pulse height analyzer
oscilloscope
- sorts and categorizes the pulses and plots them on a frequency distribution graph or volume distribution graph
RBC histogram
36-360 fL
curve should begin at baseline
smaller population normally to the right of the main population = cells that have been corrected for coincidence
PLT histogram
2-20 fL
curve of best fit overlaid on raw data
curves start and stop at baseline
WBC histogram
> 35 fL
should start or be really close to baseline
three distinct populations usually (lymphs, monos, grans)
the principle by which hematology instrumentation provides white blood cells differentials and nRBC differentiation
flow cytometry
sample types for flow cytometry
- routine = EDTA whole blood
- special heme = EFTA whole blood, LiHeparin whole blood
clinical applications of flow in special heme
- T cell enumeration: monitoring progression of diseases such as HIV, and other immunodefs
- Lymphoma/Leukemia investigations: determining origin and ID of immature/abnormal cells
- immunophenotyping for PNH and hereditary spherocytosis
what does the diluent do in beckman/sysmex?
- dilutes & stabilizes whole blood sample
- conduct aperture current
- focus sample stream
- rinse instrument between runs
hemoglobinometer in beckman
- lysing contains cyanide pigment that binds and stabilizes the free hemoglobin
- once WBC count is complete, solution passed onto spec/hemoglobinometer
- stable cyanide-hemoglobin pigment measured at 525 nm
- %transmittance is compared to reagent blank
- Hb concentration calculated using Beer’s Law and reported g/L
Beckman VCS technology
- volume analysis = low frequency
- conductivity analysis = high frequency current differentiate between insulating cell properties (nuclear, granular, chemical composition of cell interior)
- light scatter analysis = cell complexity and granularity
5 part diff reported by Beckman
neuts monos lymphs basos and eos
T or F. Beckman instruments are abl to report nRBCs based off VCS technology
T
how does the beckman instrument differentiate nRBCs from WBCs?
VCS technology
nRBCs are often mistaken as these
small lymphs (by Beckman)
supravital stain for retics
new methylene blue
what does new methylene blue do to retics?
precipitates nucleic acids inside immature RBC (increasing granularity)
T or F. More immature RBC = more residual RNA and greater volume
T! larger and higher light scatter
(appear more to the right of a dot plot)
derived parameters in Beckman
- MCV: average vol of all RBCs; fL
- RDW: coefficient of variation of RBC Vol distribution (spread of gaussian distribution form histogram); %
calculated parameters in Beckman
- Hct: relative vol of packed RBCs; Ht is roughly 3x the Hb value if RBCs are normochromic; L/L
- MCHC: ave weight of Hb in a sample
- MCH: weight of Hb in average RBC
which instrumentation uses hydrodynamic focusing?
Sysmex
- cells sheathed by diluent and passed through the aperture one at a time to prevent coincidence and recirculation of red cells
how are cells distinguished based on size in Sysmex?
floating thresholds for the cell population (determined by the instrument)
- this allows for discrimination of populations on a sample-by-sample basis
- also helps separate PLTs from small red cells
when is a fluorescent PLT done?
when there are interferences in the impedance PLT count
- fluorescent markers label PLT RNA (after perforation)
- PLTs separated using FSC and fluorescence
- this method can differentiate immature PLT and mature PLT based on RNA
flow chamber in sysmex
- enumerate WBCs, nRBCs and the 6-part diff
- sample and diluent taken into WNR channel where RBCs (including nRBCs) are lysed
- fluorescent polymethine dye added to stain nucleus and organelles of WBC w/ high intensity and stains the extruded nRBC nucleus with low intensity
T or F. Unlike Beckman, WBCs are counted as they are differentiated in the flow cell in the Sysmex
T! it’s also corrected for nRBC
