Respiratory System specimenDr. Ancent Nzioka27 / 01 / 12

Types of respiratory samples

•Sputum•Bronchial Specimens:

Bronchial Brushings Bronchoalveolar Lavage Bronchial Aspirations and Washings

•Fine needle aspirationTransbronchial Fine-Needle AspirationTransesophageal Fine-Needle AspirationPercutaneous Fine-Needle Aspiration

•Capillary Wedge Samples•Lung biopsy

sputumHemosiderin laden alveolar macrophages

•is the most readily accessible specimen for pulmonary cytology

•Screening asymptomatic smokers with sputum cytology does not decrease mortality from lung cancer.

•Sputum cytology is generally reserved for symptomatic individuals.

•Even here, with the advent of bronchoscopy and FNA, its use as the mainstay in respiratory cytology has declined significantly

Sputum collection

•The optimum number of specimens to submit to diagnose cancer is three, and to exclude cancer is five.

•An adequate cytologic examination consists of a series of three-to-five consecutive daily sputum specimens. Alternatively, a three-day-pooled-sputum collection may be done.

•Sample can be Spontaneous expectorated /induced/post bronchoscopy

Spontaneous expectorated sample •Sputum diagnosis is optimum in patients

spontaneously producing sputum, especially when it is bloody

•Early morning, deep cough specimens are preferred

Adv•Noninvasive•Easy to collect•Cost effectiveDisadv•Significant degenerative changes•Difficulty in communicating instructions

to collect satisfactory specimen•Require multiple consecutive samples

Collection of Routine, early morning sputum series (1) The patient receives a clean sputum cup the night before and is

instructed not to use it until morning. (2)The patient clear postnasal secretions and to gargleis instructed to cough deeply ("from the diaphragm") confirmed

macrophages ("dust cells") upon awakening and expectorate all sputum into a wide-mouth, appropriately labeled container, fresh or with fixative depending on laboratory policy. The patient should be encouraged to expectorate deep sputum, not saliva.

(3) The patient continues the deep coughing or for one hour.

(4) The cup should be collected by early a.m. and immediately taken to the laboratory.

(5) The procedure should be repeated once a day for three days.

• The patient must be able to produce a deep cough specimen that is by finding the presence of on microscopy

Induced sputum •may be necessary in patients not producing

sputum. •Sputum induction increases the detection of

lung cancer•Nebulizing solution stimulates secretions in

the respiratory tract.•Nebulized solutions are varied and may

include: 15% nebulized saline, 15% saline with 20% propylene glycol, heated (115oF) 3-8% saline.

•Hypertonic solutions are more successful in inducing sputum, but less well tolerated.

Collection of an induced specimen • an induced specimen may be considered If there

is no productive sputum after thirty minutes of vigorous coughing

• The respiratory therapist usually provides this service.

• A heated aerosol solution is administered by a respiratory technician and after breathing the aerosol mist for three minutes, the patient then coughs deeply.

• The expectorated material is collected in an appropriately labeled, wide-mouth container.

• This procedure is repeated for approximately 30 minutes and all material is collected

Postbronchoscopy sputum

•used in conjunction with bronchial washings and brushings for diagnosis of carcinoma.

•It may have a higher diagnostic rate than standard sputum collection specimen; It may be that the cells are loosened up by the bronchoscopic procedure, freeing them to be coughed up

Collection of Postbronchoscopy sputum • If possible, the patient is given a clean

sputum cup before the bronchoscope is withdrawn.

•The patient should cough deeply and expectorate all sputum into the cup for one to two hours.

•The cup is collected after one-to-two hours and taken immediately to the laboratory.

•The sputum series is continued the next morning, using the routine early morning sputum series, as explained in the previous section.

preservatives

•Fresh sputum submitted immediately to the laboratory is preferred.

•Refrigeration beyond 24 hours is not recommended because even with refrigeration, pathogenic organisms may multiply. This increases exposure-risk to laboratory personnel, activates degradation pathways in the cells, and contributes to background clutter of the preparation.

•Sputum that is held more than fourhours benefits from fixation.

•fixatives include 70% ethyl alcohol, or 50% ethyl alcohol with 2% polyethylene glycol (Saccomanno's fixative).

•Specimens collected after hours or on the weekend should have an equal volume of 50% alcohol added. The addition of fixative should be noted on the requisition form

SPUTUM ADEQUACY:

-alveolar macrophages must be present (some recommend 150 macrophages per slide in non-smokers and 300 in smokers)

-presence of ciliated columnar cells is not reliable as they can be from nasopharynx

sputum processing

•pick and smear” technique•Saccomanno method•Thinlayer methods(Liquid Based

Cytology-Thinprep® and Surepath®) •embedded in paraffin for cell block

sections

pick and smear” technique

•Fresh “sputum is examined for tissue fragments, blood, or both.

•Smears are prepared from areas that contain these elements and are immediately fixed in 95% ethanol.

Saccomanno method•sputum collected in 50% ethanol and 2%

carbowax The specimen is then homogenized in a blender and concentrated by centrifugation.

•Smears are made from the concentrated cellular material.

• When properly performed, the Saccomanno method specifies that direct smears should be made of blood-flecked material

•The Saccomanno method must be performed in a biologic safety hood as a result of the risks of infection from aerosolization.

-advantages:• remove debris and mucoid material • concentrates cells and may yield a higher

number of diagnostic cells than the pick-and-smear technique, fewer false-negative results

-disadvantages: • infectious of laboratory personnel, due to

aerosolization of infectious agents • small tumor cells are dispersed by the blender,

making them difficult to detect • Tissue fragments, fungal hyphae, and

secretory vacuoles disrupted

Thinlayer methods(Liquid Based Cytology-Thinprep® and Surepath®)•AdvImproved slide quality (Decreased obscuring blood,

mucus, diathesis, air drying, and smear artifact)Better cell preservation and stainingIncreased cellularity and number of diagnostic cellsDecreased screening time Better utilization of personnelAdditional Papanicolaou

slides,Histochemistry,Immunohistochemistry, Cell block preparation can be done

Disadv•Technical problems- Cellular fluids: central dropout

•Increased cost• Cytologic features altered

Loss of backgroundLoss of cell cohesionCell shrinkageArtifactual clustering

Bronchial Specimens

•Biopsies, brushings, and washings are usually done in concert with fiberoptic bronchoscopy

•The type of the specimen collected is dependent on the findings at bronchoscopy and the clinical judgement of the operator

Bronchial Aspirations and Washings•Bronchial secretions can be aspirated

directly from the lower respiratory tract through the bronchoscope

•alternative (and more common) method is to “wash” the mucosa by instilling 3 to 10 mL of saline and aspirate the washings without not wedging of the bronchiole.

•The fluid is centrifuged and the concentrate used to make smears, thinlayer preparations, or cell blocks

•These washings are usually submitted immediately to the laboratory.

•If a delay is anticipated, they may be partially fixed in an amount of 50-70% ethanol that is equal to the specimen volume, or in the proprietary transport medium supplied by one of the manufacturers of liquid based processors

Adv•Samples wider portion of the bronchial

Disadv•Blind method•Contamination with blood,debris and

inflammatory cells obscuring

Bronchioalveolar lavage•BAL is particularly useful for the diagnosis

of opportunistic infections in patients who are immunocompromised.

• It is performed by wedging the bronchoscope in a smaller airway so as to effect a seal and rinsing the distal airways with 100 -200ml of a balanced salt solution.(e.g., Ringer’s, Hank’s, RPMI) or saline then reaspirating in aliquotes of 20-60 mL

•Balanced salt solutions are preferred for the maintenance of cytologic morphology.

• The samples initially contain respiratory epithelium, but the latter portions of the aliquots are enriched for alveolar components specimens are submitted fresh and centrifuged and the concentrate used to make smears, thinlayer preparations, or cell blocks

• The volume of fluid and the extent of the lavage depend upon the suspected disease and the training and preference of the bronchoscopist, as well as the patient’s tolerance.

Adv•Wide sampling of the bronchial tree

including alveoli

ADEQUACY: -<5% bronchial epithelial cells, >90%

alveolar macrophages -more than >10 macrophages per high

power field -there should not be significant numbers of

less bronchial epithelial cells (indicating proximal airways sampled)

-should not be less frankly purulent or bloody

Bronchial Brushings• Bronchial brushing has a higher diagnostic yield

(increased sensitivity) than either bronchial washing or sputum cytology for metastatic carcinoma, peripheral tumors, and large, necrotic cancers

• A brush is applied to the surface of an endobronchial lesion, and the entrapped cells are either smeared onto a glass slide or rinsed in a collection medium for thinlayer or cell block preparation.

• If smears are made, immediate fixation (by immersion into 95% ethanol or spray fixation) of the smears is essential to preserve morphologic detail.

• if rinsed collection media used is saccomano carbowax fixative or respective LBC fixatives

• ADEQUACY: -no need to comment on adequacy with brushings

Adv•Samples fresh cells•Direct non blinding sampleing

Disadv•Limited sampling of the bronchial tree

accessible to the bronchoscope

Fine needle aspiration

Transbronchial and Transesophageal Fine-NeedleAspiration•Transbronchial fine needle aspiration biopsy

is performed during the bronchoscopic procedure to sample endobronchial or peribronchial lesions and peritracheal or peribronchial lymph nodes, usually for evaluation of malignancy.

•Transesophageal FNAB is usually performed to evaluate paraesophageal abnormalities in the chest cavity or mediastinal and lower thoracic lymphadenopathy.

• A small, sheathed needle is advanced during bronchoscopy or endoscopy, and under fiberoptic visualization is introduced into the lymph node or lesion.

• Suction is applied while vigorously sampling the site. Suction is released, the needle is re-sheathed and removed from the bronchus.

• The material is expressed onto slides and direct smears are prepared. Additionally or alternatively, the needle can be rinsed in transport medium and submitted as a liquid based sample. The needle should never be submitted

Adv•Can sample central lesions •Can sample lymph nodes

Disadv•Technically demanding•Contamination by both neoplastic and non

neoplastic cells from bronchial tree

•ADEQUACY: -diagnostic cells present (if clinically

suspicious for a tumour) -unsatisfactory if macrophages and

bronchial cells only or if extensive necrosis

. Percutaneous Thoracic Fine Needle Biopsy

•Percutaneous thoracic FNAB is performed for evaluation of any pulmonary abnormality, but is usually used for evaluation of suspected malignancy.

• Percutaneous transthoracic FNAB is usually radiologically guided. A variety of imaging modalities are used including computerized axial tomography (CT) scan, fluoroscopic guidance and ultrasound guidance.

•The lesion is entered with a hollow needle containing a stylet. The stylet is removed and the lesion is aspirated.

complications•Pneumothorax is the most significant

complication, but of those patients experiencing pneumothorax, 5-10% require treatment; most cases of pneumothorax resolve without intervention.

• Hemoptysis occurs in up to 8% of patients. •Rare complications of air embolism do occur.

•Contraindications for performing the procedure may include an uncooperative patient unable to remain still during the procedure, anticoagulation therapy or bleeding diathesis, poor lung function, pulmonary hypertension, or a suspected vascular lesion.

•Post procedural x-rays are often obtained by the attending clinician to detect pneumothorax.

Processing of FNB

Direct smears•Wet fixed smears in 95% ethanol •Air dried smearsRinse the remaining material in the

syringe/needle in saline for cytospin or LBC fixative for thinprep

Material for cell block submitted in 10% formalin

Pulmonary Microvascular Cytology• Pulmonary microvascular cytology is not commonly

used to evaluate pulmonary lymphatic carcinomatosis.

• A pulmonary artery is catheterized and the catheter is wedged into a small vessel. A blood sample from the wedged pulmonary catheter is collected into a heparinized tube.

• The heparinized blood sample is processed to separate red cells from any diagnostic cells, most frequently by gradient centrifugation.

• The residual white cell components are evaluated for the presence of carcinoma.

• Megakaryocytes, which signal an adequate specimen and which are normally seen in the pulmonary bed, must be distinguished from cancer cells.

Lung biopsy

•Fixed in 10% buffered formalin