Hematology Automation Flashcards
How is Hgb and MCH measured?
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–Coulter: Hgb from cyanmethemoglobin method (at 525, 540, or 546nm) OR
–Hgb by sodium laurel sulfate method
–MCH is calculated
Platelet number count methods?
Platelet numbers: Impedance (Coulter, CELL-DYN)
Hydrodynamic focusing and DC detection (Sysmex)
Hydrodynamic focusing, laser low and high angle scatter (ADVIA)
***All of these methods have a size limit which ranges from 30fl-70fl, known as the threshold.
This picture is a depiction of ________? Describe the priciple.
Laminar flow is used to focus the stream of cells so that only one cell passes through the aperture at a time Two fluids are used, one that passes through the other.
PROCESSING: Samples are divided and sent to different chambers for cell counting One part has the RBCs lysed – this is used for WBC counts and differentiation One part has RBCs intact – this is used for RBC and platelet counts
Abnormal Flags: Coulter parameters
Lymphocyte population does not begin at baseline
No space between lymphs and monos
No space between monos and polys (neutrophils)
Granulocytes do not return to baseline
Interference detected
RBC cell number is measured directly through which methods?
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–Coulter and CELL DYN: Impedance
–Sysmex: Hydrodynamic focusing and DC
-Hydrodynamic focusing and laser scatter
Granulocytes, Lymphocytes, Monocytes Machines and methods
Granulocytes, Lymphocytes, Monocytes:
Coulter VCS: hydrodynamic focusing forces single cells through aperture, calculating cell volume using electrical impedance, conductivity using radio frequency and light scatter, using a helium-neon laser; separates eosinophils first
Sysmex, using Direct Current/Radio Frequency
Abbott CELL-DYN, using multiple angle polarized scatter separation (MAPSS)
ADVIA, using peroxidase staining, optical scatter and absorption
Mechanical problems/Problems detecting cell size can be caused by what?
Clots,
clumped or fragmented RBCs
Nucleated RBCs
Platelet clumping, giant platelets
Describe the SLS-hemoglobin method.
•Sodium lauryl sulfate is used to measure Hgb instead of a cyanide based reagent.
–Ferrous iron is converted to ferric iron
–SLS-methemoglobin is measured via absorbance.
Describe how cells counts are done using electronic impedance.
Changes in the electrical resistance in a fluid as a cell passes through a small aperture. The change in voltage is directly proportional to the size of the cell
What are two issues with electronic impedance?
Protein buildup around the aperture can cause increased interference when the cell is pulled through the aperture.
“Coincident passage loss” occurs when more than one cell goes through an aperture causing an abnormally large spike – this is accounted for by a calculation built into instrument software
RBC Indices Calculations:
•Hematocrit / MCV (mean corpuscular volume)
–MCV is measured in fL by taking the mean of the histogram and hematocrit is calculated as %:
HCT = RBC X MCV / 10
–HCT is measured by cumulative pulse height and MCV is calculated as fL:
MCV = (Hct/RBC) x 10
MCH (mean corpuscular hemoglobin) is calculated in pg: Hgb / RBC X 10
MCHC is calculated as %
–Hgb/Hct X 100
•RDW: red cell distribution width
Calculated using the CV
SD X 100/m
What are the platelet indices?
Sizing
PDW
Describe the Cyanmethemoglobin Method.
To measure Hgb: diluting fluid contains cyanide which combines with the iron in hemoglobin and can be measured at or around 540nm.
What advantage is gained with laminar flow vs impedance?
•Avoids many of the potential problems with impedance alone
–Sheath fluid prevents protein build up on aperture
–Eliminates recirculation of cells
–Reduces pulse height irregularity since all cells pass through the center of the aperture
–Better resolution is obtained
Laminar flow is also known as?
(hydrodynamic focusing)
What are the RBC indices?
RBC indices:
Hgb
Hct
MCV
MCH
MCHC
RDW
Reticulocytes
Sysmex uses channels for differentials. Explain.
Sysmex:
In some cases, analyzers use different parts of the sample for analysis in different channels of the instrument.
This instrument has 5 channels for the differential:
—The differential channel which measures lymphs, monos, and neutrophils
—Immature cell channel – a selective reagent is used
—WBC counting channel
—Separate channels for basos and eos
What can be assessed by light scatter?
Size and granularity can be assessed by looking at the forward and side scatter, respectively.
(forward = size,
side = granules)
Eosinophils and Basophils Machines and methods:
Coulter VCS: volume, conductivity, scatter
Sysmex: Differential lysis, shrinkage and DC detection
MAPSS by CELL-DYN: Multiangle polarized scatter separation
ADVIA: Peroxidase staining and optical scatter/absorption (eos) and differential lysis, laser low angle and high angle scatter
The different methods of measuring reticulocytes are?
–Supravital stain (new methylene blue), volume conductivity, optical scanning (Coulter)
–Supravital stain (Auramine 0) and fluorescence (Sysmex)
–Supravital staining (new methylene blue) multiangle optical scanning (Abbott CELL-DYN)
–Supravital stain (Oxazine 750) with low and high angle optical scatter and absorbance (ADVIA)
Direct current is essentially the same as?
impedance
List the blood cells
RBCs
WBCs :
—Neutrophils
—Lymphocytes
—Monocytes Eosinophils
—Basophils
Platelets
How is PDW calculated?
Calculated from a plot of relative number over volume in fl.
What are some troubleshooting issue with Hem. automation?
Sample problems
-Mechanical interference
——RBC clumping / rouleaux / cold agglutinins
——-PLT clumping
—–Clots
-Sample defects
——Platelet satellites, changes in platelet size
—–RBC fragments
——Lipemia
RF (radio frequency) measures complexity (WBCs). How does iit work?
Alternating current in RF range penetrates cell, measures nucleus and granules. This allows measurement of N:C ratio (nucleus : cytoplasm)
What are some problems and interference/troubleshooting that should be considered when using Hemogliobin measuring methods?
–Lipemia can interfere with Hgb measurement
–Icterus (bilirubinemia) can falsely elevate Hgb
–RBCs resistant to lytic reagents, can falsely lower Hgb
***************Can choose not to report Hgb or, in the case of lipemic or icteric specimens, replace the plasma in a sample with saline and retest
WBC Differential determins what?
Type of WBC is determined by measuring cell size vs granularity/or complexity
How are histograms used in measuring/plotting cell count? What does it compare?
As each type of cell is counted, the distribution of sizes is charted in a histogram that compares the number of cells vs. the size
What basic principles/methods are used for cell counting and sorting?
Basic principles:
Electronic impedance
DC/RF
Laminar Flow (hydrodynamic focusing)
Light Scattering
Fluorescent flow cytometry
What methods are used for counting and sorting WBCs?
WBC counted and sorted by combinations of the following methods:
Impedance
Hydrodynamic focusing,
DC detection
Optical scatter and impedance
Hydrodynamic focusing, optical scatter and absorption
******This is determined after RBC lysis
Explain solution to sample defects: platelet satellites, changes in platelet size, RBC fragments, lipemia, icterus, and chylomicrons
Platelet satellites, changes in platelet size-mult by 1.1
———Collect in citrate, use reg tube for other counts etc
RBC fragments
——Requires smear review
Lipemia, icterus, chylomicrons
—-All spectrophotometric readings off
—-Rule of 3 is broken
—–Plasma replaced with saline
