ANNALS O F CLINICAL AND LABORATORY SCIENCE, Vol. 23, No. 2 Copyright © 1993, Institute for Clinical Science, Inc.

Artifactual Distortion of Cells Simulating Metastatic Small Cell Carcinoma in the Bone Marrow*

NORBERTO CARTAGENA Jr., M.D., SAUL SUSTER, M.D., and BERIA CABELLO-INCHAUSTI, M.D.

Arkadi M. Rywlin Department o f Pathology if Laboratory Medicine,

Mount Sinai Medical Center of Greater Miami,and

the University of Miami School o f Medicine,Miami, FL 33140

ABSTRACT

Artifactual distortion of hematopoietic elements in histologic prepara­tions and smears of bone marrow aspirates simulating metastatic small cell carcinom a has been recently described. The clinical, pathologic, and immunohistochemical features in 12 such cases have been studied by us.In all these cases, bone marrow smears and histologic sections showed m ultiple small clusters of atypical, hyperchromatic cells suspicious for metastatic small cell carcinoma. Immunohistochemical stains showed focal positivity of the suspicious cells for leukocyte common antigen (LCA), anti-hem oglobin A (Hem A) and anti-erythrocyte m em brane antigen (ERM), and negative staining for epithelial membrane antigen (EMA) and neuron specific enolase (NSE). Clinical follow-up of two years did not demonstrate any evidence of a primary small cell carcinoma in the lung or elsewhere.

Comparison of these 12 cases with four cases of transbronchial biopsy- proven metastatic small cell carcinoma to the bone marrow showed that this artifact had a tendency to be located at the periphery of the marrow particles, unlike the true metastatic carcinoma cells which were predom i­nantly found within the marrow particles and replacing the hematopoietic elem ents. The results of our immunohistochemical studies appear to indi­cate that these cells correspond to aggregated nuclei and cytoplasmic rem ­nants from erythroid as well as myeloid and lymphoid cells. The main importance of identifying this artifact lies in avoiding confusion with m eta­static small cell carcinoma, a distinction that may be very difficult to estab­lish on morphologic grounds alone. Immunohistochemical stains and a tho rough c lin ica l fo llow -up are necessary for arriv ing at the cor­rect diagnosis.

*Address reprint requests to: Saul Suster, M.D., Department of Pathology, Mount Sinai Medical Center, 4300 Alton Road, Miami Beach, FL 33140.

1300091-7370/93/0300-0130 $01.20 © Institute for Clinical Science, Inc.

CELLS SIMULATING METASTATIC SMALL CELL CARCINOMA IN BONE MARROW 131

Introduction

N u m e ro u s a r t i f a c t s h a v e b e e n described in histologic sections of aspi­rated bone marrow .3 Recently, the pres­ence of clum ped or crushed cells simu­lating a malignant small cell carcinoma in sections and smears of bone marrow aspi­ra tes has b e e n re c o g n iz e d .1,6 T hese clum ped cells have been thought to rep­resen t erythroblasts, and some of the hypotheses offered to explain this artifact include anticoagulation with ethylenedi- amine tetraacetic acid (EDTA) or hepa­rin , or th e p re s e n c e of m em b ran e- bound antibody .1,6

Twelve cases of smears and histologic sections of bone marrow aspirates show­ing clumps of hyperchromatic cells simu­lating m etastatic small cell carcinom a have been studied and have been com­pared with four cases of transbronchial biopsy-proven metastatic small cell carci­noma to the bone marrow. The results of im m unohistochem ical stains in these cases are discussed, as well as the mor­phologic features that may serve to dis­tinguish them from true metastatic small cell carcinoma to the bone marrow.

Materials and Methods

Twelve cases of bone marrow aspirates showing clumps or clusters of hyperchro­m atic cells susp ic ious for m etasta tic small cell carcinom a w ere iden tified from the Surgical Pathology files at the Mount Sinai M edical Center of Greater Miami betw een 1985 and 1988. The bone marrow aspirates were performed in the posterior iliac crest or in the sternum. Additionally, 11 gauge Jamshidi needle biopsies performed at the same time as the aspirates w ere also availab le for review in three patients (cases 8, 10, and 11). For controls, four cases of transbron­chial biopsy-proven metastatic small cell carcinoma of the lung to the bone marrow were also examined.

The bone marrow aspirates in cases 1 to 10 as well as the four cases of m eta­static small cell carcinom a w ere p ro ­cessed by a technique described by Ryw- lin et al,4 whereby smears were prepared directly from the marrow particles, and the remaining particles were placed in formalin, filtered, and processed to yield sections of concentrated bone marrow particles for conventional histology. The aspirated bone m arrow particles from case 11 were placed in EDTA, smears were then prepared, and the remaining particles were pretreated with formalin, centrifuged into a pellet, and em bedded in paraplast for sectioning. The aspirated bone marrow particles in case 12 were first allowed to clot and then fixed in for­malin for processing. In all cases, bone marrow smears were done directly from the aspirated bone m arrow and w ere s ta in e d w ith W rig h t-G ie m sa . T h e remaining bone marrow particles were em bedded in Paraplast after fixation, and 4-m icron thick sections w ere sta ined with hematoxylin and eosin (H&E) and Gomori’s Prussian blue for hemosiderin.

For immunohistochemical studies, the following antibodies were em ployed by the avidin-biotin complex im m unoper- oxidase techn ique :5 mouse anti-hum an m onoclonal a n tib o d ie s to e p ith e lia l membrane antigen (EMA) and leukocyte common antigen (LCA);* m ouse an ti­human monoclonal antibodies to erythro­cyte m em brane an tig e n (ERM ) and hemoglobin (H e);t rabbit antisera to neu­ron specific enolase (NSE), mouse anti­human monoclonal antibody to hemoglo- bin-A (Hem A), linking antibodies, and r a b b it p e ro x id a s e -a n t ip e ro x id a s e ; t mouse anti-human monoclonal antibod­ie s^ and avidin-biotin-complex kits for demonstration of mouse monoclonal anti­

* Dako, Santa Barbara, CA.t Accurate, Westbury, NY.t Biogenex, Dublin, CA.§ Cooper Biomedical, Malvern, PA.

132 CARTAGENA, SUSTER, AND CABELLO-INCHAUSTI

bodies.11 The chromogen, diaminobenzi- dine, was used for antibody localization. All s lides w ere co u n te rs ta in e d w ith hematoxylin, dehydrated, and mounted in perm ount. Positive and negative con­trols were run concurrently for all anti­bodies tested.

Results

C l in ic a l F in d in g s

Eight of the patients w ere men and four w ere wom en. T heir ages ranged from 13 to 87 years (mean, 67). The rea­son for the bone m arrow p rocedu re ranged from evaluation of anemia or neu­tropenia (eight cases) to staging for can­cer (malignant lymphoma in two, naso­pharyngeal carcinoma in one, and breast cancer in one). All patients w ere fol­lowed for a minimum of two years or until their time of death. Two of the patients expired six and 12 months following the procedure, one owing to diffuse m eta­static spread of nasopharyngeal squa­mous cell carcinoma and the other owing to heart failure. None of the remaining patients showed evidence of the devel­opm ent of a small cell carcinoma in the lung or elsewhere. Seven patients were typed and crossmatched prior to transfu­sion. None of these patients revealed a c irc u la tin g a n tib o d y , no r w ere any Coombs positive test results recorded.

P a t h o l o g ic F in d in g s

Examination of the H&E stained sec­tions of bone m arrow aspirates in all cases sh o w ed n um erous c lu s te rs of clum ped, hyperchrom atic, occasionally crushed cells contained m ostly naked nuclei with small amounts of attached cytoplasm (figures 1 A and 1 B). Identical clumps of cells were also observed in the

11 Vector, Burlingame, CA.

corresponding G iem sa stained smears prepared from the bone marrow particles, regardless of w hether or not the aspirated bone marrow material had been exposed to an anticoagulant (such as EDTA). The clum ps o f susp icious cells w ere no t present in any of the bone marrow core n e e d le (Jam shid i) b io p sie s s im u lta ­neously obtained in three of the cases. The four cases of transbronchial biopsy- proven small cell carcinoma of the lung show ed on bone marrow exam ination similar clusters of small, hyperchromatic cells with scant cytoplasm (figures 2 A and 2 B). These clusters of atypical cells were likewise only present in the Giemsa smears and aspirated particle prepara­tion, bu t were not detected in the needle core b iopsy sections. C om parison of these four cases with the 12 cases con­taining the artifact revealed subtle differ­ences. In the histologic sections, the clum ped cell artifact frequently showed a tendency to be located in the periphery of the bone marrow particles or surround­ing deposits of fibrin (figure 1). In con­trast, in the cases of metastatic small cell carcinoma, the clumps of m alignant cells showed a tendency to be present w ithin the bone m arrow particles rep lac ing hem atopoietic tissue (figure 2). In the bone marrow smears, the cells of m eta­static small cell carcinom a con tained more cytoplasm and their nuclei showed better nuclear detail with some degree of moulding. In contrast, in the clum ped cell artifact, the nuclei showed more vari­ability in size and shape as well as the presence of occasional m ultilobed nuclei showing a tendency to overlap. No dis­cernible chrom atin pattern or nuclear detail could be appreciated in these cells.

Im m unohistochem ical stains could only be performed in nine of the 12 cases. Stains for Hem A, ERM, He, and LCA showed focal positivity in several of the cellular elem ents w ithin the clum ped cell artifact. Stains for EM A and NSE were uniformly negative in these areas.

CELLS SIM ULATING METASTATIC SMALL CELL CARCINOMA IN BONE MARROW 133

Occasional cells or cell fragments, some containing m ultilobed nuclei, showed pale, finely granular positivity typical of that seen due to endogenous peroxidase activity in red cells and polymorphonu­clear leukocytes. In the cases of biopsy- proven metastatic small cell carcinoma, the lym phoid and e ry th ro id m arkers tested were negative in the suspicious clumps, and there was focal peroxidase activity for NSE in some of the cells. Stains for EMA were negative in all of the cases tested.

Discussion

The presence of clum ped cells in the bone marrow simulating metastatic small c e ll c a rc in o m a h as b e e n re c e n tly reported .1,6 A series of 12 such cases has been studied by the present authors in an effort to define better the morphologic and im m unohistochem ical features of this phenomenon. Contrary to what had been previously postulated, the presence of th is a rtifac t in the bone m arrow appears to be unrelated to the use of anti­coagulants. In our study, smears were prepared from aspirated bone marrow particles in 10 cases prior to the introduc­tion into formalin or an anticoagulant. T he su sp ic ious c lu m p ed cells w ere p re se n t in both the G iem sa sta ined smears and in the Paraplast-em bedded bone marrow sections. In one of our cases (case 1 1 ), th e asp ira ted m aterial was introduced into EDTA prior to the prepa­ration of the sm ears, as in the case reported by Vernon.6 The clum ped cells noted were identical to those observed in the non-EDTA treated cases. None of our cases were collected in heparin, as was the case reported by Krause.1

In the serum of the two previously reported cases of clum ped cells in the bone marrow ,1’6 IgG specific antibodies (C oom bs’ positive te s t resu lts) w ere detected. Upon review of the patients’ transfusion records in our cases, seven of

the 12 patients had been typed and cross­matched. In none of those seven patients were Coombs positive results reported in the routine crossmatch. Thus, red blood cell membrane bound antibodies did not appear to play a role in the induction of this phenomenon in our cases. Also, the fact that this artifact was only present in smears and sections obtained by aspira­tion and they were not seen in the core b iopsy specim ens o b ta in e d s im u lta ­neously (in three of the cases) w ould seem to suggest that this artifact may be related to the aspiration procedure itself, perhaps as a result of the shearing forces exerted on the cells as they pass through th e n e e d l e d u r i n g t h e a s p i r a ­tion procedure.

It has been suggested in previous stud­ies that the clum ped cells resem ble red cell precursors.1,6 In order to substantiate this theory, im m unoperoxidase stains were performed for red cell antigens and leukocyte common antigen. Evaluation of these stains, which are cytoplasmic and membrane markers, was made diffi­cult by the fact that the majority of the cells in the clum ped artifact consisted of naked nuclei w ithout cytoplasm; how ­ever, a few of the cells contained either fragments of or intact cytoplasm. Most of these cells show ed positiv ity for red blood cell an tigens, and a few w ere positive for LCA. Some of the clum ped cells also showed a pale, finely granular staining pattern similar to that produced by the endogenous peroxidase activity of po lym orphonuclear leukocytes. H igh power examination showed m ultilobated nuclei in some of these cells, suggesting a granulocytic lineage. Thus, imm unoper­oxidase stains and morphology appeared to indicate that the clusters of suspicious cells were composed of m yeloid and ery­throid cells admixed with lymphocytes.

Comparison of immunohistochemical staining in the group of cases containing the artifact and the cases of b iopsy- proven metastatic small cell carcinoma

134

FIGURE 1. (A) Low-power view of bone marrow aspirate showing cluster of clumped cells (center)simulating metastatic small cell carcinoma. Notice that the clumped cells appear lying free and unrelated to the bone marrow particles (xlOO); (B) Higher magnification from hematoxylin and eosin [H&E] same cluster showing overlapping cells with naked nuclei, very poor nuclear detail, and an overall “smudged”, dirty appearance (H&E, oil, x 1,000).

proved of lim ited value in differential diagnosis because of the paucity of ele­m ents d isp lay ing positive stain ing in both groups. In general, however, the

metastatic small cell carcinoma elem ents showed isolated, focal positive staining w ith NSE antibodies and w ere com ­pletely negative for the erythroid and

CARTAGENA, SUSTER, AND CABELLO-INCHAUSTI

CELLS SIMULATING METASTATIC SMALL CELL CARCINOMA IN BONE MARROW 135

FIGURE 2. (A) Low-power view of bone marrow aspirate showing cluster of true metastatic small cell carcinoma to the bone marrow (center). Notice that the cluster of metastatic small carcinoma cells is seen within a bone marrow particle and partially replacing it (hematoxylin and eosin [H&E] x 100). (B) Higher magnification of metastatic small cell carcinoma to the bone marrow showing more nuclear detail with distinct chromatin pattern, scant rims of cytoplasm, and moulding of the cells (H&E, oil, X 1,000).

lymphoid markers. The focal endogenous p e ro x id ase ac tiv ity obse rv ed in th e clum ped cell artifact, on the other hand, could be mistakenly interpreted as NSE positivity if evaluated out of context.

Also, NSE positivity may not always be expressed in small cell carcinoma, or the staining reaction may be very focal, thus limiting the usefulness of this antibody. This artifact should also be distinguished

136 CARTAGENA, SUSTER, AND CABELLO-INCHAUSTI

from a similar population of lymphoid­like cells term ed hem atogones, which may be found in bone marrows (espe­cially of children) with benign processes or w ith malignant lymphomas elsewhere, in which case they may simulate malig­nant lymphoma involvement of marrow .2 H em atogones, how ever, do not form dense accumulations and thus appear to differ from the artifact described in the present paper.

The most important aspect of this arti­fact lies in its potential confusion with metastatic small cell carcinoma. The two may be extremely difficult to separate on morphologic grounds. Some of the distin­guishing features that may be of aid in making this distinction include the topo­graphical location of the clum ped cells in histologic sections of bone marrow, and careful cytologic examination of the bet­te r p rese rv ed areas in bone m arrow smears. In our study, the clum ped cell artifact showed a tendency to be located in the periphery of the bone marrow par­ticles in histologic sections of aspirated marrow, w hile the cells of m etastatic small cell carcinoma tended to be present w ithin the bone marrow particles them ­selves replacing the native hematopoi­e tic e le m en ts . In the bone m arrow sm ears, sm all cell carcinom a usually showed a scant amount of cytoplasm with some degree of nuclear detail and dis­

crete moulding of the nuclei, whereas the c lum ped cell artifact show ed m ainly naked nuclei with only rare shreds of attached cytoplasm, and the cells showed a ten d e n c y to overlap , w ith o u t any d e g re e o f m o u ld in g or a p p re c ia b le nuclear detail. The most reliable crite­rion for diagnosis, however, was a thor­ough c lin ic o p a th o lo g ic c o rre la tio n . Crushed cells in the bone marrow should be addressed with caution by the pathol­ogist, and thorough clinical evaluation should be recom mended before a diagno­sis of m etastatic small cell carcinom a is rendered.

References

1. Kr a u s e , J. R.: Erythroblasts that simulate a metastatic tumor. Arch. Pathol. Lab. Med. 110: 94, 1986.

2. M c K e n n a , R. W., B l o o m f i e l d , C. D., and BRUNNING, R. D.: Nodular lymphoma: Bone marrow and blood manifestations. Cancer 36: 428-432, 1975.

3. R y w l in , A. M .: Histopathology o f the Bone Marrow. Boston, Little, Brown and Company, 1976, pp. 180-190.

4. R y w l in , A. M ., M a r v a n , P., and R o b in s o n , M . J.: A simple technic for the preparation of bone marrow smears and sections. Am. J. Clin. Pathol. 53:389-393, 1970.

5. S t e r n b e r g e r , L. A.: Immunocytochemistry, 2nd ed. New York, John Wiley & Sons, Inc, 1979.

6. VERNON, S. E.: Clumping of erythroblasts simulating a metastatic neoplasm. Arch. Pathol. Lab. Med. 109:569-570, 1985.