In WBC count with Turk's diluent, AAG only destroying RBC. Why it doesn't affecting WBC?
The diluent doesn't have any compound that perform isotonic environtment between diluent and the cells. why doesn't WBC swell and damaged?
Why in RBC count we need antimicrobial compound but it doesn't needed in WBC count?
Can we use other stain in WBC count beside gentian violet?
Thanks for response, sorry for stratified questions :D
Turk’s fluid Composition: • Glacial acetic acid= 1.5ml→lyses RBC • 1% gention violet= 1ml→ stain nuclei of WBC • Distilled water= upto 100ml→ solvent • Thymol= 1 pinch→prevent growth of fungus The diluting fluid is not isotonic and the very dilute acid solution lyses RBC but not WBC.
Thanks Valbona, so you mean that WBC structure doesn't affected by slightly acidic and anisotonic environtment? Why? Is that because of present of the nuclei?
Dear Anggadia,
Is that because Turk's fluid composition does not affect WBC membrane.
thank Valbona, but i still curious what part in WBC membrane that absent in RBC membrane so they have different durability to glacial acetic acid
AAG modifies the internal pH of RBC. Due to the reduced pH, the Hemoglobin structure changes and more hydrophilic groups are exposed. In conclusion, the AAG lysis of RBC is a lysis caused by similar mechanism than the isotonic environmental changes . For these reasons WBC are not involved. Gentian violet (also called crystal violet) is the better stain because it has in addition an antifungal action.
wow, so that's why....
so much thanks, Antonio, that's what i want to know
:D
Hi, can i use turk's solution instead of RBC lysis buffer during isolation of DNA from blood?
Although acetic acid is a weak acid, it is able to causes a denaturation of DNA. On WBC as well as on PLT, a fixative action allows the cellular structure to be maintained
What mean "visualization"? If your purpose is the microscopic visualization of the platelets/WBC while fresh only, you can use the Solution Turk, I think. If you need to observe these after staining, I don't know: It depends on the type of staining. Anyway you can reduce the RBC presence and increase the WBC/PLT number applying the following simple procedure:
- place a decapped whole blood tube inclined about 45 degrees and wait for 30-45 minutes until sedimentation of RBC is advanced but not conclused
- Using a plastic pipette, recovers plasma above RBC. This plasma will be enriched in PLT and WBC. If you worked well, the RBC presence could be negligible or absent
In this way you will be able to observe WBC and PLT in a "native" situation without chemical interferences.
I'm curious: please informs me later.
Two suggestions for you. First: freezes and thaws 2-3 times your whole blood sample. Then wash 2 times with distilled water ans re-suspend in saline or plasma (better). By this method the RBC lysis could be incomplete and ghosts or intact RBCs might be observed. Second: you can try by using the same reagents which are used in our haematological analyzers for WBC count. In the majority of these, different mixtures of hypotonic solutions and detergent are used. By this way, the RBCs lysis will be complete but the WBC and PLT morphology could be involved. So, the only way is to try and verify because the final result depend on the type of analyzer. You must also consider that the reagents from main counting channell must be used only because in the BASO channell of all analyzers even the WBC are lysed.
What is the effect of RBC diluting fluids on leucocytes?
[It is clear that WBC diluting fluids cause hemolysis of erythrocytes so that counting only WBCs is possible using hemocytometers.]
In the haematological analyzers RBC are counted in two different ways: (i) impedance method in which the hydrodynamic focusing avoids or minimizes the possibility of coincidence of two or more cells; (ii) optical method in which cell counting is performed by flow cytometry. Different autoanalyzers use impedence or optical methods and, in some types, both methods alternatively. In both impedance and optical channels, RBC are diluted with an isotonic solution. In addition in the impedance method the diluting reagent is an electrolitic solution that allows the transmission of the electric impulse. So, WBC are not lysed but in spite of this, the leukocytes can't influence the RCB count because a high dilution (>500 time) is used in the RBC counter channel.
Thank you so much for your response! For the automation, yes you perfectly described; however, my question is what would happen to WBCs when using the RBC diluting fluid for the manual dilution and counting?
The manual procedures for RBC count are the same (or very similar) with respect to the automatic procedures. Infact, for those, an isotonic diluent is used that not affects the WBC morphology. At the same time a normal amount of leukocytes not interferes with RBC count due to the used dilution (1:101)
Dear red blood cells are denatured because of the glacial acetic acid.