Microbiology
Meningitis: A Case Study
Rita A. Krueger, MT(ASCP)
Case History
A three-month-old infant was
brought to a clinic for pediatric
evaluation. The infant had had a mild
fever (101 °F) for the past 24 hours
and was described as very irritable and
fussy by the mother. Physical exam-
ination results were normal. A lum-
bar puncture was performed, a
complete blood cell count and blood
cultures were obtained, and urine was
collected for urinalysis and culture.
The results are given in Table I.
Results and Discussion
The laboratory data for the patient
are indicative of meningitis, which is
an inflammation of the meninges.
Meninges is a collective term for the
layers surrounding the brain and
spinal column—the pia mater, arach-
noid, and dura mater. Meningitis is
further categorized by its pathogenic
agent—bacterial meningitis (BM) and
aseptic meningitis (AM)—which in-
cludes viral, fungal, mycobacterial,
amebic, and mycoplasma etiologic
sources. Viral meningitis is the most
common type of aseptic meningitis.
Defining a viral or bacterial cause can
be determined using Gram's stain,
cultures, and a variety of rapid meth-
ods for the diagnosis of meningitis.
From Riverside Medical Center, Minneapolis, MN.
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Viral Meningitis
A 1973 CDC surveillance report
showed over 8,000 cases of aseptic
meningitis. Eighty to 85% of these
cases were caused by unidentified
agents; 15% to 20% of the 8,000 cases
were due to the enteroviruses and it
is thought that enteroviruses caused
some of the unknown cases but lacked
the culture confirmation.1 Such re-
ports have illustrated that enterovi-
ruses are the most common agent of
viral meningitis (VM). Echoviruses,
coxsackie, and polio viruses are types
of enteroviruses. The mumps virus,
arboviruses, and herpes simplex virus
(HSV) types 1 and 2 represent the
other most common viral agents. CDC
reports indicate a prevalence of en-
terovirus cases during the summer and
early autumn. However, an entero-
viral meningitis can also occur during
Table 1: Results of Pediatric Evaluation
Peripheral Blood
WBC
Hemoglobin
Differential
Band forms
Segmented
Lymphocytes
Monocytes
CSF
WBC
RBC
PMN
Monocytes
Protein
Glucose
Gram's stain: no organisms seen
LABORATORY MEDICINE • VOL. 18, NO. 10, OCTOBER 1987
other seasons of the year.2
The clinical presentation of VM
varies with the patient's age, char-
acterized by some or all of the follow-
ing symptoms: fever, headache, nuchal
stiffness, lethargy, vomiting, and sen-
sitivity to light. Infants especially tend
to present with nonspecific symptoms
that may only include irritability and
fever. When a fever of unknown ori-
gin is present, central nervous system
infection must be suspected. A lum-
bar puncture is needed to diagnose
meningitis and to clarify further a
viral or bacterial etiology. Table II
summarizes typical CSF results in VM
and BM. CSF pleocytosis with a pre-
ponderance of mononuclear cells,
marginally elevated to normal pro-
tein and normal glucose, represents
the classic profile of VM.
Studies have shown 20% to 75% of
12,000
12g/dL
12%
70%
10%
8%
330 mm 3 (330 x 106'L)
10 mm 3 (10 x 106/L)
70%
30%
50 mg/dL (50 g/L)
55 mg/dL (55 g/L)
677
 Table II: Summary of CSF Results in Viral and Bacterial Meningitis
Viral
Gross appearance Clear to hazy
WBCx10«/L 10-1,000
Major cell type Lymphocyte
Protein Normal or increased
Glucose Normal
CSF/blood glucose 0.6-0.8
VM cases to have polymorphonuclear
(PMN) leukocytosis in their first CSF
cell count.2 The chamber differential
can be difficult and has poor precision
when small numbers of cells are ob-
served. Wright's staining of spun sed-
iment has the following disadvantages:
(1) cell recovery is inconsistent, (2) cell
disruption and distortion is common
due to the high rpm, and (3) the qual-
ity of the smear is generally poor.
Filtration, sedimentation, and cyto-
centrifugation are among the meth-
ods offering improved cellular
concentration with minimal change
in appearance. Cytocentrifugation
seems the simplest technically, pro-
viding rapid concentration of cells with
good detail.3 Mengel retrospectively
reviewed 75 cases to determine if cy-
tocentrifuge data could distinguish BM
from VM. On the basis of 75 cases, he
developed the following criteria to
evaluate the data: (1) greater than 60%
PMNs is supportive evidence of and
(2) repeat LPs six to eight hours can
show a shift from neutrophils to
mononuclear cells, distinguishing AM
from BM.4 Other investigations have
likewise demonstrated that spinal taps
done six to 48 hours after the initial
one showed a significant decrease in
the percentage of neutrophils, thus
establishing a diagnosis of AM.
The diagnosis of VM is based com-
monly on viral isolation from cultures
or retrospective viral titer analysis.
Isolation of a virus from the CSF pro-
vides conclusive evidence of the etiol-
ogy of the meningitis. In addition to
CSF viral cultures, stool and throat
specimens may yield isolates in pa-
tients with meningitis. With a fecal-
oral route mode of transmission, en-
teroviruses are most commonly iso-
lated from stools. This sensitivity does
not provide concurrent specificity,
however. The interpretation of a pos-
itive viral isolate from the stool or
throat when the CSF virus culture is
negative provides only presumptive
evidence of the causal agent.5 Such
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Meningitis
Bacterial
Turbid
>S00
PMN
Increased
Decreased
<0.6
culture data need to be evaluated
against clinical presentation and cross-
checked with viral titers. In a study
by Mintz and Drew,5 CSF viral cul-
tures confirmed 43% of the VM cases.
Simultaneous throat and rectal swabs
isolated viruses for 89% and 83% of
the cases, respectively. Thus, the
probability of isolating the virus in-
creases by culturing the throat and
feces along with the CSF. The appar-
ent cause-effect relationship between
the isolate and the illness must take
into account all relevant clinical and
laboratory results. 56 The Mintz study
also found rectal swabs inadequate
substitutes for fecal specimens. They
concluded CSF and stool cultures pro-
vided maximum diagnostic informa-
tion.5
Primary monkey kidney and a va-
riety of human cell lines can support
the growth ofmost enteroviruses. The
cell lines are examined for cytopathic
effect (CPE), which provides pre-
sumptive identification. Different vi-
ruses cause characteristic CPE
changes. In some circumstances, such
as a recognized epidemic of VM, the
observation of CPE in cell cultures
could be sufficient to substantiate a
viral cause. However, if definitive
identification is desired, neutraliza-
tion tests are necessary. In neutrali-
zation techniques, the patient's viral
isolate incubates with a battery of
known immune sera. Such a scheme
represents a "rifle" approach to virus
typing and is superior to the "shot-
gun" method.6 Viral cultures do not
yield an isolate in the latter. Acute
and convalescent serum samples are
tested with the most probable viral
agents for a fourfold titer increase. For
example, over 60 types of enterovi-
ruses are known to infect the central
nervous system, but ten types are re-
sponsible for 71% of the cases.1 Neg-
ative viral titers against the ten most
common enteroviruses could mean
that the antigen pool did not contain
the particular virus that the patient
had. Unfortunately, lack of a virus
isolate may leave this hit-or-miss ti-
ter analysis as the only recourse.
HSV and arboviruses are rarely de-
tected in CSF cultures. HSV-1 usu-
ally causes meningoencephalitis (an
inflammation of both the meninges
and the brain), making brain tissue
the best source of isolating the virus.
HSV-2 is more likely to cause aseptic
meningitis and has been found in
CSF.2 Serology testing of antibody ti-
ters for herpes simplex has poor spec-
ificity. Fifteen to 20% of patients with
seroconversion do not have HSV.1 The
ratio of HSV antibody in the CSF and
serum has been used to improve spec-
ificity. This comparison is difficult to
evaluate in the early stages of infec-
tion.
Arboviruses require infant mice for
detection, so most laboratories rely on
serologic methods to diagnose them.
Over 250 types can cause AM. Epi-
demiology variables such as vector
exposure, geographic location, and
seasonal infection patterns can re-
duce the range of possible arbo-
viruses.1 These epidemiology factors,
coupled with the case numbers, show
that four arboviruses are responsible
for most cases of meningitis and en-
cephalitis in the United States—St
Louis encephalitis virus, Eastern
equine encephalitis virus, Western
equine encephalitis, and the Califor-
nia encephalitis group.2
The value of doing viral cultures is
considered academic by some since the
results usually follow bacteria cul-
tures. Chonmaitree et al7 performed
a two-year case review of CSF viral
cultures. They concluded that viral
cultures were beneficial since they al-
lowed for termination of antibiotic
therapy and shortened hospitaliza-
tion. However, they recommended CSF
viral culturing only when other di-
agnostic data support a viral etiology.
They found spinal fluids were more
likely to be positive for a virus if the
culture was set up immediately after
lumbar puncture.
To summarize, viral culturing is the
best method for diagnosing VM. A vi-
rus detected in the CSF has an un-
deniable etiologic role. Viral isolates
only from the stool or nasopharynx
need careful consideration before la-
beling them the etiologic agent. Viral
titers are most productive when a vi-
rus is isolated. When an isolate is not
 obtained, titers provide "hit or miss"
information.
Bacterial Meningitis
The spinal fluid testing is the key
to differentiating VM from BM. The
Gram's stain is the simplest tool for
diagnosing BM. On the basis of pop-
ulation studies in the United States,
the following bacteria are the most
frequent cause of BM: Hemophilus in-
fluenzae, Streptococcus pneumoniae,
and Neisseria meningitis. Among
neonates, group B streptococci and
Escherichia coli are the predominant
pathogens. The overall case fatality
rate of 26% for pneumococcal menin-
gitis ranks it the highest in compar-
ison, with 6% for H influenzae and 10%
for meningococcal meningitis. In in-
fants younger than one month, a 22%
fatality rate is involved with group B
streptococci.8 With such high risk of
death in lieu of antibiotic therapy,
clearly BM represents a medical
emergency.
The clinical presentation of BM is
variable and does not have unique
features to distinguish it from VM.
Fifty to 75% of children have a pro-
dromal upper respiratory infection.9
Several of the following factors have
been identified as prognosticators: (1)
state of consciousness, (2) age of pa-
tient, (3) seizures, and (4) another su-
perimposed disease process. Upon
hospital admission, a comatose pre-
sentation is more likely to result in
abnormal neurologic sequelae than an
irritable or lethargic state. Patients
younger than 1 year or older than 40
years are at higher risk of mortality
and morbidity. Seizures caused by
bacterial toxins or brain damage are
associated with increased chance of
hearing handicap or other neurologic
abnormality. Seizures in children oc-
cur more frequently in H influenzae
and pneumococcal cases than in those
caused by meningococci. Any under-
lying disease process weakens the pa-
tient's immune response and may
lengthen the recovery time as well as
increase the chance of post-recovery
sequelae.9
In the peripheral blood, an elevated
white count is characteristically seen.
A left shift on the differential count
is another common feature. Blood cul-
tures are drawn because bacteremia
usually accompanies BM. For the in-
fant in the case study, a urinalysis
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and culture was done to rule out uri-
nary tract infection as the cause of
fever.
CSF is normally clear and colorless,
and any haziness or turbidity indi-
cates a pathologic condition. A pleo-
cytosis of approximately 400 x 106 cells
per liter would cause a cloudy ap-
pearance. When more than 6,000 x 106
RBCs per liter are present, the CSF
appears grossly bloody. If traumatic
puncture is a possibility, an approxi-
mation of the true WBC count is to
subtract 1 WBC per 700 RBCs in the
chamber count. This correction as-
sumes that there is a normal number
of leukocytes and erythrocytes in the
peripheral blood.10
The rule of thumb, "hundreds of CSF
WBCs in VM and thousands in BM"
can be applied in most cases, but the
exceptions that occur detract from
using it as the definitive test. Fish-
bein et al11 did a retrospective study
of 50 cases of BM and found seven with
less than 7 WBCs x 106/L. Organisms
were seen on two of the initial CSF
Gram's stains, but the nearly normal
CSF protein and glucose results were
also in contrast to the "typical" CSF
findings in BM. The patients in this
study were elderly or had other de-
bilitating conditions. 11 Thus, the
screening ability of CSF cell counts,
glucose, and protein determinations
in compromised individuals can be di-
minished. PMNs, an abnormal con-
stituent of spinal fluid, are usually the
major type of leukocyte seen in the
differential count. This aspect of BM,
too, has noteworthy exceptions. In a
retrospective chart review of 103 doc-
umented cases of BM, Powers12 found
lymphocytosis in 14% of them. When
the total white cells counted was less
than 1,000 x 106/L, thirty-two percent
of the reviewed patients had more than
50% lymphocytes. Previous studies had
shown that lymphocytic responses
were most commonly caused by Lis-
teria monocytogenes or previous an-
tibiotic therapy. Of the 14 cases
reviewed, only one had Listeria iso-
lated.12 The role of the Gram's stain
and culture in these situations is fur-
ther stressed.
Hypoglycorrhachia, defined as an
abnormally low CSF glucose level, is
usually present in BM. In order to best
assess the CSF glucose, a serum glu-
cose sample should also be obtained
since all CSF glucose is derived from
LABORATORY MEDICINE • VOL. 18, NO. 10, OCTOBER 1987 6 7 9
the blood. The normal CSF/blood glu-
cose ratio is 0.6 to 0.8. The ratio can
be useful when a patient had hyper-
glycemia with apparently normal CSF
glucose. A study by Merritt and Fre-
mont-Smith found glucose levels 10 to
40 g/L in 57% of BM and less than 10
g/L in 23% of the 154 cases re-
viewed.10 Hypoglycorrhachia in CSF
with significant pleocytosis is pre-
sumptive of BM.
An elevated level of spinal fluid
protein is a key sign of disease. The
elevation indicates a pathologic con-
dition that has increased permeabil-
ity at the blood-brain barrier. In the
study by Merritt and Fremont-Smith,10
less than 2% of the cases had normal
protein levels.
Other chemical tests have been ap-
plied to the CSF in hopes of differ-
entiating VM from BM. Among these
are CSF immunoglobulins, lactate, and
creatine kinase brain isoenzyme. In-
vestigations have shown that none of
these tests consistently discriminate
between the two.213
Spinal fluid specimens should be
promptly taken to the laboratory. Re-
frigeration is contraindicated due to
the fastidiousness of H influenzae and
N meningitidis. Spun sediment is ex-
amined after gram staining; 2,500 rpm
for 15 minutes is commonly used.
Higher spin rates and membrane fil-
ters can be used to enhance the con-
centration of smaller numbers of
bacteria. Even with centrifugation,
extensive examination of the smear is
necessary. Bacteria counts below 105/
mL (108/L) are common and would re-
sult in less than one organism per oil
filed. The skill of the observer is crit-
ical to accuracy.
Rapid diagnostic methods have be-
come available in an effort to aug-
ment the Gram's stain and to preempt
culture results. The rapid techniques
include countercurrent Immunoelec-
trophoresis (CIE), latex particle ag-
glutination (LPA), coagglutination
testing (CAT), and the limulus lysate
test. In addition to enhancing diag-
nostic speed, these tests are generally
not influenced by prior antibiotic
treatment. CIE, LPA, and CAT may
also be performed on serum and urine
specimens. Despite their potential for
a speedy diagnosis, the reliability of
the rapid methods is subject to the
specificity and sensitivity of the re-
agents. Available antisera are not

HIB
V7
- \
• •
Anti-H influenzae type B with protein A of S aureus coagglutinates with homologous
antigen.
standardized and vary considerably.
Thus, a negative result cannot be con-
sidered conclusive since it could be
caused by low antigen levels or poor
antisera reactivity. Even a positive
result should have culture confirma-
tion since cross-reactions may hap-
pen. Clinicians use rapid methods to
complement the smear and culture,
though the decision to admit a patient
and the antibiotics chosen for treat-
ment are usually independent of the
rapid method result.
CIE was formerly one of the few
rapid methods available but is now
used less frequently. Newer, simpler
methods such as LPA and CAT offer
comparable or improved sensitivity
and specificity. CIE remains as the
basis for evaluating the performance
of the newer methods.
In latex particle agglutination kits,
latex particles are coated with anti-
bodies to the bacteria commonly as-
sociated with BM. In the presence of
6 8 0 LABORATORY MEDICINE • VOL. 18, NO. 10, OCTOBER 1987
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N routinely available, although it was
F
.b
F Cassady15 evaluated the test on 208
c CSF specimens collected from new-
• • Protein A
homologous antigen, strong aggluti-
nation occurs. The size of the clumps
and the speed of their development
correlate with the quantity of antigen
present. The detection of the polyri-
bose-phosphate (PRP) capsule of H
influenzae type B with LPA is supe-
rior to CIE; levels as low as 1 ng of
PRP are positive in LPA, whereas CIE
requires at least 10 ng/mL of anti-
gen.9
Staphylococcal CAT is another rapid
test available. In this test, the protein
A antigen in the Staphylococcus au-
reus cell wall is bound to the Fc por-
tion of an antibody to a specific
bacteria. The Fab portion of the an-
tibody is thus unoccupied and free to
unite with homologous antigen (Fig-
ure). When bacterial antigen is pres-
ent in the CSF, agglutination occurs.
Comparisons of CAT and CIE indi-
cate very similar sensitivities and
specificities. Webb and associates14
tested the Phadebact Streptococcus
Test and found it more sensitive in
detecting the diminishing antigen
levels following antibiotic therapy
than CIE. Antigen persistence was
detected an average of 6.3 days with
CAT versus 5.1 days with CIE.
The limulus lysate test is one not
first demonstrated as a detector of
endotoxins by Bang in 1946. Since
then, many other investigators have
documented its usefulness. As applied
in BM, it assays the CSF for the li-
popolysaccharide endotoxin found in
the cell walls of gram-negative bac-
teria. A lysate made from horseshoe
crab amebocytes is gelatinized when
bacterial endotoxin is present in the
CSF. Tubes containing the lysate and
CSF are observed and graded for al-
tered appearance at 30-minute inter-
vals with a maximum two-hour
incubation. A positive result must be
at least 2+ (increased turbidity and
viscosity) or a 3+ (solidified mass).15
The test is negative in AM and pos-
itive with toxin concentrations ex-
ceeding 0.05 ng from N meningitidis
or H influenzae type B. Dyson and
borns with suspected meningitis. Pos-
itive Limulus results were obtained
only from the six infants with gram-
negative CSF isolates. The initial
Gram's stain revealed the causative
organism in three of the six positive
Limulus specimens. The gram-pos-
itive bacteria of four infants were
negative at the two-hour readout.
Candida and Aspergillus can cause
false-positive results due to their sim-
ilar endotoxins.10 The usefulness of the
limulus test with blood has been less
clear-cut and remains difficult to in-
terpret.
After culture confirmation of bac-
terial meningitis, additional labora-
tory testing may be indicated. The
need for repeat lumbar punctures to
ensure effective antibiotic therapy has
been debated. The bulk of available
evidence suggests subsequent taps are
not necessary in most cases unless the
sensitivity pattern of the pathogen is
unusual. Children with meningitis
may develop a syndrome of inappro-
priate antidiuretic hormone secre-
tion. When this happens, the kidneys
retain water leading to serum elec-
trolyte dilution and hypoosmolality.
Due to the patient's risk of cerebral
 or pulmonary edema, the clinician may
closely monitor serum sodium and os-
molality levels. 16
For the patient in the case study,
negative CSF and blood cultures ruled
out BM. A virus culture was not per-
formed, but the infant's clinical course
was uncomplicated so a virus was the
most probable cause. Serology testing
for virus identification is most effi-
cacious when cultures are done in
parallel. BM can be diagnosed on
spinal fluid gram stains with good ac-
curacy. Rapid techniques such as LPA
and CAT are useful adjuncts to the
bacterial culture and Gram's stain.
The variability of CSF cell counts,
differential, glucose, and protein lev-
els limits their reliability in differ-
entiating VM from BM.
References
1. Rubin S: Detection of viruses in spinal fluid. Am
J Med 1983;74:124-128.
2. Ratzan K: Viral meningitis. Med Clin North Am
1985;69:399-413.
3. Krieg A: Clinical Diagnosis and Management,
ed 16. Henry J (ed). Philadelphia, WB Saunders
Co, 1979, pp 641-642.
4. Mengel M: The use of the cytocentrifuge in the
diagnosis of meningitis. Am J Clin Pathol
1985;84:212-216.
5. Mintz L, Drew W: Relation of culture site to the
recovery of nonpolio enteroviruses. Am J Clin
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6. Pearson G, Valdmanis A, Mann J, et al: The
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7. Chonmaitree T, Menegus M, Powell K: The clin-
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8. Broome C, Schlech W: Bacterial Meningitis.
Sande M, Smith A, Root R (eds). New York,
Churchill Livingstone, 1985, pp 1-10.
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10. Fishman R: Cerebrospinal Fluid in Diseases of
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11. Fishbein D, Palmer D, Porter K, et al; Bacterial
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Arch Intern Med 1981;141:1369-1372.
12. Powers W: Cerebrospinal fluid lymphocytosis in
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13. Rutledge J, Benjamin D, Hood L, et al: Is the
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Immunoelectrophoresis for detection of group B
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15. Dyson D, Cassady G: Use of limulus lysate for
detecting gram-negative neonatal meningitis.
Pediatrics 1976;58:105-109.
16. Shann F, Germer S: Hyponatremia associated
with pneumonia or bacterial meningitis. Arch
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