Troubleshoot
When a slide fails the quality check (QC), the laboratory scientist must diagnose the root cause and implement a corrective action immediately. The problem typically lies in one of three areas: the Reagents (chemistry), the Equipment (environment), or the Specimen (biology). The “Troubleshooting Matrix” guides this decision process
Reagent Troubleshooting
Chemistry issues usually manifest as poor staining, refractility, or background noise
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Problem: Refractile (Shiny) Chromosomes
- Cause: Water in the Fixative. Methanol is hygroscopic (absorbs water from air). “Old” fixative allows water to coat the chromosomes, acting as a barrier to the Giemsa stain
- Correction: Prepare Fresh Fixative: (3:1 Methanol:Acetic Acid) immediately. Centrifuge the pellet and wash it 3 times with the new fixative until the pellet is pure white. Re-drop the slide
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Problem: “Greasy” Background / Debris
- Cause: Residual lipids or Red Blood Cell (RBC) membranes (hemoglobin). This creates a dirty film on the slide that obscures the chromosomes
- Correction: More Washes. The pellet needs 2–3 more rounds of Fix/Spin/Aspirate. If the pellet is yellow/brown, it is not clean. It must be white
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Problem: “Grey” Chromosomes (Low Contrast)
- Cause: Slow Drying. If the fixative evaporates too slowly, the chromosomes flatten too much and lose their sharp edges
- Correction: Increase the evaporation rate. Use a warmer slide surface or gently blow on the slide during drying
Equipment/Environmental Troubleshooting
Environmental issues affect the physical spread (tight vs. scattered)
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Problem: Tight / Enclosed Metaphases
- Cause: Low Humidity: (<30%) or Cold Drafts. The fixative dried before the cells could explode
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Correction:
- Artificial Humidity: Drop the cells onto a slide, then immediately hold it over a \(50^\circ\text{C}\) water bath (vapor) for 2–3 seconds (“huffing”)
- Wet Slide Technique: Dip the slide in ice-cold water. Shake off excess. Drop the cells onto the thin film of water. This delays evaporation
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Problem: Scattered / Broken Metaphases
- Cause: High Humidity: (>60%). The cells absorbed too much water and burst violently
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Correction:
- Heat: Place the slide on a \(60^\circ\text{C}\) hot plate immediately after dropping
- Dry Slide Technique: Ensure the slide is bone dry and warm before dropping. Do not use water vapor
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Problem: Uneven Spreading (Rings)
- Cause: Dirty Glass. Oil or dust on the slide prevents the drop from spreading circularly
- Correction: Use pre-cleaned slides. Store slides in alcohol before use. Wipe slides with a lint-free tissue (Kimwipe) before dropping
Suboptimal Specimen Troubleshooting
Some specimens are biologically difficult (low cellularity, necrotic). We cannot change the biology, so we adapt the technique
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Problem: Low Cell Count (Sparse)
- Cause: Poor culture growth (e.g., hypocellular marrow). Finding cells is tedious
- Correction: Concentrate the Pellet. Remove all supernatant until only the tiny pellet remains. Resuspend in a single drop of fixative. Drop this entire concentrated drop onto a small area of the slide (\(10\times10\text{ mm}\)) rather than the whole slide. This creates a “dense spot” for the analyst to focus on
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Problem: Clotted / “Sticky” Pellet
- Cause: Dead cells (DNA release) or fibrin clumps make the pellet gooey. It won’t pipette
- Correction: The “flick” technique.: Vortex the tube vigorously. If large clumps remain, allow them to settle for 5 seconds, then pipette the supernatant (which contains the single cells) from the top of the fluid, avoiding the heavy junk at the bottom
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Problem: Heavy RBC Contamination (Bloody Pellet)
- Cause: Hypotonic shock failed to lyse all RBCs
- Correction: Carnoy’s Wash.: The acetic acid in the fixative is also a lysing agent. Perform multiple washes with fresh fixative. The acid will eventually dissolve the RBC membranes, clearing the pellet