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Sputum and
Bronchoalveolar
Lavage
Robin Christian G. Cao
Johanna Selina D. Lim
Jennie Q. Lingad
Anna Kamille S. Suyat
Case 1:
A 57 year old man with persistent cough was asked to collect sputum for 3 consecutive days,
the results are as follows:

Sputum
Spontaneously Produced Sputum
Spontaneously expectorated sputum has been a mainstay in the diagnosis of pulmonary lesions. While
some patients readily produce a representative sputum sample, the collection procedure can be time
consuming, uneconomical, and inefficient. Typically, three to five deep-cough specimens are obtained
on consecutive days after the patient is instructed in the proper technique for providing a satisfactory
specimen: after mouth rinsing and throat cleaning, the patient takes a deep breath, holds it for up to 2o
seconds, and then coughs. With this method material is forcefully expectorated from the airways. The
procedure should be repeated for up to 30 min to produce a sufficient quantity of a representative
specimen. 5-10 ml of specimen is usually needed.
In addition to mucus and mature and immature squamous cells exfoliated from the oral cavity, the
specimen may contain lymphoid cells from the tonsils and adenoids. A satisfactory specimen is one that
is representative of the bronchial mucosa and pulmonary parenchyma and contains macrophages
(generally carbon-bearing), derived from the alveolar spaces. Inspissated mucus from the terminal
airways (Curschmann`s spirals) and columnar cells from the bronchial tree and nasopharynx can also be
seen. The presence of only squamous cells indicates an unsatisfactory sample. However, exfoliated cells
from upper airway epithelial lesions may be seen in such samples.
Induced Sputum
Day 1
‡ Physical
‡ Slightly mucoid
‡ Microscopic
‡ Numerous
squamous epithelial
cells
‡ No organisms found
Day 2
‡ Physical
‡ Mucopurulent rusty
‡ Microscopic
‡ Gram¶s stain shows
Curshmann¶s spirals
and Charcot-Leyden
crystals
Day 3
‡ Same as day 2 but
with numerous
eosinophils
0any patients, particularly those who are asymptomatic, generally are unable to spontaneously produce
a satisfactory sputum specimen by deep coughing. These patients may be 'induced` to produce a
diagnostic sample representing the respiratory mucosal surface and associated lesions. Induction
techniques vary, but generally they involve inhalation of a preheated (37
o
C) hypertonic saline solution
or mucolytic agent (0ucomyst) for 10 to 15 min. the patient is asked to cough for up to an additional 20
min; a pooled sputum sample is then collected and submitted for examination.
Questions:
1. Classify whether the results are abnormal or normal.

Curschmann`s spirals
Curschmann's spirals refer to a finding in the sputum of several different lung diseases.



Charcot-Leyden crystals
Charcot-Leyden crystals are microscopic crystals found in people who have allergic
diseases such as asthma or parasitic infections such as parasitic pneumonia or ascariasis.
Day 1
‡ Physical
‡ Slightly mucoid
‡ Microscopic
‡ Numerous
squamous
epithelial cells
‡ No organisms
found
Day 2
‡ Physical
‡ Mucopurulent rusty
‡ Microscopic
‡ Gram¶s stain shows
Curshmann¶s
spirals and
Charcot-Leyden
crystals
Day 3
‡ Same as day 2 but
with numerous
eosinophils
They vary in size and may be as large as 50 µm in length. Charcot-Leyden crystals are
slender and pointed at both ends, consisting of a pair of hexagonal pyramids joined at
their bases. Normally colorless, they are stained purplish-red by trichrome. They consist
of lysophospholipase, an enzyme synthesized by eosinophils, and are produced from the
breakdown of these cells.


Eosinophils
Sputum eosinophilia was first described as a feature of asthma by Gollasch more than a
century ago. Thirty-years later, eosinophils were found to be a part of the pathological
process that characterises asthma . Since then, much work has been undertaken to
understand the precise role of eosinophils in mediating airway inflammation, tissue
damage and repair in asthma pathogenesis.
It has been suggested that eosinophils are important in the immunological defense
system against helminths (worms).


2. What can be the reason for the difference in the result on Day 1.
The presence of only squamous cells on the specimen collected may be due to
improperly collected specimen. The squamous cells may a product of the natural
exfoliation of the oral cavity. A satisfactory specimen is one that is representative of the
bronchial mucosa and pulmonary parenchyma and contains macrophages (generally
carbon-bearing), derived from the alveolar spaces. Inspissated mucus from the terminal
airways (Curschmann`s spirals) and columnar cells from the bronchial tree and
nasopharynx can also be seen.
3. What is the possible cause of the result obtained?
Bronchial asthma
Bronchial asthma is a condition of the lungs which is characterized by
constriction (narrowing) of the bronchi.
Bronchi and bronchioles contain thick, tenacious mucous plugs. The mucous
contains Curschmann's spirals, eosinophils and Charcot-Leyden crystals.

Bronchial Asthma
Laboratory examination of sputum for evidence of bronchial asthma is often neglected although
characteristic patterns can be seen in sputum. The sputum is usually white and mucoid and may contain
blood or pus especially during the presence of an underlying bacterial infection. Approximately one
third of all asthmatics will have sputum showing evidence of intercurrent respiratory infection. Some of
the following findings are frequently observed in sputum.
Eosinophilia. The sputum has a distincitve eosinophilic staining properties that have been
attributed to an increased number of eosinophils and to the increased accumulation of serum proteins
from the inflammation of the allergic reaction. Specifically elevated levels of eosinophil granule major
basic protein have a high degree of association with asthma. When associated with an elevated level of
Charcot-leyden crystal protein concurrent bronchopulmonary infection is usually present. Also, sputum
eosinophilia appears to be associated with a better response to corticosteroids; unless there is an
underlying infection, neutrophils are not present.
Charcot-Leyden Crystals. These are rarely found in sputum except in cases of bronchial asthma.
They may be absent in fresh sputum but make their appearance if the specimen is allowed to sit. The
crystals are colorless pointed hexagons and vary greatly in size. The average length is about three to
four times the diameter of a red blood cell. Often they appear needle-like. They are derived from the
disintegration of eosinophils; hence they stain strongly with eosinophils.
Bronchial Epithelial Cells. The epithelial cells often occur singly and show hydropic
degeneration, with poor definition of the original morphology. During acute exacerbations, these cells
gather in larger clusters, display a vacuolated cytoplasm with ciliated border and are known as Creola
bodies. They are seen in approximately one half of the cases. Also present are well preserved,
hypersecretory goblet cells occurring singly or in clusters.
Creola bodies are a histopathologic finding indicative of asthma. Found in a patient's sputum,
they are ciliated columnar cells sloughed from the bronchial mucosa of a patient with asthma.
Curschmann`s Spirals. These are found most frequently in bronchial asthma and are fairly
characteristic of the disease. Curschmann`s spirals consist of mucus of high viscosity and are formed in
the lumen of small bronchi, bronchioles, and ducts of the seromucous bronchial glands. They may be
observed in chronic bronchitis and in heavy cigarette smokers, but in these cases there is nearly always
an underlying asthmatic tendency. 0acroscopically they can sometimes be organized by the naked eye
and appear as yellow-white, mucoid, wavy threads frequently coiled into little balls. Unraveled, their
length rarely exceeds 1.5 centimeters, microscopically a central thread is seen around which mucus is
wrapped, supported by a fibril network. The central thread is formed by the shedding of the lining
epithelium. Often embedded within the mucus are eosinophils and Charcot-Leyden crystals.



Case 2:
A physician performed bronchoscopy from the lower respiratory tract of a 53 year old woman. During
culture and sensitivity, the specimen was negative for growth. Giemsa stain revealed round cysts that
resemble H. capsulatum.
Bronchoalveolar lavage
Bronchoaveolar lavage (BAL) consists of instillation of a physiological solution (normal saline,
Normosol, or Plasmalyte) into a specified area of the pulmonary parenchyma, followed by aspiration.
The material obtained represents sampling of 1 to 3 million alveoli (about 1 percent of all alveoli).
BAL samples are most useful in investigating diffuse alveolar processes, such as Pneumocystis carinii
pneumonia, viral infections, chemotherapy- or radiation-related changes, or lesions with transalveolar
spread, such as alveolar proteinosis and bronchoalveolar cell carcinoma. Additionally, BAL has been
used in the study of various immunologic inflammatory, and infectious processes occurring within the
alveolar spaces, including lymphomas, post-transplant lymphoproliferative disorders (PTLD), interstitial
lung diseases, and inhalation-related disorders. Notably, only rarely are interstitial lung diseases
diagnosed using BAL. However the specimens obtained may be useful for differential cell counts and
special ancillary investigations, including flow cytometry or immunohistochemical studies.
Questions;
1. What is the most probable condition of the patient?
The patient is suffering from Histoplasmosis. It is a disease caused by the fungus
Histoplasma capsulatum. Symptoms of this infection vary greatly, but the disease
primarily affects the lungs. Occasionally, other organs are affected; this is called
disseminated histoplasmosis, and it can be fatal if untreated. Histoplasmosis is common
among AIDS patients because of their lowered immune system.

2. How come the culture and sensitivity was negative?
Culture, which is often considered the 'gold standard,` is limited by the slow growth of
the organism, which may take more than 20 days to grow.
Histopathologic examination of BAL specimens usually reveals the diagnosis in a few
hours in those patients who have disseminated disease. Histopathologic findings may be
falsely negative, however, because of sampling error, low number of organisms, or
inexperienced cytologists. As a result, the sensitivity of cytopathologic analysis of BAL
varies among published studies, ranging from 40% to 85%. In addition, culture may
require several weeks to isolate and correctly identify the organism

3. What other tests can be done to confirm your diagnosis?
Histoplasmosis can be diagnosed by samples containing the fungus taken from sputum,
blood, or infected organs. It can also be diagnosed by detection of antigens in blood or
urine samples by ELISA or PCR. It can also be diagnosed by a test for antibodies
against Histoplasma in the blood.

Serum and urine Antigen detection
Antigens and antibodies can be found in your urine and blood. It's a quick and fairly
accurate way of detecting disseminated histoplasmosis as well as chronic or mild cases
of the disease. But false-negative results are a problem, especially in people who have
compromised immune systems or are infected with other types of fungi. The test can
also be positive in people who live in areas where histoplasmosis is common and have
had past exposure to H. capsulatum, even though their current symptoms may be due to
something else.
y Testing urine and serum for Histoplasma antigen remains the preferred
method for rapid diagnosis. In fact, the antigen is detected in 92% of
urine samples and 85% of blood samples in patients who have
disseminated disease.
y These are useful in individuals who are immunocompromised when
antibody production may be impaired.
y Detection rates in cases of acute progressive disseminated
histoplasmosis are 50% with serum assay and 90% with urine assay.
y Cross-reactivity with Blastomyces and Coccidioides species causes
false-positive results.
y Urine antigen levels may be used to follow the patient's course.
y In making the diagnosis of progressive disseminated histoplasmosis,
blood cultures, blood antigen, urine antigen, and Histoplasma
immunodiffusion and complement fixation should be obtained
Blood Cultures
This is considered the gold standard for confirming a diagnosis of histoplasmosis.
During the test, a small amount of blood, mucus or tissue from your lymph nodes, lungs
or bone marrow is placed on a medium that enhances the growth of fungus and is then
checked for the presence of H. capsulatum. The drawback is the time it takes for the
fungus to grow up to four weeks. For that reason, it's not a good choice in cases of
disseminated disease where delayed treatment may prove fatal. Examples of medium are
BHI or Brain-Heart Infusion medium and Potato Dextrose Agar.
y Blood culturing should be performed in all patients, and results are
positive in 50-70% of patients with progressive disseminated
histoplasmosis.
y Results are rarely positive in patients with other types of histoplasmosis.
Complement-fixation assay
Titer is considered positive at reciprocal dilutions greater than 1:8. A titer with
dilutions greater than 1:32 suggests active histoplasmosis infection. Cross-
reactivity with antigens from Blastomyces dermatitidis and Coccidioides
immitis may cause a false-positive test result. False positive tests may also
occur in persons with lymphoma, tuberculosis, or sarcoidosis.
Positive results are expected in 5-15% of cases of acute pulmonary infection 3
weeks after exposure. This figure increases to 75-95% at 6 weeks in cases of
symptomatic infection. Test results usually normalize over months, with
resolution of infection.
Test results may remain positive in 70-90% of cases associated with chronic
pulmonary histoplasmosis or chronic progressive disseminated histoplasmosis.














SA0PLE TEST PAPER
1. What is the volume acceptable for sputum collection following sputum analysis?
a. 5ml to 10ml
b. 15ml to 20ml
c. 25ml to 30ml
d. 35ml to 40ml

2. What causes the sputum to turn greenish after standing more than 24 hours?
a. Bilirubin
b. Biliverdin
c. Pseudomonas
d. Verdoperoxidase

3. Which of the following is a fragment of necrotic pulmonary tissue primarily seen in such
diseases as pulmonary gangrene or tuberculosis?
a. Bronchial casts
b. Broncholiths
c. Cheesy masses
d. Curschmann`s spirals

4. Which of the following is a microscopic crystal found in people who have allergic diseases such
as asthma or parasitic infections such as parasitic pneumonia or ascariasis?
a. Bronchial casts
b. Cheesy masses
c. Charcot-Leyden crystals
d. Curschmann`s spirals

5. Which of the following is found most frequently in bronchial asthma and are fairly
characteristic of the disease?
a. Bronchial casts
b. Cheesy masses
c. Charcot-Leyden crystals
d. Curschmann`s spirals

6. Which part of the aliquot in Bronchoalveolar lavage contains the largest number of cells?
a. 1
st
aliquot
b. 2
nd
aliquot
c. 3
rd
aliquot
d. 4
th
aliquot


7. How much saline is infused into the lungs following bronchioalveolar lavage collection?
a. 100ml to 150ml
b. 5ml to 10 ml
c. 75ml to 90 ml
d. 20ml to 60 ml

8. Which of the following conditions causes the sputum to be rusty in color?
a. Cardiac failure
b. Recent hemorrhage
c. Decomposed hemoglobin
d. Pulmonary infarction

9. Which of the following are histopathologic finding indicative of asthma? They are found in a
patient's sputum, they are ciliated columnar cells sloughed from the bronchial mucosa of a
patient with asthma.
a. Creola bodies
b. Goblet cells
c. Curschmann`s spirals
d. Eosinophils

10. Can Curschmann`s spirals be seen macroscopically?
a. YES
b. NO























ANSWER KEY
1. What is the volume acceptable for sputum collection following sputum analysis?
a. 5ml to 10ml
b. 15ml to 20ml
c. 25ml to 30ml
d. 35ml to 40ml

2. What causes the sputum to turn greenish after standing more than 24 hours?
a. Bilirubin
b. Biliverdin
c. Pseudomonas
d. Verdoperoxidase

3. Which of the following is a fragment of necrotic pulmonary tissue primarily seen in such
diseases as pulmonary gangrene or tuberculosis?
a. Bronchial casts
b. Broncholiths
c. Cheesy masses
d. Curschmann`s spirals

4. Which of the following is a microscopic crystal found in people who have allergic diseases such
as asthma or parasitic infections such as parasitic pneumonia or ascariasis?
a. Bronchial casts
b. Cheesy masses
c. Charcot-Leyden crystals
d. Curschmann`s spirals

5. Which of the following is found most frequently in bronchial asthma and are fairly
characteristic of the disease?
a. Bronchial casts
b. Cheesy masses
c. Charcot-Leyden crystals
d. Curschmann`s spirals

6. Which part of the aliquot in Bronchoalveolar lavage contains the largest number of cells?
a. 1
st
aliquot
b. 2
nd
aliquot
c. 3
rd
aliquot
d. 4
th
aliquot


7. How much saline is infused into the lungs following bronchioalveolar lavage collection?
a. 100ml to 150ml
b. 5ml to 10 ml
c. 75ml to 90 ml
d. 20ml to 60 ml

8. Which of the following conditions causes the sputum to be rusty in color?
a. Cardiac failure
b. Recent hemorrhage
c. Decomposed hemoglobin
d. Pulmonary infarction

9. Which of the following are histopathologic finding indicative of asthma? They are found in a
patient's sputum, they are ciliated columnar cells sloughed from the bronchial mucosa of a
patient with asthma.
a. Creola bodies
b. Goblet cells
c. Curschmann`s spirals
d. Eosinophils

10. Can Curschmann`s spirals be seen macroscopically?
a. YES
b. NO


























References:
Case 1
Henry`s Clinical Diagnosis and 0anagement by Laboratory 0ethods 18
th
Edition by John Bernard
Henry
Henry`s Clinical Diagnosis and 0anagement by Laboratory 0ethods 21
st
Edition by John Bernard
Henry
A Laboratory Guide to Clinical Diagnosis, 5
th
Edition by RD Eastham
http://www.harrisonspractice.com/practice/ub/view/Harrisons%20Practice/141270/1/asthma
http://findarticles.com/p/articles/mi_gGENH/is_20050229/ai_2699003744/
http://erj.ersjournals.com/cgi/content/full/20/6/1359
http://pathhsw5m54.ucsf.edu/case17/discussion17.html
http://www.answers.com/topic/charcot-leyden-crystals
http://www.answers.com/topic/curschmann-s-spirals-1
http://www.answers.com/topic/curschmann-s-spirals-1
Case 2
Henry`s Clinical Diagnosis and 0anagement by Laboratory 0ethods 18
th
Edition by John Bernard
Henry
Henry`s Clinical Diagnosis and 0anagement by Laboratory 0ethods 21
st
Edition by John Bernard
Henry
A Laboratory Guide to Clinical Diagnosis, 5
th
Edition by RD Eastham
http://www.aspergillus.org.uk/secure/articles/pdfs7/19375640.pdf
http://www.gen-probe.com/pdfs/pi/102962.pdf
http://en.wikipedia.org/wiki/Histoplasmosis#Diagnosis
http://emedicine.medscape.com/article/299054-diagnosis
http://www.ncbi.nlm.nih.gov/pmc/articles/P0C150317/
http://www.ncbi.nlm.nih.gov/pmc/articles/P0C2292670/











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