Student Internship Programme (SIP) HAEM

Neutrophil Alkaline Phosphatase (NAP) stain

Intended use
NAP score is used to distinguish Chronic Granulocytic Leukaemia from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.

Principle

At an alkaline pH, NAP hydrolyses substrate naphthol AS-BI phosphate to liberate napthol-AS. The liberated naphthol couples with a diazonium salt, Fast Blue BB to produce an insoluble complex which precipitates at the site of enzyme activity. The sites of alkaline phosphatase activity will appear as blue granules. The NAP score is the sum of the rating of 100 consecutive segmented and band neutrophils using a scale of 0 to 4+ according to the appearance and intensity of the precipitated dye.

Specimen collection and handling conditions

1. Fresh capillary/venous blood preparation, not anticoagulated is required.
2. EDTA blood is not recommended as enzyme activity is inhibited.

Preparation of stock substrate solution

a) Dissolve 30mg naphthol AS phosphate in 0.5ml N,N-dimethylformamide. CAUTION: N,N-dimethylformamide is toxic by inhalation. To be prepared in fume hood.
b) Add 0.2M tris buffer, pH 9.1 to bring volume to 100ml.

c) Store at 4-10 deg celcius. Stable for 2 months.

Preparation of working substrate

a) Add 10mg Fast Blue BB to 10ml stock substrate solution. Mix well. CAUTION: Fast Blue BB is carcinogenic and a possible mutagen. To be prepared in fume hood.

b) Prepare fresh when needed.

Quality control

A normal control smear must be included with every batch of stain and results should fall within tolerance limits.

Procedures

1. Fix air-dried smear in buffered 60% Acetone in Citrate brought to room temperature for at least 5 mins.

2. Rinse with tap water and dry slide.

3. Using filter paper No.41, filter the freshly prepared working substrate directly onto slides. Stain for 15 mins at room temperature. Discard working solution into the toxic waste container.

4. Wash in tap water.

5. Counterstain with 0.5% neutral red for 1 min.

6. Wash and dry.

7. Count under oil immersion 100 neutrophils. The slides are examined and counted by 2 technologists and an average of 2 results is reported.

Interpretation

Sites of enzyme activity are represented by discrete bright blue granules of varying sizes. Nucleus is stained red. The enzyme is present predominantly in segmented neutrophils. Cytoplasmic granules of eosinophils do not stain while basophils cannot be differentiated from other granulocytes.

One hundred segmented or band forms of neutrophils are rated. The sum of 100 cell ratings give a score with a possible range from 0-400.

Normal range: 30-100

Clinical significance
Chronic Granulocytic Leukaemia and Paroxysmal Nocturnal Haemoglobinuria are associated with abnormally low or absent staining. Conversely, leukaemoid reactions, polycythaemia vera and myelofibrosis are associated with markedly elevated NAP scores. High scores can also be obtained from neonates, pregnant women or women taking oral contraceptives.

Notes
1. Do not make the smear too thick to avoid inadequate fixation.
2. Fixed smears should be stored at -20 deg celsius if staining delayed for >5-6 hours.
3. Scoring of enzyme activity should be made in areas of slide with optimal cell morphology.

Answers to possible questions

Why must the working solution be prepared fresh before every staining?
Precipitation will occur over time once the Fast Blue BB is added to the stock substrate solution that will be indicated by a colour change from bright yellow to dull brown. Therefore it is important that the working solution be mixed and applied fast.


What are the various illnesses stated?
Please visit Wikipedia or other search engines to administer your enquiries. I'm sure you will learn more than what I'll be able to explain this way. :)




I've also taken some pictures and will seek to post them as soon as possible once I get relevant approval. Hope you've learnt something!

Alex Tan 0503222B TG02

Hello everyone!! It’s my turn to blog for the week. I am currently at coagulation lab. The test which I am going to share with you guys is 50%PT/APTT. I suppose you should be familiar with PT and APTT which we had done before in during haematology lab session. Does it ring a bell? To refresh your memory…

PT is used to screen for abnormalities of those factors that are involved in the extrinsic pathway (factor 5,7,10) and prothrombin and fibrinogen.

APTT is sensitive to deficiencies of factor II,V,VIII,IX,X,XI,XII( Factor 2,5,8,9,10,11,12) involved in the intrinsic pathway.


Test: PT

1) PT reagent is incubated in water bath at 37 degrees for 3mins
2) 0.1ml of test plasma is incubated in 37 degrees water bath for 3mins
3) 0.2ml of PT reagent was pipette into the 0.1ml of test plasma after the incubation period
4) start the stopwatch simultaneously and shake the test tube to mix the contents well and observed for clot

Test:APTT

1) 0.1ml of APTT reagent and 0.1ml of calcium chloride was incubated in water bath at 37 degrees for 5mins
2) 0.1ml of test plasma was added to 0.1ml of APTT reagent. Remember to shake the tube to mix the contents well
3) the tube was placed at 37 degree water bath for 4mins
4) 0.1ml of calcium chloride was pipette into the plasma and APTT reagent
5) start the stop watch simultaneously and observed for clot





Test: 50%PT and APTT correction test

Intended use:
It is used to screen for the presence of inhibitors in patient’s plasma.

Principle:

50% addition of normal plasma is sufficient to restore the deficient factor. Complete or partial correction time suggests a clotting factor deficiency, whereas failure to correct the clotting time suggest the presence of inhibitors. Normal plasma contains all coagulation factors and is mixed with patient’s plasma to detect the presence of circulating inhibitors in the patient’s plasma. If a factor deficiency is responsible for the abnormal clotting time addition of normal plasma should provide the deficient factors and correct the PT and APTT time. However if abnormal clotting time is caused by a circulating inhibitors in the patient’s plasma, the clotting time will remain prolonged even when normal plasma is added. The inhibitor inhibits factors in normal plasma as well as in the patient’s plasma.

Example: if the patient’s PT is 18secs
Normal plasma is 12secs
Average is (18+12)/2=15secs
PT is corrected if 50%PT <15secs>

that's all! feel free to ask if in doubt..

kai lin

0503211E

SIP posting

For this past few weeks I was attached to Haematology

At haematology section, I have learn various principle of the tests such as retic count, Dengue testing.

RETIC COUNT
For retic count, the analyzer used is CELL-DYN Ruby. Reticulocytes are defined as transitional red cells between nucleated red cells and the mature erythrocytes. The reticulocyte assay enables determination of the percentage of reticulocytes using a whole blood specimen. The method used is based on light scatter measurement of stained cells.

Staining Procedure:
1. Label one tube of CELL-DYN Reticulocyte Reagent(Phosphate buffered saline solution contain New Methylene Blue N) for one specimen.
2. Verify the whole blood specimen have no clotting and mixed well.
3. Pipette 20 micro-litre of whole blood specimen into the tube of reticulocyte reagent.
4. Incubate the stained reticulocyte specimen for 15 mins at room temperature.
5. Ready for sampling in CELL-DYN Ruby

Principle of Procedure:
Reticulocytes contain ribosomal RNA, this RNA can be seen by certain dyes that simultaneously stain and precipitate the polyanion to form a reticulum. The CELL-DYN Ruby reticulocyte method uses the thiazine dye New Methylene Blue N. Sample preparation is done by diluting a small volume of blood into pre-measured staining solution and incubate for at room temperature for 15 mins for the staining of reticulum to complete. The stained sample could be tested using CELL-DYN Ruby. The stained sample will be aspirated in the analyzer and diluted with the Reagent(WBC Lyse). Once diluted, the RBCs sphere due to the influence of the nonionic detergent incorporated into the staining solution. Sphering is necessary to eliminate optical orientational noise that would otherwise be introduced into the scatter measurement. The usual lytic action of the Reagent is prevented by electrolytes contained in the staining solution and the lack of the usual incubation period used in this channel during WBC analysis. Also the high New Methylene Blue concentration in the staining reagent exerts a stabilizing effects on RBCs.

During data acquisition, 0,10 and 90 degrees scatter is collected. The 0 degrees threshold is set high enough to exclude most platelets. Histogram data is used to differentiate reticulocytes, mature RBCs, platelet clumps and nucleated cells. Reticulocytes have similar 10 degree scatter to mature RBCs but differ them by exhibiting greater 90 degree scatter.

Dengue Testing using DENGUE DUO CASSETTE
The Dengue Duo Cassette is for the qualitative presumptive detection fo IgM and IgG Ab to dengue virus in human serum, plasma and whole blood. The assay can be used for the presumptive differentiation between primary and secondary infection. Positive result are presumptive and must be confirmed by virus isolation, paired serum analysis, Ag detection by immunochemistry or viral nucleic acid detection for confirmation of dengue virus infection.


Dengue, a flavivirus is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aegypto and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestation ranging from asymptomatic to fatal haemorrhagic disease.


In the Dengue Duo Cassette, IgM and IgG are determined simultaneously using a single addition of serum, plasma or whole blood. Thus, a differentiation between primary and secondary infection can be made by a single application of serum, plasma or whole blood. In primary infections, serum IgM Ab can be detected from dengue patients as early as 3-5 days after onset of fever, generally persisting for 30-90 days, although detectable levels may be present for 8 months post-infection.

Secondary infection is characterised by high IgG levels that may or may not be accompanied by elevated IgM levels. The sensitivity of this assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative. In contrast, patient with secondary infections will have a positive IgM result.

Assay Procedure
1. Add 10 micro-litre of whole blood, serum or plasma to the cirular well and allow the sample to be absorb into the specimen pad within the circular well.



2. Hold the buffer bottle vertically and 1 cm above the square well and add 2 drops of the buffer to the square well at the base of the cassette.


3. Read the result exactly after 15 mins after adding the buffer.


Princple
When present in the patient sample, dengue-specific IgM or IgG Ab bind to the anti-human IgM or IgG Ab immobilized in two lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 Ag are captured by the bound patient's IgM or IgG to give visible pink line. A procedural control is included to indicate that the assay has been performed correctly.

Interpretation of Result

Primary infection

Pink bands appear in the IgM and Control regions

The test is positive for IgM Ab and is suggestive of primary dengue infection.






Secondary infection


Pink bands appear in the IgM, IgG and Control regions.

The test is positive for the IgM and IgG Ab and is suggestive of a secondary dengue infection.

Secondary infection

Pink bands appear in the IgG and Control region.

The test is positive for IgG Ab and is suggestive of secondary infection.















Negative

A pink band appears in the control region only.

No detectable IgG and IgM Ab to dengue. The result does not exclude dengue infection. Retest in 3-4 days if dengue infection is suspected.

Invalid

No pink band appear in the control region.


The test is invalid and should be repeated.

This are the 2 tests I have learn. Feel free to ask question.
Juexiu TG02

SIP- Cytology

This month, i'm scheduled to the Cytology department in which the bulk of the specimens it deals with are mainly PAP smears, urine and body fluids (such as peritoneal, pleural and pericardial fluids, CSF).

Scope of Cytology:

• Diagnosis of malignancy
• Identify premalignant conditions
• Identify infections and associated organisms
• Evaluation of hormonal effects of estrogen and progesterone

PAP (Papanicolaou) Smearing and Staining

Purpose: Diagnosis of cervix cancer (causative agent: Human Papillomavirus).

Procedures:

1. After smearing on slides, specimems will be sent to the Cytology lab.
2. Check to make sure the patients' data on the request log tallies with that on the slides.
3. Load them into a rack then into the automated staining machine which has been programmed to perform PAP staining.
4. After staining, the slides are mounted for microscopic examination.

PAP Staining (The Regressive Method- Nuclei Overstained)

Principle: Exhibit differences in cellular morphology, maturity and metabolic activity. As intact cells in a cytologic smear tend to appear in 3-D configurations and overlap, PAP allows resultant transparency and clear definition of nuclear details.

Procedures:

1. Fix the cells in 95% ethanol for 15 minutes to allow the cells to absorb the dyes in the subsequent staining procedures which are fully automated.
2. Unacidified haematoxylin
3. Blue in water (nuclear staining)
4. Dilute hydrochloric acid (remove background staining caused by excess haematoxyin)
5. Rinse in water(3 rounds to set the haematoxylin)
6. 95% alcohol (3 rounds to remove all water as subsequent staining reagents are alcohol-based and water cannot mix with alcohol)
7. OG (Orange-G) stain (stains keratin orange)
8. 95% alcohol (2 rounds)
9. EA Polychrome stain (stains mature cells- mostly cancerous cells red and immature cells green)
10. 95% alcohol (2 rounds to provide clearer view of overlapping cells)
11. 100% alcohol (2 rounds for final dehydration)
12. Xylene (2 rounds for clearing to allow microscopic examination)

Results: Nuclei- blue or black

Cytoplasm (non-keratinised)- green or blue

Cytoplasm (keratinised)- pink or orange

Red blood cells- orange

Quality Control of PAP Staining

1. Checking of nuclear staining

• Staining is crisp and sharp
• Parachromatin is clear (not muddy)

2. Cytoplasmic counterstains

• Sharp and clean
• 3 distinct colours: red (Eosin-Y); orange (OG); green (EA)

Troubleshooting Guidelines for PAP Staining

1. Problems with absorption of haematoxylin due to inadequate removal of carbowax resulting in irregular and spotty staining (poor quality).

• Soak in 95% ethanol for complete fixation to enable the cells to have better absorption of haematoxylin

2. Lack of contrast in nuclear staining due to poor quality of haematoxylin.

• Replace water rinses used for blueing purposes
• Tap water should be slightly alkaline. Acidic tap water will result in nuclear fading while heavily chlorinated tap water will bleach out the haematoxylin.

3. Problems with cytoplasmic stains due to improper rinsing of stains after OG and EA

• Do not allow the slides to sit in alcohol solution following OG and EA stains as it can wash the stains away
• Rotate ethanol rinses as ethanol nearest the dye becomes discoloured

4. Water droplets seen on slides due to water in xylene (xylene will appear milky)

• Change the xylene
• Add Silica-Gel pellets to absolute alcohol to minimize the possibility of water contamination of xylene

That's all! Enjoy your SIP! Take care! ^_^

June Tham

TG02

0505073G

Embedding and Microtomy

Embedding

Introduction
After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding. Each batch of blocks is tallied against the listing of the blocks for the corresponding batch to ensure that no blocks are missing before embedding.

Principles
The tissue is placed in molten paraffin (56* melting point) such that after the paraffin cools, the tissue and paraffin will form a block of sufficient consistency to cut thin sections

Materials
Embedding machine
Embedding moulds
Forceps

Embedding technique
1. Open the processing cassette carefully and view the tissue.
2. Select a mould that best corresponds to the size of the tissue.
3. Partially fill the mould with paraffin.
4. With warm forceps remove the tissue from the cassette and place it at the bottom of the mould – refer to specimen orientation below.
5. Gently press the surface of the tissue against the solidifying wax to hold it in the desired position.
6.Ensure no tissue is stuck on the lid then discard the lid of the cassette and place the bottom of the cassette firmly on the top of the mould.
7. Fill the combined mould and cassette with paraffin.
8. Place the mould on ice to solidify the wax, thus separating embedding tissue-cassette from the mould.
9. The tissue and cassette forms a paraffin block ready for sectioning.

Specimen orientation
1. Tissue sections are embedded flat to ensure that complete section is obtained.
2. Orientation should be such that the resistance the tissue offers the knife proceeds from the lesser amount towards the greater amount as the block is sectioned. This prevents the harder tissue from compressing the softer tissues and produces a smoother section.
3. There should be an adequate margin of embedding medium surrounding all sides of the tissue for maximum cutting support.
4. Tubular structures such as vas deferens, veins, arteries and fallopian tubes must be embedded such that the knife cuts across the lumen. These should be placed vertically in the mould.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder, urinary bladder and uterus must be positioned such that the plane of the section is across all tissue layers. The epithelial surface should be placed such that the plane of section is perpendicular.
6. Multiple specimens should be placed side by side close together so that call pieces can be sectioned.
7. Rectangular tissues should be orientated parallel to each other and with their long axis perpendicular to the plane of section.
8. Small bisected cysts should be embedded with the cut surface down and ensure that no air bubbles are trapped in the paraffin.
9. Muscle biopsy in 2 pieces should be embedded with one piece in a longitudinal and the other in a vertical position.

Microtomy

Introduction
Paraffin blocks from the blocking room are brought to the cutting room for sectioning

Principles
Microtomy is the use of a microtome to make thin sections for microscopy. Rotary microtomes are used in the laboratory

Materials
Room Temperature floatation bath
Warm floatation bath – 48 plus-minus 4 degrees Celsius thermostatically controlled
Cryoplate
Disposable microtome blade
Microscope glass slides
Rotatry microtome
Soft pencil

Solution preparation
1. Type II Water 2000ml

Allow the water in the floatation bath to reach 48 plus-minus 4 degrees Celsius on a thermostat before sectioning. Technologist must record this temperature on the temperature chart before sectioning

2. 1% alcohol floatation bath
95% alcohol 5ml
Type II Water 500ml

This is used to float the tissue section prior to transferring on the heated floatation bath above. It is an alternative when folds on the tissue are difficult to get rid, as alcohol having low vapour pressure will increase the surface tension when transferring the tissue onto the heated floatation bath.

Microtomy Technique
1. Rough cutting – Secure the paraffin block in the block holder of the microtome. Adjust it to ensure that it clears the knife. Readjust the block holder screws to place the block parallel to the knife if necessary. During the rough-cut, while being manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Sectioning stops when the entire surface of the tissue is exposed. The block is then removed from the holder.
2. All blocks should be rough cut before sectioning because:

a.Dense and hard tissue that may cause nicks and blunt the knife more rapidly can be identified e.g. bone, cervix and thyroid. Such blocks could be separated from the rest and appropriately pre-treated before sectioning. These blocks should be sectioned last as they may still cause nicks and score lines to the blade.
b. Sutures and staples attached to the surgical specimens may not have been removed and can be detected during rough cutting.
c. Hard bone is identified and additional surface decalcification can be done, by placing the block face down in decalcifying agent e.g. RDO for further decalcification prior to sectioning.
d. Fibrous tissue e.g. fibroids can be softened in mollifex or 10% fabric softener for 5 minutes. The paraffin blocks are washed in water after the appropriate treatment porior to sectioning.
3. Chilling – the blocks are then placed face down on the cryoplate to chill the block to facilitate fast sectioning. This renders the block sufficiently hard for thin sections.
4. Sectioning – secure and adjust the block in a similar manner as rough cutting. Using the handwheel, section the block at 3-4 microns, by allowing the block to advance automatically. Some tissue biopsies are sectioned at different thickness.
5. Flotation- gradually lower the section or sections if a ribbon is cut, onto the water bath. If difficulty is encountered in spreading of the tissue, float the section on the alcohol bath first so as to increase the surface tension before transferring onto the water bath. Allow the section to remain on the water bath until it has spread sufficiently. Transfer the section onto a glass slide. The corresponding biopsy number is written on the frosted end of the glass slide.
6. Separation of slides – Slides for routine HE stain, special stains and unstained sections should be separated on different racks.

Serial Sections
First submission
Tissues less than 0.5cm in size
Gastric biopsies
i. 1 HE slide with 6 serial sections
Liver biopsies
i. Slide 1 – 2 consecutive complete sections for HE
ii. Slide 2 – 1 section for MT
iii. Slide 3 – 1 section for Ret
iv. Slide 4 – 2 consecutive complete sections for HE
Bone marrow
i. Section at 2um thick
ii. 1 HE slide with 6 serial sections for small specimen or 3 HE sections on 3 slides labelled VL1, VL2, VL3 for larger specimens and 1 slide for Retic
Renal biopsies
i. Section at 2um thick
ii. 10 slides with 3 sections each as follows:
1. HE on slide 2, 5 and 8
2. Pas on slide 1m 6 and 9
3. PaAg on slide 4
4. PgMT on slide 3, 7 and 10
TBLB – Trans bronchial lung bx
i. Section at 5um thick
ii. 10 slides with 2 sections each as follows:
1. HE on slide 2, 5 and 8
2. TB stain on slide 10 for TB cases only
Endomyocardial bx
i. Section at 5um thick.
ii. 10 slides with 2 sections each as follows:
1. HE on slide 4, 6, 10
2. MT on slide 7
Prostatic needle bx
i. 1 HE slide with 6 serial sections
Tissue biopsies less than 0.5cm in size not specificed above
i. 1 HE slide with 6 serial sections
Larger tissue biopsies
i. 1 HE slide
Request for variable levels
For small tissue approximately less than 0.5cm in size
i. Repeat 6 serial sections on 1 slide labelled VL 1 for HE
For larger tissue approximately more than 0.5cm in size
i. Repeat 3 serial sections on 3 slides labelled VL1, VL2 and VL3 respectively for HE
Request for special stains
Addition sections will be cut and stained as requested
For additional levels of routine biopsy specimens or irregular number of sections required, serial sections will be cut according to the request of the pathologist.
All personnel receiving verbal or phone orders must read back the entire order to verify accuracy and transcription.

Desmond Heng
TG02
0503179D

Student Internship Programme (SIP) HAEM

Are you surprised that haematology department also performs urinalysis? I hope you do, because I, for one, certainly did. The difference between the urine examinations in various departments lies mainly with the type of tool used for analysis. The principles are similar in that common findings such as RBCs, WBCs, epithelial cells (EC) are counted and reported. Kova glasstic 10 has 10 chambers[able to charge 10 different urine samples] and in each of the chamber, there lies 81 small grids.

Name of Test: Urine microscope examination using Kova glasstic 10 with grid slide

Principle of Test: Undiluted and uncentrifuged urine is drawn into the Kova slide 10 chamber for microscopic examination and identification

Type of Specimen: "Early morning" or random urine, midstream clean catch samples

[Morning-voided sample preferred as it is more concentrated than random samples during the day as urine could be diluted due to increased fluid consumption; giving false picture of a patient's health]

Procedures[FYI]:

1. Check that name on form tally with sample.

2. Assign a lab number and stamp for WBC, RBC, EC grid on request form.

3. Mix urine thoroughly by swirling several times before opening.

4. Record the appearance(colour/turbidity) of sample.

5. Charge 9µl urine on the Kova glasstic slide chamber.

6. By capillary action, 6.6µl of the urine will be drawn into the chamber resulting in a homogenous suspension of the sediment. (you may ask how, but there is a tiny compartment under the chamber where excess urine goes and only the stipulated amount of urine will be held in the chamber)

7. Allow 10 minutes for the cells to settle. (as urine continues to flow within the chamber)

8. Examine for urinary sediments and quantitate casts/crystals at low power 100x

9. Quantitate all cells at high power 400x. Count the cells within the lines of the small 0.33mm square grid.

a) For low cell count sample: count the total cells of each specific type contained in 36 small grids of the counting grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 36 beside the stamp grid.

b) For high cell count sample: count the total cells of each specific type contained in 10 small grids of the counting grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 10 beside the stamp grid.
c) For very high cell count sample: count the total cells of each specific type contained in 1 small grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 1 beside the stamp grid.

10. Enter the reported values and the LIS system will compute the resules and convert the values to per µl.

11. Transfer the computed values to the request form on the appropriate space.

Reference ranges:

WBC: 0-6/ µl

RBC: 0-3/ µl

-Alex Tan Tg02 0503222B

To dorothy

Flow cytometry is a technology that stimulataneously measures and then analyses multiple physical characteristics of single particles, usually cells as they flow in a fluid stream through a beam of light. The properties include the particle's size, granularity and fluorescence intensity. samples are incubated with monoclonal antibodies conjugated with flurochrome which are excited by laser.Physical properties are measured as the flurochrome tagged cells pass through the laser light.

reference range of CD4 : 28.2-50.7%
reference range of CD8: 12.5-38.5


To kangting

here's the ans

reference range if CD4 : 28.2-50.7
reference range of CD8:12.5-38.5

To phuiyuen

This test can be performed after renal transplant to see whether is there any sign of rejection by the recipient. CD4 level should be dropping and CD8 level on the rise.



To zahirah

CD4 T-cells are a type of lymphocyte (which is one type of white blood cell). They initiate the immune system’s response to viruses such as HIV. CD4 cells can be infected and killed by HIV, so the CD4 count generally declines. CD8 T-cells, on the other hand, are the immune system’s killers of abnormal or infected body cells. As HIV infection worsens, the number of CD8 cells present in the body tends to increase.

Flurochorme labeled antibodies are used such as FITC and PE.


To jiahao

Ya. it is also possible for leukaemia.

Take b-cell type Chronic lymphocytic leukaemia as an example, the cell markers are CD5, CD20,CD52,CD23,CD19 and CD37.

To Michelle


In this case, i am talking about CD4 and CD8, these two surface markers. CD4 is for T- helper cells and CD8 for T-supressor cells which both have their respectively specific functions which i mentioned in my post. As for the subgroups, CD4 and CD8 are actually subgroups of mature T lymphocytes and the surface marker for it is CD3. CD4 and CD8 are subsets of CD3.

To Yvonne

well, this test is not a confirmatory test for HIV. other tests are needed to carry out to confirm the diagnosis.

HELLO!

section posted: flow cytometry lab

Name of test: CD 4 and CD 8 count

Purpose of the test : it is used as a diagnostic and prognostic tool for clinical classification of immunodeficiency states such as autoimmune state and immune monitoring of responses to therapy such as to monitor progression of AIDS by measuring CD4 and CD 8 levels.
CD4 is expressed by T-Helper cells (collaborate with B cells promoting their proliferation and promote t cytotoxic cells development.)
CD8 is expressed by T- suppressor cells (suppress immune responses)

Principle of the test: flow cytometry has been commonly used for the measurement of surface marker antigens by fluorescent labeled monoclonal antibody which will pass through laser beam generating scattered and fluorescent signal that can be processed by computers. These surface makers are related to specific functions and subgroups.

Clinical significance: CD4 and CD 8 measurement are used to monitor progression of AIDS as CD4 cells are depleted by HIV infection whereas CD8 cells persist.

well, that's all! have fun for sip..

kai lin
0503211E
tg02

Rountine Laboratory

I was posted to the routine laboratory which deal with all body fluid, these include urine, peritoneal fluid, CSF and semen as well. Today i will be posting about semen analysis which is a pretty interesting topic itself.

Semen analysis is the testing for male infertility problems. This test provide information about the quality and quantity of the sperms. The semen sample is analysed for volume, viscosity, pH and colour of the ejaculate, sperm concentration, motility and morphology. It is also examine for presence of RBC or WB which is often an indicator of infection, inflammation.

The ejaculate should be obtained after at least 3 days of abstinence from sexual intercourse and is best produced by masturbation. The entire ejaculater is collected in a clean sterile container. Condom must NOT be used as it is spermicidal. The sample must be examined within 1-2 hours after collection.

Procedure:
a. Measure the volume received
b. Note the appearance; record and abnormalities such as change in colour, decreased in turbidity or presence of blood.
c. Note the change in viscosity.
d. SQA 11 C- P will provide information on progressive motility and normal morphology
e. Aggluntination- exmaine under microscope for clumping of spermatoza
f. viability:
1) Add a drop of seminal fluid to 1 drop of eosin stain in a 75x 10 mm test tube
2) Mix and stand for 5 minutes
3) Examine under 40 x objective for viable spermatoza. Dead spermatoza will be stained pink
g. pH - Dip a pH indicator strip into the seminal fluid and record the result using the standarh chart
h. Cell count (density)
1) Mix the seminal fluid thoroughtly and dilute fluid to a 1:20 concentration
2) Mix and count in KOVA slide
i. % motility -Mix specimen well. Place a drop into the glass slide and read microscopically. ( Note the quality of motility and progression)

Interpretation
a. Volume: norma volume ranges between 1.5-6.0ml
b. Appearance:
1) Colour- normal semen appear grey to white opalescent fluid. Different colour may indicate problem
2) Viscosity - hyper viscosity may impair sperm motility
3) Liquefaction - Normal time frame is 10-30 minutes.
c. Normal sperm count is > 20 x 10^6sperm/ml
d. Motility - sperm motility study identifies the number if motile sperm seen in an ejaculate specimen.
e. Morphology - Nomal sperm have oval head shapes, an intact central and an uncoiled single tail.
f. Aggluntination - presence of aggluntination can be caused by antisperm antibody.
g. pH - normal pH should range from 7.2-8.0

Ching Wei
0503288C

SIP online sharing

Hihi to all. Sorry for the late posting as I couldnt log in due to the cookies. It is the 6th week of SIP, time really fly. For the past 6 weeks I been went to Processing, Biochemistry and Immunoassy Section. For the biochemistry and immunoassay section, all of the tests is done by analyser. While processing is understandable is to order entry

For this sharing, I will talk more about immunoassay section. I will be sharing some guideline on HIV screening & confirmation tests and BDvacutainer uses.

In the immunoassay section, the analyser use is the Architect ci2000. This machines uses the chemiluminescent microparticle immunoassay technololgy to determine the presence of Ag, Ab and analytes in the sample.

The BDVacutainer System




Cap Colour: Red
Additives: None
Effects on Specimen: Blood clots, and the serum is separated by centrifugation
Uses: Chemistries, Immunology and Serology, Blood Bank (Crossmatch)

Cap Colour: Gold
Additives: Separating gel and clot activator
Effects on Specimen: Serum separator tube (SST) contains a gel at the bottom to separate blood from serum on centrifugation
Uses: Serology, endocrine, immunology, including HIV

Cap Colour: Light green
Additives: Plasma Separating Tube (Na Heparin)
Effects on Specimen: Anticoagulants with lithium heparin; Plasma is separated with PST gel at the bottom of the tube
Uses: Chemistries

Cap Colour: Lavender/Purple
Additives: EDTA (liquid form)
Effects on Specimen: Forms calcium salts to remove calcium to prevent clotting and platelet clumping
Uses: Hematology

Cap Colour: Light blue
Additives: Sodium citrate (Na Citrate)
Effects on Specimen: Forms calcium salts to remove calcium
Uses: Coagulation tests (PT, PTT)

Cap Colour: Dark green
Additives: Sodium heparin or lithium heparin
Effects on Specimen: Inactivates thrombin and thromboplastin
Uses: Ammonia, lactate, HLA typing

Cap Colour: Dark Blue
Additives: Sodium heparin
Effects on Specimen: Forms calcium salts. Tube is designed to contain no contaminating metals
Uses: Toxicology and trace element testing (zinc, copper, lead, mercury) and drug level testing

Cap Colour: Light Gray
Additives: Sodium fluoride and potassium oxalate
Effects on Specimen: Antiglycolytic agent preserves glucose
Uses: For lithium level, use sodium heparin. Glucose test.

Guideline to interpretation of result of HIV Screening and Western blot confirmation tests

HIV Screen: Non-reactive

Interpretation: A non-reactive HIV Ab screening test does not necessary exclude the possibility of infection with HIV. If exposure to HIV is suspected and thus serum is taken less than 3 months after this exposure, this should be retested after that time.

HIV Screen: Reactive

HIV Ab Confirmation: Negative

Interpretation: This result is commonly due to the presence of non-specific Ab, but occasionally may be seen in very early HIV infection. If exposure to HIV is suspected, 10ml blood in EDTA is send in 2-4 weeks time for repeat screening. For female patients in late pregnancy, the repeat sample is sent immediately.

(Rationale: HIV Ag is detected 1 to 2 weeks earlier than HIV Ab, thus making HIV screening test more sensitive than the western blot in the window period)

HIV Screen: Reactive
HIV Ab Confirmation: Indeterminate
Interpretation: This reactivity pattern commonly occurs in uninfected individuals due to the presence of non-specific Ab but may also be observed in early HIV infection. If exposure to HIV is suspected, 10ml blood in EDTA is send in 2-4 weeks time for repeat screening. Otherwise, another serum sample taken 6 weekslater will permit diagnosis in the majority cases.
Note: If a second sample is taken after 6 weeks and the western blot remain unchanged(still indeterminate), the following will be the interpretation applies:
- This indeterminate western blot profile has remain unchanged over 6 weeks. This may indicate a negative HIV Ab status associated with the lack of clinical signs and symptoms and/or supported by a negative history of exposure. If repeated exposure is suspected after the first sample is collected, send another sample for repeating screening.

HIV Screening: Reactive
HIV Ab Confirmation: Inconclusive
Interpretation: This is an indeterminate profile that include Ab to envelope and/or polymerase genes, which one often more sensitive indicators of earlu seroconversion. This profile may however also be due to non-specific Ab. 10ml blood in EDTA is send in 2-4weeks time to resolve the result.

HIV Screening: Reactive
HIV Ab Confirmation: Positive
Interpretation: Patient is consider HIV Ab positive

To confirm whether HIV positive or negative take a long procedure. And HIV Ab confirmation is negative does not mean negative too. As to test for HIV Ab confirmation take a period of time after exposure.

Feel free to ask qn but pls be mercy.

Juexiu Tg02