EXERCISE 7: Automated Differential MLAB 1315 Hematology

EXERCISE 7: Automated Differential MLAB 1315 Hematology

AUTOMATED DIFFERENTIAL

LAB OBJECTIVE

Dry lab

1. The student will interpret 5 differentials from the Coulter STKS, the Bayer Advia and the Abbott Cell-Dyne by examining the instrument printout of the CBC as well as the histograms and scattergrams.
2. The student will state the theory of the technology used to perform the automated differential from 4 different instruments.

Wet lab

The student will examine the scatterplots of CBCs performed at CPL and interpret the results stating whether the automated differentials are normal or abnormal.

PRINCIPLE

Coulter STKS (currently in use as a primary instrument at CPL and as a backup instrument at SetonMedicalCenter.)

The Coulter STKS performs a five-part differential consisting of monocyte, lymphocyte, granulocyte, eosinophil and basophil. The Coulter VCS established WBC differential technology uses three measurements: individual cell volume, high-frequency conductivity and laser-light scatter as explained below:

Volume Analysis -Electronic leukocyte volume analysis uses a low-frequency current measure volume of the WBCs as they impede the current when they pass through the aperture.

Conductivity - Cell walls act as conductors to high-frequency current. As the current passes through he walls and through each cell interior, it detects differences in the insulating properties of cell components. It characterizes the nuclear and granular constituents and the chemical composition of the cell interior.

Scatter - Leukocytes are hydrodynamically focused and passed in a steady stream through a sensing zone on which a laser light is focused. As each cell passes through the sensing zone of the flow cell, it scatters and reflects the focused light which is detected by a photodetector. The patterns of scatter are measure at various angles (forward scatter at 180 and right angle scatter at 90). Scattered light provides information about cell structure, shape and reflectivity. The characteristics are used to differentiate the various types of WBCs and to produce scatterplots with a five-part differential.

Bayer Advia (currently at SetonMedicalCenter and Brackenridge)

The Bayer Advia utilizes 3 technologies to enumerate and identify cells: optical flow cytometry, cytochemistry and light scattering. WBCs are fixed with formaldehyde and stained with peroxidase in the peroxidase reaction chamber. The high heat in the chamber lyses platelets and RBCs and causes the WBCs to be fixed and dehydrated. Forward-angle light scatter and tungsten light optics are used to measure WBC size and peroxidase activity. Myeloperoxidase is a granulocyte enzyme marker that is present in varying degrees in neutrophils, eosinophils and monocytes but is absent from basophils, lymphocytes and blasts. A specific basophil count is determined separately in the basophil/lobularity chamber. Whole blood is exposed to an acid buffer that selectively lyses all cells except basophils. The resulting particles are sorted and quantified by measuring the forward angle and light scattering properties. An additional category of cells is reported by the Advia as LUC (large unstained cells). This category reflects atypical lymphocytes or blasts.

Sysmex TOA

The Sysmex TOA uses radio frequency (RF) and direct current (DC) to enumerate and identify WBC populations. The RF method detects and sizes lyse-treated cells based on density and nuclear size, whereas the DC method sizes the entire cell, nucleus, and cytoplasm. Cells pulses detected by RF and DC methods are then displayed as a 3-dimensional WBC scattergram which contains information about the distribution of lymphs, monos and granulocytes. Separate histograms are generated for basophil and eosinophil populations.

Abbott Cell-Dyne (currently at Seton Northwest)

This system uses multiangle polarized scatter separation (MAPSS) flow cytometry with hydrodynamic focusing of the cell stream. The leukocyte differential is accomplished by light scatter. It features three independent measurements and focused flow impedance. Multidimensional light scatter and fluorescent detection are used as well.

SPECIMEN

EDTA-anticoagulated blood

QUALITY CONTROL

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.

REAGENTS, SUPPLIES AND EQUIPMENT

As indicated for specific instrument

INTERPRETATION OF RESULTS

LEUKOCYTE HISTOGRAM ANALYSIS

Size-referenced leukocyte histograms display the classification of leukocytes according to size following lysis. It does not display the native cell size. The lytic agent lyses the cell and the cytoplasm collapses around the nucleus, producing differential shrinkage. The histogram subpopulations reflect the sorting of the cells by their relative size, which is primarily their nuclear size. As each leukocyte passes through the aperture, it causes a change in electrical resistance which is proportional to its volume. The histogram is a representation of the sizing of the leukocytes. The differentiation is as follows:

Cell type / Size range / Cells that fall within this size range
Lymphocytes / 35-90 fL / lymphs and atypical lymphs
Mononuclear / 90-160 fL / Monos, promyelocytes, myelocytes, plasma cells and blasts
Granulocyte / 160-450 fL / segs, bands, metas, eos and basos

A valley or depression should be seen between each population. Abnormal sized cells or abnormal particles can cause abnormal patterns and the instrument will print an alert specific for the region where the abnormal pattern exists. The following table lists the region (R) flags and the abnormalities they may represent:

Leukocyte Region Codes

R Flag / Region / Abnormality
R1 / Far left / Erythrocyte precursors (NRBCs)
Cryoglobulins
Nonlysed erythrocytes
Giant and/or clumped platelets
R2 / Between lymphs and monos / Blasts
Basophilia
Eosinophilia
Plasma cells
Abnormal/variant lymphs
R3 / Between mons and granulocytes / Abnormal cell populations
Eosinophilia
Immature granulocytes
R4 / Far right / Increased absolute granulocytes
RM / Multiple flags

SCATTERPLOT ANALYSIS

Coulter Model STKS and MAXM

DF 1

Light Scatter DF 1

On a scatterplot, individual cells are depicted as points reflecting cell volume and light-scattering characteristics. Clusters of cells are identified as monocytes, eosinophils, lymphocytes and neutrophils based on their relative position on the scatterplot. Separate scatterplots comparing cell volume and conductivity properties are generated to quantify basophils.

Floating discriminators examine areas between different cell populations. Abnormalities are identified by specific region flags. The STKS flagging system is enhanced by providing specific alphanumeric codes and three types of messages including suspect, definitive and condition messages. Suspect messages flag abnormal cell populations or distributions such as platelet clumping, variant lymphocytes and immature granulocytes. These messages appear in the cell classification window on the sample analysis display and abnormalities should be confirmed by a microscopic review. On the DMS screen, you can see the results two dimensions at a time and rotate the screen to view the cells in three different ways. The density of the cells is represented by colors with yellow being the highest density followed by red, green and blue.

Bayer ADVIA

Leukocyte Differential cell identification by peroxidase content and cell size. (Technicon)

The Advia uses cytochemical as well as light scatter to differentiate WBCs. It provides two scattergrams - one for peroxidase and one for basophil/lobularity. The WBCs are lysed, fixed and stained with a peroxidase reagent. A dark precipitate forms in the primary granules of leukocytes containing peroxidase when a chromogen is added with hydrogen peroxide as the substrate. Eosinophils and neutrophils are strongly positive and monocytes are weakly positive. Peroxidase is not present in basophils, lymphocytes, blasts or large unstained cells (LUC) The clusters of dots on the scattergram are defined and analyzed, the cells are counted and classified based on information stored in the computer. This information is used to generate the total WBC and differential except for basophils.

The nuclear channel is used to measure the conformation of the nucleus of WBCs. When exposed to a surfactant at a low pH, the membranes and cytoplasm disintegrate and only the nucleus is left. The nuclear channel cytometer distinguishes leukocytes by differences in nuclear shape and counts basophils. A fixed horizontal threshold separates basophils from the nuclei of other leukocytes.

An immunoperoxidase reaction is employed for lymphocyte subtyping. Two monoclonal antibodies are added to the whole blood followed by a peroxidase reagent. Lymphocytes which have been labeled by the immunoperoxidase reaction, appear between the unlabeled lymphocyte population and the monocytes. Cells with endogenous peroxidase such as neutrophils stain intensely and appear far to the right.

Abbot Cell-Dyne

The five-part differential is performed using multi-angle polarized scatter separation technology (MAPPS). Sheath fluid surrounds the sample stream for laminar flow and hydrodynamic focusing of the cells into single file for passage through the optically clear quartz flow cell where the vertically polarized helium neo laser beam is interrupted by passage of each individual cell. The light scattered by cell passage through the laser beam is collected by sensors at 4 different angles. The four measurements from each cell are stored for step by step analysis and cell identification for the five-part differential.

AUTOMATED DIFFERENTIAL STUDY QUESTIONS

Name______

Date______

1. Draw and label R regions on a WBC histogram. (10 pt)

1. List the abnormalities that cause a flag in each region (10 pt)

1. List the cells that are located in each region of the following WBC histogram. (10 pts).

1. Locate each WBC region on the Coulter STKS and Bayer Scatterplots

Light Scatter DF 1

Leukocyte Differential cell identification by peroxidase content and cell size. (Technicon)

1. Read the article attached and answer the questions at the end.

LAB EXERCISE #7 MLAB 1315

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