AUTOMATION IN HEMATOLOGY Flashcards by Janah Belaguin

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

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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)

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Preserves cell function

Preserves cellular morphology

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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)

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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)

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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.

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is a routine test

CBC

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The automated CBC report includes:

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

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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

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PRINCIPLES INVOLVED IN AUTOMATIONELECTRONIC IMPEDANCE

-
-

Cell-Dyn 3000 (Abbott)
Sysmex NE 8000 Cell Counter (Baxter)
COBAS Cell Counter (Roche)

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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

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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

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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

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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

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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

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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

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Is the methodology used by some Coulter instruments to characterize cells for the scattergrams.

PRINCIPLES INVOLVED IN AUTOMATION

LASER SCATTERING

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___ is utilized for cell counts as well as cell characterization by FLOW CYTOMETERS.

Laser light scatter

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-
-

Technicon H-3
Cell Dyn 3000 (Abbot)
Serono Diagnostics System 9000

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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)

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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.

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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

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Is used along with light scatter.

PRINCIPLES INVOLVED IN AUTOMATION LIGHT ABSORBANCE

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Instruments using LIGHT ABSORBANCE:

Sysmex 8000
COBAS
Technicon H-3

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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

Analytical factors: - - Post-analytical factors: -

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. Principles employed in platelet counts: - -

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. Sources of falsely elevated counts (thrombocytosis) are due to: - -

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 H*1/H*2/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

AUTOMATION IN HEMATOLOGY Flashcards

(70 cards)