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Laboratory Medicine Conference :
Diagnostic Testing for
Pulmonary Conditions
Jim Holliman, M.D., F.A.C.E.P.
Professor of Surgery and Emergency Medicine
Director, Center for International Emergency Medicine
M. S. Hershey Medical Center
Penn State University
Hershey, Pennsylvania
Lab Medicine Conference :
Pulmonary Lecture Topics
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Sputum gram stain and cultures
Pleural effusion studies
Pulse oximetry
Arterial blood gases
Use of Sputum Gram Stain in
Diagnosis of Pneumonia
ƒ Identify specific bacterial agent(s)
–Allow antibiotic selection accuracy early
–Assists in admission decision (staph)
ƒ Indicate non-bacterial infectious cause
–Prompts selection of other diagnostic tests
(fluorescent stains, serologies, etc.)
ƒ Indicates non-infectious causes of infiltrates
–Allergic
–Autoimmune
Comparative Costs of Sputum
Tests for Pneumonia
ƒ Sputum smear & gram stain : $18
ƒ Sputum culture & sensitivity : $41
(includes gram stain )
ƒ Sputum culture for Legionella : $62
(includes smear)
ƒ Quantitative bronchial culture : $80
ƒ Aspirate for Pneumocystis stain and culture :
$41
Comparative Costs of Other Tests
Used in Diagnosis of Pneumonia
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Chest X-ray : one view :
$ 98
Chest X-ray : two views : $ 133
CBC
:
$ 21
Differential :
$ 26
Blood culture :
$ 56
Total diagnostic cost for pneumonia
workup (CBC, diff., CXR, sputum
culture, blood culture) would then be $
277
Steps in Performance of a Gram Stain
ƒ Swab out a thin film of specimen on glass slide ; Air dry the slide
ƒ Heat fix the slide over an alcohol lamp
ƒ Cover slide with crystal or gentian violet for 10 seconds ; Rinse
with tap water
ƒ Cover slide with Gram's iodine for 10 seconds ; Rinse with tap
water
ƒ Decolorize by flooding the slide with 95 % ethanol
ƒ Rock slide back & forth till a "swelling" of purple is seen ; Rinse
with water
ƒ Flood slide with counterstain safranin red for 10 seconds ; Rinse
with water
ƒ Blot with lens paper and air dry
Hints to Improve the Quality of a
Gram Stain
ƒ Initially always prepare 2 specimen slides
–Stain second specimen if Gram Stain of first fails
ƒ Prepare a separate smear on the same slide of your
own oral secretions
–If the Gram Stain turns out OK, this should show both
gram pos. & neg. bacteria
ƒ If ethanol - acetone used as decolorizer, limit the
contact time to avoid over-decolorization
ƒ Do not try to re-decolorize a bad slide or recounterstain
–Start over with new slide instead
Evaluating the Source and
"Quality" of a Sputum Specimen
ƒ Good-quality sputum specimens (lower
respiratory tract)
–Presence of mucus (purulent or opaque)
–Alveolar macrophages present
–> 25 neutrophils / low-power field and < 10
epithelial cells / low-power field
–> 20 : 1 ratio of neutrophils to epithelial cells
–One predominant organisim
Evaluating the Source and Quality
of a Sputum Specimen (cont.)
ƒ Poor-quality sputum specimen (upper
respiratory tract)
–Saliva clear, watery
–Alveolar macrophages absent
– < 25 neutrophils / low-power field and >
10 epithelial cells / low-power field
– < 20 : 1 ratio of neutrophils to epithelial
cells
–No predominant organism, or mixed flora
Poor quality sputum gram stain with squamous cells and no
macrophages
Helpful Diagnostic Clues from
the Gross Appearance of Sputum
Gross findings
Foul smell
Creamy yellow or salmon
color
Currant-jelly color
Raspberry-syrup color
Red color
(pseudohemoptysis)
Blood-streaked
(hemoptysis)
Yellow-green
Infectious causes
Oral anaerobes(aspiration)
Staphylococcus aureus
Pneumoccoccus ; Klebsiella
Pneumonic plague
Serratia
Klebsiella , Influenza,
Meningococcus, Pneumonic
plague
Non-infectious allergic asthma
General Interpretations of
Sputum Gram Stains
ƒ Polys, few epi. cells, one main bacteria :
bacterial pneumonia
ƒ Mixed flora of bacteria : aspiration
pneumonia
ƒ Scant polys and no dominant organism :
atypical or viral pneumonia
Other Information Obtainable from
the Sputum Gram Stain
ƒ Rough estimate of bacterial density
–10 or more organisms of one bacterial type per
6 > 10
oil immersion field correlates with
colony forming units per ml. of sputum
ƒ If predominantly monocytes :
–Legionella
–Mycoplasma
–Mycobacteria
Other Sputum Stains to
Consider
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Wright - Giemsa : (eosinophils)
Methenamine silver : (Pneumocystis)
Acid - fast : (Mycobacteria)
Direct fluorescent antibody :
(Legionella)
Gomori methenamine stain of sputum showing Pneumocystis
carinii
Use of Wright-Giemsa Sputum
Stain
ƒ Gram stain doesn't differentiate neutrophils from
eosinophils
ƒ Diagnostic considerations if mainly eosinophils
present :
–Allergic asthma
–Eosinophilic pneumonia
–Drug reactions
–Allergic bronchopulmonary aspergillosis
–Parasites : Pulmonary migration phase of nematodes ;
Lung flukes
–Sarcoidosis
Drugs That Can Cause Pulmonary
Eosinophilia
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Aspirin
Azathioprine
Bleomycin
Carbamazepine
Chlorpropamide
Cromolyn
Hydralazine
Imipramine
Isoniazid
Methotrexate
Methylphenidate
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Minocycline
Naproxen
Nitrofurantoin
Para-aminosalicylic
acid (PAS)
Penicillin
Phenytoin
Sulfasalazine
Sulfonamides
Tetracycline
Specific Diagnostic Clues From
the Sputum Gram Stain
ƒ Gram positive
–Medium - small diplococci : Strep.
pneumoniae
–Large clustered cocci : Staph. aureus
–Medium cocci in chains : Group A Strep
(virulence often inversely proportional to
chain length)
–Comma shaped : Nocardia
Specific Diagnostic Clues from
the Sputum Gram Stain (cont.)
ƒ Gram negative
–Coccabacillary (pleomorphic) :
Hemophilus influenzae
–Bacilli : Klebsiella or enterics
–Diplococci : Branhamella catarrhalis or
Neisseria meningitidis or other Neisseria
species
Sputum gram stain of Streptococcus pneumoniae
Staphylococcus aureus pneumonia in a patient with
leukemia
Sputum gram stain of Nocardia asteroides
Sputum gram stain of Strep. pneumo. and Hemophilus
influenzae
Sputum gram stain of Hemophilus influenzae
Hemophilus influenzae
Sputum gram stain of Hemophilus influenzae
Sputum gram stain of Klebsiella pneumoniae
Branhamella catarrhalis
Sputum gram stain of Branhamella catarrhalis
Sputum gram stain of Branhamella catarrhalis or Neisseria
meningitidis
Sputum gram stain of Branhamella catarrhalis
Sputum gram stain of Mycoplasma pneumoniae (note
numerous monocytes)
Candida albicans buds and pseudohyphae in the sputum of a
patient with A.I.D.S.
Limitations in Interpretation of
the Sputum Gram Stain
ƒ Only 40 to 50 % of pneumonia patients in most
series can generate an adequate specimen
ƒ Nonpathogenic carrier rate of Strep. pneumonia
is 2 to 9 % (up to 30 % in some reports)
ƒ Gram negative oropharyngeal colonization
occurs in 30 to 90 % of hospitalized or
institutionalized patients (% increases as length
of institutionalization increases)
Limitations of Sputum Cultures
for Pneumonia
ƒ Fastidious pathogens can be overgrown on
plate by oropharyngeal flora (especially if
plating delayed)
ƒ Reported incidence of negative sputum cultures
:
–Bacteremic pneumococcal pneumonia : up to 50 %
–Hemophilus influenzae pneumonia : 35 to 45 %
–Gram negative bacilli pneumonia : 30 to 40 %
(plus species from sputum and blood cultures are at
variance in 30 %)
Limitations of Blood Cultures in
Pneumonia Cases
ƒ Incidence of positive blood cultures
with proven pneumonias :
–Strep. pneumoniae : 15 to 25 %
–Klebsiella : 14 %
–Hemophilus influenzae : 2 to 12 %
–Anerobes : 4 %
Technique for Sputum Induction to
Improve Quality of Sputum Specimen
ƒ Have patient brush teeth & surface of
tongue
ƒ Rinse mouth & expectorate
ƒ Inhale nebulized 3 % saline for 10
minutes via a facemask (O2 at 8 liters /
min)
ƒ Rapid sequential coughs with
collection of sputum in specimen cup
Sensitivity of Direct
Immunofluorescence of Sputum
ƒ Legionella : 50 %
–Not useful yet for other bacterial species
ƒ Chlamydia trachomatis : 90 %
ƒ Influenza A or B : 80 %
ƒ Useful especially if lab unequipped to
do tissue cultures
"Invasive" Methods to Obtain
Sputum for Culture
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Transtracheal aspirate
Bronchoscopy with protected brush
Transthoracic needle aspirate
Needle pleural biopsy
Open lung biopsy
General Indications for Invasive
Sputum Acquisition Methods
ƒ Standard diagnostic tests negative or
inconclusive, and :
–Progressive respiratory failure despite initial
empiric therapy
–Multiple antibiotic allergies
–Suspected nonbacterial infection requiring
prolonged or hazardous treatment with other
chemotherapeutic agents
–Persisent infiltrate possibly associated with
malignancy
Pleural Effusion : Laboratory
Evaluation
ƒ Composition of normal pleural fluid
–Is an ultrafiltrate of serum
–Similar composition to serum except protein <
1.5 grams / dl.
–Clear, colorless
–Relatively acellular (< 1500 cells per microliter,
mainly monocytes)
ƒ Normal rate of production (17 ml / day in
70 Kg adult) balanced by reabsorbtion
Pleural Effusion : Diagnostic
Clues From Fluid Color
ƒ Yellow-white
–Empyema
ƒ Red
–Malignancy
–Trauma
–Postpericardiotomy syndrome
–Pulmonary embolus
ƒ White
–Chylothorax
–Empyema
Pleural Effusion : Diagnostic Clues
From Fluid Color (cont.)
ƒ Brown
–Ruptured amebic abscess
–Esophageal rupture
ƒ Black
–Aspergillus niger infection
ƒ Yellow-green
–Rheumatoid pleurisy
ƒ Green
–Bilio-pleural fistula
Pleural Effusion : Diagnostic
Tests to Routinely Order
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Gram Stain
Culture
Cell count and differential
Total protein
LDH (lactate dehydrogenase)
pH
Usually should try to get > 30 cc. for
complete analysis
Pleural Effusion : Additional Diagnostic
Tests to Consider Ordering
ƒ Glucose
ƒ Amylase
ƒ Acid - fast stain / mycobacterial culture
ƒ Fungal culture
ƒ Cytology
ƒ Creatinine
ƒ Triglycerides
ƒ Rheumatoid factor
ƒ Anti-nuclear antibody titer
ƒ Hyaluronic acid (elevated in mesothelioma)
Bloodwork to Consider Ordering
in Evaluating Pleural Effusion
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Serum total protein
Serum glucose
Serum LDH
Serum amylase
CBC & differential
Serum creatinine
Arterial blood gas ( for pH )
Initial Use of Lab Results in
Evaluating Pleural Effusion
ƒ First step : determine if effusion is exudate or
transudate
ƒ Exudate has at least one of these:
–Pleural fluid LDH : serum LDH ratio > 0.6
–Pleural fluid protein : serum protein ratio > 0.5
–Pleural fluid LDH > 2/3 upper normal limit of serum
LDH
ƒ Is a transudate then if none of these three
present
Exudative Causes of Pleural
Effusion
ƒ Collagen vascular disease
–Systemic lupus erythematosis
–Rheumatoid pleuritis
–Sjogren's syndrome
–Wegener's granulomatosis
ƒ Drug reactions
–Bromocriptine (Parlodel)
–Dantrolene (Dantrium)
–Methysergide (Sansert)
–Nitrofurantoin (Furadantin)
–Procarbazine (Matulane)
Exudative Causes of Pleural
Effusion (cont.)
ƒ GI disorders
–Esophageal perforation
–Pancreatitis
–Pancreatic pseudocyst
–Diaphragmatic hernias
–Subphrenic abscess
ƒ Neoplasms
–Mesothelioma
–Primary lung cancer
–Metastases to pleura
Exudative Causes of Pleural
Effusion (cont.)
ƒ Infections
–Pyogenic bacteria (especially
pneumococcus)
–Tuberculosis
–Viral
–Fungal
–Actinomycosis
–Nocardiosis
–Parasites (especially Entamoeba
histolytica)
Exudative Causes of Pleural
Effusion (cont.)
ƒ Miscellaneous causes
–Chest trauma / rib fractures
–Postpericardiectomy
–Post - myocardial infarction
–Aortic rupture / dissection
–Pulmonary embolus
–Meig's syndrome
–Myxedema
–Sarcoidosis
–Uremia
–Urinary tract obstruction
–Chest radiation therapy
Transudative Causes of
Pleural Effusion
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Congestive heart failure
Cirrhosis
Nephrotic syndrome
Peritoneal dialysis
Postpartum
Pulmonary embolus*
Sarcoidosis*
Myxedema*
*can also cause exudative effusion
Ranked Order of Most Common
Causes of Pleural Effusion
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CHF
Infection
Malignancy
Pulmonary embolus
Diagnostic Utility of Pleural
Fluid Analysis
ƒ Provides definitive diagnosis in 20 %
ƒ Provides probable diagnosis in 50 %
ƒ Allows partial "rule - out" diagnoses in 30
%
ƒ Some series report diagnostic in 75 %
Interpretation of RBC Counts
in Pleural Fluid
ƒ If hematocrit > 50 % of peripheral blood hematocrit
: hemothorax (as from trauma or aortic rupture)
ƒ If Hct > 1 % (> 100,000 RBC's / mm3) :
–Malignancy
–Pulmonary embolus
–Trauma
ƒ Note : only 5000 to 10,000 RBC's / mm3 impart a
red color to pleural fluid, so only 1 cc. of blood
leakage (as from the tap itself) may make the fluid
look red
Interpretation of WBC Counts
in Pleural Fluid
ƒ Transudates usually have < 1500 WBC's / mm3
ƒ If WBC > 10,000 / mm3 :
–Para-pneumonic effusion
–Pancreatitis
–Pulmonary embolus / infarction
–Collagen vascular disease
–Malignancy
–Tuberculosis
Types of Cells to Quantify in Differential
Cell Count in Pleural Fluid
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Mesothelial cells
Plasma cells
Macrophages
Lymphocytes
Neutrophils
Eosinophils
Basophils
Malignant cells
Interpretation of WBC Differential
Counts in Pleural Fluid
ƒ Predominance ( > 40 to 50 %) of neutrophils indicates :
–Pneumonia
–Pulmonary embolus
–Pancreatitis
–Intra-abdominal abscess
–Early tuberculosis
ƒ Lymphocyte predominance indicates :
–Tuberculosis, or malignancy
–Should prompt pleural biopsy
–Less likely : Lymphoma, sarcoid, rheumatoid pleurisy
ƒ Basophilic predominance : rare, but may indicate leukemic
involvement of pleura
Interpretation of WBC Differential
Counts in Pleural Fluid (cont.)
ƒ If eosinophilia (> 10 %) present :
–Most due to hemothorax (trauma)
–Pulmonary embolus / infarction
–Asbestos exposure
–Previous thoracentesis
–Parasites (paragonimiasis, hydatid cysts)
–Fungal infection (histoplasmosis)
–Drug reaction
–Hodgkin's lymphoma
Interpretation of Pleural Fluid
Protein Levels
ƒ Level > 4 g/dl common with tuberculosis
ƒ Levels from 2.5 to 6 g/dl common in
parapneumonic & malignant effusions
ƒ Level < 3 g/dl typical in CHF (rarely may
be > 4 g/dl after brisk diuresis
ƒ Pleural fluid albumin to serum albumin
gradient of 1.2 g/dl or more suggests
diuretic effect on CHF effusion
Interpretation of Pleural Fluid
Glucose Levels
ƒ Glucose < 60 mg/dl or < 1/2 of serum
glucose indicates :
–Rheumatoid pleurisy
–Parapneumonic effusion / empyema
–Less likely but possible :
ƒ Malignancy
ƒ Tuberculosis
ƒ S.L.E.
ƒ Lymphoma
ƒ Esophageal rupture
Interpretation of Amylase
Levels in Pleural Fluid
ƒ If pleural fluid amylase elevated (> serum
level) :
–Pancreatitis
–Pancreatic pseudocyst
–Esophageal rupture
–Malignancy (non-pancreatic adenocarcinoma)
–Ruptured ectopic pregnancy (rare)
ƒ If able to fractionate amylase isoenzymes,
can differentiate pancreatic versus salivary
source
Interpretation of pH Levels in
Pleural Fluid
ƒ pH < 7.3 generally found in same conditions
as low glucose level
ƒ pH < 7.2 in parapneumonic effusion
indicates need for chest tube drainage
ƒ pH < 7.3 with malignancy predicts short
survival time
ƒ pH < 7.2 with rheumatoid or malignant
effusion does not necessarily require chest
tube
Immunologic Markers in Pleural
Fluid
ƒ Pleural fluid rheumatoid factor > 1: 32
suggests rheumatoid pleurisy
ƒ Pleural fluid antinuclear antibody titer > 1 : 160
suggests S.L.E.
ƒ Presence of L.E. cells in pleural fluid is
diagnostic of S.L.E.
ƒ Complement levels (C3 & C4) are low in most
of these patients
Use of Pleural Biopsy
ƒ Presence of exudative effusion with lymphocyte
predominance of undetermined cause suggests
need for biopsy
ƒ Cope or Abrams pleural biopsy needle used
ƒ Biopsy is most likely to identify :
–Tuberculosis
–Malignancy
–Fungal infection
–Sarcoidosis
–Rheumatoid pleurisy
–Parasitic disease (hydatid cyst)
Other Diagnostic Tests to Consider
After Evaluation of Pleural Fluid
ƒ Computed tomography of chest
–Useful to identify parenchymal
abnormalities obscured by the effusion on
chest X-ray
ƒ Thoracoscopy
–90 to 100 % accuracy in identifying
tuberculosis or malignancy
ƒ Fiberoptic bronchoscopy
Typical Characteristics of
Pleural Effusion
Disease
Appearance
Tuberculosis
Serous
WBC's
(cells / mm3)
Lymphs
(< 5000)
Glucose
(mg/dl)
pH
Same or
less than
serum
< 7.4
Parapneumonic complicated
Turbid,
purulent
PMN
(> 20,000)
< 60
< 7.2
Parapneumonic -
Turbid
PMN
(< 10,000)
Same as
serum
normal
uncomplicated
Typical Characteristics of
Pleural Effusions (cont.)
Disease
Carcinoma
Pancreatitis
Lupus
Pulmonary
embolus
Uremia
Mesothelioma
Chylothorax
Appearance
Serous,
bloody
Turbid
Serous,
bloody
Serous,
bloody
Serosanguinous,
bloody
Viscous,
bloody
Milky, turbid,
serous
WBC's
(cells/mm3)
lymphs
(2000 to 4000)
PMN's
PMN's or
monos
PMN's or
lymphs
Lymphs
Monos
Lymphs
Glucose
(mg/dl)
Same as
serum
Same as
serum
Same as
serum
Same as
serum
Same as
serum
< 60
(in 70 %)
Same as
serum
pH
> 7.3
(in 70 %)
7.3 to 7.35
> 7.3
> 7.3
> 7.3
< 7.3
(in 70 %)
> 7.4
Typical Characteristics of
Transudative Pleural Effusions
Disease
Congestive
heart
failure
Cirrhosis
Peritoneal
dialysis
Nephrotic
syndrome
Appearance
WBC's
(cells/mm3)
Glucose
(mg/dl)
Lymphs
(< 1000)
Same as
serum
normal
Lymphs
(< 1000)
Same as
serum
normal
Resembles
dialysate
Monos
(< 100)
300 to 400
> 7.4
Serous
Monos
Same as
serum
> 7.4
Serous
Serous or
hemorrhagic
pH
Complications of Empyema
ƒ Mortality
–15 % even with chest tube drainage &
antibiotics
ƒ Bronchopleural fistula
ƒ Pleural fibrosis
ƒ Pleural - cutaneous fistula
Bronchopleural Fistulas
ƒ Can present as :
–Air - fluid level in undrained effusion
–Persistent air leak thru chest tube
–Production of copious sputum when patient
lying on contralateral side
ƒ Can be mimicked on CXR by :
–Gas - producing bacteria in pleural space
–Parenchymal lung abscess
ƒ May need chest CT to tell from abscess
Bronchopleural Fistulas :
Treatment
ƒ High frequency jet ventilation if large
air leak and positive pressure
ventilation needed
ƒ If small leak : antibiotics & chest tube
drainage
ƒ Surgical closure if it persists for
several weeks
Pleural Fibrosis
ƒ Commonest complication of undrained
empyema
ƒ Computed tomography needed to define the
extent of fibrosis & detect parenchymal disease
"behind" the CXR density
ƒ Pulmonary function tests useful to define
degree of functional impairment
ƒ Not all need surgical decortication (such as
elderly or inactive patients)
Summary : Cost-Effective
Pleural Fluid Analysis
ƒ First : determine exudate versus transudate
–Initially order LDH & protein only
–If transudate, usually no further tests needed
ƒ If exudate, order focused additional tests based
on suspected etiology (infectious vs.
malignancy, etc.)
ƒ Have lab save any extra fluid, so patient can be
spared repeat thoracentesis just for diagnosis