Sternheimer-Malbin Staining to Detect Decoy Cells in Urine of 213

Kidney Transplant Patients

Lizhi Yana, Hongbo Guoa, Lizhong Hanb, Hualiang Huanga, Yan Shenc, Jing Hec, and Jinlin Liuc,d,*
a
Department of Clinical Laboratory, Inner Mongolia Baogang Hospital, Baotou, Inner Mongolia, China; bDepartment of Urology, Inner
Mongolia Baogang Hospital, Baotou, Inner Mongolia, China; cDepartment of Clinical Laboratory, Zhejiang Provincial People’s Hospital,
Hangzhou Medical College, Hangzhou, China; dKey Laboratory of Tumor Molecular Diagnosis and Individualized Medicine of Zhejiang
Province, Hangzhou, China

ABSTRACT
Background. Human polyoma viruseassociated nephropathy frequently refers to
allograft failure after kidney transplant. Thus, the early detection of viral activation is
extremely important for these immunocompromised patients.
Methods. Previously, urine polyoma viruseinfected cells (decoy cells) were indicated as
the virus action, usually screened by the routine papanicolaou cytology in renal biopsy,
but these methods are complex and the positive rate is low. In this article, the direct
microscopy observation method, Wright-Giemsa staining, and Sternheimer-Malbin (SM)
staining were all used to screen the decoy cells in urine samples of 213 kidney transplant
patients who had used immunosuppressive drugs.
Results. Among them, decoy cells were detected in 40 cases (18.8%) by the direct
observation method, 44 cases (20.7%) by Wright-Giemsa staining and 49 cases (23.0%) by
SM staining. Furthermore, the most common polyoma viruses, BK and JC viruses, were
also confirmed in 41 (83.7%) cases among these 49 decoy cellepositive samples.
Importantly, compared with other decoy cell detection methods, SM staining is fast, easy
to operate, and has a high positive rate.
Conclusion. Therefore, SM staining is recommended as a fast and effective method for
screening urine decoy cells in kidney transplant patients.

W ITH immunosuppressive agents, the success rate of

renal transplantation has been greatly improved,
with better survival rates and quality of life for renal
transplant patients. However, these immunosuppressive
agents significantly increase the risk of polyoma virus
infection after surgery. When the human body is infected
with a polyoma virus, the virus can stay in the latent stage or
result in related diseases such as polyoma viruseassociated
nephropathy [1]. At present, 14 kinds of human polyoma
virus have been found, including the most common polyoma
viruses, BK and JC viruses [2]. These viruses can be latent in
renal tubular epithelial cells and urinary tract transitional
epithelial cells. When the host’s immunity becomes
compromised, these viruses can rapidly reactivate and
replicate, resulting in renal tubular epithelial cell necrosis
and a sharp decline or even loss of function in the trans-
planted kidney [3e9]. Therefore, early detection should be
considered to fight against polyoma viruseassociated
nephropathy [2].
Traditionally, an allograft needle biopsy is required for
definitive diagnosis of polyoma viruseassociated nephropa-
thy, but this is not easy for these transplant patients. Eval-
uation of the presence of urine polyoma viruseinfected cells
(decoy cells) is a valuable form of noninvasive screening
method [10]. Previously, papanicolaou cytology is widely
used to screen these cells in different laboratories world-
wide, but the protocol is complex and the positive rate is low
[5,11]. It is not suitable for large-scale and rapid screening
*Address correspondence to Jinlin Liu, MD, PhD, Department
of Clinical Laboratory, Zhejiang Provincial People’s Hospital,
People’s Hospital of Hangzhou Medical College, 158 Shangtang
Road, 310014, Hangzhou, China. E-mail: liujinlinhz@163.com

ª 2020 Elsevier Inc. All rights reserved. 0041-1345/20

230 Park Avenue, New York, NY 10169 https://doi.org/10.1016/j.transproceed.2020.01.044

Transplantation Proceedings, XX, 1e6 (2020) 1

2 YAN, GUO, HAN ET AL
of polyoma virus infection specimens [12,13]. Therefore, in
this article, 213 kidney transplant patients were enrolled.
Sternheimer-Malbin (SM) staining, Wright-Giemsa stain-
ing, and direct microscopy observation methods were all
used to screen the decoy cell in urine samples of these
kidney transplant patients who had used immunosuppres-
sive drugs.
MATERIALS AND METHODS
Patients
Two hundred thirteen renal transplant patients in Inner Mongolia
Baogang Hospital from January 2013 to June 2019 were enrolled,
including 133 men and 80 women. All patients took immunosup-
pressive agents. Two 10-mL urine samples were collected with clean
disposable urine sediment tube. One tube was used for the
screening of the decoy cells under the microscopy. Another spec-
imen was screened for viral nucleic acid detection when decoy cells
of the first urine sample were positive. The studies were approved
by the ethical review board at Inner Mongolia Baogang Hospital.
SM Staining
Ten-milliliter urine samples from kidney transplant patients were
centrifuged at 1500 rpm/min for 10 minutes, the supernatant was
discarded slowly, the bottom sediment was left at 0.2 mL, and then
mixed thoroughly. The samples were subsequently stained with SM
dye solution at the ratio of 5:1 for the sample vs SM dye solution.
After 1 minute of dyeing, 20-mL samples were added to the clean
slide and covered with a cover slide. Then the cells were observed
under a microscope with a low-power lens, and the suspicious decoy
cells under the oil microscope were confirmed by microscope
photographs.
Wright-Giemsa Staining
The urine sample was collected in a clean tube, mixed, and
centrifuged at 1500 rpm/min for 10 minutes. The supernatant was
slowly drained, and 0.2-mL sediment was left in the tubes. Then the
20-mL sediment was mixed and the smear slides were made slowly.
The slides were then slowly dried and subsequently stained with
Wright-Giemsa staining for another 10 minutes, then washed, dried,
and observed under a microscope.
Cytologic Evaluation of Decoy Cells
A light microscope was used to scan each slide methodically for
virus-infected cells, which had enlarged nuclei with a single large
basophilic intranuclear inclusion for the decoy cells. We then
generated a picture using a digital camera. In addition, the number
of decoy cells on each smear was carefully counted. The clinical
information from the patients’ medical records was also reviewed.
Nucleic Acid Detection
Human polyoma virus BK and JC nucleic acid were detected from
decoy cellepositive urine samples. Samples were mixed thoroughly
and shaken for 15 seconds. A 1-mL sample was moved to a 1.5-mL
Eppendorf tube. We then centrifuged the samples for 10 minutes at
13,000 rpm/min and discarded the supernatant. Lysis solution (50
mL) was added to extract the nucleic acid and then amplified by
polymerase chain reaction (PCR) methods. BK virus and JC virus
PCR kits were purchased from the Sinomdgene Company, Beijing,
China.
Table 1. Statistical Analysis of Decoy Cells by Different Methods
No. of Decoy Cell by Direct Decoy Cell by Wright- Decoy Cell by
Patients Observation Giemsa Staining SM Staining
213 cases 40 cases 44 cases 49 cases
Percentage 18.8 20.7 23.0
Abbreviation: SM, Sternheimer-Malbin.
RESULTS
Prevalence of Decoy Cells
We retrospectively examined 213 urine sample specimens
collected at Inner Mongolia Baogang Hospital over a 6-year
period. By the direct microscopy method, decoy cells were
observed in 40 cases, the incidence was 18.8% (Table 1).
The incidence of decoy cells in urine samples was relatively
low. Thus, 2 other methods were also used, including
Wright-Giemsa staining and SM staining. Between them, 44
cases were positive for decoy cells by Wright-Giemsa
staining, and the incidence was 20.7%. But for SM stain-
ing, 49 cases were positive for decoy cells, with an incidence
of 23.0% (Table 1). Therefore, we recommend SM staining
as an effective method for screening urine decoy cells in
kidney transplant patients.
Prevalence of BK and JC Polyoma Virus in Decoy
CellePositive Samples
Decoy cells associated with BK polyoma virus are well
known in urine, with homogeneous, hyperchromatic nuclei
mimicking cancer cells [14]. Furthermore, cells with similar
cytopathic changes have been associated with reactivation
by other polyoma viruses (JC virus) [15]. Therefore, further
identification of nucleic acid by PCR method is recom-
mended to avoid misdiagnosis. Herein, the 2 most common
polyoma viruses, the BK and JC viruses, were examined in
these decoy cellepositive samples (Table 2).
Between them, 16 BK positive polyoma virus cases were
found, with an incidence of 32.7%. For the JC polyoma
virus, 20 positive cases were found, with an incidence of
40.8%. For the BK and JC double positive cases, 5 cases
were found, with an incidence of 10.2%. In total, 41 poly-
oma virus cases were confirmed in 49 decoy cellepositive
samples (Table 2).
Typical Cytology of Decoy Cells by Direct Observation
Method
When renal tubular epithelial cells or urinary epithelial cells
are infected with polyoma virus, the virus is replicated in the
cells, destroying the structure of the nucleus and causing the
significant characteristic changes of the nucleus. Previously,
Table 2. Statistical Analysis of Viral Nucleic Acid Positive
Samples in Positive Decoy Cell Cases
BK and JC
BK Positive JC Positive Double Positive Total
49 decoy cell samples 16 20 5 41
Positive rate 32.7% 40.8% 10.2% 83.7%
DECOY CELLS IN KIDNEY TRANSPLANTATION 3

Fig 1. Direct observation method for the decoy cells. (A) The cell and the nucleus are large (1000). (B) The ratio of nucleus to cyto-
plasm increased and the nuclear membrane became thicker (1000). (C) The chromatin structure was destroyed with coarse granular
shape and intranuclear inclusions as indicated by the red arrow (1000).

papanicolaou cytology was widely used to screen the decoy
cells in different laboratories worldwide, but the protocol is
complex and the positive rate is low [5,11]. First, we eval-
uated the direct observation method for decoy cells in urine
samples with centrifuge. Under microscopy, decoy cells have
a larger body, higher ratio of nucleus to cytoplasm, larger
nucleus, thicker nuclear membrane, and even naked nucleus
in the direct observation method without the staining (Fig
1AeC). But compared to other staining methods, the
direct observation method has unclear cell structure, and
when the number of decoy cells is small, it is easy to miss
these cells.
Typical Cytologic Findings of Wright-Giemsa Staining
Thus, 2 other staining methods were used for the decoy cell
evaluation. In Wright’s staining, we found that decoy cells
are characterized by large cell body, incomplete parts of the
cell body, sometimes naked nucleus, light purple-red cyto-
plasm, increased nuclear-cytoplasmic ratio, large nucleus,
coarse granular or massive chromatin, and nucleoli that are
not obvious or even disappear (Fig 2AeC). Compared with
SM staining, Wright-Giemsa staining requires the smear
method. In the process of smearing, decoy cells may be
destroyed, making the cell incomplete or unable to maintain
the original state of cells. Thus, the characteristic changes
are not obvious, especially when the number of decoy cells is
small, and the decoy cells are easy to miss.
Typical Cytologic Findings of SM Staining
We then used SM staining to identify the decoy cells. These
cells are characterized by large volume, different cell size,
irregular shape, and coarse granular chromatin, and some
virus-infected cells display inclusion body. Some cells even
display prominent features, including large nucleus, nuclear
deviation, thickening of nuclear membrane, markedly
higher nuclear-cytoplasmic ratio, destruction of nuclear
chromatin structure with coarse granular or massive feature,
vacuole-like changes in the nucleus, nucleolus disappearing
or not obvious, incomplete cell bodies, nuclear exfoliation,
and sometimes only naked nucleus (Fig 3AeG). Addition-
ally, in all urine decoy cellepositive cases, about 40% of the
patients had different proportions of phagocyte (Fig 3H, I).

Fig 2. Wright-Giemsa staining for the decoy cells. (A) The cell is large and incomplete, the nucleus is large, and the nucleolus disap-
pears (1000). (B) The ratio of nucleus to cytoplasm increased, the cytoplasm is purple red, and the binuclear decoy cells can be
observed (1000). (C) The ratio of nucleus to cytoplasm is high, some cells are fragmented, and the chromatin is coarse granular
(1000).
4 YAN, GUO, HAN ET AL

Fig 3. Sternheimer-Malbin (SM) staining for the decoy cells. (A) Case 1: Cell volume increased after human polyoma virus infection. (B)
Case 2: The size of cell body varies with different volume, the structure of nuclear chromatin destroyed, the nucleus is vacuolar and the
nuclear membrane is obviously thicker. (C) Case 3: The cell volume is irregular. (D) Case 4: The cell volume was larger and the ratio of
nucleus to cytoplasm was high. (E) Case 5: Binuclear decoy cells with thickening nuclear membrane. (F) Case 6: Cell denucleation with
incomplete cell structure, and some decoy cells even display naked nucleus. (G) Case 7: Higher cell nuclear-cytoplasmic ratio with
significantly thicker nuclear membrane, and some cells even display visible larger nuclear inclusion body. (H and I) Case 8, 9: The decoy
cells were accompanied by phagocytes, which engulfed the human polyoma viruseinfected cells. All (1000).

DISCUSSION urine by PCR usually precedes renal biopsy. Although PCRs

The effect of polyoma virus infection on a transplanted
kidney has been clear. A large body of literature has re-
ported that renal transplant patients with long-term use of
immunosuppressive agents are susceptible to polyoma virus
infection or viral replication in latent infection state,
resulting in polyoma viruseassociated nephropathy. Current
guidelines recommend that virus reactivation should be
monitored by regular screening for viral nucleic acids in
blood or decoy cells in urine [16,17]. Determination of
urinary release of decoy cells and viral load in plasma and
have better positive predictive value and are more accurate
than urine cytology to predict polyoma virus infection, they
are costlier and not universally available [18].
For the decoy cell, routine papanicolaou cytology
commonly was used to screen decoy cells worldwide. This
method requires many steps, such as preparing, fixing, and
staining. The operation is relatively tedious, and the
detection rate of positive cells is relatively low [11]. Chakera
et al reported that decoy cell positivity occurred in 56 of 313
patients (17.9%) [18]. Poloni et al reported the rate of
DECOY CELLS IN KIDNEY TRANSPLANTATION
bright field microscopy to detect decoy cells is 14.7% of
patients (15/102 patients) [19]. Additionally, decoy cells
were observed in 237 of 1138 urine samples (21%) [17].
In addition, many laboratories use phase contrast
microscopy to examine decoy cells, but the characteristics
of these decoy cells frequently change, and it is not easy to
accurately detect them [12]. In our study, the direct
observation method has unclear cell structure, so when the
number of decoy cells is small or there is no significant
morphologic change, it is easy to miss these cells. For
Wright-Giemsa staining, decoy cells may be destroyed
during the smear process, which makes it impossible to
maintain the original state of cells. Importantly, SM
staining is a kind of in vivo staining method in which the
dye is added directly to the urine sediment. The main
components are yellow sand and violet crystal. Despite the
different chemical nature of urine cells, the staining cells
remain in the original state. In addition, this method is easy
and fast and can be observed within 1 minute, so it is not
easy to miss these decoy cells even though the urinary
sediment SM staining was affected by urine pH. The
staining cells were slightly different, but it did not affect the
decoy cell morphology. Additionally, in the process of
urine decoy cell classification, 40% of the cases were
accompanied by the increase of phagocytes in different
proportions. Originally, the phagocytes originated from
monocyte macrophage system. They could be increased in
urinary tract diseases such as bacterial or viral infection. In
these urine samples, we found that phagocytes could
swallow the leukocyte, erythrocyte, or even the decoy cells
could be swallowed.
Viral nucleic acid detection is the best method for
detection of polyoma virus. BK and JC viruses infect the
urinary tract, establishing lifelong persistent infections that
usually are asymptomatic. JC viruseassociated nephropathy
is considered rare in comparison with nephropathy caused
by the BK virus [17]. Therefore, the BK virus more
frequently triggered the appearance of decoy cells than did
JC virus at equivalent viral titers [17]. However, in this
article, results showed that the JC virus triggered more
decoy cell than the BK virus (40.8% vs 32.7%). Addition-
ally, in 16.3% of the decoy cellepositive cases, the detection
of BK and JC virus nucleic acid was within the normal
range, which may be due to the cellular change of the urine
cells by the latency of polyoma virus in the body. In addition,
other kinds of polyoma viruses can also result in the
appearance of decoy cells, but most hospitals only check for
BK and JC viral nucleic acids. Other polyoma viruses could
also result in this cellular change for the decoy cell forma-
tion [20]. But other unknown viral subgroups and adeno-
virus infection can also lead to decoy cell formation in the
urine.
At present, the use of urine cytology automated micro-
scopy could significantly improve the efficiency of decoy cell
screening [13]. Moreover, if the SM staining method were
also used combined with automated urine screening, decoy
cell detection would be more efficient and accurate. Early
5
clinical intervention can reduce the recurrent polyoma virus
replication, stabilize the function of the transplanted kidney,
and improve the survival rate of these patients.
REFERENCES
[1] Korneffel K, Gehring B, Rospert D, Rees M, Ortiz J. BK
virus in renal transplant patients using alemtuzumab for induction
immunosuppression. Exp Clin Transplant 2019. https://doi.org/10.
6002/ect.2019.0041.
[2] Demey B, Tinez C, Francois C, et al. Risk factors for BK
virus viremia and nephropathy after kidney transplantation: a sys-
tematic review. J Clin Virol 2018;109:6e12.
[3] Her T, Schutzbank TE. Evaluation of the Luminex
ARIES(R) system for the detection and quantification of BK virus
(BKV) DNA in plasma samples from kidney transplant recipients.
Diagn Microbiol Infect Dis 2019;94:129e34.
[4] Zhou Y, Yao L, Yu Z, et al. 多瘤病毒在肾移植受者中的感
染特征 [Characteristics of BK polymavirus infection in kidney
transplant recipients]. Nan Fang Yi Ke Da Xue Xue Bao 2019;39:
120e4 [in Chinese].
[5] Freedman BI, Kistler AL, Skewes-Cox P, et al. JC polyoma
viruria associates with protection from chronic kidney disease
independently from apolipoprotein L1 genotype in African Amer-
icans. Nephrol Dial Transplant 2018;33:1960e7.
[6] Saribas AS, Coric P, Bouaziz S, Safak M. Expression of novel
proteins by polyomaviruses and recent advances in the structural
and functional features of agnoprotein of JC virus, BK virus, and
simian virus 40. J Cell Physiol 2019;234:8295e315.
[7] Skulratanasak P, Mahamongkhonsawata J, Chayakulkeereeb M,
Larpparisutha N, Premasathiana N, Vongwiwatana A. BK virus infec-
tion in Thai kidney transplant recipients: a single-center experience.
Transplant Proc 2018;50:1077e9.
[8] Dao M, Pecriaux A, Bessede T, et al. BK virus-associated
collecting duct carcinoma of the renal allograft in a kidney-
pancreas allograft recipient. Oncotarget 2018;9:15157e63.
[9] Munoz-Gallego I, Moral N, Pascual C, Alonso Y,
Folgueira L. BK virus viral load: analysis of the requests received by
the microbiology laboratory and clinical involvement of the issued
results. Eur J Clin Microbiol Infect Dis 2019;38:1969e73.
[10] Chen TW, Chen CY, Lin NC, et al. How to improve the
positive predictive value of urinary decoy cell surveillance for pol-
yomavirus BK-associated nephropathy in kidney transplant pa-
tients. Transplant Proc 2016;48:924e8.
[11] Pillai KR, Jayasree K, Pisharody R, Abraham EK. Decoy
cells in the urine cytology of a renal transplant recipient: an
immunohistochemical study. Indian J Pathol Microbiol 2010;53:
347e50.
[12] Yamada Y, Tsuchiya T, Inagaki I, Seishima M, Deguchi T.
Prediction of early BK virus infection in kidney transplant recipients
by the number of cells with intranuclear inclusion bodies (decoy
cells). Transplant Direct 2018;4:e340.
[13] D’Alessandro M, Poli L, Lai Q, et al. Automated intelligent
microscopy for the recognition of decoy cells in urine samples of
kidney transplant patients. Transplant Proc 2019;51:157e9.
[14] Kimura M, Hayashi T. Significance of decoy cells for indi-
cating viral cytopathic effect in urine cytology. Cytopathology
2016;27:220e1.
[15] Funahashi Y, Iwata S, Ito Y, et al. Multiplex real-time
PCR assay for simultaneous quantification of BK polyomavirus,
JC polyomavirus, and adenovirus DNA. J Clin Microbiol 2010;48:
825e30.
[16] Nankivell BJ, Renthawa J, Jeoffreys N, et al. Clinical utility
of urinary cytology to detect BK viral nephropathy. Transplantation
2015;99:1715e22.
[17] Funahashi Y, Kato M, Fujita T, Ishida S, Mori A, Gotoh M.
Association between the polyomaviruses titers and decoy cell positivity
rates after renal transplantation. Transplant Proc 2016;48:921e3.
6 YAN, GUO, HAN ET AL

[18] Chakera A, Dyar OJ, Hughes E, Bennett S, Hughes D,
Roberts IS. Detection of polyomavirus BK reactivation after renal
transplantation using an intensive decoy cell surveillance program is
cost-effective. Transplantation 2011;92:1018e23.
[19] Poloni JA, Pinto GG, Giordani MS, et al. Bright field mi-
croscopy to detect decoy cells due to BK virus infection in the fresh
and unstained urine sediment in kidney allograft recipients. J Clin
Lab Anal 2016;30:1044e50.
[20] Assis P, Carvalho CE, Silva MS, Ribeiro B, Carvalho MDG.
JC and BK virus DNA detection in archival slides of urine cyto-
spin from renal transplant patients. Transpl Infect Dis 2018;20:
e12901.