Lab 2 Flashcards
(23 cards)
What is the purpose of estimating leukocyte functionality?
To determine if isolated leukocytes are alive and functional.
What was the historical test used to measure leukocyte activity?
The leukocyte alkaline phosphatase (LAP) test.
What does alkaline phosphatase (ALP) do?
ALP removes phosphate groups from nucleotides, proteins, and alkaloids.
Why is dephosphorylation important in the immune system?
It could be an important signaling step in the development of cellular responses.
What were the LAP test results in malignant neutrophils?
ALP levels are either low or absent.
What conditions showed increased LAP scores?
Polycythemia vera, thrombocythemia, acute lymphatic leukemia, and pernicious anemia during reticulocytic crisis.
Why was the LAP test deemed too subjective?
It required the technician to find 100 neutrophils and was scored based on stain intensity and granules.
What has replaced the LAP test for diagnosing CML?
More accurate and advanced genomic tools.
What is the staining principle of the LAP test?
ALP converts naphtol AS-BI phosphate into phosphate and an aryl naphtholamide, which forms an insoluble dye.
What color stain is visualized in neutrophilic leukocytes?
Purple stain.
What is the purpose of the fast blue working dye solution?
It is used in the staining procedures for preparing slides.
What is the first step in the staining procedure?
Prepare three smears: two for positive staining and one as a negative control.
How should the smear be prepared?
Put 5 µl of pig bone marrow cells on the glass slide and make a smear. Then, dry the smear for 10 minutes.
What is the purpose of the citrate-acetone-formaldehyde fixative?
To fix the cells for 30 seconds.
What is the next step after draining off the fixative solution?
Wash the slides for 3 minutes with distilled water.
What should be done for the negative control after washing?
Place the negative control slides into the jar of acid-stopping solution inside the 37°C water bath and incubate for 10 minutes.
What is the purpose of the reaction buffer?
To equilibrate the smear for 2 minutes.
What is the composition of the dye-mixture solution?
Each tube contains Fast blue working dye solution, enhance solution, and ion solution.
What should be done after incubating the dye-mixture solution?
Add 50 µl of substrate solution and filter the mixture using a syringe filter.
What is the next step after draining off the equilibration buffer?
Put the slides into the staining chamber.
How long should the smear be covered with the substrate-dye-mixture solution?
Incubate for 30 minutes at room temperature in the dark.
What should be done after incubating the slides?
Wash the slides briefly with three changes of acid stopping solution.
What is the final step in the staining procedure?
Rinse the slides briefly with distilled water and put them into a normal petri dish.
