EXERCISE #6: Automated CBC MLAB 1315 Hematology

EXERCISE #6: Automated CBC MLAB 1315 Hematology

AUTOMATED CBC

(not including automated differential)

LAB OBJECTIVE

Dry lab

1. The student will identify and interpret CBC parameters and histograms from previously run CBCs as well as correlate CBC results with various disease states.
2. The student will state methods of measurement and derivation of the CBC parameters.

Wet lab at CPL

1. The student will observe the Coulter STKS in operation and identify the different components of the instrument.
2. The student will observe the specimen as it travels through the instrument and explain the processes occurring.
3. The student will perform 10 automated CBCs on the Coulter STKS and interpret the results. 7 tests will be done using the primary sampler and 3 tests will be done using the secondary sampler.

PRINCIPLE

The counting of the cellular elements of the blood (erythrocytes, leukocytes, and platelets) is based on the classic method of electrical impedance*. The aspirated whole blood specimen is divided into two aliquots and mixed with an isotonic diluent. The first dilution is delivered to the RBC aperture bath, and the second is delivered to the WBC aperture bath. In the RBC chamber, both the RBCs and the platelets are counted and discriminated by electrical impedance as the cells are pulled through each of three sensing apertures. Particles between 2 and 20 fL are counted as platelets, and those greater than 36 fL are counted as RBCs. A reagent to lyse RBCs and release hemoglobin is added to the WBC dilution before the WBCs are counted by impedance in each of three sensing apertures in the WBC bath. After the counting cycles are complete, the WBC dilution is passed to the hemoglobinometer for hemoglobin determination (light transmittance read at a wavelength of 535 nm). The electrical pulses obtained in the counting cycles are sent to the analyzer for editing, coincidence correction and digital conversion. Two of the three counts obtained in both the RBC and the WBC baths must match within specified limits for the counts to be accepted by the instrument.

Following are the parameters of the CBC and their method of measurement when using the Coulter instruments:

(Parameter = a statistical term that refers to any numerical value that describes an entire population.)

Parameters and their derivation

Parameter / Unit of reporting / Derivation / Calculation
WBC / n x 103 cells/μL / # of leukocytes measured directly, multiplied by the calibration factor / NA
RBC / n x 106 cells/μL / # of red cells measured directly, multiplied by the calibration factor / NA
Hemoglobin / g/dL / A beam of white light shines through the lysed WBC solution and then through an optical filter. The transmittance of light (525 nm) as compared to a reagent blank is converted to absorbance, then converted to g/dL using a calibration factor / NA
MCV / fL / Derived from the RBC histogram by multiplying the # of RBCs by the size of RBCs and multiplied by a calibration constant. / NA
Hematocrit / % / Calculated / RBC x MCV
10
MCH / pg / Calculated / Hgb X 10
RBC
MCHC / g/dL / Calculated / Hgb X 100
Hct
RDW / % / Derived from RBC histogram / NA
Platelet / n x 103 cells/μL / # of platelets derived from the Plt histogram and multiplied by a calibration constant / NA
MPV / fL / Derived from the platelet histogram / NA

*Principle of electrical impedance

This method of cell counting was originally developed by Coulter Electronics and is referred to as the Coulter principle. Cell counting and sizing is based on the detection and measurement of changes in electrical impedance (resistance) produced by a particle as it passes through a small aperture. Particles such as blood cells are nonconductive but are suspended in an electrically conductive diluent. As a dilute suspension of cells is drawn through the aperture, the passage of each individual cell momentarily increases the impedance (resistance) of the electrical path between two submerged electrodes that are located on each side of the aperture.

The number of pulses generated during a specific period of time is proportional to the number of particles or cells. The amplitude (magnitude) of the electrical pulse produced indicates the cell’s volume.

The output histogram is a display of the distribution of cell volume and frequency. Each pulse on the x axis represents size in femtoliters (fL); the y axis represent the relative number of cells.

NOTE: Instruments vary in their method of counting and measuring cells in the CBC. For this exercise, we will refer to the Coulter method. Instrument manufacturers and their websites are listed below:

Manufacturer / Instruments available / Web address
Abbot / Cell-Dyn 1200, 3200, 4000 / abbot.com
Bayer Diagnostics / Advia 60 and 120 / bayerdiag.com
Beckman-Coulter / STKS, Gen-S / beckmancoulter.com
Sysmex / SE-Series / Sysmex.com

SPECIMEN

EDTA-anticoagulated blood

QUALITY CONTROL

1. Commercial low, normal and high controls

Monitor the CBC and differential parameters. Latron controls monitor the performance of the volume, conductivity and light scatter for the automated differential. Control values are stored in the instrument computer and can be monitored with the generation of a Levey-Jennings graph for each parameter.

1. XB analysis

Patient results are monitored with continuous XB analysis (weighted moving averages), which uses the patient’s own data to monitor population values and instrument performance. Batches of 20 samples are used to track MCV, MCH, and MCHC values. This method can be used to detect changes in sample handling, reagents, or instrument performance. Batches of 20 are automatically printed out. The analyzer is considered to be in control when the MCV, MCH and MCHC determined on a batch of 20 patients by use of the XB algorithm are within 3% of the expected mean indices of the population.

1. Delta Checks

Another method of quality control is the use of delta checks (delta means “difference”) which compares a patient’s own values with their most previous results. If the difference between the two is greater than laboratory-set limits, the current result is immediately flagged for review. These can only be used if the instrument is interfaced with a host Laboratory Information System (LIS).

1. Mode to Mode

A selected specimen is run in both primary and secondary mode and results must fall within a specified limit.

REAGENTS, SUPPLIES AND EQUIPMENT

Isoton III

Lyse S III diff

Clenz

Coulter Scatter-Pak (contains Erythrolyse II and StabiLyse)

Coulter STKS instrument

Gloves

COULTER STKS SYSTEM COMPONENTS

Main Unit

Sample handlerAutomated, cassette-based transport for Primary mode

Open-vial secondary mode

Bubble/blood detector

Bar code reader

DiluterPrimary mechanical operating unit - aspirates, pipets, dilutes, mixes, lyses and senses

Control ModuleControls the timing and sequencing of the operating cycles. As it receives pulses and raw data from both CBC and VCS (diff) diluters, it counts, measures and computes parameters and then sends the information to the DMS (Data Management System)

Electronic Power SupplySupplies the necessary power for all instrument functions.

Pneumatic Power SupplySupplies all air pressures and vacuums needed to operate the system

PrinterPrints results in the format designated by the user

Reagent System

IsotonDilutes the whole blood sample

Stabilizes cell membranes

Conducts aperture current

Carries and focuses the sample stream in the flow cell to enable the WBC differential measurements

Rinses the system between samples

Lyse III diffAllows leukocytes to be counted because it disrupts erythrocytes, frees the hemoglobin and reduces the size of cellular debris

Erythrolyse IIDilutes the blood samples

Rapidly lyses red cells

Reduces cellular debris to an insignificant level

StabiLysePreserves the leukocytes in near-native state so they can be differentiated into their subpopulations through the volume, conductivity and light-scatter measurements

Coulter ClenzCleans and rinses Diluter parts. Using it daily prevents protein buildup and eliminates routine aperture bleaching.

OPERATION OF INSTRUMENT

At the beginning of the day, Daily Startup must be performed. During this operation, Coulter Clenz left from Shutdown is flushed out and replaced with Isoton. At the end of startup, background counts for WBC, RBC and PLT are performed and must be within acceptable limits. Electronic checks are also performed. Both background and electronic checks are automatically printed out at the end of the startup cycle. Acceptable background counts must be as follows:

WBC0.4

RBC0.4

Hgb0.1

Plt3.0

After performing daily startup and controls, the instrument is ready for operation.

Operating cycle

Load blood tubes into the cassette and place the cassette into the loading bay. They are automatically transported, mixed, aspirated and analyzed. Sample tubes can be identified by bar codes. Cassettes are also identified by bar codes. Place cassettes in the right stack, then press [START/CONT] on the Diluter keypad. The cycle begins.

Transport

The right lift platform beneath the stacked cassettes rises and the bottom cassette is deposited on the transport. The platform lowers the cassette to the level of the rocker bed. The cassette is then moved onto the rocker bed where it is rocked back and forth, mixing the samples. The cassette continues to move toward the sensing station until it reaches the tube sensor. When the first tube is sensed, the stripper plate locks onto the tube. After at least 14 rocks from the time the cassette was loaded, the rocker bed locks in a 45 forward position.

At the sampling station the tube is locked in position and the piercing needle rotates upward. The tube ram pushes the tube out from the cassette causing the needle to pierce the tube stopper. The bar-code reader passes down over the tube to identify the patient. An aliquot is aspirated from the tube for analysis. After aspiration, the piercing needle is flushed and the next sample is aspirated in the same way. This continues until all tubes in the cassette have been sampled. The cassette then moves to the left side of the instrument where it can be removed. Results appear on the computer screen and are printed as they become available.

Red and White Blood Cell Counting

Each bath has one aperture. A precise volume of suspension is drawn through each aperture by the regulated vacuum. At each aperture, the unit counts cells in three sequential periods of 4 seconds each. During each counting period, the analyzer gathers and amplifies the cell pulses. It also checks that WBC and RBC data accumulations are above a predetermined low cut-off value.

Extended Counting

If accumulations are too low, the unit extends the sensing period. This ensures that the size-distribution curves accurately reflect the true cell population.

Coincidence Correction

Occasionally, more than one cell goes through the aperture at the same time. When cells coincide, however, the analyzer counts only one pulse. As the frequency of coincidence is proportional to the actual count, the system easily corrects results for coincidence.

Sweep Flow

The sweep flow is a steady stream of diluent that flows behind the RBC aperture during sensing periods. This keeps cells from swirling back into the sensing zone. Because these swirling cells would be peripherally sensed, their pulse height would be similar to Plt pulse heights.

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LAB EXERCISE#6 MLAB1315*

EXERCISE #6: Automated CBC MLAB 1315 Hematology

INTERPRETATION OF RESULTS

Histogram characteristics

Histograms are graphic representations of cell frequencies versus size. In a homogeneous cell population, the curve assumes a symmetrical bell-shaped or gaussian distribution. A wide or more flattened curve is seen when the standard deviation from the mean is increased. Histograms provide information about erythrocyte, leukocyte and platelet frequency and distribution as well as depict the presence of subpopulations. Shifts in one direction or another can be of diagnostic importance.

Red cell histograms are derived by plotting the size of each red cell on x axis and the relative number on the y axis. They are used to determine the average size, distribution of size, and to detect subpopulations. This histogram represents a normal red cell distribution. The small tail to the right of the curve represents coincidence, multiple cells passing through the aperture at the same time.

Erythrocyte histogram

This reflects the native size of erythrocytes or any other particles in the erythrocyte size range. The RBC histogram displays cells as small as 24 fL, but only those between 36 and 360 fL are counted as RBCs. The extension of the lower end of the scale from 36 to 24 fL allows for the detection of erythrocyte fragments, leukocyte fragments and large platelets. Normal quantities of leukocytes are present in the RBC bath and are included in the RBC count, but they are not significant. Only if the leukocyte count is markedly elevated will the histogram and count be affected.

Larger than normal cells will cause the histogram to shift to the right and smaller than normal cells will cause the histogram to shift to the left. A bimodal peak illustrating a dimorphic RBC population (camel humps) can be seen in such situations as cold agglutinin disease, after transfusion of red blood cells into a person with abnormally sized RBCs, treated iron deficiency anemia, as well as other conditions.

RDW

The RDW (red cell distribution width) is a measurement of the width of the bases of the RBC histogram with exclusion of the extreme ends. It is calculated by dividing the standard deviation by the mean of the red cell size distribution and is expressed as the coefficient of variation percentage. The RDW is increased in treated iron deficiency, vitamin B12 deficiency, folic acid deficiency, post-transfusion.

Plt histogram

Platelet histograms

Platelet counting and sizing in both the electrical impedance and optical systems reflect the native cell size. In the electrical impedance method, counting and sizing take place in the RBC aperture. In the electrical impedance system, the analyzer’s computer classifies particles that are greater than 2 fL or less than 20 fL as platelets, The raw data is sorted and histograms are then smoothed (smooth curve) and tested against mathematical criteria that eliminate nonplatelet particles and finally fitted to a log-normal distribution curve. This distribution curve has a range of 0 to 70 fL (fitted curve). The final platelet count is derived from the integrated area under this “best fit” log-normal curve.

The expected cell coincidence error (more than one cell passing through the aperture at a time) is corrected based on mathematical probability. On the Coulter, a minimum of 400 particles per aperture must be detected. If an insufficient number of particles are present in the 2–20 fL range, a “no-fit” condition is reported. An alert is generated if the three generated histograms do not agree or if the results are not within the range of 3 to 15 fL.

Particles within the platelet size range can interfere with the platelet count and histogram. Small particles, such as bubbles or dust, can overlap at the low end of the histogram. Microcytic erythrocytes can interfere at the upper end. If the histogram does not return to the baseline at both the right and left of the peak, either there is severe thrombocytopenia or nonplatelets are being counted. Either erythrocyte or leukocyte fragments may be responsible. Is such cases, the platelet count and derived parameters of MCV and PDW are not reliable.

MPV

The MPV is a measure of the average volume of platelets in a sample and is analogous to the erythrocytic MCV. In EDTA, platelets undergo a change in shape by swelling. This alteration causes the MPV to increase about 20% during the first hour. Afer an hour, the size is stable for at least 12 hours, however MPV values should be based on specimens that are between 1 and 4 hours old.

In normal patients, there is an inverse relationship between platelet count and size. The volume increases as the platelet count decreases. Because of this inverse relationship, the MPV and the platelet count must be considered together. No single normal range exists. Patients with a lower platelet count normally have a high MPV and patients with a high platelet count have a lower MPV. Young platelets are larger and cause an increased MPV. Analysis demonstrates that an MPV between 7.4 and 10.4 fL is in the normal range if the platelet count is normal.

Various disorders are associated with altered MPV values. The MPV is often decreased in aplastic anemia, in megaloblastic anemia, or as the result of chemotherapy. Hypersplenism is associated with an MPV that is inappropriately low for the platelet count. Platelet destruction associated with disseminated intravascular coagulation causes an increase in the MPV proportional to the severity of thrombocytopenia. The MPV is often increased in patients with myeloproliferative disorders or heterozygous thalassemia.

NORMAL VALUES

REPORTING RESULTS

Parameter / NormalRange
WBC / 4.8-10.8 x 103/μL
RBC / Male4.7-6.1 x 106/μL
Female 4.2-5.4 x 106/μL
Hemoglobin / Male 14-18 g/dl
Female 12-16 g/dl
Hematocrit / Male 42-52%
Female 37-47%
MCV / Male 80-94 fl
Female 81-99 fl
MCH / 27-31 pg
MCHC / 32-36 g/dl or %
RDW / 11.5-14.5%
Platelets / 150,000 - 450,000/μL
MPV / 7.4-10.4 fl

LINEARITY LIMITS

The instrument will be accurate as long as the results fall within a certain range known as the linearity range. Linearity ranges very by instrument.

LinearityRange Table

Parameter / Coulter STKS / Bayer Advia
WBC / 0-99.9 x 103/μL / 0.02-400 x 103/μL
RBC / 0-7.0 x 106/μL / 0.0-7.0 x 106/μL
Hgb / 0-18 g/dL / 0-22.5 g/dL
MCV / 50-200 fL / NA
Plt / 0-999 x 103μL / 5.0-3500 x 103μL
MPV / 5-20 fL / NA

INSTRUMENT CODES