Intratracheal Sampling Device

Abstract

An intratracheal sampling device comprising an inner collection tube coextensively and retractably mounted within a hollow probe for insertion within the larynx of a subject animal with the collection tube in a retracted position. Depression of a plunger mounted at the external end of the probe extends the collection tube within the animal's trachea and at the same time activates means for supplying wash fluid to the probe as well as vacuum means communicating, via a sample collection vessel, with the collection tube. Wash fluid is ejected in a jetting action through an annular clearance between the distal end of the probe and the extended collection tube. The ejected fluid, together with contained exfoliated cells from the tracheal walls, is recovered in the sample collection vessel after returning through the collection tube.

Description

BACKGROUND OF THE INVENTION

The invention relates generally to cell sampling apparatus and more particularly to an intratracheal sampling device which uses washing fluid under pressure to collect exfoliating cells from the tracheal walls. It was made in the course of, or under, a contract with the U. S. Atomic Energy Commission.

Several animal models suitable for experimental induction of bronchiogenic carcinoma have been developed in recent years. These open the way to study the etiology, pathogenesis, and morphogenesis of neoplastic diseases of the respiratory tract, and should ultimately result in refinements of existing diagnostic and therapeutic procedures and the development of new ones.

Exfoliative cytology of the respiratory tract has become an indispensable diagnostic tool for malignant and other diseases of the airways. The usefulness of existing lung cancer models would be increased if efficient means were developed for obtaining exfoliated cells from the respiratory tracts of various laboratory animals, such as rats and hamsters, used in studies of respiratory carcinogenesis. Such means would help bridge the gap between cytological and histological findings during development of lung cancer in man, and should be useful in gaining a better understanding of the sequence of events during the morphogenesis of cancer by aiding in clarifying the relationship between metaplasia, atypia, and malignancy, and separating reversible from irreversible "precancerous" lesions.

The success of diagnostic studies of the above type will depend largely upon the existence of techniques and apparatus for efficiently and repeatedly collecting exfoliated cells from the lower respiratory tract. Existing equipment and methods for lung lavage do not fully meet such requirement, however. Existing methods involve sequentially filling one lung with wash liquid and then removing the liquid while oxygen is supplied to the remaining lung. Such methods are slow, complicated, yield an excessively dilute suspension of cells, and produce adverse side effects in the animal being tested.

It is, accordingly, a general object of the invention to provide apparatus for efficiently collecting exfoliated cells from the lower respiratory tract.

Other, more particular objects of the invention will become apparent upon examination of the specification and appended drawing.

SUMMARY OF THE INVENTION

In accordance with the invention, an intratracheal sampling device is provided for collecting exfoliated cells from the lower respiratory tracts of laboratory animals. The sampling device comprises a flexible inner collection tube coextensively and retractably mounted within an elongated hollow probe suitable for insertion within the larynx of the animal from which a sample is desired. A manually operated plunger is mounted within the probe handle for extending and retracting the collection tube and for actuating means for supplying wash fluid to the probe as well as vacuum means communicating, via a collection vessel, with the collection tube. Wash fluid is ejected in a jetting action through an annular clearance between the distal end of the probe and the extended collection tube. The ejected fluid, together with exfoliated cells from the tracheal walls, is recovered in the sample collection vessel after passing through the collection tube. Use of such apparatus to obtain cell samples is fast, simple, yields a relatively concentrated suspension of cells, and causes a minimal trauma to the subject animal.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to the drawing, a sampling device made in accordance with the invention is shown in its operating mode. The sampling device includes a hollow elongated probe 1 having a handle portion 2 and a tapered extension 3, a manually actuable plunger 4 slidably mounted within handle portion 2, and a flexible sample collection tube 5 extending axially through the plunger and tapered extension 3. Sample collection tube 5, which is fixed to plunger 4 and movable therewith, is slidably traversible through a slide seal 6 provided between handle portion 2 and tapered extension 3 to prevent leakage of wash fluid into the handle portion. As shown, the sample collection end 7 of sample collection tube 5 extends from the distal end of tapered extension 3 when plunger 4 is depressed. A sleeve 8 of polytetrafluorethylene is provided at the distal end of extension 3 for insertion into the animal's larynx up to the point shown by broken line 9. Sleeve 8 and collection tube 5 are sized to form a liquid supply and retrieval nozzle wherein wash liquid jetted outwardly through the annular passage between the sleeve and collection tube is retrieved through the end 7 of the collection tube extending beyond the end of the sleeve.

A plunger extension 11 extends axially along the outside of handle portion 2 in alignment with switch 12 which is fixed to the handle. Depression of plunger 4 causes plunger extension 11 to close switch 12, thereby activating respective vacuum and wash liquid injection means 13 and 14. Timer 15 terminates the vacuum and injection of wash liquid after a suitable interval. Wash liquid injection means 14 supplies wash liquid to the interior of tapered extension 3 by means of a flexible conduit 16. The wash liquid thus supplied travels to the distal end of extension 3 where it discharges, in a jetting action, through the annular spacing between sleeve 8 and extended sample collection tube 5. The wash liquid thus discharged is recovered through tube 5 as indicated by the flow arrows in the drawing. Vacuum means 13 communicates, via vacuum conduit 17 and sample collection vessel 18, with end 19 of sample collection tube 5, thereby facilitating recovery of wash liquid and exfoliated cells suspended therein. Sample collection vessel 18 acts as a trap where a sample 20 of wash liquid and exfoliated cells is recovered for study.

In operation, the animal from which the trachael washing is to be taken is anesthetized, placed in an inclined position with its mouth held open and its larynx brought into view. Sleeve 8 is then inserted into the upper part of the larynx and plunger 4 depressed to extend collection tube 5 about 15 mm out of the end of the sleeve. At the end of the depression stroke of plunger 4, switch 12 is closed and vacuum and wash fluid injection means 13 and 14 activated, causing, in a 5-second interval, 2 ml of wash liquid to be jetted through sleeve 8 and then recovered through tube 5. Shearing forces caused by the jetting motion of the wash liquid causes exfoliated cells to be swept into the wash liquid, aspirated into collection tube 5, and delivered to sample collection vessel 18. The vacuum is maintained at a constant value which is sufficient to remove all wash liquid delivered but insufficient to cause a negative pressure buildup in the lung. Conventional specimen fixing procedures as described below are applied to the cells collected in the trap.

After each sampling procedure, the sampling device is removed from the animal being tested and 6 ml of prefixative is aspirated through collection tube 5 to wash off any cells remaining in the tube and transfer them to collection vessel 18. The prefixative is a solution of 2 ml of melted Carbowax (Carbowax R1540, polyethylene glycol available from Union Carbide Corporation, New York, N. Y.) in 100 ml of 50 percent ethanol. The upper perforated stopper 21 can be transferred to another collection vessel for further use after addition of the prefixative.

The recovered specimen is prefixed in the 50 percent ethanol -- 2 percent Carbowax solution for at least 2 hours and then centrifuged for 5 minutes at 240 g and 25 minutes at 960 g to collect the cells therein on a coverslip at the bottom of the collection vessel. Most of the supernatant is pipetted off and the coverslip allowed to dry while the Carbowax forms on opaque, waxy, protective film over its surface. The specimens are then postfixed in 95 percent ethanol for 1 hour after which they are placed in running tap water for 5 minutes to remove the Carbowax, stained, and mounted face down on microscopic slides. Before use, the coverslips are washed thoroughly until they are coated with water, and a thin layer of albumin-glycerin is rubbed on the surface facing the specimen.

Proper operation of the subject sampling device is achieved only when the wash liquid is returned through a centrally located tube 5 as previously described in reference to the drawing so that a maximum shearing action is provided by the jetted wash liquid. In addition, collection tube 5 must be more flexible than sleeve 8 and retractable therein. End 7 of collection tube 5 must be extendable beyond the end of sleeve 8 a limited distance so that flooding in the region of liquid discharge does not occur. An extension distance of 15 mm has been found effective in practice.

EXAMPLE I

Tracheal washings were obtained from rats and hamsters from 1 to 5 times at intervals of 1 to 14 days. Consistently, 1-2 .times. 10.sup.5 cells were obtained from those animals when 2 ml of wash liquid and short methoxyflurane narcosis were used. Cell yield appeared to depend on a number of variables such as the amount of washing fluid used or the type and duration of anesthesia. The cell types obtained included ciliated cells, goblet cells, clusters and sheets of respiratory cells, alveolar cells, polymorphonuclear leukocytes, and squamous cells. The relative frequency with which the respective cell types appeared is shown in the following table.

TABLE

Frequency of Appearance of Different Cell Types in Tracheal Washings from Rats and Hamsters

Relative Frequency (%).sup.a Cell Type Rats Hamsters Ciliated cells 13 (.+-.3.1) 6.8 (.+-.1.7) Goblet cells 0.2 (.+-.0.2) 0.1 (.+-.0.2) Unidentified columnar cells 10 (.+-.1.7) 4.7 (.+-.2.3) Alveolar cells 25.6 (.+-.5.2) 41 (.+-.5.4) Polymorphonuclear leukocytes 1.1 (.+-.1.1) 5.3 (.+-.2.5) Unidentified noncolumnar cells 27.4 (.+-.4.5) 12.6 (.+-.2.5) Nuclei 23.1 (.+-.3.9) 28.2 (.+-.7.4) Squamous cells 0.3 (.+-.0.6) 1.4 (.+-.1) .sup.a A total of 200 cells were counted from each tracheal washing. Samples were obtained from 10 rats and 10 hamsters. Figures in parentheses indicate the standard deviation of the percentage.

To determine whether the intratracheal washings could be performed repeatedly on a single animal without damaging the tracheal epithelium or initiating an inflammatory response, histological examinations of the respiratory trace tissues were made after single or repeated intratracheal washing. No signs of inflammation or injury of lung or tracheal tissues were found. More than 1000 cytology samples were obtained in rats and hamsters without any deaths occuring as a result of the sampling. Even those animals bearing lung tumors tolerated repeated washings without apparent ill effects.

EXAMPLE II

The feasibility and usefulness of the subject sampling device in obtaining samples for studies of lung tumor development were demonstrated by following the cytological changes observed in the tracheal washings of a hamster which had been intratracheally injected with a massive dose of carcinogen.

The exfoliative cytology obtained from this hamster at 1.5, 5, and 6 months after the start of the experiment were correlated with the histopatholgical findings. At 6 weeks (after 5 weekly intratracheal injections of benzo a pyrene) many dyskaryotic squamous cells appeared in the tracheal washing. The cell size varied considerably and was sometimes two- to three-fold larger than normal. The cellular shape was elongated, polygonal, round, or quite irregular, and the cytoplasm stained red or orange. The variation in size and shape and the hyperchromasia of the nuclei were very pronounced. These dyskaryotic cells persisted in all the following tracheal washings and might have shed from the squamous cell carcinomas found at autopsy 4-5 months later. Five months after the experiment began, another cell type appeared in the tracheal samples. These cells were round or cuboidal and showed occasionally beaded arrangement. The large nuclearcytoplasmatic ratio, the coarse chromatin arrangement of the hyperchromatic nuclei, and the variation in nuclear size were also characteristic of this cell type. In the ninth tracheal washing at 6 months (immediately before the hamster was killed), cells and clusters were found that closely resemble the morphological characteristics of the tracheal sarcoma. The cells were typically spindle-shaped and their cytoplasm stained light purple. The elongated nuclei varied in size and showed dark chromatin clumps. The clusters had a whorled arrangement of cells that corresponded to the histological pattern of the sarcoma.

Claims

What is claimed is:

1. Apparatus for collecting cells from animal respiratory tracts comprising:

a. an elongated hollow probe having a handle portion and a tapered extension portion for insertion into the respiratory tract to be sampled, said extension portion terminating in a wash liquid discharge end;

b. a flexible sample collection tube having first and second ends, said sample collection tube being slidably mounted within said probe and selectively extensible from said wash liquid discharge end thereof;

c. means for selectively extending said first end of said sample collection tube from said wash liquid discharge end;

d. means for ejecting wash liquid from said wash liquid discharge end of said probe;

e. means, in fluid communication with said second end of said collection tube, for maintaining reduced pressure in said tube so as to cause ejected wash liquid and exfoliated cels contained therein to enter said first end of said tube and flow therethrough; and

f. a collection chamber in fluid communication with said second end of said collection tube for receiving sample material removed through said collection tube.

2. The apparatus of claim 1 wherein said wash liquid discharge end of said probe comprises a polytetrafluorethylene sleeve having an internal diameter which is greater than the external diameter of said sample collection tube, said sample collection tube extending coaxially through said sleeve, wash liquid being ejected through the annular clearance between said sleeve and said collection tube.

3. The apparatus of claim 1 wherein said means for selectively extending said first end of said sample collection tube from said wash liquid discharge end comprises a plunger fixed to said tube and slidably disposed within said handle portion of said probe.

4. The apparatus of claim 3 wherein said plunger activates said means for ejecting wash liquid from said wash liquid discharge end and said means for maintaining reduced pressure in said sample collection tube.

5. The apparatus of claim 4 further including a timer for shutting off said means for ejecting wash liquid from said wash liquid discharge end and said means for maintaining reduced pressure in said sample collection tube.