CELL-DYN
®
3200 System Operator’s Manual
14-3
9140181H—October 2001
Section 14
Reticulocyte Package
Principles of Operation
The Reticulocyte Package software is designed to configure the instrument to
process stained, diluted specimens. When the Reticulocyte Package is turned ON,
the instrument automatically selects the appropriate configuration file and adjusts
the instrument settings to the values in this file. This configuration is retained until
the Reticulocyte Package is turned OFF. The instrument then returns to the
Standard Hematology settings.
Reticulocytes are defined by the National Committee for Clinical Laboratory
Standards (NCCLS) as transitional red cells, between nucleated red cells and the
so-called mature erythrocytes
1
. In contrast to mature RBCs, reticulocytes contain
ribosomal RNA. This RNA can be seen with certain supravital, cationic dyes that
simultaneously stain and precipitate the polyanion to form a network or reticulum.
The CELL-DYN 3200 System reticulocyte method uses the thiazine dye New
Methylene Blue N. The reticulocyte assay is performed in the WOC channel of the
instrument. Sample preparation is performed manually by dispensing 20 µL of
blood into a tube of CELL-DYN Reticulocyte Reagent. At room temperature,
staining of reticulum is complete within approximately 15 minutes. The stained
sample is aspirated in the Open Mode. After the stained sample is aspirated, it is
diluted approximately 50-fold with WBC Lyse Reagent. Once diluted with WBC
Lyse, the RBCs sphere due to the influence of the nonionic detergent incorporated
into the staining solution. Sphering is necessary to eliminate optical orientational
noise that would otherwise be introduced into the scatter measurements. The usual
lytic action of the WBC Lyse is prevented by electrolytes contained in the staining
solution and the lack of the usual incubation period used in this channel during
WBC analysis. In addition, the high New Methylene Blue concentration in the
staining reagent exerts a stabilizing effect on RBCs.
During data acquisition, 0 degree, 10 degree, and 90 degree scatter are collected for
up to 30,000 events. The 0 degree threshold is set high enough to exclude most
platelets. Histogram data are used to differentiate reticulocytes, mature RBCs,
platelet clumps, and nucleated cells. Reticulocytes have 10 degree scatter that are
similar to the scatter for mature RBCs, but differ from them by exhibiting greater
90 degree scatter. Reticulocytes are reported in percent. The instrument will
automatically calculate the Reticulocyte Absolute value if an RBC count is entered.
The RBC value may be obtained from the Standard Hematology Data Log, or it
may be entered by the operator directly on the
RETIC PATIENT SPECIMEN
screen.
