THE WHITE IRIS^®
Leukocyte Differential Analyzer     

General Overview

The following overview depicts the process used to generate a white cell differential.

The White IRIS:

• 1. Mixes an EDTA anticoagulated whole blood specimen on its autosampler;
  2. Reads the specimen's bar code label for positive tube identification;
  3. Closed-tube samples the specimen;
  4. Separates the red cells from the white cells leaving a leukocyte rich plasma;
  5. Stains the white cells using a supravital stain;
  6. Transmits the stained white cells, by hydrodynamic focusing, through a flowcell which is coupled to a microscope with a video camera;
  7. Captures images of the white cells as they pass through the flowcell; and, finally...
  8. Classifies each white cell as a granulocyte, lymphocyte, monocyte, eosinophil, basophil, variant lymphocyte, metamyelocyte, myelocyte, promyelocyte, or blast.
  

• Then a pre-classified pictorial summary of each specimen is presented for a medical technologist or pathologist to confirm or edit each cell's classification.
  
  After the pictorial has been accepted by the individual performing the review, a final percentage leukocyte differential is then computed based on the approved classification of each individual cell. At this stage a printout can be obtained and the results are archived for future review.

  Sample Preparation for Imaging Flow Microscopy

  Imaging Flow Microscopy works best if the specimen composition and population density are optimized. Since whole blood typically contains between 500 to 1000 times more red cells than white cells, any attempt to use whole blood directly in performing a leukocyte differential would place an inordinate burden on the image processor.
  
  In currently available automated differential hematology analyzers, the lytic agent used to remove red blood cells creates specimens with considerable stroma and debris. Because this debris and stroma interferes with the clarity of the leukocyte images stained with the metachromatic cytoprobe used on The White IRIS, it is necessary to remove the red blood cells rather than lyse them.
  
  This is accomplished by a unique enhanced gravity fractionation process developed at International Remote Imaging Systems (IRIS).
  

  

  The enhanced gravity fractionation process used in The White IRIS to separate red cells from leukocytes is accomplished by the addition of a patented combination of synergistic agents. As shown in Figure 4, the enhanced method is about twice as fast as the standard polymer method yielding sufficient fractionation within a few minutes.
  
  Once the red cell fraction settles, the upper leukocyte-rich plasma fraction is aspirated. This fraction now only contains about 2 to 5 times more red cells than white cells. The remaining red cells are then removed by lysis, yielding a final product with typically less than 2% erythrocytes and a leukocyte density of approximately 1000 x 10⁶ per liter. This achieves a 50,000 fold relative concentration of white to red blood cells and only a six-fold dilution based on a population density of 6000 x 10⁶ per liter in whole blood. Imaging Flow Microscopy easily accommodates leukocyte populations 100 times more dense. Thus, unlike all other hematology analyzers which require external dilutions for leukemic specimens exceeding 100 x 10³ leukocytes per microliter, The White IRIS can accommodate them without predilution.
  
  

  Staining the Leukocytes with 2-MPM

  
  The 2-MPM cytoprobe used in The White IRIS to stain leukocytes produces a distinct metachromatic expression for each cell type. The richness of expression allows variations within individual cell types to be easily identified.
  
  This unique single-component stain offers instantaneous differential staining of mature cells in suspension, and this is ideal for rapid automated flow analysis.
  

  Characteristic Staining

  
  The following are descriptions of the characteristic staining pattern and visual appearance for each of the different cell types evaluated by The White IRIS.
  
  Neutrophil: The cytoplasm appears tan to light brown, and may have occasional red dot like structures. In neutrophils with toxic granulation there may be many red to red-orange dots, imparting an overall orange color to the cell. The nucleus appears unstained, and may show interruptions or breaks in the continuity of the nucleus, corresponding either to segments or to crossing of the nucleus by another nuclear segment.
  
  
  Band: The cytoplasm appears tan to light brown, and in young bands may display a cluster of red to red orange dots. Usually, this cluster is localized in one area of the cell, but in some bands, may appear dispersed throughout the cell. The nucleus shows a contiguous light tan color, without the apparent interruptions or breaks found in neutrophils. Sometimes, the nucleus is serpentine and seems to coil around the interior of the cell, or to be compressed in one segment of the cell.
  
  Lymphocyte: The cytoplasm appears creme to very light beige in color, and dot-like structures are not visualized. The nucleus is slightly darker in appearance.
  
  Clustered Lymphocyte: The cytoplasm appears creme to very light beige and contains a single or cluster of red dots. In most, this cluster is sharply localized in one area of the cell. In others, one or more granules are dispersed throughout the cytoplasm. The nucleus is slightly darker in appearance.
  
  Monocyte: The cytoplasm is abundant, pinkish in color, and may show frayed edges, as well as, pseudopodia. There may also be magenta dots scattered randomly throughout the cytoplasm. The intensity of the pink color may vary and in some monocytes it may be a bright pink or rose color. In a single sample, both pale and intensely pink stained monocytes can occur. The nucleus displays an irregular lobulated configuration and is unstained.
  
  Eosinophil: The overall appearance of the eosinophil is dark mahogany-brown, with granules that appear as numerous large dots that stain dark brown. The nucleus is unstained, and is usually bilobed. If the dots obscure the nucleus, it's bilobed features may not be discernible.
  
  Basophil: The overall appearance of the basophil is deep magenta to purple with numerous plum colored granules. It's nucleus is unstained and may be obscured by the overlying plum colored dots.
  
  Metamyelocyte: The cytoplasm appears light brown to beige and contains a mixture of distinct yellow, red, and orange-brown dots, imparting an overall reddish-brown color to the cell. Some of the red and orange-brown dots are clustered in one area, whereas others appear dispersed. The nucleus is large, beige, appears smooth or irregular, and occupies approximately 30-40% of the cell volume.
  
  Myelocyte: The cytoplasm appears light brown to beige and contains many red, brown, and magenta colored dots that are not quite as distinct as they are in meta-myelocytes. These dots often overlie and partially obscure the nucleus. The nucleus is unstained and is round to oval. The nucleus occupies approximately 50% of the cell volume.
  
  Promyelocyte: The cytoplasm is light brown to beige in appearance, with an overall magenta-plum hue containing a cluster of magenta-plum colored dots. In early promyelocytes, this cluster is small. In older promyelocytes, the cluster may be large. Often, promyelocytes contain many magenta-plum dots, and the entire cell appears magenta-plum, particularly when the dots overlie the nucleus and obscure its details and configuration. The nucleus is unstained, may contain one or more yellow gray colored nucleoli, and occupies approximately 75% of the cell volume.
  
  Blast: The cytoplasm is scant, agranular, and stains creme to pale yellow. The nucleus is pale yellow, contains one or more sharply demarcated nucleoli that stain yellow gray, and occupies approximately 80% or more of the cell volume.
  
  Type II Blast: The cytoplasm is scant, appears pale yellow, and contains a single magenta dot or a cluster of several dots localized in one area of the cell. The nucleus stains pale yellow to apple green, contains one or more yellow to yellow gray sharply demarcated nucleoli, and occupies approximately 80% or more of the cell volume.
  
  Three populations of cells (monocytes, clustered lymphocytes, and blasts), initially seem very similar in appearance to the untrained eye, so the following table is provided to help differentiate between them.
  
  The remaining immature cells are easily differentiated, one from another, by color and appearance.
  

  CELL	NUCLEUS	NUCLEAR VOLUME	NUCLEOLUS	CYTOPLASM
  Monocyte	irregular, lobulated	30 - 40% of cell	not seen	abundant, pale pink to intense rose pink, magenta dots
  Clustered Lymphocyte	round to oval, looks flat	90% of cell	rarely seen	creme to light beige color, single red dot or cluster of red dots
  Blast	round to oval, looks 3-D	80% of cell	usually seen - may be multiple	pale, may contain small cluster of magenta dots

  
  

   

   

   

   

   

  
  Table 1 - Highlighted difference between lymphocytes, monocytes, and blasts

  
  Imaging Flow Microscopy

  
  The heart of the Automated Intelligent Microscope (AIM) flow imaging system is the laminar flowcell (Figure 2) designed to provide microscopic imaging of individual cells. Cells are advanced and directed by hydrodynamic focusing through a plane 50 micrometers deep, which is coincident with the microscope objective focal plane. As the cells pass through this focal plane, stroboscopic illumination allows the camera to capture each cell's image electronically for classification and later review.
  

  
           

  Figure 2 - Laminar flowcell in which cells are hydrodynamically focused through the objective focal plane.
  

  Specimen Review

  
  When performing a manual differential, a smear is first characterized using a high power field scan. To retain this global perspective, The White IRIS first presents the reviewer with a summary screen of all captured cells - computer sorted by leukocyte class. And, unlike other automated differentials, The White IRIS can classify immature white cells, as well as present them to the operator for confirmation.
  
  With each cell class occupying a fraction of the display area and approximately proportional to the percentage of the cell-type in the differential, the eye-brain combination immediately recognizes whether it is a normal or abnormal distribution. The presence or absence of any abnormal morphology is also immediately apparent. This whole presentation corresponds very closely to the high power scan of a smear.
  
  If no abnormalities are observed and the reviewer agrees with the differential summary screen presented, the reviewer accepts the results by touching a single button and the screen advances to the next differential summary screen. Elapsed time or this process is typically 10 seconds. If the reviewer determines some editing is required, he or she simply touches the edit button. The computer then presents each cell class (i.e., PMNs, lymphs, etc.), one at a time, to be reviewed and edited. After the editing is complete, the computer redisplays the summary screen with the new percentages computed. Because of the high number of cells used (500 to 2000) in computing The White IRIS differential, it takes a substantial change from editing to alter the overall differential percentages.
  
  If the computer's algorithms can not sort some cells due to severe abnormal morphology, it will place them into the "Unclassified" category and alert the reviewer to audit the specimen. In a sense, this is equivalent to flagging present on most automated hematology analyzers, except on The White IRIS, the cells in question can be visually reviewed. Competent review of these specimens can typically be completed in about 1 minute.
  
  

  Benefits/Limitations

  Impedance counters and flow cytometers, on-the-other-hand, count a large number of cells in a very short period of time, but they measure very few cell parameters and the cells used in deriving the differential can not be visually reviewed for accuracy in sorting. Automated slide readers typically count about 100 white cells and have slow processing rates - limited by stage motion and the continuous focusing required to isolate and identify individual cell images.
  
  The AIM flow imaging technology utilized in The White IRIS, however, combines the best features of a slide reader (cell image visualization) with that of a flow cytometer (large number of cells counted quickly). It separates the white cells from the red cells, stains them, and then presents them (500 - 2000) to its camera (hydrodynamically focused) for electronic image capture and classification.
  
  Such images, aside from providing an enormous source of measurable parameters, allow operating modes simply not available in other flow systems. In particular, they include:
  

  • 1. Combined human/computer determinations in which the computer performs the initial white cell differential, and the human makes the final decisions on non-conforming images... without the need for additional specimen preparation;
    
    2. Singular determinations (human or computer) in which all cell classifications are made by the computer and accepted by the human, or all cells are classified and accepted by human observation and identification; and
    
    3. Cell image archiving, which enables the captured cell images to be used for teaching, side-by-side comparisons with other cells, and retrospective studies.
    

  Clearly, the intellect of the technologist or pathologist is most preferable in interpreting non-conforming abnormal cell images. And, specimen preparation and data collection are best automated to ensure reproducibility, accuracy, and to minimize biohazard exposure.
  

  	Pattern Recognition -Based Slide Reader	Impedance- Based Counting	Light Scatter- Based Flow Cytometry	Imaging -Based Flow Microscopy
  Rate of analysis (cells per second)	2	5000	2000 - 5000	20 - 2000
  Capability to display images	Yes	No	No	Yes
  Number of parameters measured	Many	1	Up to 6	Many
  Limiting technology	Slide movement and image processing	Single file particle flow	Single file particle flow	Planar particle flow and image processing

  
  Table 2 - Comparison of technologies for cell analysis.
  

AUTOMATED URINALYSIS SYSTEMS

AUTOMATED BLOOD ANALYSIS SYSTEMS


AUTOMATED CYTOGENETIC WORKSTATIONS

CYTOLOGY/BODY
FLUID PRODUCTS

SAMPLE PREPARATION CENTRIFUGES

SAMPLE COLLECTION/PREP PRODUCTS