Blast-Like Cells in Cerebrospinal Fluid of Neonates
Possible Germinal Matrix Origin
JOHN R. FISCHER, M.D., DIANE D. DAVEY, M.D., MARGARET L. GULLEY, M.D., AND JAMES A. GOEKEN, M.D.
Primitive cell clusters (PCCs) composed of immature blast-like
cells were observed in Wright's-stained cerebrospinal fluid (CSF)
cytocentrifuge specimens from four infants over an 18-month
period. All of these patients had hydrocephalus; in three this
was secondary to subependymal germinal matrix intraventricular
hemorrhage (IVH) associated with prematurity. The fourth was
associated with Arnold-Chiari malformation, Chiari type II. In
the CSF samples from the patients with IVH, hemosiderin-laden
macrophages were also prominent and, in some cases, were intimately admixed with the PCCs. Immunoperoxidase staining of
cytocentrifuge preparations from one of the patients revealed
that the PCCs stained with neuron-specific enolase (NSE) but
not with pan-leukocyte antibodies. Cells with similar morphologic
characteristics to PCCs in the CSF of infants have been infrequently illustrated in the literature and thought to be of hematopoietic origin. However, the immunohistochemical findings and
the clinical presentation suggest that PCCs are most likely germinal matrix cells. (Key words: Germinal matrix cells; Cerebrospinal fluid cytology; Cytospin; Immunoperoxidase; Intraventricular hemorrhage) Am J Clin Pathol 1989;91:255-258
EXAMINATION OF WRIGHT'S-STAINED cytocentrifuge preparations of cerebrospinal fluid (CSF) specimens is a routine procedure in our laboratory. Occasionally we have noted clusters of immature cells in CSF specimens from neonates that raised the question of
malignancy on morphologic grounds. Such cells have been
previously described as "undifferentiated leptomeningeal
cells."' This study is an attempt to further characterize
the nature of these cells by immunohistochemical and
clinical methods.
Methods
All CSF specimens with immature cells are reviewed
by a pathologist in the hematology laboratory at the University of Iowa Hospitals. By this review, four patients
with primitive cell clusters (PCCs) over an 18-month period in 1986 and 1987 were identified and charts reviewed.
CSF specimens were cytocentrifuged at 100 X g for 10
Received April 26, 1988; received revised manuscript and accepted
for publication August 1, 1988.
Presented in part at the annual meeting of the United States and Canadian Academy of Pathology, Washington, D.C., March 1988.
Address reprint requests to Dr. Goeken: Department of Pathology,
University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242.
255
Department of Pathology, University of Iowa Hospitals and
Clinics, Iowa City, Iowa
minutes (Shandon, Inc., Pittsburg, PA), and a Wright's
stain or immunostains were applied.
The immunostains were performed after acetone fixation by the avidin-biotin-peroxidase technique with the
use of the following primary antibodies: (1) polyclonal
neuron-specific enolase (NSE) (Biogenex Laboratories,
Dublin, CA); (2) pan-leukocyte cocktail composed of 25
nL each of the following monoclonal antibodies in 500
nh TRIS buffer: T29/33 mouse anti-common leukocyte
antigen (Hybritech, Inc., San Diego, CA); HB12 pan-leukocyte antibody (prepared locally from the American
Type Culture Collection clone GAP 8.3); DAKO LC
mouse anti-leukocyte common antigen (DAKO Corporation, Santa Barbara, CA); and (3) normal rabbit and
mouse serum to serve as a negative staining control.
Primary staining for 60 minutes was followed by secondary staining with biotinylated anti-Ig (BioGenex) for
30 minutes, labeling with peroxidase-conjugated biotinavidin complexes (BioGenex) for 30 minutes, and color
development with 3-amino-9-ethylcarbazole (AEC; prepared just before use by 1:150 dilution in sodium acetate
buffer of stock 3 g/dL AEC in N,N dimethyl formamide).
Positive control staining on frozen section tissue was assured for each primary antibody. The slides were counterstained with Mayer's hematoxylin and coverslipped
with aqueous mounting media (Aqua-mount®, Lerner
Laboratories, New Haven, CT).
Results
The clinical characteristics of the four patients and
chronology of their CSF problems are listed in Table 1.
Patients 1-3 were premature males who had respiratory
distress syndrome and intraventricular hemorrhage. Patient 4 was a term male with Arnold-Chiari malformation,
Chiari type II (hydrocephalus, absent corpus callosum,
and small cerebellum), a lumbosacral meningomyelocele,
and bilateral clubbed feet. An ultrasonic examination on
patient 4 showed no evidence of intraventricular hem-
256
A.J.C.P. • March 1989
FISCHER ET AL.
Table 1. Characteristics of Patients with Primitive Cell Clusters in CSF
IVH
Gestational Age
(weeks)
Birth Weight
Patient
(g)
IVH*
(day noted)
(grade)
Hydrocephalus
(day noted)
PCCf
(day[s] noted)
VP Shunt*
(day placed)
1
2
3
28
32
28
1,240
1,770
1,140
8
1
4
II
III
IV
21
11
4
24
29
_
4
39
3,260
—
—
1
• Intraventricular hemorrhage. Grade I: hemorrhage confined to the germinal matrix; grade
II: intraventricular hemorrhage: grade HI: intraventricular hemorrhage with ventricular dilation:
and grade IV: intraparcnchymal hemorrhage.7
orrhage. The CSF cell counts and chemistry values were
compatible with what one would expect in hydrocephalus
after intraventricular hemorrhage (patients 1-3) or hydrocephalus alone (patient 4).8 No patients had a malignancy develop on follow-up examination (10-24 months).
Primitive cell clusters consisted of cohesive cells with
scant to moderate amounts of basophilic cytoplasm. Nuclei were round to oval with even, dispersedfinechromatin
and small single nucleoli (Fig. 1). Occasional nuclei were
indented. Hemosiderin-laden macrophages with abundant
cytoplasm and kidney-shaped nuclei were usually present
on the cytospin (Fig. 2) and frequently intermixed in the
PCCs. Immunoperoxidase staining of the CSF cytospin
preparations from patient 2 revealed peripheral cytoplasmic staining of the PCCs by anti-NSE, which is present
in high concentrations in neurons and neuroendocrine
cells. The pan-leukocyte cocktail did not stain the PCCs.
Lymphocytes and histiocytes on the CSF cytospin preparations served as internal positive controls for the panleukocyte cocktail.
Discussion
Kolmel has illustrated clusters of cells from the CSF of
infants with subarachnoid hemorrhage that are morphologically quite similar to PCCs. He designates them "undifferentiated leptomeningeal cells," believing that they
represent promonocytes derived from pluripotential cells
in the reticuloendothelial tissue of the leptomeninges.3
The relationship of intraventricular hemorrhage secondary to rupture of a subependymal germinal matrix
hemorrhage in low birthweight premature newborns with
respiratory distress syndrome is well recognized.4 The
common location of these subependymal matrix hemorrhages is in the region of the terminal vein between the
thalamus and the candate nucleus.2,6 Germinal matrix
tissue persists as subependymal deposits in the cerebrum
of the fetus and premature infant. It is most pronounced
in fetuses of six to eight months gestational age and diminishes with further maturation. The matrix serves to
give rise to cells required in the formation of the cerebral
cortex, basal ganglia, and other neuronal assemblies of
the forebrain.9
41
56
7
43
100
10
t Primitive cell clusters in cerebrospinal fluid.
t Ventriculoperitoneal shunt.
Given the characteristic clinical presentation, morphologic characteristics, NSE positivity, and pan-leukocyte
negativity of the PCCs, we believe that these cells may
represent subependymal germinal matrix cells. NSE has
been carefully investigated by both enzymatic and immunohistochemical methods and has been shown to be
present in high concentration in both normal and neoplastic neuroectodermally derived cells. It has also, however, sporadically been found in a variety of nonneuroectodermal tumors including both carcinomas, T-cell
leukemias and lymphoblastoid B-cell lines, so that it cannot be considered to be truly "neuron specific."5 In our
study, lymphoid origin of the blast-like cells could be ruled
out because these should also have been pan-leukocyte
antibody positive.
As is shown in Table 1, the PCCs were never seen on
initial lumbar puncture but usually appeared at variable
intervals after development of hydrocephalus was noted
by head ultrasound. We have no explanation for the timing of the appearance of these cells. Also, the PCCs were
noted before surgical placement of a ventriculoperitoneal
shunt when possible trauma to the ependyma might be
expected.
Care should be taken to avoid confusing PCCs with
two broad categories of cells: first, cells that are normal
components of the central nervous system such as choroid
(Fig. 3) or ependymal cells'; and, second, neoplastic cells
including lymphoblasts (Fig. 4), neuroblastoma (Fig. 5),
or medulloblastoma cells. In the former category, choroid
plexus cells are distinguished by their small bland uniform
nuclei and low nuclear-cytoplasmic ratios. Lymphoblasts
may have similar cytologic features to the cells of PCCs
but are not as cohesive. Medulloblastoma and neuroblastoma cells are usually larger than cells in PCCs but
otherwise are similar.
A larger panel of immunohistochemical markers in addition to NSE and pan-leukocyte antibodies would be desirable to characterize these cells optimally. However, the
PCCs were usually few in number and volumes of CSF
were insufficient for performance of an exhaustive immunohistochemical study. Indeed, PCCs were present on
the Wright's stain preparation in case 4, but none was
Vol. 91 • No. 3
BLAST-LIKE CELLS IN CSF
FIG. 1 (upper, left). PCCs from CSF of patient 3 showing cohesive cells, high nuclear cytoplasmic ratios, fine chromatin,
and nucleoli. Wright's stain (X250).
FIG. 2 (upper, right). A cluster of hemosiderin-laden macrophages from the CSF of patient 3. Wright's stain (XI32).
FIG. 3 (lower, left). Choroid plexus cells in CSF. Wright's stain (X250).
FIG. 4 (lower, right). Blast cells of acute lymphoblastic leukemia in CSF. Wright's stain (X250).
257
258
A.J.C.P. • March 1989
FISCHER ET AL.
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who share a fairly common clinical presentation. The
morphologic characteristics of these cells may suggest a
diagnosis of neoplasia; however, correlation of the morphologic characteristics with a clinical picture of neonatal
subependymal germinal matrix hemorrhage and/or hydrocephalus will aid in the recognition of the benign nature of these cells. Immunohistochemical studies may be
helpful if sufficient cells are recovered.
Acknowledgments. The authors thank Linda Schneekloth for secretarial
support and Joel Carl for his aid with the illustrations.
References
1®
L
W*
^j?
FIG. 5. Neuroblastoma cells in CSF. Wright's stain (X132).
recovered on additional immunohistochemical cytospin
preparations.
In summary, we have described NSE-positive, pan-leukocyte-negative cell clusters found in the CSF of neonates
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