot. This is because, at times, the tissues are not on the same level in the wax. This is probably due to the tissues not being pressed down towards the mould during embedding. When the tissues are not on the same level, much shaving needs to be done to expose every part of the tissue. When this happens, the risk of losing parts of the tissues increases.


Nurathirah
Tg01
0606561I

11th Week of SIP

Name: Hardina Hamzah

Topic: Microbiology

Title: Processing of Urine Specimens

Urine culture is commonly done to determine whether the patient has urinary tract infection. A urinary tract infection is a bacterial infection that affects any part of the urinary tract. When bacteria get into the bladder or kidney and multiply in the urine, they cause a UTI. Enterobactericeae species such as, E.coli, Kleibsiella pneunomiae, Proteus mirabilis and Pseudomonas aeruginosa, cause most of the urinary tract infection.

Retrieved from: www.pharmacy-and-drugs.com

There are two types of media used and there are TSA with sheep blood agar and MacConkey agar. TSA (Trypticase Soy Agar) with sheep blood agar is useful for the cultivation of a wide variety of fastidious microorganisms. TSA is a general purpose media produced via enzymatic digestion of soybean meal and casein and TSA is frequently the base media of other agar plate type. In this case, it is the base media for blood agar plate. MacConkey agar is a culture medium to grow Gram-negative bacteria and stain them for lactose fermentation. It contain bile salts (to inhibit Gram-positve bacteria), crsytal violet dye (also inhibit Gram-negative bacteria), neutral red dye (to stain microbes fermenting lactose), lactose and peptone.

TSA with Sheep Blood Agar

Retrieved from: www.madsci.org

MacConkey Agar

Retrieved from: www.madsci.org

The urine specimen need to be processed immediately as if prolong, the results may not be accurate. The urine is mixed well before inoculating it onto agar plate. 1ml calibrated disposable loop is used and the loop will be immersed into the urine specimen bottle. The sample will be inoculated onto Blood agar plate first and then MacConkey agar plate by making a straight vertical line down the centre of plate and will be followed by series of close perpendicular streaks through the original line. The plates will then be incubated at 35°C aerobically overnight. At the same time, microscopy cell count of the fresh urine specimen has to be done. The urine sample will be transferred to the notch on the slide chamber of KOVA GLASSTIC SLIDE 10 with grids. The urine sample will be transferred by using the capillary tube and the urine sample will be drawn into the chamber resulting in a homogenous suspension of sediment. The cells to be counted are epithelial cells, white blood cells and red blood cells. When there are a lot of white blood cells (eg. >100 cells) that are counted; this can indicate that the patient may be having urinary tract infection.

KOVA GlASSTIC SLIDE

Retrieved from: http://www.cenmed.com/productDetail.asp?productid=17291&catID=&category=5617&mainCat=&cat=5305

After office hours or during weekends/public holidays or even there is a delay in transporting urine specimen, the dipslide will be used. The dipslide has three types of media which are Cysteine Lactose Electrolyte Deficient (CLED) agar, MacConkey agar and E.coli agar. CLED agar is a valuable non-inhibitory growth medium used in the isolation and differentiation of urinary organisms. Being electrolyte deficient, it prevents the swarming of Proteus species. Lactose fermenters produce yellow colonies on CLED agar; non-lactose fermenters appear blue. It has a pH of approximately 7.3. The staff nurse will dip the dipslide into the container containing patient’s urine and then seal the dipslide into the bottle. The staff in the laboratory will help to incubate overnight. One disadvantage of using this method is that there will be no urine microscopy cell count.

Uricult Trio Dip Slide

Retrieved from: http://www.oriondiagnostica.com/files/odextra/Clinical%20Microbiology%20brochure/782-03GB_Uricult.pdf

The cultures and dipslides which are made on the previous day will be read on the next day. Usually when there is a mixed growth of bateria (different types of organisms growing), it is considered insignificant and will be recorded as negative. However, when there is predominant growth of only one type of bacteria, it is considered as significant and will be recorded as positive. Enterobacteriaceae species can be identified easily by seeing the morphology of the colonies and to confirm the species, indole test can be done. For example, when E.coli is suspected to grow, indole test is done on the spot to confirm. A small filter paper is used and a drop of indole is placed on the filter paper. Then, the filter paper is rubbed against one of the colonies. When the filter paper turns blue, it indicates positive and it is proven that it is an E.coli. Then the culture will be sent for further test which is sensitivity testing. However, when there is a doubt on the identity of the organism, the organism will sent for identification and sensitivity test.

week 10!

Subject title: Clinical Chemistry
Name of test: Urine Microalbumin Qualitative Test


Yay! 10 more weeks to go =)


Principle:

• Urine Microalbumin Qualitative Test is a screening test to determine the presence of albumin and creatinine in urine. An albumin-to-creatinine ratio is also determined since the amount of albumin in the urine and the concentration of urine varies throughout the day, hence if we were to take the albumin results, it would not be very accurate. Creatinine on the other hand is excreted out on a consistent basis and its level is relatively stable in the urine. This test can be considered semi-quantitative, however it is not a confirmatory test.
  

• This test involves plastic strips which contain two reagent areas that test for albumin and creatinine. The strips are then read instrumentally using CLINITEK STATUS®

• Normally, albumin is present in urine at concentrations of less than 20mg/L. Measurements of 20-200mg/L indicates microalbuminuria whereas results of >200mg/L indicate clinical albuminuria. It is abnormal to have protein in your urine as the glomerular basement membrane is actually poorly permeable to it. Microalbuminuria can be an indication of glomerular damage.

• Test principle for albumin: It is based on dye binding using a high affinity sulfonephthalein dye. If albumin is present, the colour of the reagent area that test for albumin will change to blue. The colour ranges from pale green to aqua blue.
  

• Test principle for creatinine: It is based on the peroxidase-like activity of a copper creatinine complex that catalyzes the reaction of diisopropyl-benzene dihydroperoxide and 3,3,'5,5'-tetramethylbenzidine. The colour ranges from orange through green to blue.

Materials:

1. Patients' urine


2. CLINITEK® Microalbumin Reagent Strips

Retrieved August 31,2008, from http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay~q_catalogId~e_-111~a_catTree~e_100001,1015867~a_langId~e_-111~a_productId~e_172993~a_storeId~e_10001.htm

3. CLINITEK® STATUS analyser

Retrieved 31 August, 2008, from http://diagnostics.siemens.com/webapp/wcs/stores/servlet/ProductDisplay?productId=172991&storeId=10001&langId=-111&catalogId=-111&catTree=100001,1015867

Method:

1. Check labels on the urine container to ensure the name of the patient tally

2. Key in the patient's lab no in the analyser

3. Dip a reagent strip into the sample making sure both reagent areas are wet

4. Remove the strip and drag the edge of the strip against the rim of the urine container to remove excess urine

5. Place the reagent strip with the reagent areas facing up, onto the analyser test table. Press START and the table will be automatically pulled into the analyser where the strip will be read.

6. Record the results displayed on the analyser

Results:

• The results are determined by the Albumin-to-Creatinine Ratio (A:C) which is calculated automatically by the analyser
• The results are recorded as NEGATIVE when the A:C displayed is <30mg/g>
• The results are recorded as ABNORMAL when the A:C displayed is 30-300mg/g
• The results are recorded as HIGHLY ABNORMAL when the A:C displayed is >300mg/g

Note:

• Both ABNORMAL and HIGHLY ABNORMAL samples will be confirmed with the quantitative test which can be carried out in the machines COBAS or MODULAR P used in our lab
  

Nur Farhana Binte Ramlan
0604834B

9th week

hello everyone..
this is the 9th week of our SIP..
how's things going?
it has been a busy week for both me and my buddy as we have started embarking on our major project.
we have been dividing our time between benchwork and MP during this time.

for the past weeks, i have been posted to different department which includes urine section, order entry and chemistry section. in urine section, i was introduced to the routine work. basically it the same routine work that has been mentioned by dyana in her latest entry. beside doing urinalysis, the med tech there also have to perform any arterial blood gas (ABG) request. i was only allowed to observe how they process the test as it require the id of the person performing the test. it was rather an easy test to do as they just have to load the syringe into the machine and the machine will produce the result.

i was then attached to order entry section the week after. as i had been there before, i was already familiarise with the work flow there. only that this round i was station to do the order entry itself. there was so many codes to remember. but thankfully there was people around to help me out. there was also files which contain the codes placed near the counter. we can always refer to it. apart from having to know the codes for the different test, we must also know how to labeled the tubes for the different test.
EDTA tube are commonly used for FBC and GHB.
HEPARIN tube can be use for most of the tests. this applies to GEL tube as well as PLAIN tube. FLUORIDE tube are used mainly for glucose, particularly fasting.
if the blood are less than half of the tube, we hav to transfer to aliqoute it out to a secondary tube before performing the test. if the blood is too little, another tube will be use called the hitachi cup.
after labelling, we will then give the tubes to the different sections.

chemistry section was fully automated. we just have to load the specimens into the machine and the machine will produce the result. and as a med tech, we will have to anaylse the results and validate them. however there are certain things that we have to take note when loading the tubes into the machines.
there are 3 machines that are used in chemistry. they are MPA, SWA and COBAS.
MPA is the machine that consists of centrifugation, decapping of the tube's cap and aliquoting the specimen according to the test requested.
in MPA we cannot load citrate, edta and fluoride tube. the tubes in the same rack has to be of the same height. we can load 5 tubes on 1 rack.
SWA and COBAS are those that performed the tests.
there are tests that are only available for COBAS and some that are only available for SWA. we have a list of those tests placed near the chemistry processing table which we can refer to if we are not sure which machine to use. those test that are not listed can be loaded into either one of the machines.
there are times wen the MPA will be fully loaded with samples. so, for urgent cases we usually have to spin the sample manually. we called it 'offline'. whenever we are told spin 'offline', it means that we have to use the secondary tube for the sample.
sometimes, it gets pretty confusing.

i guess that's all i have to share for this entry.
thanks for reading.

sutiana
tg01
0604651j

8th week

for the past 3 weeks, i have been posted to the cytology lab. the atmosphere in the cytology lab is definitely much cosier, probably because the specimens received in the cyto lab is much lesser as compared to the amount of specimens received in the histo lab.

In cyto lab, specimens are categorized into two fields; gynaecological specimens and non-gynaecological specimens. gynaecological specimens are cervico-vaginal smears while non-gynaecological specimens are sputum, urine, CSF and other fluids(peritoneal fluids etc).

1. The gynaecological specimens are received along with the requisition forms.
a) Ensure that the identification number on the slides tally with the number of the forms.
2. The specimens are then fixed in 95% alcohol.
3. The specimens are then loaded into a rack to be placed in the pap stain machine.

Principles of pap stain.
The papanicolaou stain method is a polychrome staining reaction. It is used to portray the variations of cellular maturity and metabolic activities. This method can be used for cervico-vaginal smears and smears from the different bodily secretions which includes respiratory and digestive, to detect the presence of cancer cells. The pap stain involves 3 different types of stains; the Harris haematoxylin, Orange G and EA50. The harris haematoxylin will stain the nuclei blue. EA 50 is made of three elements. The different elements are eosin, Bismarck brown and fast green. The combination of Orange G and EA50 is responsible for the different range of green, blue and pink hues to the cell cytoplasm according to the degree of keratinization of the cell. The cytoplasm of non-keratinized, normal superficial and intermediate squamous cells are stained green. However when the cells are keratinized, the staining becomes orange and pink.

Note: The slide with the specimens smeared in a thin, monolayer manner will give the best staining results. This is because the thickness of the section will affect the intensity of the staining.


4. The stained slides are then placed in a container filled with xylene.

5. The slides are mounted manually.
a) Just enough depex is filled on a cover slip.
b) Take a slide from the rack in the xylene and place it on the cover slip.
c) Ensure that there are no bubbles present between the slide and slip.
d) If bubbles are seen, press the slip gently to allow the bubbles to escape.
e) If there are too many bubbles seen, the slide should be remounted.
f) To remount the slide: Place slide in a container with xylene until the cover slip dissociates
and until the mounting medium is removed. Then, mount as usual.

6. The mounted slides are left to dry.
7. Label the slides according to the lab number.
8. The slides are ready to be screened.


Nurathirah
0606561I
Tg 01