peripheral blood candidosis infection leading to spurious platelet and white blood cell counts

Peripheral blood candidosis infection leading to spurious

platelet and white blood cell countsJ. F. LESESVE*, M. A. KHALIFA†, R. DENOYES†, F. BRAUN†

CLINICAL OBSERVATION

We report a 65-year-old man diagnosed 7 months ago

with a disseminated adenocarcinoma of the stomach.

At the time of this report, he had received chemo-

therapy treatment for several weeks and had an intra-

vascular catheter. He was admitted to the hospital

with a fever of 39.5 �C related to sepsis. A first blood

cell count was performed using an ADVIA 120 auto-

mated analyzer (Bayer Healthcare, Tarrytown, NY,

USA; blood extracted through a catheter). The haemo-

globin count was 9.1 g/dl, red blood cells (RBC)

2.95 · 1012/l, haematocrit 28.3%, mean cell volume

95.9 fl, corpuscular haemoglobin content 30.8 pg,

mean corpuscular haemoglobin concentration 32.2

g/dl, RBC distribution width 17.8%, reticulocytes

62 · 109/l, white blood cell (WBC) counts 8.05 · 109/

l (for the basophil channel) but 4.14 · 109/l (for the

peroxidase channel) with 2.21 · 109/l granulocytes,

5.18 · 109/l lymphocytes and 100 · 109/l platelets

with mean platelet volume 9 fl (Table 1). The same

sample was then analysed using a Sysmex XT 2000i

counter (Sysmex, Kobe, Japan). While the RBC series

was unchanged, WBC counts showed 8.84 · 109/l

(WBC/baso channel) and 4.16 · 109/l for DIFF chan-

nel. No WBC differential was offered. Platelets were

*Laboratory of Haematology,

Universitary Hospital, Nancy,

France†Laboratories of Haematology,

Universitary Hospital, Thionville,

France

Correspondence:

Jean Francois Lesesve, Laboratory

of Haematology, CHU Brabois,

Vandoeuvre 54511, Nancy, France.

Tel.: 33 383 153 757;

Fax: 33 383 153 789;

E-mail: [email protected]

doi:10.1111/j.1751-553X.2008.01069.x

Received 7 November 2007;

accepted for publication 10 March

2008

Keywords

Haematology analyzer, automated

counts, artefact, platelet count,

Candida sp.

SUMMARY

We report a patient with thrombocytopenia secondary to disseminated

stomach adenocarcinoma and sepsis whose platelet and white blood

cells were falsely enumerated by two automated haematology analyz-

ers. The cause of the spurious counts became obvious when numerous

yeast forms were observed on the peripheral blood smear. Artefactual

automated analyzer results are detailed.

SHORT REPORT INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

� 2008 The Authors

572 Journal compilation � 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 572–576

91 · 109/l for impedance count and 71 · 109/l for

optical count. On both analyzers, platelet and WBC

data were flagged. The platelet volume distribution

curve was irregular and did not return to baseline for

volumes >20 fl (Figures 1 and 2). Abnormal dots were

detected in the WBC distribution histograms

(Figures 3 and 4). Platelet clumps and nucleated RBC

(NRBC) flags were raised by both analyzers. Surpris-

ingly, on the same day, the patient had sudden

modifications of his cell counts. As compared with the

days before (baselines counts for platelet and WBC

being 50 and 4 · 109/l, respectively), the decrease of

platelet count and the increase of WBC count were

confirmed on a direct venepuncture (second sample).

Examination of the smear under light microscopy

revealed the presence of yeast forms either isolated

(approximate diameter 2–3 lm) or clumped in small

aggregates of 2–5 micro-organisms (5–15 lm,

Figure 5). Some of the yeasts were phagocytosed into

polymorphonuclears or monocytes. Thrombocytopenia

was confirmed by manual technique (platelet count

estimated at 30 · 109/l) and no aggregates were

observed. WBC count and differential were estimated.

To determine whether the source of the yeast was a

true infection or a collection tube contamination, a

third extraction (venepuncture) under sterile condi-

tions was performed. Positivity for Candida spp. was

demonstrated, then these organisms were identified as

Candida glabrata. Despite intensive management, the

patient deceases rapidly.

DISCUSSION

Observation of micro-organisms on the peripheral

blood smear associated with positive blood culture is a

rarity even in septic patients (Zandecki et al., 2007).

Table 1. Manual and automated

counts for white blood cells and

platelets

Microscope ADVIA 120 XT 2000i

Leucocytes (·109/l) 4.1 8.05/4.14 8.84/4.16

Neutrophils (%) 70 27.4 –

Lymphocytes (%) 22 64.4 –

Monocytes (%) 2 6.4 –

Eosinophils (%) 1 0.3 –

Basophils (%) 1 0.3 0.6

Large unstained

cells (%) (flags)

4 1.5 –

yeasts NRBC blasts, lysis

resistant RBC

NRBC, abnormal

scatter lysis

resistant RBC

Platelets (·109/l) (flags) 30 100 91/71

– Clumps,

large platelets

Clumps, abnormal

distribution

RBC, red blood cell; NRBC, nucleated RBC.

Figure 1. Bayer ADVIA 120 histograms for platelets.


Journal compilation � 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 572–576

J. F. LESESVE ET AL. PERIPHERAL BLOOD CANDIDOSIS INFECTION 573

Organisms typically detected on these stained periph-

eral smears included Histoplasma capsulatum, Candida

sp., Plasmodium sp. and Staphylococcus sp. (Marshall,

Theil & Brandt, 1990; Molero, Lemes & De la Iglesia,

2000). As in our case, most patients suffered from an

immune deficiency syndrome, had intravascular cath-

eters and were in critical conditions.

Previous studies documented that the presence of

micro-organisms in the peripheral blood can result in

spurious platelet and WBC counts or electronic differ-

entials (Marshall, Theil & Brandt, 1990). Spurious

enumeration of yeast forms as platelets is an extremely

rare artefact (Arnold, Jowzi & Bain, 1999). Less than

10 cases have been reported but without full details on

abnormal changes generated by the haematology

analyzers used or were related to analyzers from

previous generations (Vincenot-Blouin et al., 2007).

Nevertheless such situations are probably underesti-

mated (two similar observations occured in France

during the preparation of this manuscript implicating

Figure 3. Bayer ADVIA 120 histograms for white blood cells.

Figure 2. Sysmex XT 2000i histograms for platelets.



574 J. F. LESESVE ET AL. PERIPHERAL BLOOD CANDIDOSIS INFECTION

Candida parapsilosis and albicans). Given that fungi may

have the same size as platelets, their presence could

lead to increased platelet counts in thrombocytopenic

patients infected by Candida (Latif et al., 2003). On the

ADVIA 120 analyzer, platelet count is performed

according to cell size (from 0 to 60 fl, large angle scat-

tering) and structure (refraction index between 1.35

and 1.45, small angle scattering), which allows for dis-

tinction between small RBC and platelets. This count

is performed on the XT 2000i analyzer using the

impedance (‘I’) method (thresholds: 2–40 fl) or an

optical (‘O’, flow cytometric-based) technique after

staining of the cells by polymethine (a DNA/RNA fluo-

rochrome that discriminates platelets from RBC). Both

analyzers falsely identified yeast cells as platelets,

resulting in an overestimation of the platelet number.

The identification of large platelets on the histograms

coupled with their absence on the smear without

visible platelet aggregates suggested the presence of

‘particles’ in the sample (yeasts falsely identified as

platelets). This resulted in a decrease of the thrombo-

cytopenia level (platelet estimated count 30 · 109/l).

The XT 2000i flagged counts as a result of suspicion

of aggregates (DIFF channel) and abnormal platelet

Figure 5. Light microscopy image of the Candida (May Grunwald Giemsa slide stain ·1000). Yeast were either iso-

lated, aggregated or phagocytosed by polymorphonuclears or monocytes. See platelets for comparison.

Figure 4. Sysmex XT 2000i histograms for white blood

cells.



J. F. LESESVE ET AL. PERIPHERAL BLOOD CANDIDOSIS INFECTION 575

distribution. Optical (cytometric) count was partially

decreased as compared with the impedance count but

was still overestimated.

Increased WBC counts, along with abnormal WBC

histograms with alert messages can be indicative of a

bacterial, fungal or malarial parasites presence, partic-

ularly if the infective bodies are clumped together.

For the ADVIA 120, the perox channel provides a bi-

dimensionnal analysis (volume and peroxidase activ-

ity) after selective lysis of RBCs and platelets. The

flags in our analysis concerned fragments, aggregates

and erythroblasts, and abnormal dots in the lympho/

monocytes areas were also observed. The baso chan-

nel gives the reference count for WBC (enumeration

of nuclei after cytoplasmic stripping) and classifies the

cells according to chromatin density and lobulation.

Baso channel histogram showed abnormal dots (with

lysis resistant particles) and an aborted threshold

between monocytes and polynuclears. For the XT

2000i, the DIFF channel provides a three-dimensional

analysis after a moderate lysis of the cytoplasm and

DNA/RNA staining by polymethine dye. The abnormal

DIFF histogram demonstrated loss of the discriminat-

ing thresholds and an extra dot area ranging from

debris to lymphocytes leading to NRBC and aggregates

flags. In the WBC/baso channel, cells are analysed

according to volume and structure after cytoplasm

stripping (only basophils are preserved). The yeast

cells filled the ghosts’ area and were counted as WBC,

resulting in the RBC R+ lysis flag. No discrimination

was possible between ghosts and leucocytes and the

DIFF/BASO ratio increased to 0.41 (the DIFF channel/

baso ratio is a research parameter that is needed

to verify the calibration between DIFF channel and

WBC/baso channel; the reference value is 1; it is

used for the management of the RBC lyse resistance

flag).

In conclusion, we would like to point out that

while dissemination of measurable circulating micro-

organisms is extremly rare, it should be considered

when automated analyzers report spurious counts

along with abnormal platelet or WBC flags. Although

the automated analyzers were not able to discriminate

the yeasts, the incoherent flagged results made it

impossible to ignore this artefact. Laboratorians

reviewing slides should be aware of the phenomenon

that yeast may cause spurious automated platelet and

white cell counts.

ACKNOWLEGEMENTS

The authors are indebt to Drs N. Chioukh, E. Andre-

Kerneis and M. Zandecki (Belfort, Meaux, Angers

hospitals) for their comments related to similar situa-

tions. Thanks to Mrs Emmanuelle Schindler for

reviewing the manuscript.

REFERENCES

Arnold J.A., Jowzi Z. & Bain B.J. (1999)

Candida glabrata in a blood film. Brit-

ish Journal of Haematology 104, 1.

Latif S., Veillon D.M., Brown D.,

Kaltenbach J., Linscott A.J., Oberle A.

& Cotelingam J.D. (2003) Spurious

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Marshall B.A., Theil K.S. & Brandt J.T.

(1990) Abnormalities of leukocyte

histograms resulting from microorgan-

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Molero T., Lemes A. & De la Iglesia S.

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International Journal of Laboratory

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peripheral blood candidosis infection leading to spurious platelet and white blood cell counts

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