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 infectionPink 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
9 comments:
Hello!
May i know how does the New Methylene Blue in the staining reagent "stablilize" the RBCs??
Kangting
0503331A
TG02
hihi kang ting, sorry delay as I was asking my colleauge regarding this qn.
As I was mention the lytic action of the reagent(WBC lyse: it is use to dilute the stained sample when the sample is aspirated in CELL-DYN Ruby). This lytic action will be prevented by the electrolytes contain in the staining solution, which is the retic solution(containing New methylene blue, potassium oxalate, n-dodecyl and beta-D-maltoside). And due to New methylene blue will stain the RBC thus stabilize RBCs.
Hopefully this will clear your doubt.
Juexiu
tg02
Hi Juexiu
Can i ask how many days can the blood be kept in the fridge in order to ensure its accuracy?
and for my lab, 1% brilliant cresyl blue stain, which does not contain fixatives like methanol and will ensure viability of cells, is used. does the Methylene Blue that u used similar to my lab?
Chaur Lee
Hello!
Why is that secondary infection results are either be IgM & IgG positive and IgG positive only? Does that mean that some patients will not produce IgM against dengue during the secondary infection? Thanks!
Charmaine Tan
TG01
Hi juexiu,
I understand that there is 4 serotypes for dengue, if the patient is infected with one serotype before and get infect by another serotype would it consider primary or secondary infection?
thank you!!
Avery
TG02
hey,
do u know how will dengue affect the results of FBC? except the platelet count.
why do blast normally occur?
elaine
hi juexiu
my lab also use the kit but i think they way we determine and report is quite different. may i ask there are some situation whereby the IgG and IgM is weakly positive, do your lab counter check with other parameter? what parameters? or still report as Positive?
Lizzie
hi there
what of curiousity... why do we need to carry out retic count?
cass
hihi,
Chaur Lee:
For all sample that is send to lab will kept for 2 weeks b4 is discart. You ask me regarding accuracy of the result relate to number of days the blood is kept, in regard of this question, it depend what test is running. Therefore, I am wondering which test u mentioning?
For retic count:
Specimen can run up to 8 hours without refrigerating it. While in another situation, specimen older than 8 hours and up to 72 hours after collection can be processed if the specimen is stored in the refrigerator during this time.
For dengue:
The blood specimen, the serum should be separate asap and stored in freezer(less than or equal 20 degrees celscius) or refrigerator(2-8 degrees celscius)if not have to tested within 2 days.
Actually for dengue accuracy is depend on the time sample is collect following the onset of fever. For accuracy result, blood specimen should be collecte between 6 to 14days following the onset of fever.
Another question you ask is regarding whether the 1% brilliant cresyl blue stain, which does not contain fixatives like methanol and will ensure viability of cells, that your lab used similar to Methylene Blue. The retic reagent that my lab does not contain methanol as well. Both served the same purpose which is to stained the retic for actual count.
Hope this will clarify ur qn
Charmaine Tan:
For secondary infection result is to be either IgM & IgG +ve or IgG +ve. As we have learn IgM is the first Ig to produce for infection. When there is a secondary infection IgG is produce, thus for a secondary infection, IgG must be +ve. However, that does not mean
patients with secondary infection will not produce IgM against dengue. It will still produce IgM with high IgG. Is just that in sceondary infection, there is much higher IgG than IgM.
Avery:
If the patient infect by another serotype, it consider as primary infection. As Ig is against the first serotype not another serotype.
Cass:
the retic count is a blood test performed to assess the body's production of immature red blood cells (reticulocytes). THis test is usually performed when patients are evaluated for anemia and response to its treatment.
This test show the rate of bone marrow is producing red cells. A normal count means that the production is adequate; a decreased count means it is not. It determine whether a lack of red cells in an anemic person is caused by a bone marrow problem, by excessive bleeding, or by red cell destruction.
It is also used to monitor the response of bone marrow response to treatment for anemia. The retic count rises within days if the treatment is successful. It is also used following bone marrow transplant to evaluate the new marrow's cell production.
Lizzie:
If the IgM and IgG is weakly +ve in patient that is not diagnose with dengue. A retest will be perform. And will also look into the FBC result, for parameter that will be affect if the patient has dengue.
If IgM and IgG is weakly +ve in dengue patient, it will be alright as since the patient is diagnose with dengue. Weakly +ve IgM and IgG is due to the patient is recovering. And will check with the FBC result whether WBC and platelet had been rising or not.
Elaine:
Patient with dengue will affect the FBC result. It affect the WBC and lower the platelet count and Hct will rise.
Hope this will clarify all doubt.
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