506
WALKER ET AL.
sistently negative in all areas and all patients. The prostate
carcinoma, therefore, is more like oral cavity epidermoid
carcinoma than papillary transitional cell carcinoma of
the urinary bladder in regard to its loss of blood group
antigens.
These observations suggest another possible use for the
SRCA technic in prostatic pathology. The prostatic lesions
that are borderline between hyperplasia and carcinoma
present a difficult clinical problem. It is essentially impossible to predict which patients subsequently will develop an invasive prostatic adenocarcinoma and which
ones will not. It is possible that the SRCA test might help
to differentiate this group of lesions, so-called atypical
hyperplasia, in much the same way that it separates the
borderline urinary bladder and oral cavity lesions. In patients with borderline oral cavity lesions or grade I transitional cell carcinoma of the urinary bladder, a negative
SRCA test predicts an increased risk of recurrence. We
currently are examining cases of atypical prostatic hyperplasia using the SRCA technic in order to determine
A.J.C.P. • April 1984
if this test can predict patients who will develop a malignant neoplasm.
References
1. George DI, Burzynski NJ, Miller RL: Reactive properties of oral
lesions to the specific red cell adherence test. Oral Surg 1979;
47:51-57
2. Gupta RK, Schuster, R, Christian WD: Loss of isoantigens A, B
and H in Prostate. Am J Pathol 1973; 70:439-448
3 Lange PH, Limas C, Fraley EE: Tissue blood group antigens and
prognosis in low stage transitional cell carcinoma of the bladder.
J Urol 1978; 19:52-55
Silverberg E: Cancer statistics, 1983. CA 1983; 33:9-25
Weinstein RS, Coon J, Alroy J, Davidson I: Tissue associated blood
group antigens in human tumors, Diagnostic immunohistochemistry. Edited by RA DeLellis. New York, Masson Publishing,
1981, pp 239-261
Yamase HT, Powell GT, Koss LG: A simplified method of preparing
permanent tissue sections for the erythocyte adherence test. Am
J Clin Pathol 1981;75:178-181
Young AK, Hammond E, Middleton AW: The prognostic value of
cell surface antigens in low grade, non-invasive transitional cell
carcinoma of the bladder. J Urol 1979; 122:462-464
Electronic Counting of Spinal Fluid Cells
INGEBRIGT TALSTAD, M.D.
An analysis of the electronic counting of leukocytes in cerebrospinal fluid (CSFLpc) was made theoretically in models and in
patients. At spinal fluid dilutions of 1/500, 1/50, 1/25, and
1/2, linearity was obtained by electronic counting down to 1,000,
100, 20, and 2 Lpc (106/L), respectively. The electronic particle
counters produce satisfactory results at Lpc > 100 (106/L) but
need modifications to produce satisfactory results at Lpc < 100
(106/L). It is theoretically possible to reduce the variation nine
times by electronic counting as compared with microscopic
counting. A method for the correction of blood admixture at
traumatic spinal puncture by electronic counting was shown to
be satisfactory. (Key words: Spinal fluid; Electronic counting)
Am J Clin Pathol 1984; 81: 506-511
MICROSCOPY is at present the routine method for spinal
fluid cell counting, however, the precision is poor at low
cell numbers. The precision is improved by ultrafiltration
of a large volume of spinal fluid,1 but this method is
cumbersome in routine use. While the electronic counting
Received March 25, 1983; received revised manuscript and accepted
for publication October 24, 1983.
Address reprint requests to Dr. Talstad: 5016 Haukeland Hospital,
Bergen, Norway.
Hematological
Division, Haukeland Hospital,
University of Bergen, Norway
of blood cells is a routine method, no similar method is
yet available for spinal fluid cell counting. The present
study demonstrates a theoretic approach to spinal fluid
cell counting and studies the capacity of electronic particle
counters to measure the low cell concentrations that may
occur in spinal fluid.
Materials and Methods
Theoretical Approach
The standard deviation (SD) in microscopic or electronic counting equals the square root of the number of
cells counted (Poisson's law).2 The relative precision of
electronic counting theoretically is improved compared
with microscopic counting by using a lower dilution of
the spinal fluid and/or by counting a larger volume
(Table 1).
507
BRIEF SCIENTIFIC REPORTS
Vol. 81 -No. 4
Table 1. A Theoretic Approach to Electronic Counting of Spinal Fluid Cells
Expected Variation
Counting Method
M
Microscopy
A
B
C
D
E
F
Electronic
Electronic
Electronic
Electronic
Electronic
Electronic
Sample Volume
Dilution
3.2 ^L
1:1
20 tiL
1:500
1:50
1:25
1:2
1:20
1:2
200/»L
400 ML
5,000 nL
500 ML
500 nL
No. of Cells
Counted
CV
3.2 nL
3.2X
100/\/l2X
500 ML
500 nL
500 nL
500/xL
5,000 fiL
500 nL
X
10X
20X
250X
250X
250X
Counting Volume
cvr =
100/\fr
IOO/VIOX
100/V20X
100/V250X
100/V250X
100/V250X
CVE/CV M
1.78
0.57
0.40
0.11
0.11
0.11
Model Experiments
Microscopic CSFLpc Counting
Mononuclear cells (MC) and polymorphonuclear cells
(PML) were separated from the peripheral blood using
two gradients: Lymphoprep® (Nyegaard and Co, Norway)
and Granuloprep at 400 X g.3 After being washed two
times, the cells were suspended in cell-free spinal fluid
(5,000 X g, 30 minutes) to obtain a stock suspension that
was counted electronically. Various cell concentrations
of MC and PML were obtained by dilution with cell-free
spinal fluid (theoretic cell concentrations); this method
is more exact than other counting methods at low cell
concentrations.
A Fuchs Rosenthal's counting chamber was used in
which leukocytes in 3.2 j*L spinal fluid were counted (X):
Clinical Material
The spinal fluid from 33 consecutive patients with
meningitis, 20 serous meningitis, and 13 bacterial meningitis, was examined within 30 minutes of being obtained
by microscopy as well as by electronic counting.
Electronic Counting of Cerebrospinal Fluid (CSF)
A ZF-model (Coulter Electronics, Harpenden, England)
is a semiautomatic particle counter in which the sum of
erythrocytes and leukocytes (Epc + Lpc) were counted
at the calibration used for erythrocytes; Lpc were counted
at the calibration used for leukocytes after lysis of erythrocytes with Zaponin® (Coulter Electronics); Epc then
could be calculated.
The electronic counting Methods A, B, and C consist
of the use of dilutions of 20, 200, and 400 yL spinal fluid
in 10 mL Isotone® (Coulter Electronics). Method D consists of centrifuging 5 mL spinal fluid (1,500 rpm, 10
minutes), decanting the supernatant, and remixing the
residue in 10 mL Isotone. Method E consists of diluting
500 fiL spinal fluid with 10 mL Isotone and by 10 times
repeated counting (Table 1).
The automatic electronic particle counter Hemalog
6000® (Technicon) and the S-plus II® (Coulter Electronics)
gave Epc and Lpc directly.
CSFLpc = (X/3.2)10 6
Correction at Traumatic Spinal Puncture
The admixture of leukocytes from blood at traumatic
spinal puncture can be corrected as follows:
CSFLpc = CSFLpc (total) - Lpc (admix)
The number of leukocytes counted in the spinal fluid
(CSFLpc [total]) include leukocytes in the spinal fluid
(CSFLpc) as well as the admixture of leukocytes from
blood (Lpc [admix]).
Lpc (admix) = BLpc • CSFEpc/BEpc
Lpc (admix) is obtained by electronic counting of leukocytes in blood (BLpc), erythrocytes in the spinal fluid
(CSFEpc), and blood (BEpc).
Results
The linearity of electronic counting methods was studied in models. Linearity was found down to 1,000, 500,
and 20 Lpc (106/L) at dilutions (1/500, 1/50, and 1/25,
respectively (Fig. 1). Dilution 1/2 (Table 1, D) was obtained by concentrating cells from 5 mL spinal fluid; high
precision (r = 0.96, N = 29) and linearity (Fig. 2) was
obtained down to 2 Lpc (10 6 /L).
Another method (Table 1, E), which theoretically gives
the same precision as Method D, was studied by ten times
repeated counting, obtaining a counting volume of 5,000
fiL. Satisfactory linearity was found down to 2 Lpc (X106/L)
and the coefficient of correlation was r = 0.98 and r = 0.97
for PML and MC, respectively (Fig. 3).
In spinal fluid from patients (Fig. 4), a comparison was
made between an extensive microscopic counting method
and electronic counting using Method C above and
TALSTAD
508
CSF-LPC (x10 6 /l)
(Coulter Counter)
AJ.C.P. • April 1984
1 5 0 0
125
150
100
I
K)
100
KX»
»
CSF-LPC ( x 1 0 6 / l )
(theoretical)
FIG. 1. Electronic CSFLpc Counting (models): PML suspended in cell-free spinal fluid (abscissa), were compared with the concentrations
measured by electronic counting (ZF-model, Coulter Electronics) at dilutions 1:500, 1:50, and 1:25 (ordinate) (Methods A, B, and C).
Method D below Lpc 50.106/L (r = 0.99, N = 33). There
was no significant difference between microscopic and
electronic counting in bacterial meningitis {P > 0.10),
but there was a significant difference in serous meningitis
(P < 0.025).
CSF-LPC
Corrections at traumatic spinal puncture: A high degree
of correlation was obtained between the theoretic and
measured CSFLpc (r = 0.98, N = 18), after correction
for the admixture of leukocytes from blood (Fig. 5).
The Hemalog 6000 showed linearity in counting of
(x106/l)
(Coulter Counter)
40
Me
30-
FIG. 2. Electronic CSFLpc
Counting (models): PML diluted in
cell-free spinal fluid (abscissa) were
compared with concentrations measured by electronic counting (ordinate) after the concentration of cells
from 5 mL spinalfluid(Method D).
20
10-
-i20
0
10
CSF-LPC ( x 1 0 6 / l ) (theoretical)
20
-r40
Vol. 81 • No. 4
BRIEF SCIENTIFIC REPORTS
509
CSF-cells(x10'/l )
FIG. 3. Electronic CSFLpc
Counting (models): PML and MC
were suspended in cell-free spinal
fluid (abscissa), and low cell concentrations were obtained by dilution. Using dilution 1/20, half of the
cells were counted by repeated
counting of 500 ^L X 10 (ordinate),
using the ZF-model (Coulter Electronics) (Method E).
10
20
( Theoretical)
PML or MC down to 100 LPC (X106/L), however, by
increasing the sample size from 0.35 to 0.70 mL, linearity
was obtained down to 10 Lpc (X106/L). The S-plus II
showed linearity of PML and MC down to 200 Lpc
(X106/L)(F,g. 6).
30 0
10
30
40
50
CSF • cells (10'/1 )
Discussion
The advantage of electronic counting is that more cells
can be counted within seconds than within minutes by
microscopy. However, the main problem with light mi-
CSF-LPC(x10 6 /l)
Coulter Counter
10000
1000
FIG. 4. Comparison of microscopic and electronic CSF Counting in 20 patients with serous
(O) and 12 patients with bacterial (•) meningitis: An extensive microscopic counting
method, using 8 Fuchs Rosenthals counting
chambers < 25 and 1 chamber > 25.106
Lpc/L (abscissa), was compared with electronic
counting using Method C > 50 and Method
D < 50.10* Lpc/L.
100
10-
10
100
1000
CSF-LPC U 1 0 6 / l )
Microscopic counting
10 000
TALSTAD
510
A.J.C.P. • April 1984
CSF-LPC ( 1 0 6 / l )
(Coulter Counter)
10 0 0 0 r
1000
FIG. 5. Corrections at traumatic spinal puncture (model study): Series of PML and MC
were prepared in cell-free spinalfluidand mixed
with various quantities of blood. Comparison
between the theoretic (abscissa) and the corrected CSFLpc obtained by electronic counting
(ordinate) are shown.
100
100
1000
10 0 0 0
CSF-LPC ( 1 0 6 / l )
(Theoretical)
CSF
(Hemalog 6000)
3000
2000
1000-
500
1000
I500(x!0 6 /l)
CSF (theoretical)
1000
2000
3000(xl0 6 /D
CSF (theoretical)
FIG. 6. Automatic counting of spinal fluid cells (model study): PML and MC were suspended in artificial spinal fluid (abscissa) and compared
with cell concentrations measured by Hemalog 6000 or Coulter S-plus II (ordinate).
Vol. 81 -No. 4
BRIEF SCIENTIFIC REPORTS
croscopy as well as with electronic counting is that of
obtaining satisfactory precision at low cell numbers. The
interference from noise in electronic counting makes the
precision at low cell concentrations less than that theoretically calculated. The present study shows that the precision in electronic counting was improved greatly at low
cell concentrations when a low dilution of spinal fluid
and a large sample size were used. A particularly high
precision was obtained by the concentration of cells from
5 mL spinalfluid.However, this method is not particularly
practical. Nor is it practical to improve the precision by
performing repeated counting with an electronic particle
counter. It is necessary to modify the electronic particle
counters before they can produce satisfactory results for
the counting of spinal fluid cells. This can be done by
using a relatively large sample size (0.5 mL) and by prolonging the counting time 10 times, i.e., counting half of
the cells present after dilution (Method E). Method F
(Table 1) shows that the same precision as seen with
Methods D and E is obtained theoretically by using a
low dilution of spinal fluid, by counting cells in half of
this volume, and by using the electronic separation of
cells; Method F may have an advantage over Method E
in that a shorter counting time is needed. Whether the
precision by electronic compared with microscopic
counting may reach that theoretically evaluated, depends
511
on how satisfactorily the electronic separation of cells is
carried out.
The comparison of microscopic and electronic counting
in patients showed a high degree of correlation (r = 0.99).
However, the occasionally lower values seen with microscopic than with electronic counting was most likely due
to so few cells being counted by microscopy, since similar
deviations did not occur in the model studies.
Another important problem is the detection of meningitis at traumatic spinal puncture; this has not been
satisfactorily solved previously.4 The present study shows
that correction for the admixture of leukocytes from blood
was possible. It may be possible to include this correction
automatically by modifying the electronic particle counters.
References
1. Burechailo F, Cunningham TA: Counting cells in cerebrospinal
fluid collected directly on membranefilters.J Clin Pathol 1974;
27:101-105
2. Goldstein A: Biostatistics: An introductory text. Third edition. New
York, MacMillan, 1965, pp 117-124
3. Talstad I: Simultaneous separation of monocytes, lymphocytes and
polymorphonuclear cells from the peripheral blood. Int Soc Haematol, European and African Division, Sixth Meeting (Athens)
1981, p408
4. Wilson JW, Stevens JB: Effects of blood contamination on cerebrospinal fluid analysis. J Am Vet Med Assoc 1977; 171:256258
Wright's Stain in Rapid Diagnosis of Pneumocystis carinii
JAVIER DOMINGO, M.D. AND HARLAN W. WAKSAL, M.D.
The conventional Hematology Wright's stain has been used in
touch preparation from open lung biopsies to identify Pneumocystis carinii. A 100% positive correlation has been found
using this rapid and readily available technique when compared
to the conventional silver stain and permanent histologic sections.
(Key word: Pneumocystis carinii; Wright's stain) Am J Clin
Pathol 1984;81:511-514
THE INCREASE in incidence of Pneumocystis carinii
in association with acquired immunodeficiency syndrome
(AIDS), as well as its prevalence in other immunocompromised hosts, have made apparent the necessity for a
Received June 21, 1983; received revised manuscript and accepted
for publication September 13, 1983.
Address reprint requests to Dr. Domingo: Department of Pathology,
Long Island College Hospital, 350 Henry Street, Brooklyn, New York
11201.
Department of Pathology, State University of New York,
Downstate Medical Center, 450 Clarkson Avenue,
Brooklyn, New York
rapid, sensitive, and specific stain for its diagnosis. These
cases usually are rushed requests following an open-lung
biopsy in a patient with diffuse, progressive pulmonary
infiltrates.5 The usual stains for Pneumocystis carinii,
which included Grocott's original silver method,3 Giemsa's, Gram-Weigart, and toluidine blue stains1"6 have required either two to three hours for completion, special
staining set-ups, or the skills of experienced technicians.
More recently a 10-minute silver stain,4 a modified Rapid
Grocott's methanamine-silver nitrate stain,2 have been
developed with satisfactory results. However, these procedures also have required special stain set-ups to be