Subject: MMIC
Topic: Methicillin Resistant Staphylococcus Aureus (MRSA) Rapid Screening

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body. It's tougher to treat than most strains of staphylococcus aureus -- or staph -- because it's immune to some commonly used antibiotics.

Recent research suggests that in the identification of MRSA, it is more accurate to either directly detect the gene encoding the methicillin resistance determinant (mecA) or its product, penicillin-binding protein 2' (2a), or PBP2' (PBP2a), which is found in the cell membrane of MRSA. However, as nucleic acid hybridization and DNA amplification techniques such as PCR for detecting the mecA gene are expensive and technically demanding, simple and more inexpensive techniques are required for routine use. MRSA-Screen was developed expressly for this purpose, providing results in 15 minutes with minimal labor and no specialized equipment.

Principle
MRSA Screen is a rapid slide latex agglutination assay which detects the penicillin binding protein 2 (PBP2’) of methicillin-resistant S.aureus. Latex particles are sensitized with a monoclonal antibody against PBP2’ and will specifically react with MRSA to cause agglutination visible to the unaided eye. It will therefore enable the rapid and appropriate antimicrobial therapy for serious MRSA infections.

Equipment & Materials
NMRSA Screen kit
Sensitised Latex 2.5 ml x 1
Control Latex 2.5 ml x 1
Extraction Reagent 1 10 ml x 2
Extraction Reagent 2 5 ml x 1
Test card 55
Mixing stick 220
BAP
Micropipette and tips (50ml)
A beaker, tripod, wire net and a burner for boiling the specimen


Procedures
A. Sample Preparation
1) Colonies believed to be S. aureus taken from BAP or other media, after incubating for 18-24 hours at 35°C are used.

B. PBP2’ Extraction
2) Add 4 drops of Extraction Reagent 1 into a test tube.
3) 4 drops of Extraction Reagent 1 are added into a test tube.
4) A loopful of cells are taken using a platinum loop or other appropriate device and suspended in the test tube.
5) The test tube is placed into a boiling water bath or heating block and heated for 3 minutes.
6) The test tube is removed and allowed to cool to room temperature.
7) A drop of Extraction Reagent 2 is added into the test tube and mixed well.
8) The test tube is centrifuged at 1500 xg for 5 minutes.
9) The supernatant is used as specimen.

C. Latex Agglutination
10) Circles on test card are labeled with Sensitised latex and Control latex for each specimen.
11) 50 ml of specimen is placed onto the test circles
12) Sensitised latex or control latex is added into each respective test circles.
13) They are mixed well using a mixing stick
14) The test card is rotated by hand for 3 minutes and agglutination patterns are observed.

Interpretation
Agglutination is seen with Sensitized but not Control Latex: PBP2’ positive (MRSA)
No agglutination is seen with either latex reagent : PBP2’ negative (MRSA)



Boon Ching

Cytogenetics

For the past 4 weeks, I was attached to Cytogenetics Laboratory. In this entry, I will discuss about peripheral blood culture for chromosome studies.

Peripheral Blood Culture

Specimen Requirement

• 3 - 5ml of peripheral blood in sterile tube wuth sodium heparin as anticoagulant.
• Sterile techniques must be observed when collecting blood.
• The sample is sent to cytogenetics lab as soon as possible.

Clinical Significance

1. T-lymphocytes present in peripheral blood are exposed to a mitogen (e.g. PHA) to stimulate cell divison. They are then arrested at metaphase with the addition of colcemid. Chromosomes for analysis are obtained upon harvesting.
2. Clinical uses of constitutional karyotype obtained from blood cultures include:

• Genetics diagnosis/genetic counselling
• Determine the constitutional karyotype of families of individuals with chromosome abnormalities
• Determine carrier status
• Comparison of lymphocyte karyotypes with those of other tissue e.g. skin, AF or CV
• Confirmation of structural and numerical abnormalities or mosaisim
• Determine the constitutional karyotypes of patient with haematologic malignancies or solid tumors
• Aid in the diagnosis of chromosome breakage syndromes e.g. Fanconi Anaemia
• To provide material for metaphase FISH

Method

1. Thaw 1 tube of complete Rpml medium and 1 tube of complete M199 medium in water bath at 37ºC.
2. Add 0.1ml PHA into each tube.
3. Centrifuge blood specimen at 1200rpm for 10 minutes. Record volume of blood received and note down whether there is haemolysis, jaundice, clot etc. in worksheet.
4. With a sterile transfer pipette, gently pick up »1ml of buffy coat together with some plasma from the blood.
5. Invert transfer pipette and mix the sample thoroughly in the bulb. Distribute the sample into the culture tubes.
6. Tighten caps and mix well by inverting the culture tubes several times.
7. Place the culture tubes in a slant test-tube rack. The slant is at approximately 45º angle and the purpose is to increase the surface area of the culture for gaseous exchange. Leave the rack in a 37ºC incubator for 48 hrs.
8. Invert the tubes every morning to re-suspend pellet.
9. 48 hrs after culture set-up, add 50μl of MTX x 10-5 m working solution into each tube. Mix well by inverting the tubes and re-incubate at 37ºC for another 18 hrs.
10. After 18 hrs, add 50μl of thymidine to each tube.
11. Mix well and incubate at 37ºC for 4 hrs.
12. Proceed to harvesting.

Note: MTX is to block cells in the S (DNA synthesis) phase of cell cycle. Thymidine is to promote longer and better G-banded chromosomes in routine blood cultures.

Harvesting of Peripheral Blood Cultures

Harvest Process

1. Centrifuge tubes at 1200 rpm for 10 minutes. Aspirate supernatant.
2. Loosen pellet gently. Add approximately 8-10ml warmed hypotonic solution into each tube; invert tubes 2-3 times to ensure the suspension is well mixed.
3. At the beginning of handling the first tube, start a timer on 5 minutes counting down for every batch of »12 tubes. Leave the tubes at room temperature for the whole period of 5 minutes.
4. At the end of the 5 minutes, transfer tubes unto a covered centrifuge buckets and spin tubes at 1500 rpm for 6 minutes.
5. Aspirate and discard supernatant.
6. Repeat step 2 and 3.
7. At the end of the 5 minutes, add 1 pipetteful of fixative and invert tubes 2-3 times to mix well.
8. Repeat step 4 and 5.
9. Re-suspend cell pellet by flicking tubes until no pellet clumps are visible.
10. Add another 5 pipetteful of fixative. Invert tubes 2-3 times to mix well.
11. Stand the tubes at room temperature for at least 15 minutes. Aspirate and discard supernatant.
12. Centrifuge the tubes at 1000 rpm for 10 minutes.
13. Re-suspend pellet in 4 pipetteful of fixative. And repeat step 12. check that the suspension is clear after centrifugation, if not repeat this step again especially for clumpy and jelly-like pellets.
14. Re-suspend pellet in 3 pipetteful of fixative and store in refrigerator at 4ºC until ready to make slides. Proceed to slide making.
    

After slide making and staining, the slide is ready for chromosome analysis.
That’s all for this entry. Enjoy reading. Feel free to ask questions. =)

Doreen (tg 01)

p.s. for the interview question, the amniocytes are not lysed. Fixation can strengthen cell membranes, improve chromosome morphology, and rid the sample of debris such as lysed red cells. Hence, after fixation, the cells are ready for slide making and staining.

SIP

Subject: Blood Banking

Topic: Tranfusion Reaction Investigations

Transfusion reaction is any adverse outcome associated with the infusion of blood or blood components. The most common signs & symptoms are chills, fever & urticaria. Patients can show these symptoms during or within several hours after transfusion.

Require:

• 5 ml clotted blood (purple capped tube)
• 5 ml plain blood (yellow capped tube)
• Pre-tranfusion specimen (taken for crossmatching before transfusion reaction)
• Donor pilot tube & segment tube* from blood pack

*pilot tube - blood segment taken from blood pack before transfusion
*segment tube - taken from blood pack after transfusion

1. Exclude clerical errors
The transfused blood pack and patient sample are examined for the following to exclude possible clerical errors in patient or blood identification in the lab:

• Patient identification (name & IC no.)
• Unit ID on both blood bag & label
• Pre-reaction records on patient's buff card (ABO/Rh typing, antibody screening & compatibility results)
• Computer record of both patient & blood unit

2. Visual check
Visual check is done on both the Post I (blood sample) & Post II (blood & urine samples) for hemolysis. (E.g. Haemoglobinaemia, haemoglobulinuria & spherocytosis)

3. Exclude serological errors
a) ABO blood grouping and Rh typing are performed on the following for evidence of a mismatched ABO/Rh or gross incompatibility:

Patient's
- Pre transfusion sample
- Post transfusion sample I (Post I)
- Post transfusion sample II (Post II)

Donor's
- Pilot tube
- Blood pack/segment

b) Direct Antiglobulin Test (DAT) is performed on patient's sample (pre, post I & post II) & donor's sample (pilot tube & segment).
- To demostrate antibodies/complement bound to red cells in vivo.

c) Compatibility testing between patient's samples (Pre, Post I & II serum) & donor's cells are repeated.

d) Antibody screening on the patient's samples (Pre, Post I & II serum) for clinically significant Ab are repeated.

4. Biochemical analysis
Plain blood (yellow cap) sample is sent for biochemical investigations of:

• Serum bilirubin
  (High level of bilirubin -> haemolysis)
• Haptoglobulin
  (Low level of haptoglobin –> increased intravascular haemolysis)

5. Immunohaematological investigation (HLA Ab test)
The test is done when the patient has any 1 of the symptoms (chills, rigors, elevated temperature)and suspected of non-haemolytic febrile transfusion reaction.

6. Bacteriological investigation
The blood pack is sent for sterility testing.
- Perform gram stain & culture the donor's unit to check for bacterial contamination.

Classification

Type of Reaction	Positive Results
Acute Haemolytic Reaction	- Visual changes suggestive of hemolysis - Mismatched ABO/Rh typing - Missed antibody - Missed inappropriate crossmatching - Biochemical hemolysis
Reaction due to Bacterial Contamination	- Visual changes suggestive of bacterial contamination - Positive culture result
Non-Haemolytic Febrile Transfusion Reaction	- Presence of HLA antibodies
Allergic Transfusion Reactions	- Presence of IgA antibody

dorothy
Tg01

SIP

Subject: histopathology
Topic: special stain

Special stains such as PAS for carbohydrate, Gomori’s Methenamine silver nitrate for fungus and Gordon and sweet’s silver impregnation for reticulum fibres can be performed by an automated staining machine. The automated stainer used in the lab is Ventana NexES.(The procedure had been introduced by Sharon and June’s blog so I am not going to repeat) Some special stains can only be performed manually as the kit for that particular stain is not available or temporally out of stock. Perl’s reaction and von kossa’s stain are example of special stains that can be perform manually.

Perl’s reaction

Function: demonstration of iron and haemosiderin

Principle: Dilute hydrochloric acid is used to release haemoglobin or myoglobin in red cells that is tightly complexed with ferric iron. An insouluble blue compound, ferric ferrrocyanide will be formed once the ferric iron reacts with potassium ferrocynade solution.

Chemical required: 20% aqueous hydrochloric acid
20% aqueous potassium ferrocynanide
Nuclear fast red

Procedure:
1) Dewax, and section to water.
2) Prepare perl’s reagent. (equal part of 20% aqueous hydrochloric acid and 20% aqueous potassium ferrocynanide)
3) Treat with perl’s reagent for 20 minutes.
4) Rinse with distil water.
5) Counterstain with nuclear fast red
6) Wash, dehydrate, clear and mount

Results: iron----dark blue
Nuclear----red

Von Kossa’s stain for calcium

Function: demonstrate calcium deposits.

Principle: tissue on the slide is treated with Silver nitrate solution. Under the presence of ultra-violet light, calcium salt is reduced and replaced by silver deposits, and will be appeared as metallic silver.

Chemical required: 10% aqueous silver nitrate
Nuclear fast red

Procedure:
1) Dewax and section to water
2) Soak slides with 10% aqueous silver nitrate and leave under UV light for 3-4 hours
3) Rinse in water
4) Counterstain with nuclear fast red for 1-3 minutes.
5) Wash, dehydrate, clear and mount in DPX.

Results: calcium----black

Wing Fat
Tg01