Clinical Diagnosis B

FLUIDS AND SPUTUM

Sources: 2020 PPT – Dr. Maybelle Cabungcal-Buenvenida, Henry’s Clinical Diagnosis and Management by Laboratory Methods

PLEURAL FLUID PLEURAL EFFUSION: RECOMMENDED TESTS

o Pleural Cavity – potential space lined by mesothelium of the visceral and parietal
pleura; It contains a small amount of fluid that facilitates movement of the two
membranes against each other.
o Pleural Fluid is plasma filtrate derived from capillaries of the parietal pleura
o It is produced continuously at a rate dependent on capillary hydrostatic pressure,
plasma oncotic pressure, and capillary permeability.
o It is reabsorbed through the lymphatics and venules of the visceral pleura.

 EFFUSION – accumulation of fluid; it results from an imbalance of fluid

production and reabsorption.
▪ Serous Effusion – fluid accumulation in the pleural, pericardial and
peritoneal cavities.

PLEURAL FLUID: SPECIMEN COLLECTION

o THORACENTESIS
▪ for any undiagnosed pleural effusion
▪ for therapeutic purposes in patients with massive symptomatic effusions
o Tubes to be used:
▪ Heparinized tubes – to avoid clotting PLEURAL FLUID: GROSS EXAMINATION
▪ EDTA tube – for total and differential cell counts
o Approximately 15% of transudates are blood-tinged
▪ Blood culture media – inoculated at bedside, for aerobic and anaerobic
o Bloody pleural effusion – hematocrit > 1%
bacterial cultures
▪ Trauma
o All remaining fluid (>/= 100mL) should be submitted.
▪ Malignancy
▪ If malignancy, fungal or mycobacterial infection is suspected.
▪ Pulmonary infarction
o pH measurements - Fluid should be collected anaerobically in a heparinized syringe
and submitted on ice TRAUMATIC TAP HEMOTHORAX
o Fresh specimen for cytology – stored up to 48 hours in the refrigerator. Uneven blood distribution Pleural fluid hematocrit >50% of the blood
Fluid clearing with continued aspiration hematocrit
CLASSIFICATION OF PLEURAL EFFUSION: TRANSUDATIVE vs EXUDATIVE Formation of small blood clots

TRANSUDATE EXUDATE o Turbid, milky and/or bloody specimens - centrifuged and supernatant examined:
GROSS EXAMINATION o CLEAR SUPERNATANT = due to cellular elements or debris
Clear, pale yellow to straw-colored Cloudy or turbid o TURBIDITY PERSISTS = chylous or pseudochylous effusion
Odorless Feculent odor (anaerobic infections)
Does NOT clot Often CLOT if not heparinized CHYLOUS vs PSEUDOCHYLOUS EFFUSION
CAUSES / ETIOLOGY
Increased Hydrostatic Pressure Increased Capillary Permeability TRUE CHYLOUS PSEUDOCHYLOUS
Decreased Plasma Oncotic Pressure Decreased Lymphatic Resorption Leakage from the thoracic duct Breakdown of cellular lipids in
Congestive Heart Failure Infections resulting from obstruction by long-standing effusions such as
Hepatic Cirrhosis ▪ Bacterial pneumonia CAUSES
lymphoma, carcinoma or rheumatoid pleuritis, tuberculosis
Hypoproteinemia (e.g., nephrotic syndrome) ▪ TB, other granulomatous diseases (e.g., traumatic disruption or myxedema
sarcoidosis, histoplasmosis) Sudden ONSET Gradual
▪ Viral or mycoplasma pneumonia Milky white or yellow to bloody APPEARANCE Milky or greenish, metallic sheen
Neoplasms
Lymphocytosis Mixed cellular reaction
Bronchogenic carcinoma MICROSCOPIC
Cholesterol crystals
Metastatic carcinoma
≥ 110 mg/dL < 50 mg/dL
▪ Lymphoma TRIGLYCERIDES
(≥ 1.24 mmol/L) (< 0.56 mol/L)
▪ Mesothelioma (increased hyaluronate
Chylomicrons present LIPOPROTEIN Chylomicrons absent
content of effusion fluid)
ELECTROPHORESIS
Noninfectious inflammatory dse involving pleura
▪ Rheumatoid disease
(low pleural fluid glucose in most cases) o Laboratory Criteria for Pleural Exudate
▪ Systemic lupus erythematosus
(LE cells are occasionally present)
Pulmonary infarct
Light’s
(may be associated with hemorrhagic effusion)
Criteria
FLUID FROM EXTRAPLEURAL SOURCES
Pancreatitis (elevated amylase activity in effusion fluid)
Ruptured esophagus (elevated amylase activity and low pH)
Urinothorax (elevated creatinine and low pH)

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CD B: Fluids and Sputum
o Light’s Criteria PLEURAL FLUID: CHEMICAL ANALYSIS
▪ Exudates meets one or more of the following criteria: o PROTEIN
 Pleural fluid / Serum protein ratio > 0.5 ▪ Total protein or albumin has little clinical value
 Pleural fluid / Serum LD ratio > 0.6 ▪ Combined with other parameters, differentiate exudates from transudates
 Pleural fluid LD level > 2/3 of the serum upper limit of normal ▪ Protein electrophoresis – shows pattern similar to serum, but with higher
▪ Sensitivity = 98% proportion of albumin
▪ Specificity = 80% o GLUCOSE
▪ Glucose level of normal pleural fluid, transudates and most exudates is similar
PLEURAL FLUID: MICROSCOPIC EXAMINATION to serum levels
o TOTAL CELL COUNT ▪ DECREASED GLUCOSE:
▪ Manual hemocytometer methods ▪ < 60 mg/dL or < 3.33 mmol/L
▪ Automated cell counts ▪ Pleural Fluid / Serum Glucose Ratio < 0.5
o DIFFERENTIAL LEUKOCYTE COUNT AND CYTOLOGY ▪ Rheumatoid pleuritis, Grossly purulent parapneumonic exudates,
▪ Prepared by cytocentrifugation, smear is air-dried and stained with malignancy, TB, non-purulent bacterial infection, lupus pleuritis &
Romanowski stain esophageal rupture
▪ Filtration or automated concentration methods with Papanicolau stain o LACTATE
▪ Automated WBC differential counts ▪ Useful adjunct in the rapid diagnosis of infectious pleuritis
▪ Liquid-based thin layer methods ▪ Higher levels in bacterial and tuberculous pleural infections
o CELL TYPES ▪ Moderate elevations in malignant effusions
MESOTHELIAL CELL MALIGNANT CELLS o ENZYMES
▪ AMYLASE
 Elevations above serum level (usually 1.5 – 2.0 or more)
⎯ Pancreatitis
⎯ Esophageal rupture Elevated salivary
▪ Mesothelial Cells - common in pleural fluids from inflammatory process ⎯ Malignant effusion isoform of amylase
▪ Malignant Cells ▪ LACTATE DEHYDROGENASE (LDH)
▪ Neutrophils – pleural inflammation  Used in separating exudates from transudates
▪ Lymphocytes – most are small, but medium, large and reactive (transformed)  Levels rise in proportion to degree of inflammation
variants may also be seen ▪ ADENOSINE DEAMINASE (ADA)
▪ Plasma Cells  Particularly rich in T lymphocytes
▪ Low-grade Non-Hodgkin Lymphoma & Chronic Lymphocytic Leukemia – may  Significantly increased in tuberculous pleuritis
be difficult to distinguish from benign lymphocyte-rich serous effusion o INTERFERON – γ (GAMMA)
▪ Eosinophils – eosinophilic effusion (10% or more eosinophils), air or blood ▪ Significantly increased in the pleural fluid of patients with tuberculous
in the pleural cavity pleuritis, also in patients with lymphocyte-rich pleural fluid from
▪ Mast Cells or Basophils – often accompany eosinophils nontuberculous causes
▪ Charcot Leyden Crystals o pH
▪ In assessing the prognosis of parapneumonic (pneumonia-related) effusions.
CELLULAR DIFFERENTIATION OF PLEURAL EFFUSIONS  pH > 7.30 = resolves with medical therapy alone
 pH < 7.30 = complicated parapneumonic effusion (loculated or
associated with empyema), requires surgical drainage
 Borderline complicated exudates (pH 7.20 – 7.30) = close monitoring
with repeat measurements
o LIPIDS
▪ Triglycerides and LPP Electrophoresis – helpful in identifying chylous effusion
▪ Cholesterol – useful in separating transudates from exudates
o C-REACTIVE PROTEIN (CRP)
▪ Diagnosis and assessment of severity of parapneumonic effusion
▪ Clinically useful screening test for:
 Organ Disease
 Index of disease activity
 Measure of response to therapy
o TUBERCULOSTEARIC ACID (10-METHYLOCTADECANOIC ACID)
▪ First isolated from M. tuberculosis
▪ Fatty acid; structural component of mycobacteria
▪ Not normally present in human tissue
▪ Uses gas chromatography / mass spectroscopy (GC-MS)
▪ Measured in sputum, bronchial washing and pleural fluid from PTB patients
o TUMOR MARKERS
▪ Not recommended as a routine test
▪ Useful adjunct in enigmatic non-inflammatory exudates with (-) cytology
▪ CEA, CA-153, CA-549, CA-724, CFRA-211
▪ CEA – adenocarcinomas, PSA – metastatic prostate cancer

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CD B: Fluids and Sputum
PLEURAL FLUID: IMMUNOLOGIC STUDIES TRANSUDATIVE vs EXUDATIVE
o RHEUMATOID FACTOR – pleural effusions associated with seropositive RA o Light’s Criteria
o ANA – effusions due to lupus pleuritis ▪ Exudates meets one or more of the following criteria:
o COMPLEMENT  Pleural fluid / Serum protein ratio > 0.5
▪ Decreased Levels (CH50 < 10 U/mL) (C4 Level < 10x55 U/g protein)  Pleural fluid / Serum LD ratio > 0.6
 In most patients with rheumatoid or lupus pleuritis  Fluid LD level > 200 U/L

PLEURAL FLUID: MICROBIOLOGIC EXAMINATION PERICARDIAL EFFUSION: ROUTINE TESTING

o Bacteria most commonly associated with Parapneumonic Effusions are: ▪ Cell Count ▪ LD
o Staphylococcus aureus ▪ Glucose ▪ Bacterial Culture
o Streptococcus pneumoniae ▪ Total Protein ▪ Cytology
o β-hemolytic group A streptococci o Other more specific tests – appropriate only when there is a high clinical suspicion
o Enterococci of unusual causes of pericardial effusion
o Gram-negative bacilli
o Anaerobic bacteria PERICARDIAL FLUID: MICROSCOPIC EXAMINATION
o Both anaerobic and aerobic cultures should be performed. o HEMATOCRIT and RBC COUNT
o Gram Stain – sensitivity is approximately 50% ▪ Document the presence of a hemorrhagic effusion
o Cytocentrifugation – increases the sensitivity of gram stain ▪ Of limited value for differential diagnosis
o Resin containing blood culture bottles – improve the isolation of certain bacteria in o TOTAL LEUKOCYTE COUNT
partially treated patients ▪ > 10,000 /uL = bacterial, tuberculous, malignant pericarditis
o For patients with suspected Mycobacterium tuberculosis: o LEUKOCYTE DIFFERENTIAL COUNT
▪ Acid-fast staining ▪ To determine the cause of pericardial effusions
▪ Culture ▪ To evaluate for atypical of malignant cells
▪ Pleural Biopsy – rapid presumptive diagnosis of TB by histopathologic o CYTOLOGY
demonstration of granulomas or acid-fast bacteria ▪ Identification of malignant cells
▪ Metastatic CA of lungs and breasts – most frequently observed in malignant
pericardial effusion
PERICARDIAL FLUID ▪ Sensitivity = 95%
o NV: 10 – 50 mL ▪ Specificity = 100%
o Produced by a transudative process
PERICARDIAL FLUID: CHEMICAL ANALYSIS
o PROTEIN
▪ TOTAL PROTEIN = has no discriminating power in pericardial diagnosis
o GLUCOSE
▪ < 40 mg / dL (< 2.22 mmol/L) = bacterial, tuberculous, rheumatic, malignant
o pH
▪ Markedly decreased (< 7.10) = rheumatic, purulent pericarditis
▪ Moderately decreased (7.20 – 7.30) = malignancy, uremia, TB, idiopathic
o LIPIDS
▪ Separation of true chylous from pseudochylous effusions is facilitated by:
 TAG measurement
 Cholesterol measurement
 LPP electrophoresis for chylomicrons
o Fluid may be obtained by: o ENZYMES
▪ Pericardiotomy following limited thoracotomy ▪ LDH > 200 U/L = cutoff for pericardial exudates
▪ Pericardiocentesis (Sterile needle aspiration) ▪ LD and CK (in post-mortem pericardial fluid within 48 hours of death) – useful
in establishing acute myocardial injury
PERICARDIAL FLUID: GROSS EXAMINATION ▪ CK-MB, Myoglobin, Trop-I (post-mortem pericardial fluid) – significantly
o Pale yellow & Clear = Normal increased in patients with myocardial injury
o Turbid = Infection or Malignancy ▪ ADA Activity – useful adjunctive test for tuberculous pericarditis (in suspicious
o Clear & Straw-colored = Uremia cases with negative acid-fast stains)
o Large Effusions (> 350 mL) = Malignancy, Uremia, Idiopathic o INTERFERON - γ (GAMMA)
o Milky = Chylous or Pseudochylous effusion ▪ Increased Levels – tuberculous serous effusions / pericarditis
o Blood-like fluid obtained by pericardiocentesis might represent: o POLYMERASE CHAIN REACTION (PCR)
▪ Sensitive technique
INADVERTENT ASPIRATION OF BLOOD ▪ More specific than ADA in the diagnosis of tuberculous pericarditis
HEMORRHAGIC EFFUSION
FROM THE HEART ▪ Negative test does NOT rule out tuberculous pericarditis
Hematocrit is LOWER than that of blood Hematocrit COMPARABLE with that of blood
Blood does NOT clot Blood CLOTS PERICARDIAL FLUID: IMMUNOLOGIC STUDIES
Blood gas analysis results similar to venous or
o ANA – (-) test makes dx of lupus serositis highly unlikely; high titers in pericardial
arterial blood
effusions lack specificity (ex: 1:5120); Unexplained high titers = malignancy

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CD B: Fluids and Sputum
PERICARDIAL FLUID: MICROBIOLOGIC EXAMINATION ▪ <15 mL of gross blood can be aspirated
o GRAM STAIN ad CULTURE  Infusion of 1 L of saline or Ringer’s solution (20 mg/kg in children)
▪ Important aerobic bacteria:  Retrieving the fluid by gravity drainage
 S. aureus ▪ At least 600 mL is needed
 S. pneumoniae ▪ Repeated in 2 to 3 hours if initial results are negative or indeterminate.
 S. pyogenes
 Gram-negative bacilli
▪ Major anaerobic organisms:
 Bacteroides fragilis group
 Anaerobic streptococci
 Clostridium spp.
 Fusobacterium spp.
 Bifidobacterium spp.
o Viruses are rarely isolated from pericardial fluid.
▪ (e.g., coxsackieviruses, influenza virus, mumps)
o Obtaining acute & convalescent sera for antibody response to suspected viral ▪ Suggested modifications of DPL criteria:
pathogens may help support the diagnosis. (1) WBC count ≥ RBC count ÷ 150
o ACID-FAST STAIN and CULTURE  where RBC is 10 × 104/mm3 or greater
▪ Sensitivity is about 50% for tuberculous pericarditis (2) cell count ratio >1.0
o PCR-based assays = aid in the rapid diagnosis of a variety of bacterial and other  cell count ratio – ratio between WBC & RBC counts in the lavage
infections in the pericardial space. ÷ WBC/RBC ratio in the peripheral blood
▪ Specificity = 97% for hollow organ injury
(if performed at least 3 hours following injury)
PERITONEAL FLUID ▪ Other applications: Acute peritonitis, Pancreatitis
o ASCITES – pathologic accumulation of excess fluid in the peritoneal cavity  WBC count of 200 cells/mm3 – 99% probability of acute peritonitis
▪ > 50 mL of fluid
▪ Produced as an ultrafiltrate of plasma dependent on vascular permeability and o PERITONEAL DIALYSIS
on hydrostatic and oncotic starling forces. ▪ Dialysate fluid from renal patients undergoing chronic ambulatory peritoneal
dialysis – should be checked for infection

o PERITONEAL WASHINGS
▪ Performed intraoperatively
▪ Document early intraabdominal spread of gynecologic and gastric carcinomas
▪ For cytologic examination

PERITONEAL EFFUSION: RECOMMENDED TESTS

o PARACENTESIS – performed in patients with:

▪ New ascites
▪ Changes in clinical picture of old ascites
 Rapid fluid accumulation PERITONEAL FLUID: GROSS EXAMINATION
 Fever development
o Perforation of the GI or biliary tract:
o 30 mL = minimum amount needed for complete evaluation
▪ Presence of food particles
o 100 mL (at least, if possible) = for cytologic examination
▪ Presence of foreign material
o EDTA tube = for cell counts
▪ Green-yellow bile-staining in a DPL specimen
o Blood culture bottles = at bedside (10mL per bottle)
o Greenish discoloration - Acute Pancreatitis & Cholecystitis
o Blood-tinged or Grossly bloody – must be distinguished from a traumatic tap
o DIAGNOSTIC PERITONEAL LAVAGE
▪ Bright red opaque color – 15 mL blood / L fluid; require cell count
▪ No longer recommended as a routine technique for the evaluation of
o Bloody Ascites – Malignancy and tuberculosis
abdominal trauma; Oversensitive, Nonspecific
o Milky fluid (that does not clear with centrifugation) – Chylous / Pseudochylous
▪ CT & Ultrasound – rapid screening for significant abdominal hemorrhage in
▪ True pseudochylous peritoneal effusions – significantly less common; caused
hemodynamically unstable patients; evaluation of hollow viscous injuries
by disruption or blockage of lymphatic flow by trauma, lymphoma, CA, TB,
▪ A catheter is placed through a small incision into the abdominal cavity
other granulomatous dse, hepatic cirrhosis, adhesions, parasitic infestations

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CD B: Fluids and Sputum
PERITONEAL FLUID: MICROSCOPIC EXAMINATION o CREATIINE AND UREA
o TOTAL LEUKOCYTE COUNT ▪ To differentiate between peritoneal fluid and urine
▪ Useful in distinguishing ▪ Urinary Bladder Rupture
 Ascites due to uncomplicated cirrhosis  Elevated peritoneal fluid urea nitrogen and creatinine
 Spontaneous bacterial peritonitis (SBP)  Elevated BUN but Normal serum creatinine
⎯ Caused by migration of bacteria from the intestine to ascitic fluid  Due to back-diffusion of urea
⎯ Leukocyte count >5000 /uL (>50% of which are neutrophils) o BILIRUIN
▪ Vary with fluid shifts ▪ Choleperitoneum from a ruptured gallbladder:
▪ Ascitic Fluid Total Neutrophil Count – PREFERRED method for SBP diagnosis  Ascitic fluid bilirubin > 6 mg/dL
 Cutoff values of 250 and 500 neutrophils /uL have been recommended  Ascitic fluid/Serum bilirubin ratio > 1.0
▪ EOSINOPHILIA (>10%) ▪ Ratio of >/= 0.6
 Chronic inflammatory process associated with chronic peritoneal  Additional marker for an exudative process
dialysis (most common)  Accuracy is not as high as that of SAAG
 CHF, Vasculitis o pH
 Lymphoma ▪ Helpful in diagnosis of SBP in patients with cirrhotic ascites
 Ruptured hydatid cyst (especially in conjunction with leukocyte count)
o CYTOLOGY ▪ pH < 7.15 have a poor prognosis
▪ Overall sensitivity of 40 – 65% for malignant ascites ▪ low pH = malignant and pancreatic ascites and tuberculous peritonitis
▪ Sensitivity of over 95% for peritoneal carcinomatosis o CHOLESTEROL
o LIQUID-BASED THIN-LAYER PREPARATIONS ▪ Moderately useful index in separating malignant ascites (>45 to 48 mg/dL)
▪ Of peritoneal effusions and pelvic washings – superior to standard cytologic from cirrhotic ascites.
preparations in the detection of carcinoma o INTERLEUKIN-8
o IMMUNOCYTOCHEMICAL STAINS ▪ Significantly higher in SBP compared with sterile ascites
▪ Useful in characterizing atypical cells in equivocal cases ▪ 100 ng/L cutoff – sensitivity & specificity were both 100% in cirrhotic patients
o TUBERCULOSTEARIC ACID (10-METHYLOCTADECANOIC ACID)
PERITONEAL FLUID: CHEMICAL ANALYSIS ▪ Measured by quantitative chemical ionization GC-MS
o PROTEIN ▪ Tuberculous peritonitis, tuberculous meningitis (spinal fluid), pneumonia
▪ Serum-Ascites Albumin Gradient (SAAG)– superior to total protein content in (pleural fluid)
o TUMOR MARKERS
differentiating cirrhosis from other causes of peritoneal effusion
▪ SBP: Low total protein (<3 g/dL), High SAAG (>1.1g/dL) ▪ Measurement is of little value; low sensitivity & specificity
▪ Vary with extracellular fluid shifts ▪ Useful in selected cases, such as:
 Following a patient’s response to therapy
o GLUCOSE
▪ Of little value because the sensitivity and specificity are generally too low to  Early detection of tumor recurrence
be of practical value.  Cytology is negative but suspicion of malignant ascites is high
▪ DECREASED VALUES: Tuberculous peritonitis, Abdominal carcinomatosis
o ENZYMES ▪ PSA - malignant effusions due to prostate cancer
▪ CEA - suggest a poor prognosis in gastric carcinoma
▪ AMYLASE – similar to plasma levels in normal peritoneal fluid; NOT
recommended in the routine evaluation of ascites; ▪ CA-125 - epithelial carcinomas of the ovary, fallopian tubes, or endometrium
 HIGH LEVELS in pancreas-related ascites (Acute pancreatitis,  Non-malignant conditions (CV diseases & chronic liver disease)
o DNA PLOIDY ANALYSIS
Pancreatic pseudocyst), blunt and penetrating abdominal trauma,
Gastroduodenal perforation, Acute mesenteric vein thrombosis, ▪ by flow cytometry or image analysis; Useful in cases with equivocal cytology
intestinal strangulation, necrosis, non-pancreatic malignancies results when the malignant cells carry an aneuploid karyotype.
▪ ALKALINE PHOSPHATASE (ALP)
PERITONEAL FLUID: MICROBIOLOGIC EXAMINATION
 HIGH LEVELS: Hollow visceral injury (>10 U/L), Secondary
peritonitis (significantly higher mean ALP levels than SBP) o The bacteria in SBP are most often normal intestinal flora
▪ LACTATE DEHYDROGENASE (LDH) – for early diagnosis of SBP o >92% are monomicrobial
 INCREASED: Malignant effusions (High serum & peritoneal fluid ▪ Aerobic gram-negative bacilli (e.g., E. coli, K. pneumoniae) - 2/3 or more
LD levels in Ovarian Cancer ▪ S. pneumoniae, Enterococcus spp., Anaerobes
▪ TELOMERASE – a specific discriminatory marker in malignant ascites o Gram stain – sensitivity of 25% in SBP
▪ ADA – used in endemic areas to identify patients with tuberculous peritonitis o Cultures – positive in only about 50% of cases
o FIBRONECTIN ▪ Inoculation of blood culture bottles at bedside
▪ Reliable in differentiating malignant from sterile ascites – using a cutoff value ▪ Concentration of large volumes of fluid can improve sensitivity
of 85 ug/mL (85 mg/L). ▪ Use of resin-containing blood culture bottles
o LACTATE o Total neutrophil count – preferred method for the diagnosis of SBP
▪ To differentiate SBP from uncomplicated ascites. o PCR – detection of bacterial DNA in culture-negative ascitic fluid
▪ HIGH LEVELS: o In situ hybridization – performed on leukocytes suspended in peritoneal fluid; used
 Malignant and Tuberculous ascites to detect bacteria in SBP
 Hollow viscous perforation o Mycobacterium tuberculosis
 Gangrenous intestine ▪ Acid-fast stains – Sn is 20-30%, Cultures – Sn is 50-70%
 Peritonitis ▪ PCR – to detect M. tb DNA; negative result does not exclude the diagnosis.
 Intraabdominal abscess ▪ Laparoscopic examination with biopsy – high clinical suspicion for
tuberculous peritonitis

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CD B: Fluids and Sputum
SPUTUM
o Types: Expectorated, Induced, Bronchoscopic
o For optimal sample:
▪ Expectorated sputum is collected early in the morning before eating
▪ Rinse the mouth with water
▪ Expectorate a specimen resulting from a deep cough (preferably 5–10 mL)
o For persons with nonproductive coughs, a specimen may be induced.
▪ Breathe aerosolized droplets of a solution of 15% sodium chloride & 10%
glycerin for about 10 minutes or until a cough reflex is initiated
o Deliver sputum specimens promptly to the laboratory.
o Refrigerate for a short time if a delay is unavoidable.

SPUTUM: SPECIMEN PROCESSING

o Screen the sputum to determine whether they are representative of lower respiratory
secretions or of saliva.
▪ >10 epithelial cells / lpf – significant contamination saliva & should be rejected
▪ <25 epithelial cells & >25 neutrophils / lpf – probably acceptable
o Prepare a smear from a portion of the specimen consisting of purulent material.
o Stain with Gram stain.
o Examine the acceptable gram-stained smears under OIO to determine the relative
amounts of organisms.
▪ Kind of Bacterium
 gram-positive cocci in pairs, chains, or clusters
 gram-positive bacilli
 gram-negative diplococci
 gram- negative rods
▪ Quantity of Organisms (Rare, few, moderate, many)
▪ Note whether or not they are intracellular
o Inoculate the portions of acceptable specimens containing purulent material.
▪ Cystic fibrosis = also inoculating a medium selective for Burkholderia cepacia
is recommended

▪ Legionnaires’ disease
 Legionella culture
⎯ Selective & nonselective buffered charcoal yeast extract agar
⎯ Use of selective agar inhibits the growth of other respiratory flora
 Rapid, direct test (fluorescent antibody on a respiratory specimen or
Legionella antigen on a urine specimen)
 Direct fluorescent antibody (DFA) staining
 PCR

▪ Mycobacteria
 Collection of 3 samples
(at least one of which is an early morning specimen is recommended)
 Sputum & other respiratory secretions must be decontaminated – to
prevent normal respiratory flora from overgrowing the slower-growing
mycobacteria
 All specimens submitted for mycobacterial stain (AFB), culture &
molecular testing should be processed in a biological safety cabinet,
preferably in an isolated room with negative air pressure (level 3
laboratory)

▪ All specimens submitted for fungal culture:

 Should also be handled as described for mycobacteria
 Gram stain – to determine the quality of the specimens
 Culture – acceptable expectorated sputum & induced sputum for
recovery of fungi
⎯ Media with & without blood enrichment & media containing
antimicrobial agents should be used

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