A Supravital Cytodiagnostic Stain

for Urinary Sediments

Richard Sternheimer, MD

A mixture of aqueous solutions of National fast blue, a copper-phthal- in their size and form.
ocyanine dye, and pyronin B, a red xanthene dye, when added to fresh uri- Modified techniques of ordinary
nary sediment, supravitally stains benign or malignant cells and the various light microscopy,2 phase-contrast mi¬
types of casts and their inclusions. The stain facilitates identification of the croscopy,'-' interference microsco¬
formed elements and particularly aids in the differentiation of polymorpho- py,"7 and fluorescence microscopy""
nuclear leukocytes from lymphocytes, histiocytes, plasma cells, and renal have offered but limited assistance in
tubular cells. A variable staining of casts and their inclusions has been ob- solving these problems and have not
served. Tumor cells may be recognized by nuclear abnormalities or, in case obviated the need for staining meth¬
of hyperchromatic tendency, by a very rapid and early uptake of dye preced- ods. Where a more detailed structural
ing that of the surrounding cells. analysis of cells becomes crucial, as in
The staining method is rapid and simple enough for routine urinalysis and the diagnosis of bladder tumors,1" of
screening procedures. tubular necrosis, or of rejection of
(JAMA 231:826-832, 1975) renal transplants,1112 staining of air-
dried and fixed sediments has re¬
mained the method of choice. Unfor¬
(
tunately, these laboratory procedures
EVALUATION of microscopic uri¬ dergo changes in size, structure, and not infrequently entail the loss of cel¬
nary findings rests on adequate rec¬ transparency that add to the already lular elements during preparation
ognition of cellular elements and existing cytodiagnostic uncertainties and are too time-consuming for rou¬
casts. This has always been a difficult created by the poor optical contrast tine use. In contrast, wet stains offer
task since the cells may originate between cells and the liquid medium. readily available results. Separate
from diverse tissues such as epithelial Thus, differentiation, particularly supravital stains have been devised
layers of the urinary and genital of the small cells (like those cast off for red blood cells (RBCs),12 for white
tracts, blood and connective tissue from renal tubules, the deeper muco- blood cells (WBCs),141s for tumor
sources, or tumors, and are likely to sal layers, or the prostate) from cells,1"17 and for casts.18 Some stains
show variable degrees of degenera¬ polymorphonuclear leukocytes, lym¬ that facilitate recognition of a larger
tion, viability, permeability, and den¬ phocytes, histiocytes, plasma cells, or range of formed elements have been
sity when shed into the urine. Subse¬ tumor cells, has remained a frustrat¬ used more widely,19-20 but "none has
quently, by being exposed for varying ing and largely unsolved problem in supplanted the direct examination of
periods of time to different os- ordinary light microscopy.1 Obviously, the unstained sediment for routine
molarities, to fluctuations of pH, and the same predicament exists in the use."21
to enzymatic, toxic, or possibly bacte¬ identification of cellular elements and It is safe to say, therefore, that
rial agents in the urine, they also un- their decomposition products that are there exists a need for a procedure
enmeshed in urinary casts. Finally, suitable for routine urinalysis that
From the departments of medicine and pa-
the casts themselves are often diffi¬ aids in the differentiation of cells, be¬
thology,Michael Reese Hospital and Medical cult to visualize on account of the nign or atypical, as well as in the rec¬
Center, Chicago. variable optical and chemical charac¬ ognition of casts and in the character¬
Reprint requests to Michael Reese Hospital teristics of the cast matrix—hyaline ization of their inclusions. This article
and Medical Center, 2900 S Ellis Ave, Chicago,
IL 60616 (Dr. Sternheimer). vs waxy casts—and the wide variety reports such a staining method.

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 GENERAL CONSIDERATIONS prominently represented in the avoided, since pyronin has a moderate
The stain used is a mixture of a matrix of casts.28-2" affinity to Tamm-Horsfall protein
copper-phthalocyanine dye, National It was realized that these consid¬ and in sufficiently high concentration
fast blue, and a xanthene dye, pyro- erations, some of which were derived may compete with the blue dye in the
nin B. The choice of the dye mixture from observations on fixed tissues un¬ staining of hyaline casts.
was made on the basis of the follow¬ der controlled conditions of pH, The following spectrophotometric
ing data: might have only limited validity for data may serve as a more precise
The copper-phthalocyanines used in supravital staining. The approach had guideline. The stain mixture should
histology, National fast blue, alcian to be empirical and the tentative in¬ be made up to meet the following cri¬
blue, and astrablau, are dyes of high terpretations presumptive, or at best teria: When 0.1 ml of the stain is di¬
molecular weights-990, 1,341, and inferred with a "confidence mitigated luted to 100 ml deionized water, the
1,267, respectively. They differ from by prudence" (Lison).30 absorbance (from pyronin B) at ap¬
each other mainly in the side chains proximately 553 nm is 1.81±10%. The
PREPARATION OF THE STAIN
attached to the copper-phthalocya¬ ratio of the absorbance at approxi¬
nine chromophore (dimethylhydrazine As the copper-phthalocyanine dye, mately 553 nm to the absorbance
for National fast blue, isothiouronium National fast blue was preferred, be¬ (from the blue dye, alcian blue or as¬
for alcian blue, and acetate for cause it has greater potency per unit trablau) at approximately 622 nm is
astrablau). All appear to be high¬ of weight, offers greater color con¬ between 2:1 and 4:1.
ly aggregated in aqueous solutions trast, and proved to be quite stable. Such a stain mixture, when tested
(dimers, trimers, or polymers). They 1. National fast blue (Allied Chem¬ on a sediment of a urine with low
are strongly basic dyes, but are dis¬ ical Corp, No. 1946 P) was prepared in protein content, should stain hyaline
tinct from other cationic dyes (1) by a 2% aqueous solution. casts bright blue and the cytoplasm of
being precipitated and irreversibly 2. Pyronin B (Matheson Coleman & the epithelial cells in a pink color.
adsorbed to the living outer cell mem¬ Bell No. Pbl7). In view of the great
brane (as observed in ^Imoeoa),22 (2) variations in dye content of the com¬ STAINING THE SEDIMENT
by diffusing slowly through gels and mercially available pyronin, which Sediment samples were prepared in
matrices and by staining tissues may vary from lot to lot, it appeared the usual manner, by centrifuging a
slowly,23 (3) by displaying no tend¬ advisable to purify the dye by alcohol 10- to 15-ml sample of urine in a stan¬
ency to avid staining of nucleic extraction, whereby dextrin and salts dard centrifuge tube at 1,500 to 2,000
acids,2425 and (4) by prominently are removed.31 After evaporation of rpm for five minutes and discarding
staining acid mucopolysaccharides. the alcohol, dye crystals separated the supernatant, leaving only about 1
In contrast, pyronin B is a basic from the gummy residue were dis¬ to 2 drops of it to mix the sediment
dye of low molecular weight (359) and solved to form a 1.5% aqueous solution by agitation or finger flipping. Via
relatively faster diffusion rate that of relatively constant staining capac¬ a rubber-topped Pasteur pipette, 2
interacts strongly with polynucleo- ity. drops of the sediment mixture were
tides, particularly ribonucleic acids.24 3. Both solutions were filtered and transferred into a small tube and 1
These contrasting characteristics equal parts of each dye solution were drop of stain added. After shaking,
were believed to be advantageous in a mixed and the resulting mixture kept 1 drop of the stained sediment was
supravital stain, where differences in in a dropper bottle. The dispensing placed on a glass slide and mounted
the rate of permeation by the two capillary consisted of the lower end of by a cover glass for immediate micro¬
dyes could be observed directly and a Pasteur pipette fitted into the rub¬ scopic examination.
possibly correlated with membrane ber bulb of the bottle cap. Individual Staining started promptly and in¬
permeability, the physicochemical batches of dye solutions and the mix¬ creased in intensity particularly dur¬
state of cellular matrices, and the via¬ ture appeared stable for many ing the first five to ten minutes; from
bility of cells. It also appeared likely months. then on, progression of staining was
that the remarkably low affinity of If alcian blue or astrablau is used as slow and diminishing. Observation
the phthalocyanine dye for cytoplas- an alternate dye for National fast during the initial staining period,
mic ribonucleic structures2526 would blue, a stronger concentration of the however, proved extremely valuable
favor their preferential staining by blue dye is required in the stain mix¬ since the rate of staining of individ¬
pyronin. This in turn might contrast ture to achieve comparable contrasts. ual cells appeared related to their via¬
with nuclear staining by the blue dye, Depending on the purity and the man¬ bility or, as in some malignant cells,
which even under conditions of low ufacturing source of the blue dye,32 to a tendency towards hyperchroma-
pH has been found as "hard to pre- its ratio in percent weight per volume sia. Overstaining of ordinary cell
vent."26-27 Finally, the marked sensi¬ to that of the red dye may vary be¬ components occurred infrequently,
tivity of acid mucopolysaccharides to tween 2:1 and 4:1. High concentration and the stained sediment mixture
the copper-phthalocyanine suggested ratios of blue dye to red dye tend to could be viewed after standing for
the use of this dye for the staining of produce a more violet staining of the many hours. The effects of differing
casts, based on the demonstration cellular cytoplasm. On the other hand, protein and electrolyte concentra¬
that Tamm-Horsfall protein, a sial- a proportionately high pyronin con¬ tions in the urine and of the vari¬
ic-acid-containing glycoprotein, was centration in the mixture should be ations in cell content of the sediment

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 Fig 1.—Finely and coarsely granular cast; Fig 2.—Variable staining of WBCs, "glitter Fig 3—Histiocytes, WBC in late
"glitter cells" with dye precipitated on cells," and round cells (original pyelonephritis (original magnification
their surface (original magnification X400). magnification x 400). x 400).

Fig 4—Plasma cells (oil immersion). Fig 5.—Epithelial cell casts. Fungi. (Septic Fig 6.—Hyaline cast with enmeshed or
arthritis, diabetes mellitus, antibiotic attached tubular cells (original
therapy) (original magnification x400). magnification x400).

Fig 7—Cast containing tubular epithelial Fig 8.—Renal tubular cell cast. Nephrotic Fig 9.—Renal tubular cast. Nephrotic
cells. Renal amyloid (oil immersion). syndrome (oil immersion). syndrome (oil immersion).

Fig 10.—Squash preparation from Fig 11.—Epithelial cells. Note variable Fig 12.—Caudate transitional epithelial
prostate, obtained at autopsy, supravitally sizes and staining of nuclei (original cells. Note nuclear chromatin (oil
stained (original magnification x400). magnification x400). immersion).

Fig 13.—Bladder epithelial cells, obtained Fig 14.—Bladder epithelial cells, Fig 15.—Degenerative epithelial cell
by catheterization, following surgery of catheterized specimen (original inclusions; atypical transitional epithelial
ureteral stricture (original magnification magnification x400). cell (original magnification x400).
x 400).

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 Fig 16.—Multinucleated giant epithelial Fig 17.—Hyaline casts, short, long, spiral Fig 18.—Hyaline casts (original
cell(oil immersion). forms (original magnification x100). magnification x400).

Fig 19.—Variably stained casts. Late Fig 20.—Waxy cast. End-stage Fig 21 .—Hemoglobin cast, rust-brown;
pyelonephritis (original magnification pyelonephritis (original magnification angular cast with RBC: acute glomer-
x 400). X100). ulonephritis (original magnification X400).

Fig 22.—Red blood cell cast. Chronic Fig 23.—Oval fat body (oil immersion). Fig 24.—Bacteria (original magnification
glomerulonephritis (original magnification X400).
x 400).

Fig 25.—Suspected tumor cells, found in Fig 26.—Same patient, following cystos- Fig 27.—Tumor cells, RBCs, same patient
routine urine specimen (original copy: transitional cell carcinoma of blad¬ (oil immersion).
magnification x400). der. Tumor cells, RBCs (oil immersion).

Fig 28—Transitional cell carcinoma of Fig 29—Papillary transitional cell Fig 30.—Tumor cells, same patient (oil
bladder (original magnification x 400). carcinoma of bladder. "Tadpole" cell; immersion).
degenerative inclusion cells (oil
immersion).

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 mixture on the rate of staining hours thereafter, or there may be histiocytes could be identified by their
seemed to be minimized by the rela¬ gradual shift to a blue staining of the eccentric, kidney-shaped nuclei and
tively high dye concentrations used. nuclei, which is then irreversible. On their vacuolated cytoplasm (Fig 3).
The description of the staining other occasions deep blue nuclear and Cells resembling plasma cells with
characteristics is based on observa¬ red cytoplasmic staining are present eccentrically located, oval-shaped nu¬
tions and photographic records of the from the outset and will remain so in¬ clei, whose chromatin was ar¬
urinary sediments of 230 patients. definitely. The three stages of stain¬ ranged in a wheel-spoke pattern, and
The sediment samples were chosen ing may be found on different leuko¬ where the cytoplasm exhibited one or
from routine laboratory urine speci¬ cytes in the same microscopic field, more pyronin-stained inclusions,
mens whenever increased cellular or indicating that the variable nuclear were (Fig 4).
also observed
cast contents were found on ordinary staining reflects progressive degrees However, recognition of renal tu¬
examination. Clinical records of pa¬ of individual cellular damage rather bular cells on morphological grounds
tients were made available for corre¬ than changes in the composition of alone seemed to be a most difficult
lation. The observations extended the urine (Fig 2). A simple experi¬ task and fraught with uncertainty.
over a 2%-year period. In a number of ment supports this concept: If the Their appearances and staining char¬
cases, follow-up examinations were buffy coat of blood treated with cit¬ acteristics seemed to be not uniform;
performed at intervals. Many normal rate is stained wet, the WBCs will one felt more certain of their identity
sediment samples were examined as show diffuse staining by pyronin if they were found within casts (Fig 5
controls. The pH of the urine speci¬ alone. If, however, a smear is made of through 9). More often than not, the
mens varied between 5.0 and 6.5; os- the same buffy coat and stained after cytoplasm of the renal tubular cells
molarity, generally between 200 and drying, the cell nuclei will appear was ovoid or polygonal rather than
450 mOsm/liter; and albuminuria, sharply outlined in deep blue, the round; their nuclei were either vesicu¬
from trace to 4 +. cytoplasm red. lar and indistinct or sharply outlined
The results of the cell-dye inter¬ when stained dense blue. Fatty de¬
RESULTS
action are remarkably different for generation, if present, was clearly
The dyes of the mixture have a the two dyes. Pyronin can be washed visible. Not infrequently, the differ¬
tendency to combine selectively with away easily by distilled water, both entiation from the small round or
the various structural components of from the blood smear as well as from cuboidal cells of the deeper mucosal
the cells. Preferential staining of the the urinary sediment. In contrast, the layers of the urinary tract was tenta¬
cytoplasm by pyronin is quite ob¬ blue dye always remains irreversibly tively made by taking into account
vious. Cell nuclei are predominantly bound. This difference can be ob¬ the milieu of the accompanying cellu¬
stained by the blue dye, but red stain¬ served directly on the stained sedi¬ lar or formed elements. In these
ing of the nucleus can also be ob¬ ment under the microscope by adding doubtful situations, consultation of
served. These differences in nuclear distilled water below the cover glass the clinical records was helpful in try¬
staining are most obvious in the vari¬ to flood the preparation by capillary ing to corroborate or weaken a pre¬
able staining of urinary leukocytes; it action. sumptive diagnosis. A strong support
was this observation that offered a Polymorphonuclear leukocytes in for their renal origin, for instance,
clue to the mode of action of the dyes. the urinary sediment were identified was obtained by clinical data showing
Immediately after addition of the with relative ease. More difficult was a sudden rise in blood urea nitrogen

dye mixture to the sediment, leuko¬ the differentiation between mononu- and creatinine levels, oliguria, and
cytes may appear unstained and re¬ clear WBCs and the small round epi¬ symptoms suggesting tubular necro¬
sist permeation by either dye for a thelial cells derived either from renal sis. Conversely, in some instances, a
prolonged period. This is especially tubules or from the lining epithelia of marked increase in presumably renal
true for the oversized, swollen "glit¬ the remainder of the urinary tract. epithelial cells in the sediment pre¬
ter cells." When they begin to stain The size of these epithelial elements ceded the ensuing azotemia and
subsequently, the red dye always pre¬ varied not only by shrinking or swell¬ served as an early warning to the
cedes the blue and the blue will fol¬ ing with changing urine osmolarities clinician. The advantage of such rap¬
low after a varying interval, trans¬ but also in connection with fatty de¬ idly obtainable information for both
forming the red nuclear staining into generation or as a result of inflam¬ clinician and morphologist is obvious.
blue, while leaving the cytoplasm red. matory changes. Nevertheless, cer¬ Squash preparations were obtained
One reason for the delayed entry of tain distinguishing features were from autopsy material from kidney
the phthalocyanine dye may be its observed. cortex, medulla, and pelvis, as well
visible initial clumpy precipitation on as from ureters and urinary blad¬
Cells
the leukocytic cell surface, which is der, and supravitally stained with the
analogous to the precipitation ob¬ Lymphocytes, as a rule, appeared dye mixture for a direct comparison
served in Amoeba (Fig 1). comparatively smaller than renal of identically stained tissue and
In other instances, both nuclei and tubular cells or leukocytes and were sediment cells. Disregarding the
cytoplasm are immediately dyed by recognized by their densely blue nu¬ structural pattern of the cellular ar¬
pyronin only, and this appearance cleus surrounded by a small rim of rangements in the tissues, the differ¬
may remain unchanged for many cytoplasm. Small- and medium-sized entiation by morphological appear-

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 anees alone of individual small cells or blue-violet tinges. Most remark¬ teria (Fig 24). Fungi remained un¬
exfoliated from the various sources able was a changein color and ap¬ stained (Fig 5); their differentiation
seemed uncertain. As shown in pearance of the matrix of broad and from RBCs presented no problem.
Fig 10, the distinct columnar appear¬ of waxy casts in instances of marked Trichomonas also seemed to resist
ance of prostatic cells within the tis¬ proteinuria, especially nephrotic syn¬ staining, or took on a slightly bluish
sue structure cannot be recognized on dromes, mesangioproliferative glo- hue.
the separated individual cells. Small, merulonephritis, and in later stages
caudate transitional epithelial cells, of pyelonephritis. These casts stained Amorphous Mucus
however, were identifiable both in the reddish violet, showed markedly in¬ Amorphous mucus was stained in¬
preparations and in the urine. creased density and, at times, almost tensely by the blue dye, which at
The cytoplasm of the squamous epi¬ complete loss of transparency (Fig 7 times was disturbing when it became
thelial cells from bladder, urethra, or through 9 and 19, 20). Differences in superimposed on the formed ele¬
vagina found in the sediment stained staining pattern and texture seen in ments. It did not, however, render
preferably with pyronin, but various broad and waxy casts might well rep¬ cellular differentiation impossible as
tinges of violet also occurred. The resent changes within the cast matrix it would in an unstained sediment.
dense pyknotic nuclei of the large su¬ itself, since it was noted in some but The occurrence of increased mucus
perficial keratinized cells were always not all of the casts in the same micro¬ excretion was most notable in lower
stained blue, while the larger vesicu¬ scopic field. These findings offer an urinary tract infection or following
lar nuclei appeared blue or red (Fig interesting parallel to recent data, prostatic resections.
11). Under oil immersion the deep¬ obtained by immunofluorescent meth¬ There is a marked tendency of the
ly blue stained strands of nuclear ods, indicating differences in the stained mucus to aggregate. The
chromatin were clearly visible (Fig chemical composition of hyaline casts slightest pressure on the cover glass
12). The well-described wide variation and their granular inclusions35 and or a shearing motion can produce
in shape and size of the transitional also between hyaline casts, as pro¬ cast-like artifacts that, however, are
epithelial cells was distinctly visu¬ duced by certain diuretics, and the easily recognizable by their streak¬
alized. They were pear-shaped or casts excreted by patients'suffering like, regular, diagonal distribution
spindle-shaped, caudate or polygonal, from renal disease.'" However, no over the whole visual field. For this
and varied in size from small conclusions warranted before a
are reason, and as a standard precaution,
mononuclear cells to very large mul- thorough comparative analysis of the the cover glass should always be
tinuclear giant epithelial cells (Fig 12 results obtained by the two methods dropped gently on the stained sedi¬
through 16). Cells exhibiting spheri¬ has been performed. ment and no further corrections made
cal, crescent-like, or bar-shaped pyro¬ Hemoglobin casts appeared rust- thereafter.
nin-stained intracytoplasmic inclu¬ brown in color (Fig 21). Red blood cell
sions and containing either an intact casts were easily identifiable (Fig 22). Malignant Cells
blue nucleus or disintegrating and ec¬ Examples of the previously men¬ Inview of the great variability ob¬
centrically located nuclear fragments tioned wide variation in size and served in form and structure of the
were found in low-grade bladder in¬ staining appearance of renal tubular renal tubular cells, a cautious ap¬
fections or in association with tumor cells, enmeshed in or adherent to the proach to the interpretation of atypi¬
cells. Their appearance resembles casts, are given in Fig 5 through 9. cal and especially of malignant cells
that of cells described as containing Fatty degeneration of these cells can appeared indicated. Variations in size
eosinophilic cytoplasmic inclusions be recognized within and outside of and shape or alterations of the nu-
(Fig 15).33-34 the casts. Fatty-degenerated "oval clear-cytoplasmic ratio of these cells,
fat bodies" were also clearly visual¬ therefore, were considered less sig¬
Casts
ized (Fig 23). nificant than the evidence of irregu¬
Casts stained promptly and in¬ Red blood cells are distinctly out¬ lar nuclear outlines, of coarse clump¬
tensely. Pure hyaline casts appeared lined in various shades of pink, their ing or strand formation of the
in bright blue colors; their great avid¬ rims sometimes studded with tiny red chromatin material within the nu¬
ity for the phthalocyanine dye is in droplets. A tendency to agglutination cleus, of condensation of chromatin
keeping with the concept of the muco- has been observed, and, in some near the nuclear membrane, and of
protein nature of the cast matrix. instances, hemolysis of individual hyperchromatic staining. Observation
Their characteristic shapes, either erythrocytes after prolonged observa¬ of the sediment during the initial
short and plump, or long and narrow, tion (15 to 20 minutes) of the stained staining period was especially valu¬
with rounded or straight ends, in tor¬ sediment. These are obviously lim¬ able in hyperchromatic cells because
tuous or spiral forms, were distinctly iting factors in the evaluation of they stained earlier than the remain¬
visualized (Fig 17 and 18). In finely hematuria. ing cells. Thus, they could be singled
granular casts, the granular inclu¬ out from the start in a search for fea¬
sions nearly always appeared bright Microorganisms tures of malignancy. In order to ac¬
red within the blue matrix (Fig 1 and Bacteria normally appeared centuate the staining difference and
19). Coarse granules within casts stained bright red; the stain has a to prevent overstaining, the stain
tended to stain more in reddish-violet tendency to precipitate moving bac- mixture in these instances was some-

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 times diluted by one third to one half are given in Fig 25 to 30. Figure 25 the stain as a routine screening pro¬
with distilled water. is a reproduction of abnormal cells cedure for malignant cells, a much
Only 12 cases of clinically estab¬ found in a routine urine specimen of greater familiarity with the staining
lished bladder tumors have been ex¬ a patient admitted for eye surgery. characteristics, as well as controls
amined so far. In two, the diagnosis The recommended cystoscopic exam¬ by established cytological procedures
was made initially from a routine ination showed a transitional cell car¬ and confirming urological findings,
urine specimen with surgical confir¬ cinoma of the bladder. Following are needed to assess the diagnostic
mation. The remaining ten urine cystoscopy an increased number of validity of the procedure. Such a
specimens stemmed from patients malignant cells appeared in the sedi¬ study is now being conducted in our
with known bladder tumors. Exam¬ ment (Fig 26 and 27). Cytology Department.
ples of malignant cells found in in¬ While the staining procedure is
stances of carcinoma of the bladder simple enough to suggest the use of

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plantation:
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