Clin Chem Lab Med 2012;50(2):357–360 © 2012 by Walter de Gruyter • Berlin • Boston. DOI 10.1515/CCLM.2011.

766

Evaluation of nucleated red blood cells in the peripheral

blood of hematological diseases

Paolo Danise1, Mariacaterina Maconi2,*, Introduction

Fabio Barrella1, Anna Di Palma1, Daniela Avino1,
Adele Rovetti1, Maria Gioia3 and Giovanni Amendola4
1 cytes normally present in the peripheral blood of neonates,
Department of Oncology Hematology, ASL Salerno 1,
Salerno, Italy
2 pregnancy (1–3). In all the other conditions the presence of
Department of Clinical Pathology, ASL Alessandria,
Alessandria, Italy
3 the “blood-producing mechanism” (4–6).
Department of Clinical Pathology, “V. Cervello”
Hospital, Palermo, Italy
4 NRBCs in the peripheral blood is associated with some dis-
Department of Pediatrics and Intensive Care Unit, Nocera
Inferiore Hospital, Salerno, Italy
Abstract
Background: Nucleated red blood cells (NRBCs) are pres-
ent in the peripheral blood of several hematological and
non-hematological conditions, usually associated with bad
prognosis. The lack of an easy, rapid and reliable NRBCs
count method did not allow one to know the incidence of
NRBCs and to quantify them: the count was usually done
during the microscopic revision of a blood smear; this is the
reason we found few studies on NRBCs automated count in
the literature. The aim of this study was the evaluation of the
presence and the quantification of NRBCs in some onco-
hematological disorders.
Methods: This study analyzed 478 patients with the auto-
mated hematology analyzer Sysmex XE2100. The range of
NRBCs were calculated in the peripheral blood at diagnosis,
at hematological remission and during therapy.
Results: NRBCs are present in the peripheral blood of a
high number of hematological diseases and are related to
ineffective erythropoiesis or stress erythropoiesis or primary
alterations of hematopoiesis. NRBCs were found in nearly
all onco-hematological diseases at diagnosis, but not in all
patients. NRBCs were frequently found during chemotherapy
and absent at remission.
Conclusions: To the authors’ knowledge, this is the first study
that gives a range for NRBCs count in the peripheral blood of
these diseases.
Keywords: blood cell count; hematological diseases; nucle-
ated red blood cells; peripheral blood.
Nucleated red blood cells (NRBCs) are immature erythro-
but usually disappear in the first few days of life and during
NRBCs in the peripheral blood could indicate a disorder in
It has been known for over a century that the presence of
eases (7–10), mostly hematological disorders, but in this field
the first clinical application of the NRBCs counts was pub-
lished only in 1969 (11).
NRBCs are seen as a reflection of extreme increases in eryth-
ropoietic activity (e.g., in hemolytic acute episodes and severe
hypoxic stress) or as a result of damage to the bone marrow
microenvironment (for example in myelofibrosis, leukemia or
cancer) (12). NRBCs can be present in numerous conditions
in adults: thalassemic syndromes, myeloproliferative diseases
(specifically myelofibrosis), bone marrow metastases of solid
tumors, extramedullary hematopoiesis and other conditions
of hematopoietic stress (e.g., septicemia, massive hemorrhage
and severe hypoxia) (13, 14). In these situations, their presence
is correlated with the severity of the prognosis. Recently, it has
been observed that the entity and the duration of the presence of
NRBCs in peripheral blood is associated with a poor prognosis
in several non-hematological diseases (15).
For a long time, NRBCs were counted using manual micro-
scopic methods, but the limited number of cells counted and
the variability of individual skills restricted the accuracy and
precision of these counts. In addition, such counts were hard-
work and expensive. Many efforts have been made to develop
automated methods for NRBCs counting (16–18). The first
generations of automated hematology analyzers did not have
the ability to signal the presence of NRBCs; further advances
in technology have resulted in the ability of generating a “sus-
pect flag” and, only recently, automated hematology analyz-
ers give us the possibility to count NRBCs quickly and with a
high level of accuracy and precision (19–22).
The aim of this study was the evaluation of the presence
and the quantification of NRBCs in some hematological dis-
orders. NRBCs values were analyzed at diagnosis, during
treatment and at remission.

*Corresponding author: Mariacaterina Maconi, MD, Department

of Clinical Pathology, ASLAL P.zza Cavallotti, 7, 15057 Tortona,
Alessandria, Italy
Phone: +39-3407053321, Fax: +39-0131-865688,
E-mail: mmaconi@aslal.it
Received August 22, 2011; accepted October 3, 2011;
previously published online October 25, 2011
Materials and methods
Study population
The patient population included 478 patients (220 males and 258
females, mean age 57, range 15–95) affected by hematological diseases.

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358 Danise et al.: Nucleated red blood cells and hematological diseases
All patients were analyzed at the diagnosis and in different stages
of the disease: hematological remission, during chemotherapy or
treatment with tyrosin kinase inhibitors in the patients affected by
chronic myeloid leukemia (CML) or during treatment with erythro-
poietin and/or 5-azacytidine in patients with myelodysplastic syn-
drome (MDS).
Methods
A peripheral blood sample was collected in K3 EDTA tubes
(Vacutainer® Beckton-Dickinson) and analyzed on Sysmex XE-2100
(Sysmex Corporation, Kobe, Japan) hematology analyzers within
6 h after collection.
The Sysmex XE-2100 analyzer makes an accurate NRBC analy-
sis with a very good correlation to the microscope count and a flow
cytometry reference method (23, 24). A specific reagent (fluoro-
chrome–polymethine dye) completely lyses the red blood cell (RBC)
and contemporarily enucleates, shrinks and slightly stains the nuclei
of NRBC. The lysing reagent maintains the shape of the white blood
cell (WBC) while intensely staining their intra-cytoplasmic organ-
elles and nuclei. These different staining intensities between NRBC
nuclei and WBCs as well as their differing volumes are detected
by a semiconductor laser using forward-scattered light and fluores-
cence intensities. Clear separation of the two cell populations occurs
(24). Results are reported as proportion of NRBCs per 100 WBCs
(NRBCs/100 WBC) and as absolute NRBC count NRBC #, ×109/L).
Statistical analysis
Data were given as mean and as minimum and maximum value.
The statistical analysis was carried out on a personal computer with
the software Statistical Package for Social Science (SPSS 11.5,
Chicago, IL, USA).
The authors confirmed in writing that they have complied with the
World Medical Association Declaration of Helsinki regarding ethical
conduct of research involving human subjects and/or animals.
Results
The incidence and the amount of NRBCs at diagnosis in
onco-hematological diseases and monoclonal gammopathy
of uncertain significance (MGUS) are shown in Table 1.
We found NRBCs at diagnosis in almost all onco-hemato-
logical diseases, but not in all patients. A high percent of MDS
patients (62.5%) and idiopatic myelofibrosis (IM, 86.6%)
showed NRBCs; in these latter patients mean absolute values
(445) were the highest ones, lower only than in patients with
thalassemia, as described in a previous study (25).
Patients with polycythemia vera (PV), multiple myeloma
(MM), MGUS and Hodgkin’s lymphoma (HL) did not show
NRBCs in the peripheral blood at diagnosis.
In some diseases we observed a different behavior of
NRBCs at diagnosis, during therapy and in the course of
hematological remission; data are shown in Table 2.
During chemotherapy NRBCs were often detectable in
peripheral blood, both in diseases in which they were present
at the diagnosis and in diseases (MM and HL) in which they
were absent at diagnosis. NRBCs are always absent in onco-
hematological patients in hematological remission.
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In all the patients we did not find any correlation between
NRBCs and hemoglobin levels (data not shown).
Discussion
The possibility to detect and count NRBCs in the peripheral
blood quickly, with accuracy and precision, allows us to use
this analysis routinely in the study of hematological disorders
(26, 27).
Our results confirm the presence of NRBCs in hematologi-
cal diseases, determine the percent of incidence in the single
diseases and finally determine the range and the mean value
of the NRBCS in the disorders associated with the presence
of NRBCS.
Depending on the hematological disease, NRBCs incidence
in peripheral blood can range between 0% and 100%. In CML
NRBCs were always found at diagnosis, but absent during
treatment and remission. Only a few with essential thrombo-
cythemia (9.3%) showed NRBCs. These data could be useful
in the differential diagnosis with IM (86% positive). It was
very surprising that we did not find NRBCs in the peripheral
blood of patients with PV: in fact this is a disease character-
ized by clonality and in all the other myeloproliferative dis-
eases we found NRBCs.
We did not find NRBCs in the peripheral blood in patients
with MM and MGUS. So, this is no help for discrimination
between the two diagnoses.
The NRBCs behavior in lymphomas was not homoge-
neous: we did not find NRBCs at diagnosis in HL. In non-HL,
34% of patients have NRBCs at diagnosis; no correlation
was found between presence of NRBCs and bone marrow
infiltration. Also for HL this is no help for discrimination
between this diagnosis and the diagnosis of other hemato-
logical diseases that showed NRBCs in the peripheral blood
at diagnosis.
In all patients we did not find any correlation between
NRBCs and hemoglobin levels: this allowed us to exclude
anemia as a cause alone of NRBCs in the peripheral blood.
Conclusions
The data obtained showed the presence of NRBCs in almost
all hematological disorders: NRBCs were present more-
over in a changeable percentage of patients with a defined
disorder.
During chemotherapy the presence of NRBCs was likely
related to the damage and the subsequent resumption of the
erythropoiesis; on the other hand the hematological remission
was always associated with the disappearance of NRBCs.
In conclusion, the presence of NRBCs in peripheral blood
can be due, in terms of pathophysiology, to the following
three different pathogenetic conditions:
i. ineffective erythropoiesis (e.g., thalassemia syndromes
and myelodysplastic syndromes);
ii. stress erythropoiesis (e.g., erythropoietic resumption after
chemotherapy in onco-hematological diseases); and
 Danise et al.: Nucleated red blood cells and hematological diseases 359

Table 1 Incidence, percent of positive patients, mean values and range of NRBCs at diagnosis in onco-hematological diseases and monoclonal
gammopathy of uncertain significance (MGUS).

Disease Positive/total Positive, % Mean, NRBC values Range; NRBC values

patients absolute count, 109/L absolute count, 109/L

Idiopathic myelofibrosis 13/15 86.6 0.445 0.020 – 0.840

Essential thrombocythemia 4/43 9.3 0.112 0.040 – 0.260
Polycythemia vera 0/30 0 0 0
Chronic myeloid leukemia 30/30 100 0.210 0.040 – 0.430
Myelodysplastic syndromes 30/48 62.5 0.166 0.020 – 0.780
Acute myeloid leukemia 20/40 50 0.105 0.020 – 0.260
Acute lymphoid leukemia 7/21 33 0.064 0.020 – 0.160
Chronic lymphocitic leukemia 4/37 11 0.200 0.020 – 0.490
Non-Hodgkin’s lymphoma 32/92 34.8 0.091 0.020 – 0.950
Hodgkin’s lymphoma 0/35 0 0 0
Plasma cell leukemia 2/3 66.6 0.114 0.020 – 0.170
Chronic myelomonocytic leukemia 3/4 75 0.165 0.020 – 0.560
Multiple myeloma 0/47 0 0 0
MGUS 0/33 0 0 0

Table 2 Incidence of NRBCs at diagnosis, during therapy and at References

hematological remission.
1. Hermansen MC. Nucleated red blood cells in the fetus
Disease Positive at Positive Positive at and newborn. Arch Dis Child Fetal Neonatal Ed 2001;84:
diagnosis, % during hematological F211–5.
therapy, % remission, % 2. Hanlon-Lundberg KM, Kirby RS, Gandhi S, Broekhuizen FF.
Nucleated red blood cells in cord blood of singleton term neo-
Chronic myeloid 100 0 0
nates. Am J Ostet Gynecol 1997;176:1149–58.
leukemia
3. Purwosunu Y, Sekizawa A, Farina A, Okai T, Takabayashi H,
Myelodysplastic 62.5 59.1a 0
Wen P, et al. Enrichment of NRBC in maternal blood: a more
syndromes
feasible method for noninvasive prenatal diagnosis. Prenat Diagn
Acute leukemia 45 39.3 0
2006;26:545–7.
Non-Hodgkin’s 34.8 42.1 0
4. Schwartz S, Stansbury F. Significance of nucleated red blood
lymphoma
cells in peripheral blood: analysis of 1496 cases. J Am Med
Hodgkin’s lymphoma 0 35 0
Assoc 1954;154:1339–40.
Multiple myeloma 0 52.6 0
5. Lesesve JF, Bordigoni P, Maurer P, Lecompte T. Persistent nucle-
a
Erythropoietin and/or 5 azacytidine therapy. ated red blood cells in peripheral blood is a poor prognostic
factor in patients with multiple organ failure. Clin Lab Haematol
2006;28:145.
iii. primary abnormalities of hematopoiesis (e.g., acute myel- 6. Otsubo K, Kaito A, Asai O, Usui N, Kobayashi M, Hoshi Y.
oid leukemia, acute lymphoid leukemia, chronic myeloid Persistent nucleated red blood cells in peripheral blood is a poor
leukemia and idiopatic myelofibrosis). prognostic factor in patients undergoing stem cell transplanta-
tion. Clin Lab Haematol 2005;27:242–6.
To our knowledge, this is the first study that gives a range 7. Da Costa JC Jr. Clinical hematology: practical guide to the exam-
for the NRBCs count in the peripheral blood of these diseases, ination of the blood with reference to diagnosis. Philadelphia,
because previous studies give data only for a single patient PA: P. Blakeston’s Son & Company, 1901:143.
or for a single disorder. Our data can help to understand the 8. Groen J, Godfried EG. The occurrence of normoblasts in the
peripheral blood in congestive heart failure: an indication of
meaning of the presence of NRBC counts in the peripheral
unfavorable prognosis. Blood 1948;12:1445–52.
blood of some hematological diseases.
9. Frumin M, Mendell TH, Solomon SM, Novack P, Faulk AT.
Nucleated red blood cells in congestive heart failure. Circulation
1959;20:367–70.
Conflict of interest statement 10. Clifford GO. The clinical significance of leucoerythroblastic ane-
mia. Med Clin N Am 1996;50:779–90.
Authors’ conflict of interest disclosure: The authors stated that there 11. Piomelli S, Danoff S, Becker M. Prevention of bone malforma-
are no conflicts of interest regarding the publication of this article. tions and cardiomegaly in Cooley’s anemia by early hypertrans-
Research funding: None declared. fusion regime. Ann NY Acad Sci 1969;165:427.
Employment or leadership: None declared. 12. D’Onofrio G, Zini G, Tommasi M, van Hove L. Integration
Honorarium: None declared. of fluorescence and hemocytometry in the CELL-DIN 4000:

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Authenticated
Download Date | 5/26/15 9:12 AM
360 Danise et al.: Nucleated red blood cells and hematological diseases

reticolocyte, nucleated red blood cell and white blood cell viabil-
ity. Lab Hematol 1996;2:131–8.
13. Schaefer M, Rowan RM. The clinical relevance of nucleated red
blood cell counts. Sysmex J Int 2000;10:59–63.
14. Burkett LL, Cox ML, Fields ML. Leukoerythroblastosis in the
adult. Am J Clin Pathol 1996;44:494–8.
15. Stachon A, Holland-Letz T, Krieg M. High in-hospital mortality
of intensive care patients with nucleated red blood cells in blood.
Clin Chem Lab Med 2004;48:933–8.
16. Cornblett PJ, Myrick D, Levy R. Evaluation of the coulter STKS
five- part differential. Am J Clin Pathol 1993;99:72–81.
17. Savion S, Carp H, Shepshelovich J, Irlin J, Kostykov M, Fein A,
et al. Use of the antibodies against the human antigen of erythro-
blasts for the detection of nucleated erythrocytes in the maternal
circulation. Biol Neonate 1997;1:126–30.
18. Sher G, Viitisallo B, Schisano T, Pantalony D, Van Hove L.
Automated NRBC count, a new parameter to monitor, in real
time, individualized transfusion needs in transfusion-dependent
thalassemia major. Lab Hematol 1997;3:129–37.
19. Carcamo C, Ferriz C, Martin P, Fernandez de Castro M.
Improvement in the diagnostic performance of the Coulter STKS.
Lab Hematol 1997;3:264–70.
20. Hrisinko MA, Curcio K, Gupta R. A comparative evaluation of
the Beckman Coulter LH750 and Sysmex XE2100 analyzer with
regard to identification of NRBC’s [abstract]. Int J Lab Hematol
2007;29(Suppl 1):52–3.
21. Ruzika K, Veitl M, Thalhammer-Scherrer R, Schwarzinger I. The
new hematology analyzer Sysmex -2100:performance evaluation
of a novel white blood cell differential technology. Arch Pathol
Lab Med 2001;125:391–6.
22. Rolfo A, Maconi M, Cardaropoli S, Biolcati M, Danise P, Todros
T. Nucleated red blood cells in term fetuses: reference values
using an automated analyzer. Neonatology 2007;92:205–8.
23. Briggs C, Harrison P, Grant D, Staves J, MacHin SJ. New
quantitative parameters on a recently introduced automated
blood cell counter--the XE 2100. Clin Lab Haematol 2000;22:
345–50.
24. Gulati G, Behling E, Kocher W, Schwarting R. An evaluation
of the performance of Sysmex XE-2100 in enumerating nucle-
ated red blood cells in peripheral blood. Arch Pathol Lab Med
2007;131:1077–83.
25. Danise P, Amendola G, Di Concilio R, Cillari E, Gioia M, Di
Palma A, et al. Nucleated red blood cells and soluble trans-
ferrin receptor in thalassemia syndromes: relationship with
global and ineffective erythropoiesis. Clin Chem Lab Med
2009;47:1539–42.
26. Li J. The preliminary study of nucleated red blood cell count-
ing by automated haematology analyzer. Sysmex J Int
2004;14:13–7.
27. Fernandes B, Bergeron N, Hamaguchi Y. Automated nucleated
red blood cell counting in the perinatal period. Lab Hematol
2002;8:179–88.

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