PMID:37889357 | DOI:10.1007/s10565-023-09835-4
PubMed articles on: Cancer & VTE/PE
PMID:37866514 | DOI:10.1016/j.jtha.2023.10.012
PubMed articles on: Cancer & VTE/PE
Catheter Directed Thrombectomy and Other Deep Venous Interventions in Cancer Patients
Tech Vasc Interv Radiol. 2023 Jun;26(2):100900. doi: 10.1016/j.tvir.2023.100900. Epub 2023 Aug 5.
4. The mucolytic reagent can be used within 24 hours of
preparation, if stored at 2–8oC.
1. Take approximately 2.5 mL of the specimen in a clean
sterile 15–25 mL universal container.
2. Add 2.5 mL of the mucolytic Reagent and close the
container tightly with a screw cap fitted with an intact
3. Mix well by gently vortexing at every 5 minutes interval
4. After 20 minutes, unscrew the cap of the container
carefully and add 5 mL of reagent C.
5. Close again the container tightly as in step 2.
6. Mix well and centrifuge for 25 minutes at 3000–4000 g.
7. After centrifugation unscrew the cap of the container
with the content carefully and discard the supernatant
gently in an activated 2% glutaraldehyde solution,
taking care as not to disturb the pellet at the bottom.
8. To the pellet at the bottom, add 1 mL distilled water
9. Use this suspended material for microscopy (acid fast
Collect specimen directly into
bedpan or plastic wrap stretched
Microbiology and Bacteriology 849
Sterile plating loops (10 µL), biosafety hood with
Bunsen burner, centrifuge at 3000–4000 g, activated 2%
glutaraldehyde solution. 5 mL measuring cylinder, vortex
mixer, 1 mL micropipette, 15–25 mL universal container.
Collect specimen prior to use of antimicrobial agent.
Wherever possible, indicate clearly that patient is on
antitubercular drugs. Sputum: Collect 5 to 10 mL in a
sterile container from an early morning specimen of
deep productive cough. For induced specimen use sterile
saline. Have patients rinse mouth with water to minimize
specimen contamination with food particles, mouthwash,
The procedure mentioned below is for 2.5 mL of the
sputum sample. In case of variation in quantity of
specimen used, process using proportionate amounts of
reagent, mucolytic and disinfection reagent.
Preparation of Mucolytic Reagent
The mucolytic reagent must be prepared just prior to use.
1. Bring the reagents to room temperature.
2. Add one scoop full (~12 mg) of reagent B to 2.5 mL of
simultaneously eliminating all unwanted microorganisms.
Since mucous is sticky, acid fast bacilli trapped in mucoid
portion of sputum are released by mucolytic action of N-acetyl
L-cysteine. NaOH decontaminates other microorganisms,
pretreatment and disinfection with LYFECTOL increases
relative acid fast bacilli concentration and ensures its more
sensitive detection during acid fast bacilli staining and
1. Store the LYFECTOL kit at 2–8oC, away from light.
2. Stability of the LYFECTOL kit is as per the expiry date
Smear on slides Smear specimen over 1.5 by 1.5
slide container taped closed and
Sputum 5–10 mL in sterile wax-free
disposable container. Collect an
least 3 consecutive days. Do not
of patients on therapy, collect
at weekly intervals beginning 3
weeks after initiation of therapy
nasopharyngeal discharge. Have
patient rinse mouth with water
with food particles, mouthwash
or oral drugs, which may inhibit
For induced sputum, use sterile
hypertonic saline. Indicate on
848 Concise Book of Medical Laboratory Technology: Methods and Interpretations
MUCOLYTIC, DISINFECTANT, SPECIMEN
PRETREATMENT AND BUFFERING SYSTEM
(Courtesy: Tulip Group of Companies)
Infection with Mycobacterium tuberculosis remains a major
public health problem. The epidemic of tuber-culosis and
multidrug resistant tuberculosis reflects the failure of public
health and social programs towards prompt treatment
of infected cases and screening of high-risk population.
Culture, isolation and sensitivity of Mycobacterium
tuberculosis from patient groups using standard methods
remain the gold standard for Mycobacterium tuberculosis
detection and effective and swift treatment worldwide.
LYFECTOL is a reagent for laboratory use only. LYFECTOL
is provided as a three component reagent.
a. Reagent A (2% NaOH solution)
b. Reagent B (N- acetyl L-cysteine)
c. Reagent C (Phosphate buffer pH 6.8).
Accessories: Spatula for approximate weighing (12 mg)
LYFECTOL is used for decontamination and concentration of specimen containing normal microbial flora
such as sputum as per international recommendation.
Proper decontamination and concentration of specimen
containing normal microbial flora such as sputum are
crucial in detecting Mycobacterium tuberculosis.
LYFECTOL provides a liquefaction-decontamination
Gastric lavage fluid > 5–10 mL in sterile container.
Collect in the morning soon after
the patient awakens in order to
obtain sputum swallowed during
days. Use sterile saline. Adjust to
neutral pH with 10 mg of sodium
carbonate immediately following
Lymph node Node or portion on sterile
Collect aseptically, and avoid
caseous portion if available. Do
not immerse in saline or other
Specimen submitted in formalin
Skin lesion Submit biopsy specimen in
sterile container without fixative
or preservative. Submit aspirate
acceptable only if biopsy sample
or aspirate is not obtainable. For
cutaneous ulcer, collect biopsy
sample from periphery of lesion,
container or syringe with Luer tip
into SPS blood collection tubes
Disinfect site with alcohol and
Bone Bone in sterile container without
— Specimen submitted in formalin
Bone marrow As much as possible in SPS
blood collection tube or 1.5 mL
Collect aseptically. Mix SPS tube
contents immediately following
> 5 mL in sterile containers Avoid contaminating
produce false positive culture or
Since mucous traps AFB and protects other organisms
from effective decontamination a combination of 2% NaOH
(decontaminant) and 0.5% N-acetyl-L-cysteine (mucolytic
agent) is preferably employed. Neutralization of strong
decontaminating solutions before using the sample
for AFB stain and culture is usually accompanied with
sequential buffered wash of the concentrated sample
because if the pH of the concentrate remains alkaline
or acidic it can destroy the culture medium and prevent
the growth of mycobacteria and staining efficiency of the
AFB smears. The buffered wash also helps in reducing
the specific gravity of specimen and sediments the
Mycobacterium more effectively.
Another important aspect post-decontamination is
the specimen concentration and relative centrifugal force
applied to the specimen. Improvement in correlation
between specimen showing a positive smear for AFB and a
positive culture has been demonstrated by increasing the
centrifugal force applied to pellet the specimen.
Microbiology and Bacteriology 847
Recommendations for sample collection for mycobacterial isolation and acid fast staining
Specimen type Specimen requirements Special instructions Unacceptable specimen
Abscess contents aspirated fluid As much as possible in syringe
Cleanse skin with alcohol before
may provide 7H9 broth/Kirchner
Blood 10 mL SPS (yellow top) blood
collection tube or 10 mL isolator
blood culture. Mix tube contents
SPS is preferred anticoagulant
Blood collected in EDTA, which
chain mycolic acids, unique to the mycobacterial cell wall
• Failure to react with Gram stains
• Resistance to the actionof antibodies andcomplement.
The four species in the Mycobacterium tuberculosis
complex are M. tuberculosis, M. microtic, M. africanum
and M. bovis. Laboratories can use biochemical tests for
differentiation between isolated strains.
Diagnosis of Mycobacterium Tuberculosis
The diagnosis of tuberculosis is often made on the basis
of clinical symptoms, chest X-ray and sputum AFB, since
available tests based on immunological principles for
associated with them. For the time being, speedy and
appropriate laboratory diagnosis of tuberculosis infection
through AFB staining, culture and sensitivity have more
and more important role to play in sensitive detection
and appropriate treatment of patients with tuberculosis.
However, sample collection, preparation, processing
techniques and detection methods employed have a
profound effect on the sensitivity and specificity of the
results for the detection of Mycobacterium tuberculosis
infection by AFB and culture methods.
A critical factor in the ability of laboratories to isolate
Mycobacterium tuberculosis is obtaining appropriate
specimen for AFB smear and culture. Approximately 85%
of the TB cases are pulmonary. However, many patients
cannot produce sputum spontaneously and alternative
respiratory tract specimens such as induced sputum,
gastric lavage or fiberoptic bronchoscopy may be needed.
As the proportion of patients with extrapulmonary form
of tuberculosis is increasing, adequate specimen from
extrapulmonary sites need to be provided.
Sample Concentration and Decontamination
Specimens obtained from sterile sites such as CSF,
peritoneal or pleural fluids do not require decontamination.
However, most specimens for AFB smear and culture are
from the respiratory tract and do contain mixed microbial
flora. Successful recovery of mycobacteria depends
Humans are very susceptible to the tuberculosis
infection but are remarkably resistant to the tuberculosis
disease; which is dependent largely on the state of the
hosts immune system. Of all the mycobacterial species,
846 Concise Book of Medical Laboratory Technology: Methods and Interpretations
Mycobacterium tuberculosis remains the most common
cause of pulmonary tuberculosis and remains the most
virulent of all the mycobacterial species.
The disease, as now well known, is highly contagious.
Although the disease involves all susceptible individuals,
the incidence is higher among disadvantaged minorities.
Industrialization, increased crowded housing and
nutritional deprivation have influenced the spread.
third world countries but is also resurging in the Western
world. According to World Health Organization (WHO)
reports, each year an estimated eight million new cases
of tuberculosis occur, leading to three million deaths; and
almost a third of the world’s population is infected by the
causative organism, Mycobacterium tuberculosis.
According to a study, in India, the number of tuberculosis
patients is increasing at the rate of 1.5 million per year, and
a quarter of these are sputum positive. Thus, about 4% of all
Indians are infected with Mycobacterium tuberculosis.
With the emergence of the multiple drug-resistant
strains due to poorly administered therapeutic measures
and patient non-compliance, Mycobacterium tuberculosis
is challenging its containment, on the basis of empirical
The genus Mycobacterium is composed of slow growing
organisms, which are “acid fast”. Currently about 55 species
of Mycobacteria are recognized. They are non-motile,
slightly curved or straight rods (0.2–0.6 × 1–10 µm) and
may occasionally demonstrate branching. The organisms
are aerobic and have a gram-positive cell wall, although
Acid-fast bacilli can frequently be found in the mouths
of water taps, but some at least seem to be affected by 6%
H2SO4 when an attempt is made to grow them, but survive
Many of the so-called anonymous strains are classified by
their reaction to light. Photochromogens are those which
produce orange colonies when they are exposed to light
(day-light/artificial light). Scotochromogens are those
which produce colored colonies even in the dark. It is
therefore, important in dealing with this classification not
to grow them in an incubator that is opened frequently,
not to let them stand on the laboratory bench unless the
observation is actually being made. The yellow-orange
pigment develops after exposure to light in 6–24 hours. The
scotochromogens produce a yellow growth but this turns
more orange on exposure to light. The Battey type either
does not change, or if it does, the change is very slow.
Sometimes for final identification it is necessary to
inoculate an animal to see if the organism will produce
disease. The human strain of M. tuberculosis will produce
disease in both guinea pigs and rabbits, but the rabbit will
show little sign of tuberculosis. With the bovine strain,
however, the rabbit is also highly susceptible. In some
specimens, the material may be directly inoculated, in
other, only after a primary culture has been grown.
OVERVIEW OF M. TUBERCULOSIS: DIAGNOS- TIC APPROACH, AFB STAINING, CULTURE AND
This is an organism found in rats in a disease somewhat
resembling leprosy. It can be passed on experimentally to
other animals of the same species.
The smegma bacillus is an acid-fast bacillus found in the
smegma secretion around the genital and anal parts of human
beings and in some animals such as dogs. It can also be found
in other parts of the body, e.g. in the ear. The bacillus may be
found in the urine, but if the urine is carefully collected after
cleansing the external parts it can usually be avoided.
It is generally shorter and thicker than the tubercle
bacillus, and many strains of the smegma bacillus are
decolorized by alcohol, which distinguishes them from the
tubercle bacillus. They grow rapidly in culture.
The butter bacillus. These bacilli may be found in grass,
This is also known as bacillus of Johne’s disease and is
the cause of a chronic enteritis in cattle and sheep. The
primary isolation is difficult and medium has to contain
mycobactin—an extract from other mycobacteria.
This produces a chronic or subacute ulceration in both the
skin and the adjacent subcutaneous tissue, particularly of
legs and arms. Incubation temperature should be between
25 and 35°C and the best growth is at 33°C. It grows on
This bacillus has been isolated from swimming pools and
produces ulcerative lesions on the extremities. It grows
more rapidly than M. ulcerans, but will not grow above 35°C.
This is also known as Hansen’s bacillus and causes leprosy.
Smears are made from a scraping from the skin of suspected
lesions and from nasal smears. It has been found in sputum.
The usual method is skin clips from the affected areas.
Films are stained by Z-N stain, but it is customary to
use 5% sulfuric acid for decolorizing as M. leprae is not so
strongly acid fast as M. tuberculosis, but stained M. leprae
bacilli may resist decolorization with 20% sulfuric acid. The
bacilli are usually present in large numbers (in lepromatous
leprosy) and are generally found in packets like cigar
bundles within phagocytic cells called lepra cells. They
may stain uniformly but there is often marked beading. The
bacilli may also be stained fairly easily by Gram’s method.
Until recently, no claims of culture were substantiated,
This is primarily used for laryngeal swabs, but is rather
unreliable in that it does not completely ensure the
destruction of untoward organisms.
The swab is simply left in oxalic acid for
30 minutes, and then smeared on the egg medium.
This, too, is less preferable than Petroff’s method, because
it needs much longer incubation periods.
An equal quantity of sample and 10% trisodium
phosphate are incubated for 24 hours, after which the
container is centrifuged for 30 minutes and the supernatant
The deposit is neutralized with 8% HCl using
This bacillus has been isolated from soil and from
suppurative infections in men and animals and also in
Microbiology and Bacteriology 845
glandular infections. It grows rapidly and is not pathogenic
2. Other unwanted bacteria are killed, thus allowing the
tubercle bacillus to grow in pure culture.
This is considered to be one of the most reliable methods.
1. Half fill a universal container with the sputum (or other
material) and add an equal quantity of 4% NaOH (For
lesser samples a proportionately equal quantity should
2. Invert the closed container twice or three times and
place in the incubator for 30 minutes, inverting it every
3. Centrifuge at 3000 rpm for 30 minutes (The centrifuge
must be completely at a standstill before opening it
again after centrifuging tubercular samples.
5. Add a few drops of neutral red indicator to the deposit.
