Ann Lab Med.  2012 Nov;32(6):392-398.

Correction of Pseudoreticulocytosis in Leukocytosis Samples Using the Sysmex XE-2100 Analyzer Depends on the Type and Number of White Blood Cells

• Kim A
• Park J
• Kim M
• Lim J
• Oh EJ
• Kim Y
• Park YJ
• Han K

Abstract

BACKGROUND
The reticulocyte count is a good marker of erythropoietic activity of the bone marrow. In the mid-1990s, automated flow cytometric analysis replaced microscopy for the quantification of reticulocytes. Leukocytosis cases with an erroneously high reticulocyte count and a high immature reticulocyte fraction (IRF) have been reported. In this study, we analyzed reticulocyte counts in leukocytosis samples, in an effort to identify a correction method.
METHODS
The study comprised of 21 samples from 16 leukocytosis patients. Results of reticulocyte analyses obtained using a XE-2100 hematology analyzer (Sysmex, Japan) were compared with those obtained using the supravital staining technique, which is a reference method. If the samples showed erroneously high reticulocyte counts and IRF, they were reanalyzed after serial dilution with isotonic solution.
RESULTS
Five samples from 4 patients showed erroneously elevated reticulocyte counts and/or IRF on the XE-2100 analyzer. They displayed abnormal reticulocyte scattergrams, with 4 of 5 cases indicated by a flag. The white blood cell (WBC) fractions overlapped with the reticulocyte regions, especially with the IRF. Diagnoses and blast counts were variable when such errors occurred; WBC counts varied from 218.19x10(9)/L to 725.14x10(9)/L. The errors were corrected by simple dilution with isotonic solution. However, the corrective WBC counts differed according to individual cases.
CONCLUSIONS
When leukocytosis samples exhibit an abnormal reticulocyte scattergram with a flag, or an abnormally high IRF, we recommend the dilution of the sample with isotonic solution to a WBC count of about 100.00x10(9)/L, followed by reanalysis of the reticulocyte count and reticulocyte scattergram.

Keyword

Reticulocytes; Leukocytosis; Reticulocyte count

MeSH Terms

Adolescent
Adult
Aged
Automation
Child, Preschool
Female
Flow Cytometry/instrumentation/*methods
Humans
Indicator Dilution Techniques
Infant, Newborn
Leukocyte Count
Leukocytes/cytology
Leukocytosis/*diagnosis
Male
Middle Aged
Reticulocyte Count
Reticulocytes/cytology
Young Adult

Figure

• 

  Fig. 1 Reticulocyte scattergrams (left side of each panel) and reticulocyte extended scattergrams (right side of each panel) of sample no. 1 generated by the Sysmex XE-2100 hematology analyzer. (A) Sample no. 1 with no dilution (WBC count, 725.14×109/L) showing abnormal scattergrams. The WBC fractions seem to overlap with reticulocyte regions, especially with the IRF. (B) After 1:2 dilution (WBC count, 380.55×109/L), the reticulocyte scattergram normalized and the overlap between the reticulocyte and WBC regions disappeared. In the reticulocyte extension scattergram, the WBC region shifted to the right, because the fluorescence of WBCs increased. (C) After 1:3 dilution (WBC count, 273.18×109/L), the WBC region shifted further to the right. Regions: blue, mature red blood cells; pink, low fluorescent reticulocytes; red, immature reticulocytes; white, WBCs; green, UPP, upper particle portion. Abbreviations: RET, reticulocyte; RET-EXT, reticulocyte extended; WBC, white blood cell; IRF, immature reticulocyte fraction.

• 

  Fig. 2 Reticulocyte scattergrams (left side of each panel) and reticulocyte extended scattergrams (right side of each panel) of sample no. 5 generated by the Sysmex XE-2100 hematology analyzer. (A) Sample no. 5 with no dilution (WBC count, 218.19×109/L) showing abnormal scattergrams. The WBC fractions seem to overlap with reticulocyte regions, especially with the IRF. (B) After 1:2 dilution (WBC count, 118.53×109/L), the scattergrams were still abnormal. However, in the reticulocyte extended scattergram, the WBC region shifted to the right, because the fluorescence of WBCs increased. (C) After 1:3 dilution (WBC count, 78.56×109/L), the scattergrams were still abnormal. (D) After 1:10 dilution (WBC count, 25.86×109/L), the scattergrams were normalized. Regions are the same as described in Fig. 1. Abbreviations: RET, reticulocyte; RET-EXT, reticulocyte extended; WBC, white blood cell; IRF, immature reticulocyte fraction.

• 

  Fig. 3 Reticulocyte scattergrams (left side of each panel) and reticulocyte extended scattergrams (right side of each panel) of samples nos. 5, 6, and 13 generated by the Sysmex XE-2100 hematology analyzer. Sample nos. 5, 6, and 13 had similar WBC counts (~100.00×109/L), but showed normal or abnormal scattergrams. (A) After 1:2 dilution of sample no. 5 (WBC count, 118.53×109/L), scattergrams were abnormal. (B) Normal scattergrams of sample no. 6 (WBC count, 108.32×109/L). (C) Normal scattergrams of sample no. 13 (WBC count, 101.29×109/L). Regions are the same as described in Fig. 1. Abbreviations: RET, reticulocyte; RET-EXT, reticulocyte extended; WBC, white blood cell.

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