CYTOMETRY GROUP
JULY 1996
RECOMMENDED GUIDELINES FOR
THE DETECTION OF RETICULOCYTES
Ms Helen Hanlin, Mr Peter Hobson,
Ms Jennifer Staplton
CONTENTS
Controls for Reticulocyte Analysis
Appendix 1: Example of Reticulocyte Analysis
Appendix 2: Log Book Forms
INTRODUCTION
These recommendations are presented
with a view to being a minimum standard and should not be seen
to restrict the ability of any individual. This document will
be reviewed regularly to ensure that these recommendations embrace
current accepted laboratory practices.
Reticulocytes are immature red blood
cells which have shed their nucleus, but which still retain residual
nuclear material. Clinically, the reticulocyte percentage is a
useful indicator of erythropoiesis. In cases of anaemia, an elevated
reticulocyte count is indicative of normal marrow function, whilst
a decreased result may be more consistent with impaired erythropoiesis.
Traditionally the reticulocyte percentage was estimated by precipitating
the residual RNA with a dye and counting the stained cells as
a percentage of 1000 red blood cells using a microscope. This
method is well known to be imprecise and open to subjective interpretation
by the technologist.
Automated reticulocyte counting methods
overcome most of the problems of manual counting, i.e. small sample
sizes and interobserver variation. The use of an automated system
such as a flow cytometer for estimating reticulocyte percentages
has the advantage of allowing a large number of cells to be analysed
rapidly, objectively, and simply, greatly increasing accuracy
and precision.
These guidelines are written in broad
terms as a gesture to indicate and not to dictate.
Each laboratory will adopt internal
procedures and policies for the safe handling of biological specimens.
Following is a set of minimum standards which should be adopted.
1. Use universal precautions1
with all specimens.
2. Develop appropriate internal procedures
to cope with accidents such as spillage.
3. Handle and manipulate specimens in
a safe biological confinement area wherever possible.
4. Samples should be capped once removed
from the safe handling area.
5. The reagents used in the detection
of reticulocytes on the flow cytometer are to be handled with
caution, as they are potentially carcinogenic and teratogenic.
6. Appropriate safety devices such as
gloves, gowns, goggles, centrifuge carriers, automatic pipetting
are recommended whenever handling and processing specimens. Use
disposable plastic equipment wherever possible.
7. Wash hands with medicated soap after
working with specimens, removing gloves, or when leaving the laboratory,
and as in accordance with usual local laboratory policy and universal
precautions.
8. For decontamination of flow cytometers
refer to the instrument manufacturer's recommended procedures.
9. Liquid waste should be treated with
sodium hypochlorite. Solid waste should be handled carefully in
appropriate robust containers.
10. Laser safety: Most benchtop flow
cytometers use visible lasers which pose very little risk of injury
to the operator. Operators should be aware of the potential dangers
of lasers and the need for safety devices such as shields and
goggles in given circumstances. The operator is referred to the
manufacturer of the instrument and to AS 22111 with
regard to safety of lasers.
1. Universal precautions1
should be strictly observed when collecting and handling blood
samples.
2. Each specimen should be labelled
with the patient name or a unique patient identifier, and the
date and time of collection. If a preprinted label is used, the
signature or initials of the collector should appear on the label
to verify that the information relates to the patient from whom
the blood was collected.
3. Each specimen should be accompanied
by a test requisition which should include the patient name or
unique identifier, date and time of collection, age, sex, pertinent
medication and presumptive diagnosis of the patient, name of requesting
physician, and address for return of results.
4. The request form and specimen tube(s)
should carry identical patient information. Both should be checked
on receipt in the laboratory; in case of discrepancy or doubt,
a clear, documented protocol approved by the Director/Scientist-in-Charge
of the laboratory should be followed. Unlabelled samples and
forms should be discarded.
5. A red blood cell count should be
performed on each specimen.
6. As EDTA is the anticoagulant of choice
for full blood estimations, it is also the most suitable one
for reticulocyte analysis, reducing the need for a second specimen.
However, any anticoagulated blood is suitable.
7. Specimens are suitable for analysis
for up to 48 hours when stored at room temperature, or for up
to 4 days at 4°C.
1. Packaging, labelling, and transport
of specimens should comply with all current local, state, national,
and international regulations for the regions through which the
specimens will pass.
2. Specimens may be maintained at room
temperature if they will be tested within 48 hours, or at 4°C
if testing will be delayed.
3. Temperatures below 4°C
or above 37°C
must be avoided.
1. Visually inspect the specimen for
clots, haemolysis, or container defects. Re-collect the sample
if the specimen shows any visual signs of deterioration.
2. Specimens which have been collected
or transported outside these guidelines may be processed by
the laboratory according to a local approved, documented policy.
The deficiencies in the sample should be noted and the report
should reflect the effect that these deficiencies may have on
results.
1. Ideally, a red blood cell (RBC) count
should be performed on each specimen in order to calculate an
absolute number of reticulocytes. The operator must be aware
of falsely elevated reticulocyte counts where it may be necessary
to perform a full blood count or an examination of a blood film
to verify the accuracy of the reticulocyte percentage (see Limitations
of the Method).
2. The flow cytometric estimation of
reticulocytes is dependent upon the binding of suitable fluorescent
dyes to residual erythrocyte RNA. To be suitable, a dye must
have high sensitivity and specificity for RNA and should permeate
cell membranes easily and give a very stable fluorescence signal. Thiazole
orange, which is excitable at 488 nm, gives high resolution between
reticulocytes and background red blood cells. The staining
is stable for up to 4 hours at room temperature when protected
from light. Auramine O offers many of the same properties, but
requires excitation at 435 nm. Thiazole orange is therefore the
dye of choice for most routine laboratories.
3. Each laboratory should establish
suitable storage conditions and times for specimens prior to processing,
and minimum and maximum times for incubation of stained samples,
under normal operating conditions.
CONTROLS FOR RETICULOCYTE ANALYSIS
1. Several commercially prepared and assayed
control materials are available for evaluating the accuracy and
precision of reticulocyte analysis. Ideally each laboratory should
include these with each patient run. In addition, samples which
have been previously analysed can be stored at 4°C
and restained and reanalysed with subsequent batches, to examine
day-to-day variation.
2. The level of background fluorescence
should be estimated with each batch using a small aliquot of blood
in a volume of buffered saline with no added fluorescent dye.
This tube is used to exclude autofluorescence and instrument noise.
3. Each laboratory should determine
its own reference range 2-6 for reticulocytes using
its particular preparation method and instrumentation, because
significant laboratory-to-laboratory differences related to these
variables have been reported7.
4. Instrument quality control, including
laser/PMT calibration, should be performed daily (see Optical Alignment Log).
1. Sample order: Run background and
all control specimens first and then, according
to laboratory priority, run the patient samples. If processing
large batches, run controls at intervals.
2. A minimum of 10,000 events per sample
should be collected to allow for accurate assessment of the reticulocyte
population.
3. Specimens should be acquired on LOG
settings for forward and side scatter and fluorescence parameters.
The flow cytometer will detect and quantify the cells which have
bound the fluorescent dye.
4. The background (unstained) sample
should be run first. A gate should be set around the main population
of red blood cells, based on forward versus side scatter, excluding
platelets. A histogram of the fluorescence properties of the
gated events should then be displayed and a marker set outside
the main population of unstained cells. The fluorescent events
in this area represent autofluorescence and instrument noise.
This background fluorescence should then be subtracted from the
total fluorescence seen with the stained red blood cells of controls
and samples to give a corrected number of reticulocytes.
5. The controls and samples should be
gated using the method described above. Most of the red cells
in a patient sample will not take up the dye and will appear as a
large peak at the left of the fluorescence intensity plot. A
marker should be set to the right of this peak; the reticulocytes
and other cells which have stained with the fluorescent dye will
lie to the right of this marker (see Appendix 1.)
6. If an automatic program is in use,
check:
6.1 each graph to make sure you agree
with the placement of the red cell gate and marker. If you consider
that the automatic program has selected an inappropriate position for
the gate or marker, reanalyse using an alternate program.
6.2 the appearance of the graph. If
there is a large peak at 103, this may indicate a high
WBC or other cause of interference (see Limitations of the Method).
7. Results should be correlated with
clinical information, FBC results, and film observations. Any
value which is unexpected should be further investigated with
reference to the limitations of the method.
Since the dyes used for flow cytometric
analysis of reticulocytes are nucleotide markers, there are a
number of factors which, if present in a given blood specimen,
may cause falsely elevated reticulocyte estimations. The following
may require examination of a blood film, manual gating of the
red blood cell population, adjustment of automatically set markers,
or manual estimation of the reticulocyte percentage using alternate
methods:
1. Abnormally high white cell counts,
>20 x 109/L.
2. Patients with CLL with large numbers
of small lymphocytes (8-10 microns in diameter), which will appear
in the RBC gate.
3. Cases of acute leukaemia with WBC
fragments which may be included in the RBC gate.
4. A high proportion of macrothrombocytes.
5. Howell-Jolley bodies present.
6. RBC inclusions that give rise to
basophilic stippling.
7. Malarial parasites.
8. High nucleated RBC counts.
9. Unstable haemoglobins (eg. Hb Köln,
which may show autofluorescence).
There are a number of causes of aberrant
results which are not attributable to clinical conditions.
1. Possible causes of a reduced result:
1.1 Thiazole orange in aqueous solution
is quite unstable and may deteriorate rapidly. Each laboratory
should assess the stability of all working reagents under their
normal operating conditions. Control results should be closely
monitored, as reagents may deteriorate prior to the expiry date.
1.2 The proportion of cells to reagent
may be too high, quenching fluorescent tag. Prepare a fresh sample
for analysis using a higher dilution.
2. Possible causes of an increased
result:
2.1 Contamination - the background tube
containing no stain should produce virtually no fluorescence signal.
A high background may indicate contamination of the flow cytometer
or the PBS solution used to prepare the staining solution, and
thus false elevation of the control and patient samples. The
cytometer should be cleaned in accordance with manufacturer's
instructions, and a fresh background prepared in a clean tube.
The run must then be repeated.
1. The possibility of patients' contesting
the diagnostic implications derived in part from flow cytometric
testing makes it incumbent upon the laboratory to be able to demonstrate
and verify the process used in arriving at the reported test results.
2. Where possible, all listmode data
on samples should be archived for the minimum period of time required
by state and federal regulations for retention of raw data.
3. Retain all primary files, worksheets,
and final results for the minimum duration required by state and
federal regulations. These regulations may vary and each laboratory
will need to remain informed of the current requirements.
1. Report all unique patient identifiers.
2. Reticulocytes may be reported as
a percentage and/ or absolute number of the red blood cell population.
3. Reticulocytes should ideally be reported
in conjunction with a full blood count.
Each laboratory should evaluate the
quality of reagents and control specimens on a regular basis and
at least when changing to new lot numbers or preparing new solutions6.
Should external quality assurance programs for reticulocytes
become available, it is recommended that each laboratory participate.
1. Universal precautions: There appears
to be no reference that addresses the specific needs of flow cytometry.
Readers are advised to refer to the following documents:
Australian Standard AS 2211-1991, Laser
Safety.
Australian standard AS 2243.3 - 1991,
Safety in laboratories, Part 3: Microbiology.
NCCLS M29-T, Protection of laboratory
workers from infectious disease transmitted by blood, body fluids
and tissue.
MMWR 1988: 37(24): 377-82, 387-88.
CDC Update: Universal precautions for the prevention of transmission
of human immunodeficiency virus, hepatitis B virus, and other
bloodborne pathogens in health care settings.
2. Winkel, P., and Statlan, B.E. Reference
values. In Clinical Diagnosis and Management by Laboratory
Methods (ed. J.B. Henry), Philadelphia, W.B. Saunders Co.,
1979, pp29-52.
3. Martin, H.F., Gudzinowicz, B.J.,
Fanger, H. Normal Values in Clinical Chemistry, New York,
Marcel Dekker, 1975, pp102-236.
4. Henry, R.J., Cannon, D.C., Winkelman,
J.W. Clinical Chemistry. Principles and Technics, New
York, Harper and Row, 1974, pp 343-371.
5. Edward, B.S., Altobelli, K.K., Nolla
H.A., et al. A comprehensive quality assessment approach for flow
cytometric immunophenotyping of human lymphocytes. Cytometry
10:443-441, 1989.
6. McCarthy, R.C., and Fetterhoff, T.J.
Issues in quality assurance in clinical flow cytometry.
Arch.Path.Lab.Med. 113: 658-666, 1989 (in Press).
7. Keren, David (ed) et al. Flow Cytometry
and Clinical Diagnosis, 1994, ASCP Press, Chicago.
APPENDIX 1: Example of Reticulocyte Analysis
An example of a reticulocyte analysis
showing the red blood cell gate, the positioning of the marker
for estimation of the count.
Back to standards and regulations
Back to consensus documents and ring trials
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