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Methylene blue stained reticulocytes in a Giemsa film--a case study.


Sentinel node biopsy is a commonly performed as a minimally invasive means to stage cancer. The procedure involves injecting the tumour with a blue dye, such as isosulphan blue or methylene blue to mimic the spread of metastatic cancer cells (1,2). This case study illustrates an example of a female breast cancer patient who was injected with methylene blue during a sentinel node biopsy. The dye entered the circulation and stained her reticulocytes, which were then visible in a Giemsa stained blood film. The differential diagnosis included other red cell inclusions such as Pappenheimer bodies and Howell Jolly bodies.


A 69-year-old female breast cancer patient was deteriorating on the ward post-biopsy. A complete blood count (CBC) and various chemical pathology tests were sent to the laboratory to investigate the cause and guide treatment. The CBC was analysed on our automated haematology analyser (Sysmex XE5000) and a rise in mean cell volume (MCV) to 104 fL from 94 fL previously was found (reference interval: 80-99fL). To investigate the increased MCV a reticulocyte count was performed, which was raised at 140x109/L (reference interval: 20-100x109/L). The patient was anaemic, with a haemoglobin of 86g/L (reference interval: 115-155g/L).

On these indications, a blood film was made. At the time of film review, no chemistry results were available, due to the longer processing time required. The blood film was leucoerythroblastic with occasional circulating nucleated red blood cells and myelocytes. The polychromatic red cells showed fine basophilic stippling, randomly distributed throughout the cell, with variable numbers of blue inclusions within each cell (Figures 1-4). The inclusions were morphologically distinct from both Pappenheimer bodies (Figure 5) and Howell Jolly bodies (Figure 6), being finer in appearance, spread throughout the cell, and exclusively found within the polychromatic cells.

In order to determine the cause of the blue inclusions, the film was sent to be reviewed by the haematologists and was considered by the wider haematology team.

Chemical pathology results were fairly normal and did not add much to the interpretation of the film. Further investigation of the clinical situation revealed that the patient was post-sentinel node biopsy, and that methylene blue had been used to visualise the lymph node. The haematologists concluded that the methylene blue had entered the circulation and behaved in a similar fashion as a new methylene blue supravital stain, staining the reticulocytes in vivo. These inclusions were then visible in the Giemsa stained film that was prepared in the laboratory.


Methylene blue is used for sentinel node biopsies as it carries a lower risk of allergic reaction than the alternative blue dyes (1,2). The procedure itself is a minimally invasive way of staging the axilla in breast cancer, as metastases occur in an orderly pattern. The sentinel node is defined as any lymph node(s) receiving direct lymphatic drainage from the primary tumour. [1] Methylene blue is injected into the tumour, then follows lymphatic tracts into the lymph node(s). The sentinel node will then be stained blue and can be visualised and removed for staging. The vital dyes enter the circulation and can interfere with pulse oximetry, as well as making patients appear cyanotic. Patients undergoing the procedure need to be warned that the dye is excreted in the urine post operatively (1).

Methylene blue is a compound of dark green powder, which yields a deep blue colour in solutions of water or alcohol. It stains negatively charged components, such as DNA, and can be used to treat both cyanide poisoning and low levels of methaemoglobin (3). Methylene blue has been used to stain reticulocytes in the past, but was quickly replaced by more intensely staining compounds such as the chemically distinct new methylene blue (4). The procedure for reticulocyte staining in the laboratory requires a mixture of blood and stain that is incubated at 37[degrees]C (4,5). Post sentinel node biopsy, the methylene blue stays in the circulation until it is removed by the kidneys (1). Theoretically this would be sufficient time for methylene blue injected into the patient to stain reticulocytes in the bloodstream.

The differentiation between the inclusions found in this film and other more well documented red cell inclusions was solely based on the morphological characteristics. The inference that it was the internal contents of the reticulocyte staining and not anything more sinister was supported by both the high reticulocyte count and the fact that the inclusions were only found in the polychromatic cells. Performing an iron stain would have excluded Pappenheimer bodies from the differential diagnosis, as they contain iron (4,5). Due to the age of the sample, this was not done. Differentiation between reticulofilmaentous material and other red cell inclusions is a difficult task. Dacie and Lewis note that Pappenheimer bodies usually present as single small dots, less commonly as multiple dots, and tend to stain a darker colour (5). However, these observations are based on a new methylene blue stained film and so may not be applicable to this case.

Howell Jolly bodies should also be considered in a differential diagnosis. These are DNA remnants seen in asplenic or hyposplenic patients (6). However, they are morphologically dissimilar from the inclusions in this case in that they are round, larger, and stain intensely in a giemsa film (5). The inclusions in this case were very fine granules and there were many in each cell, making the differential diagnosis of Howell Jolly bodies very unlikely.

In the haematologist report for the film, the inclusions were reported as basophilic stippling. Traditionally basophilic stippling is caused by denatured RNA fragments and associated with heavy metal poisoning, haemoglobinopathies, severe infections, sideroblastic anaemia and megaloblastic anaemia (6). These conditions did not fit in with the clinical picture of our case but without extensive testing it is hard to distinguish between stippling of the cell and staining of the internal contents of the reticulocyte. A point in the favour of reticulocyte staining is that the inclusions were only present in the polychromatic cells of the film, whereas basophilic stippling would be visible in polychromatic and normochromic red cells.

A follow up slide made on day-old blood showed a large reduction in the numbers of inclusions present and it was considered insufficient for an iron stain to be helpful. This raises the possibility that the red cells were actively clearing the dye as they aged in the collection tube, and were only visible in the film for a short amount of time. In this instance, it was the last sample received in the laboratory before the patient passed away, negating any possible follow-up.

This case study is interesting because it is unsupported in the literature. A few papers document the use of methylene blue in sentinel node biopsy, but none were found describing the appearance of blue inclusions post-procedure. This may be because it is fairly rare to look at blood films on post-biopsy patients, as most of the changes to the complete blood count can be explained by the post-operative inflammatory status. It could also be attributed to the short time frame of when these inclusions are visible in the film. In this case study, films made the next day on the same blood sample had far fewer inclusions present. This could mean that many films made on post biopsy patients may be too late to visualise the inclusions. A further point to note is that the appearance of these inclusions could be particular to individuals and simply not there in most patient films. Our patient also had a high reticulocyte count, which may have made the inclusions more abundant and therefore more obvious in the film.


Red cell inclusions have a diverse range of causes. Each situation needs to be evaluated independently to determine the reasons for the inclusions and any associated pathology. This case highlights the importance of relevant clinical information for the diagnostic laboratory, as full details are essential for interpreting the blood film. In this example, blue inclusions inside the polychromatic cells appear to be caused by circulating methylene blue after a sentinel node biopsy. The inclusions were visible in a Giemsa-stained film and were time-dependent, only lasting for a few hours.


Leah Pringle, BMLSc, Medical Laboratory Scientist Southern Community Laboratories, Dunedin

Correspondence: Leah Pringle. Email:


(1.) Somasundaram SK, Chicken DW, Keshtgar MR. Detection of the sentinel lymph node in breast cancer. Br Med Bull 2007; 84: 117-131.

(2.) East JM, Valentine CS, Kanchev E, Blake GO. Sentinel lymph node biopsy for breast cancer using methylene blue manifests a short learning curve among experienced surgeons: a prospective tabular cumulative (CUSUM) analysis. BMJ Surg 2009; 9: 2.

(3.) National Centre for Biotechnology Information. PubChem Compound Database; CID=6099, https://

(4.) Dacie J, Lewis S. Practical Haematology--Eighth Edition. Longman Group Limited, New York, 1995.

(5.) Rodak B, Fritsma G, Doig K. Haematology--Clinical Principles and Applications. Saunders Elsevier, Missouri 2002.

(6.) Adewoyn AS, Nwogoh B. Peripheral blood film--a review. Ann Ib Postgrad Med 2014; 12:71-79.

Leah Pringle

Southern Community Laboratories, Dunedin

Caption: Figure 1. Blood film at 40x magnification.

Caption: Figure 2. Blood film at 100x magnification. The stippled polychromatic cell is clearly seen in the middle.

Caption: Figure 3. Blood film at 100x magnification

Caption: Figure 4. Blood film at 100x magnification.

Caption: Figure 5. Pappenheimer bodies at 100x magnification.

Caption: Figure 6. A Howell Jolly body at 100x magnification.
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Title Annotation:CASE STUDY
Author:Pringle, Leah
Publication:New Zealand Journal of Medical Laboratory Science
Article Type:Case study
Date:Aug 1, 2018
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