AUTOMATION IN HEMATOLOGY Flashcards
Fresh whole blood anticoagulated with EDTA (ethylene diaminetetracetic acid) [purple/lavender top tube]
The tube should be ___ full
The tube should be mixed/inverted _ to _ times to adequately mix the blood with anticoagulant and hence inhibit clotting.
Storage: Refrigerated at __ until needed and allowed to reach room temperature before testing.
6 to 8
1/3 full
4°C
Plasma should be ____ and fairly ___
If the plasma is ___, ___ ___ (hemolysed), this will interfere with accurate testing and must be redrawn or dealt with accordingly in testing.
Some hematology tests require ___ blood () or __ ()
pale yellow. clear
dark yellow, milky or red
heparinized (green top) serum (red top)
-
Preserves cell function
Preserves cellular morphology
For accurate counts, the test should be done within __, because in time, cells begin to change.
If the patient’s cells are altered by EDTA, ____ can be used if the blood is tested within a few hours.
2 hours sodium citrate (blue top)
Mission of the Hematology Laboratory
Provide ___
test results that reflect the condition within the body (in vivo), and not the changes that took place once the blood was removed from the patient and placed in a glass tube (in vitro)
Mission of the Hematology Laboratory
Correlate ____
results from all test information (puzzle pieces) available to provide the primary health provider with a clear and accurate picture of the patient’s state of health.
is a routine test
CBC
The automated CBC report includes:
- R
- T
- A S
- A P
- P
- H
- H
- R
- R
- M
Red blood cell count with a histogram and morphologic indices Total WBC count with a histogram A scattergram for differentiating leukocytes A platelet count with a histogram Platelet morphologic index Hemoglobin level Hematocrit RBC indices Reference ranges Markers/Flags for out of range values
Each laboratory must establish its own reference ranges due to variation in instrumentation, procedures, and local populations which may render the generic normal values less accurate for the specific laboratory involved in the testing
Each laboratory must establish its own reference ranges due to variation in instrumentation, procedures, and local populations which may render the generic normal values less accurate for the specific laboratory involved in the testing
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
-
-
Cell-Dyn 3000 (Abbott)
Sysmex NE 8000 Cell Counter (Baxter)
COBAS Cell Counter (Roche)
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
Dilution of the __ whole blood specimen in an electrolyte solution such as __. The __ suspended in this fluid are POOR conductors of electricity, whereas the __ is a GOOD conductor of electricity. The dilution must allow an adequate number of cells to count without a concentration so high that more than once cells goes through the aperture at once (_____). If more than one type of cell (___) is present in the sample, it may be necessary to __ or __ them to get the accurate count of the other.
Electrodes are located on either side of the counting orifice or path. An electrolyte solution is placed between the electrodes and a flow of electrical current is established.
EDTA saline cells solution coincidence error particle lyse or remove
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
A specific amount of solution is pulled through the aperture. Any particle (cell) that passes through the aperture will momentarily____the resistance of (or interrupt) the electrical flow between the electrodes generating a __, which can be counted, measured, and displayed on a screen.
The size of the resistance is proportional to the size of the cell. From this information the counter computer can provide an accurate count and size of the particle.
A background (quality assurance) check is obtained by first counting any miscellaneous particles within the vial of saline that may be erroneously counted as cells. This background should be VERY LOW.
Controls or standards must also be counted to ensure accuracy of the data generated. This should be a specimen of known value.
All specimens should be run at least in duplicate and most instruments do three to five determinations on each specimen to ensure the precision of the data generated
INCREASE
PULSE
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
_______ is proportional to the size of the cell. From this information the counter computer can provide an accurate count and size of the particle.
The size of the resistance
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
A ______ is obtained by first counting any miscellaneous particles within the vial of saline that may be erroneously counted as cells. This background should be ___.
background (quality assurance) check
VERY LOW
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
___________ to ensure accuracy of the data generated. This should be a specimen of known value.
Controls or standards must also be counted
PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE
Basic Procedure:
___________ and most instruments do ___ to ___ determinations on each specimen to ensure the precision of the data generated
All specimens should be run at least in duplicate
three to five
Is the methodology used by some Coulter instruments to characterize cells for the scattergrams.
PRINCIPLES INVOLVED IN AUTOMATION
LASER SCATTERING
___ is utilized for cell counts as well as cell characterization by FLOW CYTOMETERS.
Laser light scatter
-
-
Technicon H-3
Cell Dyn 3000 (Abbot)
Serono Diagnostics System 9000
PRINCIPLES INVOLVED IN AUTOMATION LASER SCATTERING
The sample is diluted in the instrument into a stream of fluid containing the cells to be counted in a single cell flow
(HYDRODYNAMIC FOCUSING)
PRINCIPLES INVOLVED IN AUTOMATIONLASER SCATTERING
The cells pass through a ___ on which a light is __. As the cell passes through the light path, it scatters the light in all directions.
A ______ senses and collects this light “scatter information” and transforms it into digital information, which provides “characteristic information about the specific cell as it passes through the flow cell.”
Traditional flow cytometers uses _____ and _____ to differentiate__ ___ ___
flow cell
focused
photodetector
FORWARD LIGHT SCATTER (0 degrees) and ORTHOGONAL LIGHT SCATTER (90 degrees)
lymphocytes, monocytes, and granulocytes.
PRINCIPLES INVOLVED IN AUTOMATIONLASER SCATTERING
The ____ uses two additional dimensions of light scattering to accurately separate the cell characteristics.
This is called _______ and involves the use of __________ (10 degrees) to resolve ___ and ___ (90 degrees) to resolve ____.
This eliminates the need for cytochemical staining or monoclonal tagging to identify these cells.
CELL-DYN 3000
MULTIANGLE POLARIZED SCATTER SEPARATION (MAPSS)
narrow-angle light scatter (10 degrees)
BASOPHILS
depolarized light scatter
EOSINOPHILS
Is used along with light scatter.
PRINCIPLES INVOLVED IN AUTOMATION LIGHT ABSORBANCE
Instruments using LIGHT ABSORBANCE:
Sysmex 8000
COBAS
Technicon H-3
PRINCIPLES INVOLVED IN AUTOMATIONLIGHT ABSORBANCE
______ which requires a _____ to count and classify leukocytes.
Specifically stains the cells of interest
darkfield optical system
Procedure is similar to light scatter, but the detection system is different.
LIGHT ABSORBANCE
Light is scattered through the opening around a darkfield disk as cells passes through the sensing zone, one at a time. The light scatter is measured by a photodetector and is related to the cell number.
If the cells is stained, some of the light will be ______________. The use of various specific cell stains, a differential count can be done.
LIGHT ABSORBANCE
absorbed proportional to the amount of staining
Used to identify an alteration in cell number, size, and some internal characteristics.
provide the number of cells in one axis and the cell size on another axis.
Leukocytes of histograms and scattergrams
provide the number of cells in one axis and the cell size on another axis.
HISTOGRAMS
plot various degrees of light scatter on the vertical and horizontal axes to classify cells based on the nuclear and cytoplasmic characteristics of light scatter.
SCATTERGRAMS
The ___ are obtained by plotting the cell size, nuclear structure and surface, and internal characteristics within a three dimensional matrix.
scatterplots
The ___ is NOT A FACTOR in the scatterplot information BUT RATHER ___ and ANY ALTERATION IN SPECIFIC CELL CHARACTERISTICS.
CELL DISTRIBUTION
CELL IDENTIFICATION
plot various degrees of light scatter on the vertical and horizontal axes to classify cells based on the nuclear and cytoplasmic chaacteristics of light scatter.
SCATTERGRAMS
LEUKOCYTESOF HISTOGRAMS AND SCATTERGRAMS
__________ if the automated count reports any unusual cells or an abnormal cell count.
The____ and ______ are important to correlate with the automated histogram or scattergram in order to note morphologic alterations that may indicate a specific disorder
A manual evaluation of the blood smear is indicated
Differential count and peripheral blood smear evaluation
Correlation factors
The WBC Count should correlate with a WBC estimate from looking at the peripheral blood smear made on the specimen.
If a specific cell characteristic is not seen, such as with blast cells, _____ must be used to differentiate a myeloblast from a lymphoblast
cytochemical stains
Correlation factors
If the WBC evaluation indicates increased ____, characteristics such as ___ _____ and _____ would help associate this elevation with a ____ or ______.
neutrophils
toxic granulations, Dohle bodies, and vacuoles
Correlation factors
If the WBC count demonstrates increased ____, the presence of REACTIVE LYMPHOCYTES would support a _____.
Its absence may otherwise indicate a ____.
lymphocytes
viral infection
malignancy
Sources of Error
Pre-analytical factors: - - - -
Presence of clots
Wrong testing temperature
Tube inappropriately filled
Hemolysis, lipemia,and cold agglutinins
-
Increased incidence of two cells passing through the aperture because of a high count.
Presence of other “interfering cells” such as pyknotic cells, nucleated RBCs, basket and smudge cells, and micromegakaryocytes
Wrong encoding
The specimen for hemoglobin is divided into three samples and diluted with a ___ reagent which lyses the red blood cells and forms a “color compound in proportion to the amount of hemoglobin present.”
cyanmethemoglobin
This specimen in triplicate, is measured by a ___ method to determine the amount of color change that provides the hemoglobin value.
spectrophotometric
The erythrocytes being counted are measured for their size and this information is _____ and ______.
The number of cells and the size of the cells are used for the computation of hematocrit.
The manual hematocrit may be ___higher than the automated hematocrit due to the plasma trapped between the cells tested.
recorded for calculation of the hematocrit and generation of the histogram.
2%
Indices are values calculated from the erythrocyte count, the hemoglobin, and the hematocrit.
RBC Indices
Provides the average size of red blood cells
If there is an equal proportion of large and small erythrocytes, the average would be ___.
The ______ is the standard deviation of the red cell size divided by the average red cell size (MCV), hence is an accurate measure of _____.
MEAN CORPUSCULAR VOLUME
normal
random distribution width (RDW)
anisocytosis
Provides the average hemoglobin per cell.
This does not however take into account an abnormal cell size.
MEAN CORPUSCULAR HEMOGLOBIN
Factors in the hematocrit in order to account for cell size and provide a more accurate value of hemoglobin level of each cell.
MEAN CORPUSCULAR HEMOGLOBINCONCENTRATION
CORRELATION FACTORS
Many of the results will appear within the normal reference ranges and, after being reviewed for appropriateness and validity, can be verified and sent to the patient’s chart.
If there is a special mark or “flag” near a result, other test should be considered to support and validate this result.
Example:
RBC indices VS peripheral smear morphology evaluation
RBC count VS hemoglobin and hematocrit (i.e. if the cells are microcytic and hypochromic, the hemoglobin should be ___ or if the rbc is low, the ___ should also be low)
low
hematocrit
Platelets
Because even relatively inexpensive whole blood counters now incorporate platelet counting capabilities, stand alone platelet counters are not often used.
-
Electrical Impedance
Optical/Light Scattering
PLATELETS Sources of Errors
Whenever the platelet count is in question, the ____ must be checked to corroborate the count and to detect abnormalities in platelets or other blood elements that may give a false value.
-
blood film
Small fragments of leukocyte cytoplasm that are numerous in leukemias Turbid plasma (Cantero, 1986)
Sources of falsely low counts (thrombocytopenia) are due to:
Adherence of platelets to neutrophils (satellitism)
Platelet clumping due to agglutinins, spontaneous aggregation, or incipient clotting due to faulty blood collection.
Always remember that among all hematologic parameters, the _____ is the LEAST REPRODUCIBLE result so the medical technologist should be very vigilant.
platelet count
Was the first one to utilize electronic impedance in the early 1960s.
Employs ____, an __ solution that maintains the integrity of the cells and conducts electricity.
Employs several dilutions:
___ and ____, which are tested in triplicate
___ which counts particles within a size range of 2 to 20fL and are counted in a 64-channel pulse analyzer (___)
____ which is determined first by lysing the cells in a solution, converting the latter into hemoglobincyanide which is measured spectrophotometrically at 530nm.
___ and ___ are derived from the rbc histogram
____ is calculated by MCV times the RBC count.
Coulter Hematology Systems
ISOTON isotonic
Erythrocyte and Leukocyte count
Platelet count
Channelizer
Hemoglobin
RBC indices and RDW
Hematocrit
A flow cytometer based model that incorporates the VCS technology combining simultaneous volumetric impedance measurements (V), cell conductivity (C) determined by electromagnetic probing of the cell and scatter (S) from a laser light source.
Coulter STKS
WBCs: ____ cells are analyzed or 20 second counting.
Cell volume: Impedance
_____: Conductivity that provides chemical composition, nuclear characteristics, and granulations (especially useful in differentiating basophils and lymphocytes)
____ is employed in the determination of eosinophils
Coulter Hematology Systems
8000
Differential count
Laser light scatter
Incorporates reticulocyte count
GEN*S
Uses a hydrodynamically focused flow cytometer with direct current electronic impedance and radio frequency conductivity to differentiate leukocytes.
Basophils and Eosinophils are selectively counted in two separate channels after ___ or shrinkage of other cell types
_____ are mathematically computed based on basophils and eosinophils
Disadvantage is the _____ (Buttarello, 1992) which is considerably lowered because of neutrophilic degranulation.
Performs __ samples per hour
____ _____ are usually reported as blasts.
TOA SYSTEMSSYSMEX NE 8000
Neutrophils
lysis
monocyte count
120
Atypical lymphocytes
Has an additional immature cell channel for counting blasts
TOA SYSTEMSSYSMEX NE 8000
___ is a fairly small, single-sample analyzer with separate components for the benchtop analyzer that tests 80 CBC+Platelet count/hour or 60 test/hour including Differential count
H*1 uses a ____ light source and _______ that is first heated at 75°C
BAYER TECHNICON INSTRUMENTS
H1/H2/H*3
H*1
tungsten halogen
cytometer for peroxidase activity
BAYER TECHNICON INSTRUMENTS
__ processes tests much faster at 100 tests/hour including the differential count
__ adds an automated reticulocyte analyzer at 100 tests per hour
H*2
H*3
Performs 120 CBC/Diff count per hour or 74 samples/hour if reticulocyte count is also selected.
Employs “___” that incorporates large, clear plastic fluidics circuit that minimizes tubings and eliminates most “pinch” values. (Thus reduces maintenance)
Includes a used friendly rack system
BAYER TECHNICON INSTRUMENTS
ADVIA 120
Unifluidics
Is a multiparameter hematology analyzer that uses a technology called _____ (MAPSS) to perform the Differential count
CELL-DYN 3000
Multi-Angle Polarized Scatter Separation
Smaller in size and more quieter in operation
Employs a unique “___” to prevent interference with lytic resistant rbcs commonly seen in neonatal samples
The impedance aperture also has a unique ___ plate to prevent RBC/PLT from reentering the sensing zone after passing through the aperture
CELL-DYN 3500
“extended lyse mode”
von Behrens
Utilizes an ___ and ___ to differentiate nucleated rbcs from leukocytes.
Utilizes a _____ that lyses rbc and stains both bare nRBCs and other nonviable WBCs with propidium iodide
CELL-DYN 4000
argon-ion laser
flow cytometric analysis
novel leukocyte reagent
During the mid-1980s, ABX was owned and distributed by Roche Diagnostics under the ___ name
In 1996, ABX along with Horiba, purchased Roche and COBAS was subsequently renamed as ___.
ABX-HORIBA DIAGNOSTICS
COBAS
PENTRA
Complete CBC, 5-part Diff count, and 27 parameters at 60 samples per hour
PENTRA 60
38 parameters including 12 reticulocyte parameters at 120 samples per hour
PENTRA 120
29 Parameters at 120 samples per hour
PENTRA 120 RETIC
Uses hydrodynamic flow for cell analysis with electrical resistance for RBCs, WBCs, and platelets
ABX VEGA
ABX VEGA
Light diffraction and absorbance, together with ____ for eosinophils, are used for the identification of NRBCs and staining of eosinophil granules by Eosinofix reagent.
Sudan Black
Basophils are counted by electrical resistance by lysing the other cells using the ___ reagent.
ABX VEGA
Basolyse
