T U B AMKHICAN JOURNAL or CLINICAL PATHOLOGY
Vol. U, No. 1, pp. 8-14
January, 1U84
Copyright © l'.IO-l bv Tlip Willmms & Wilkius Co.
Printed in U.S.A.
CRYSTALS OF CALCIUM OXALATE IN THE HUMAN KIDNEY
STUDIES BY MEANS OF ELECTRON MICROPROBE AND X-RAY DIFFRACTION
JAMES L. BENNINGTON, M.D., SETH L. HABER, M.D., JOSEPH V. SMITH, P H . D . , AND
NANCY E. WARNER, M.D.
Departments of Pathology and Geophysical Sciences, The University of Chicago, Chicago, Illinois
Crystals of calcium oxalate have been
identified in the human kidney, myocardium, thyroid gland, and brain. They
have been reported in various instances of
glycol nephrosis,8' !0, 12 primary oxalosis,1' 4i
Ci 9
and chronic renal disease with accompanying uremia.2'3' 5
After observing crystals of calcium oxalate
in the renal tubules of patients who died
without renal disease or terminal renal
failure, we searched for such crystals in the
kidneys of 500 consecutive patients who
were studied post mortem (in 1961 to 1962)
at the University of Chicago Hospitals, in
order to determine the incidence of this phenomenon.
Ka radiation and a cylindrical camera with
a 3-cm. radius. Both stationary and rotating
crystal technics were used.
Unstained sections of formalin-fixed
kidney were attached to aluminum disks
with vacuum grease, and then examined in
an ARL electron microanalyzer fitted with
an electron beam scanner. As the beam,
which was 1.0 n in diameter, scanned the
surface of the section over an area 3G0 by
360 ix, the oscilloscope beam moved synchronously. In response to modulation with
back-scattered electrons, the number of
which depends upon the atomic number of
the sample, the brightness of the picture on
the oscilloscope screen varied with the
atomic number of the elements on the surface
scanned. Because calcium is higher in the
periodic table than the elements in tissue,
the deposits were visible as bright areas on
the screen. The electron beam was fixed on
1 of the crystals and a wave-length scan
was made on the x-rays excited by the electron beam. Only the wave lengths of calcium
were found. The percentage of calcium was
estimated, with calcium carbonate and calcium oxalate as reference standards.
METHODS
Crystals to be analyzed were identified in
routine histologic sections by .means of
plane-polarized light (Fig. 1). Solubility of
the crystals was determined on formalinfixed, unstained, mounted sections.
Representative crystals were dislodged
from unstained, mounted sections with a
micromanipulator for analysis by means of
x-ray diffraction. Diffraction photographs
of 3 crystals (each approximately 0.1 mm.
in diameter) were prepared, using copper
RESULTS
Clinicopathologic Data
Crystals of calcium oxalate were present
in the kidneys of 32 (6.4 per cent) of 500
consecutive autopsied patients. Eighteen
were male, and 1-1 were female, with ages
ranging from 3 to 82 years (mean of 55
years).
Renal disease. Fourteen of the 32 patients
(44 per cent) had clinical or anatomic
evidence of renal disease (Table 1). This is
not significantly different from the incidence
of renal disease in 100 randomly selected
control autopsy patients.
Hepatic, damage.. Ten of the 32 patients had
some form of hepatic damage (Tabic 2)..This
Received, March 15, 19G3; revision received,
June 5, accepted for publication August 29.
Dr. Bennington is Chief Resident, and Dr.
Habcr is Staff Pathologist in the Department of
Pathology.,Jiaiser Foundation Hospital, Oakland,
California; Dr. Smith is Professor of Mineralogy
anil Crystallography, University of Chicago; and
Dr. Warner is Associate Professor of Pathology,
and Director, Laboratory of Surgical Pathology,
University of Chicago Hospitals and Clinics.
This study was supported (in part) by means of
research fellowships, CF9010 and H-3944, United
States- Public Health Service. The ARL Microprobe >vas purchased with National Science
Foundation grant 9192S0, awarded to J. V. Smith
and H. Ranibcrg.
8
Jan. 196%
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RENAL CALCIUM OXALATE CRYSTALS
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Fic. 1. Characteristic birefringence of crystals of calcium oxalate in renal tubules, viewed with
partially extinguished plane-polarized light. Hematoxylin and eosin. X 250.
is not significantly different from the
incidence of hepatic damage in 100 randomly
selected control autopsy patients.
Neoplasia. Twenty-three of the 32 patients
(72 per cent) had a malignant neoplasm
(Table 3). This is greater than the incidence
of malignant neoplasms in 100 randomly
selected control autopsy patients, and the
difference is significant at the 2.5 per cent
level.
Transfusion. At least 7 patients had been
recently treated with transfusions of whole
blood, and 1 with plasma. No statistical
correlation was attempted with reference to
the administration of transfusions and the
presence of crystals of oxalate in the kidneys.
of calcium oxalate are summarized in Table
4.
Although stationary photographs revealed
incomplete rings composed of spotty arcs,
rotational photographs revealed almost
complete diffraction rings (Fig. 2). The
intensities and spacing of the lines were in
agreement with those for calcium oxalate in
the X-ray Powder Data File.13
Organic compounds were not detected,
inasmuch as the electron microprobe can
not detect elements below sodium in the
periodic table, unless very special technics
are used. The electron microanalysis indicated a concentration similar to, but slightly
lower than that of pure calcium oxalate.
DISCUSSION
Characteristics of Crystals of Calcium
Oxalate
A high incidence of deposition of
The morphology, staining characteristics, crystalline calcium oxalate in the kidney has
and physical-chemical properties of crystals. been reported on the basis of studies
10
BENNINGTON ET AL.
Vol. 41
TABLE 3
TABLE 1
T Y P E S O F R E N A L D I S E A S E IN 14 O F 32 A u T o r s v
PATIENTS
WITH
RENAL
OXALATE
CRYSTALS,
TYPES
O F N E O P L A S M S IN
PATIENTS
WITH
RENAL
23
O F 32
OXALATE
AUTOPSY
CRYSTALS,
AND IN 42 O F 100 RANDOMLY SELECTED C O N T R O L
AND IN 45 O F 100 RANDOMLY SELECTED CONTROL
AUTOPSY P A T I E N T S
AUTOPSY PATIENTS
Patients with
Renal Oxalate
Crystals
Biliary nephrosis
Ischemic nephrosis
Renal vein thrombosis
Hydronephrosis
Chronic glomerulonephritis
Uric acid calculi
Pyelonephritis
Necrotizing arterionephrosclerosis
Other*
3
3
2
2
1
4
4
3 •
0
0
1
1
1
1
IG
1
0
13
14
44%
Total
Percentage
Patients
42
42%
* Nephrocalcinosis 4, m e t a s t a t i c carcinoma 3,
< intercupillary glomerulosclerosis 2, p r i m a r y carcinoma 1, polycystic disease 1, renal infarct 1,
paroxysmal nocturnal hemoglobinuria 1.
TABLE 2
T Y P E S O F H E P A T I C D I S E A S E IN 10 O F 32 A U T O P S Y
PATIENTS
WITH
RENAL
OXALATE
CRYSTALS,
AND IN 29 O F 100 RANDOMLY SELECTED C O N T R O L
Type of Neoplasm
Breast
Esophagus
Ovary
Pancreas
Lung
Uterus
Leukemia
Vagina
Neuroblastoma
Mesothelioma
Angiosarcoma
Prostate
Bladder
Thymoma
Hodgkin's disease
Other*
Total
Percentage
Patients with
Renal Oxalate
Crystals
3
2
2
2
to to
Type of Renal Disease
2
1
0
23
72%
Control
Patients
5
0
0
3
6
0
4
C
1
0
0
4
2
1
2
17
45
45%
* Colon 4, squamous carcinoma of skin 3,
lymphosarcoma 2, malignant melanoma 2, liver
2, kidney 1, adrenal gland 1, osteosarcoma 1,
unknown 1.
AUTOPSY PATIENTS
Type of Hepatic Disease
Cirrhosis
Neoplastic obstruction
of bile ducts
Hepatitis
Obstruction of porta
hepatis
Other*
Total
Percentage
Patients with
Renal Oxalate
Crystals
Control
Patients
4
3
10
8
2
1
7
0
0
4
10
31%
29
29%
* H e p a t i c abscesses 2, cholangitis 1, polycystic
disease 1.
limited to instances of chronic renal disease.
On the other hand, it should be recognized
that this approach is statistically biased,
and tends to overemphasize the relation to
chronic renal disease.
Crystals of calcium oxalate in renal
tubules (Fig. 3) were found in 32 of 500
consecutive patients on whom autopsies
were performed at the University of Chicago
Hospitals. Only 14 of the 32 patients (4.4. per
cent) had any evidence of renal disease. It
may be emphasized, therefore, that the
presence of crystals of calcium oxalate is
not solely dependent on, or diagnostic of,
chronic renal disease. The 18 patients who
were free of renal disease and had crystals
of oxalate in their kidneys had no other
demonstrable pathologic condition or metabolic defect in common.
The incidence of renal and hepatic diseases
was not significantly different in 32 autopsy
patients with crystals of oxalate in their
kidneys than in 100 randomly selected
control autopsy patients. On the other hand,
the incidence of malignant neoplasms in the
32 autopsy patients with crystals of oxalate
Jan.
1964
RENAL CALCIUM OXALATE CRYSTALS
TABLE 4
SUMMARY OF THE MORPHOLOGY, STAINING
CHARACTERISTICS, AND PHYSICAL-CHEMICAL
PROPERTIES OF CALCIUM OXALATE CRYSTALS
I. Morphology
A. Golden-brown in hematoxylin and eosin
sections; stained green in biliary
nephrosis.
13. Rosette- and fan-shaped under unpolarized light. Up to 0.1 mm. in diameter
(Fig. 3).
C. Under polarized light the majority of the
birefringent crystals were complex and
ellipsoidal to spherical (Fig. 1). Occasional crystals with straight, sharp edges
extinguished light uniformly. Koscttcshaped crystals revealed a radiating black
cross as the crystal was rotated on the
stage. Intermediate crystals had irregular
surfaces.
II. Staining
A. Crystals not stained with hematoxylin
and eosin and von Kossa stains.
B. Variable, laminated matrix stained blue
with PAS-Alcian blue stain and Schiff
stain without oxidation (Fig. 4).
HI. Physical-Chemical Properties
A. Soluble in 0.1 N HC1, or in concentrated
H2SO4. Insoluble in 10 per cent NaOH, 10
per cent NH4OH, ether, alcohol, or chloroform.
B. Calcium oxalate demonstrated by x-ray
diffraction pattern (Fig. 2).
C. The crystal was identified as SO per cent
calcium oxalate by electron niicroprobe
determination of calcium content.
in their kidneys (72 per cent) was greater
than in the control group (45 per cent). The
difference, as calculated by means of the
chi-square method, is significant at the
2.5 per cent level.
The reason for the correlation between
malignant neoplasms and renal oxalatecrystalluria is not readily apparent. Bennett
and Roscnblum3 reviewed the evidence that
all of the oxalate in the urine is endogenous,
and that the increased production of oxalate
in primary oxalosis may result from the
absence of an enzyme that degrades glycine
to formic acid. A search for abnormalities
of metabolism of glycine in patients with
carcinomas may prove to be rewarding.
With polarized light, some crystals
11
manifested a black cross, indicating radial
or tangential growth. Our studies by means
of x-ray diffraction confirm that the calcium
oxalate complex grows as a multitude of
small crystals only partly oriented with each
other, rather than as a single crystal. There
is apparently considerable variation in t)ie
pattern of growth, and also a tendency for
the later portions of the crystal to adopt
the configuration of the earlier parts. In the
3 crystals examined, the angular spreads
ranged from 20 to 50 degrees. Because
nucleation is difficult, one would expect
additional material to be deposited on an
already formed crystal, rather than to form
another one. Possibly the growth of the
earlier crystals is blocked by a matrix, in
such a manner that renucleation is necessary.
This concept is supported by the presence of
stainable, laminated material within the
crystals (Fig. 4). Perhaps, the low content of
calcium is partially a reflection of an error
in analysis, inasmuch as no attempt was
made to prepare a flat surface of the
reference standards for calcium oxalate or
carbonate.
The electron microprobe will undoubtedly
prove to be of considerable value in the study
of biologic material. Data virtually unobtainable by means of conventional analytical
technics are easily provided with this
instrument. The distribution of such
elements as chromium, iron,7 calcium, and
potassium11 can be readily determined and
related to different parts of the biologic
microstructure. The ARL microprobe is
especially suited to this type of study,
because it can be fitted with a transmitted
light accessory that permits viewing of the
tissue in either ordinary or plane-polarized
light during analysis.
SUMMARY
Five hundred consecutive autopsies at the
University of Chicago Hospitals were
reviewed in order to determine the incidence
of crystals of calcium oxalate in the kidneys.
Fifty-six per cent of the patients with renal
crystals had no underlying renal disease.
This finding does not support previous
reports of the invariable association of
crystals of calcium oxalate in the kidneys
12
BENNINGTON ET AL.
Vol. 41
.^"
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*
FIG. 2. Rotational x-ray diffraction photograph of a representative renal
tubular crystal. The pattern is characteristic of calcium oxalate.
with chronic renal disease. No statistical
correlation between the presence of crystalline deposits of oxalate in the kidneys and
hepatic disease or renal disease was observed;
however, there was a higher incidence of
malignant neoplasms in the autopsy patients
with renal crystals of oxalate than in the
control group. A possible relation to altered
metabolism of glycine is discussed.
The crystals of calcium oxalate in the
kidneys were examined by means of x-ray
diffraction, electron microanalysis, polarizing microscopy, special staining, and solubility studies. The presence of a matrix in
the crystals is thought to be the basis for
their complex crystalline pattern.
SUMMARIO IN IXTERLINGUA
Esseva revistate le reportos de 500 consecutive necropsias effectuate al Ilospitalcs
del Universitatc Chicago, con le objectivo
de determinar le incidentia de crystallos de
Jan.1964
"V-
13
RENAL CALCIUM OXALATE CRYSTALS
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f
' * -
•
•
-
*
*
F i o . 3 (upper). Radial ami fan-shaped orientation of the crystals of calcium oxalate
in renal tubules under nonpolarized light. Hematoxylin and eosin. X 030.
F I G . 4 (lower). Concentric deposition of a t least 3 layers of matrix, which is positive
with SchitT's reagent, around partially oriented crystals of calcium oxalate within a
renal tubule.
oxalato dc calcium in le rcn human. Cinquanta-sex pro cento del subjcctos eon tal
crystallos renal non habeva un subjacentc
morbo renal. Tste constatution non supporta
previc reportos dc un invariabile association
dc chronic morbo renal con le presentia, in
le rencs, dc crystallos de oxalato de calcium.
Ksscva trovatc nulle correlation statistic
14
Vol. 41
BENNINGTON ET AL.
4.
inter 1c prcsentia de depositos crystallin de
oxalato in le renes e morbo hepatic o renal.
Tamen, esseva notate un plus alte incidentia
5.
de neoplasmas maligne in le necropsiate
subjectos con crystallos renal de oxalato que
in le gruppo de controlo. Un possibile relation con alterationes in le metabolismo de 0.
glycina es discutite.
7.
Le crystallos de oxalato de calcium in le
renes esseva examinate per medio de studios 8.
de diffraction de radios X, de microanalyse
electronic, de microscopia polarisante, de 9.
tinctuarationes special, e de solubilitate. Es
opinate que le presentia de un matrice in le
crystallos es le base pro lor complexe con- 10.
figuration crystallin.
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