AFCG Clinical Guidelines: Reticulocytes



JULY 1996




Ms Helen Hanlin, Mr Peter Hobson, Ms Jennifer Staplton



Laboratory Safety

Specimen Collection

Specimen Transport

Specimen Integrity

Specimen Processing

Controls for Reticulocyte Analysis

Sample Analysis

Limitations of the Method


Data Storage

Data Reporting

Quality Assurance


Appendix 1: Example of Reticulocyte Analysis

Appendix 2: Log Book Forms


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.


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

Home Page Table of Contents Sponsors E-Mail Archive Web Sites

CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(765) 494-0757; FAX (765) 494-0517; Web, EMAIL