HEM Automated Testing Flashcards
Principle of electronic impedance in cell counting. What is it based on?
- cells suspended in conductive liquid (Isotonic diluent) that acts as electrical insulators
- flow of current is established between two submerged electrodes
- cells pass through an aperture by gentle vacuum/hydrodynamic focusing* = pulses and resistance to electrical flow can be counted and sized as a particle
*NOTE: Beckman vs Sysmex
“Number of voltage pulses” represents which hematologic parameter ?
Cell count
“Height of voltage pulses” represents which hematologic parameter ?
Cell volume
Define “co-incidence” in hematology
- 2 or more cells pass the aperture at the same time = 1 pulse counted as 1 cell
- cell count is falsely decreased and pulse height is falsely increased
What is “aperture voting ?”
- Values counted in triplicate must match
- If 2/3 match = partial vote out
- If 2 or all 3 values do not match = total vote out
Describe RBC histograms
x- axis: cell size (fL)
y-axis: cell count
- begins at baseline
- one population with a gaussian distribution
How is Hb value determined on Beckman Coulter ?
- lysing agent added to WBC dilution = lyses RBCs + release Hgb
- AFTER WBC COUNT, sample is taken to the SPECTROPHOTEMETER where Hgb is measured at 525nm
How is Flow Cytometry used to study cells ?
Forward light scatter: cell size
Side scatter: cell complexity/ granularity
VCS in Beckman Coulter analyzers
- differentiates nRBCs from WBCs based on
(V)olume: cell size
(C)onductivity: nuclear, granular, and chemical composition of cell interior
Light (S)catter: flow cytometry correlates forward and side scatter
Which WBCs are included in the 6-part differential on the Sysmex ?
- neutrophils
- lymphocytes
- monocytes
- eosinophils
- basophils
- myeloids
How do Sysmex analyzers differentiate WBCs ?
- fluorescent FLOW CYTOMETRY differentiates WBCs
- FLUORESCENCE = RNA/DNA content
- Forward Scatter = cell size
- Side Scatter = granularity/cell complexity
- polymethine dye is used to stain WBCs
- 6-PART DIFFerential includes: neutrophils, lymphocytes, monocytes, eosinophils, basophils, and immature granulocytes /myeloids
Significance of increased retic count
Anemia due to red blood cells being destroyed earlier than normal (hemolytic anemia)
How do Beckman Coulter determine retic count ? What parameters are used and how are they measured ?
- sample in stain chamber = NEW METHYLENE BLUE precipitates DNA in immature RBCs (increases granularity)
- VCS technology differentiates other cells from retics (decreased size and granularity)
How do Sysmex analyzers determine retic count ? What parameters are used and how are they measured ?
- RET chamber = lysis reagent perforates RBCs, WBC and PLTs
- FLUORESCENT markers label nucleic acids in cell
- Flow cytometry (SS and FS) differentiates RBC from retics (increased size and decreased granularity)
What stain is used for manual retic counts ? What does this stain do to retics ?
New methylene blue; causes clumping and staining of residual nucleic acid present in immature cells.
MCV (fL)
HCT/RBC x 1000
WIP When would a manual retic count be indicated instead of an automated count ?
MCHC (g/L)
HGB/ HCT
MCH
HGB/ RBC
WBC estimate when 35 WBC /3 lpf and WBC field factor = 4
2.9 x10^9/L
Formula: WBC estimate (x10^9/L)
total WBC / 10 lpf / 4
NOTE: take average of (10) fields at low power, divide by 4 (field factor)
PLT estimate when 260 PLT /10 hpf and PLT field factor = 14
364 x10^9/L
NOTE: take average of (10) fields at hpf, multiply by 14 (field factor)