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Should your lab perform tumor immunology?

Should your lab perform tumor immunology?

Tumor immunology is quickly gaining recognition as a valuable adjunct to routine laboratory testing. Enhancing the efficacy of treatment and deriving insight into the progression of disease are among the professional benefits to be gained from incorporating this procedure into your laboratory.

Certain substantial drawbacks have prevented the techniques from achieving wide clinical application. These include cost, the difficult technical aspects involved in the procedures, the absence of a large clinical data base, and the need to establish normal values. Nevertheless, none of these problems is insurmountable. * Teamwork. Setting up a unit to perform tumor immunology requires extensive consultation with your physician staff. Before setting up any meetings, though, do your homework. Compile a bibliography and make copies of articles containing pertinent information to distribute.

Ask some of the physicians who use your facility the following questions: Are they familiar with these tests? If you offered them, would they order them? Could comparable information be obtained by doing less labor-intensive and costly tests? Can the results be used to extend information obtained from more conventional tests? Is your laboratory capable of performing these tests?

If you make it over these hurdles, approach your hospital administration to discuss finances. Produce a balance sheet containing estimates of specimen load, cost per test, and potential revenues. Remember that this venture may be considered an independent unit of your organization and will be expected to produce accordingly.

One way to begin a tumor immunology program relatively painlessly is to accept specimens - properly attended during transport, of course - from other facilities. Another point to make is that your laboratory might be an ideal place to investigate new procedures and to collect clinical data to further cancer research. * Testing procedures. Various tests have been developed to assess the status of the immune system in the cancer patient, a topic of growing importance and concern. Most of these tests are still in preclinical stages and require further investigation. The assays discussed below are among those offered in clinical settings using data found in research manuals for comparison. They are still under development, however, and are not yet routinely used.

All the following assays require a sufficient number of lymphocytes. Check the patient's white blood count and lymphocytes before doing a cell separation on a ficoll-hypaque density gradient, a substance used to separate white blood cells from whole blood. Reject any specimens with a white count of fewer than 1,000 cells per [mu]l; these specimens are inadequate for testing purposes.

Doing cell separations on density gradients is an art as well as a science. Not only do recovery results vary; some cells have anomalous shapes or densities. Perform washing steps carefully to prevent further alteration of the cells and to avoid reducing the cell yield at each step.

Natural killer (NK) cell assay. Collect 50 ml of whole blood in heparinized tubes from the patient's peripheral blood system. Separate the white cells on a ficoll-hypaque density gradient. Incubate these cells for four hours with K562 (human myelogenous leukemia) cells that have been loaded with [51.sup.Cr]. During the incubation period, the NK cells will attack and lyse the tumor cells. This process releases radioactivity into the surrounding medium.

Spin down the cells. Measure the radioactivity in the supernatant to assess the extent of lytic activity by the NK cells; the greater the amount of radioactivity released, the greater the degree of killer activity assumed. An increase in NK activity may indicate an improvement in ability to combat tumor growth and proliferation. Computational programs are used to calculate the percentage of [51.sup.Cr] released with varying effector/ target cell ratios.

Mitogen stimulation assay. Collect 10 ml of whole blood and separate the white cells as explained above. Incubate the white cells for various lengths of time (some for up to three days) with the following mitogens: phytohemagglutinin, concanavalin A (con A), and staphylococcal phage lysate. The incubation medium contains tritiated thymidine. As the cells proliferate, the tritiated thymidine is taken from the medium by the dividing cells and incorporated into their genetic material. Measuring the radioactivity in these cells yields an index of the proliferative response of the patient's cells to a mitogenic challenge.

T-cell suppressor assay. Draw 40 ml of heparinized blood and separate the white cells as above. Culture the white cells - you are specifically interested in examining the lymphocytes - both with and without con A, which promotes the proliferation of T suppressor cells. After a sufficient incubation period, expose fresh lymphocytes (responder cells) to lymphocytes cultured with and without con A. Pulse the cells with tritiated thymidine and incubate them overnight.

Measure the DNA synthesis of responder cells after exposing them to lymphocytes cultured with and without con A. The degree of suppression of the immune system equals one minus the ratio of suppressed responder cells to control responder cells multiplied by 100 per cent. High suppressor activity may indicate disease progression caused by blockage of the immune system. * Making headway. Lymphokine activated killer cell therapy (LAK therapy), a new technique that involves autologus lymphocyte therapy of advanced malignancies, has gained considerable attention in the last several years. Lymphocytes are removed from the patient, stimulated by biological response modifiers, and reinfused into the same patient. By being made more active, the white cells of certain subpopulations are better able to fulfill their "seek and destroy missions" against tumor cells.

In the tumor immunology lab, these white blood cells are assayed with a variety of sophisticated cell culture techniques before, during, and after the initial course of therapy. In some assays, results may be seen in cultured cells before overt reactions are evident in the patient, thus providing the physician with more information on which to base subsequent therapy. Because the work is done in vitro, many novel drugs, biological response modifiers, and physical modalities may be rapidly tested in response to physician request. * Attitude and training. Tumor immunology is not for the faint of heart. All the tests are laborious cell culture procedures adapted from research literature. Each takes six to nine hours of effort, depending on specimen load, and requires an almost fanatical dedication to quality control and pipetting techniques. These procedures will not please technicians who prefer to utilize push-button tests that have been extensively debugged in a fully automated lab. The new techniques should also be avoided by laboratory staff members who are easily frustrated or who are unaccustomed to performing tests that require extensive cell manipulation.

Most of the technicians at our laboratory have bachelor's degrees in biology, microbiology, or chemistry. A background in immunology is also ideal for doing this sort of work. Medical technologists who have inquiring minds and enjoy a research-like environment with non-standard, ever-changing tasks tend to enjoy tumor immunology. Our laboratory director, coauthor Wass, has had three years of postdoctoral work in tumor cell biology as well as extensive experience in cell culture work. Basic science and medical technology backgrounds work well in this area. Medical technicians need additional training in sterile procedure. * Financial considerations. A substantial cash outlay is needed to set up a tumor immunology laboratory (Table I). Supplies are also costly: Most flasks, plates, and pipets are made from petroleum-based plastics. Also needed are centrifuge tubes, freezing vials, ficoll-hypaque density gradient, cell culture media (alpha mem), and fetal bovine serum.

Other considerations include space renovations and the costs of light, heat, power, and salaries. At least two technicians are needed to carry out the procedures, depending on the workload. Our laboratory started with a director and one medical technician. We now have three full-time and three part-time medical technicians as well as an investigator with a doctoral degree who serves as a consultant on quality control.

Table : Table I

Cost of equipment
 for a tumor
 immunology lab
Instrument cost
Laminal flow hood $ 8,000
[CO.sub.2], water-jacketed incubator 5,000
Water baths, shakers, rockers 1,000
Inverted microscope 4,500
Cryostat, liquid nitrogen tank 12,000
Pipet guns 800
Micropipets, adjustable 500
Beta counter 17,000
Gamma counter 7,000
Cell harvester 5,000
Heater 500
pH meter 500
Osmometer 900
Total $ 62,700

* Clinical use. Although it is generally agreed that cancer tends to be accompanied by detectable defects in the functioning of the immune system, the mechanisms have yet to be fully explored. Furthermore, the assays we have outlined are relatively new to the clinical arena. Extensive studies to correlate clinical data with laboratory findings began only within the last several years.

Although using these assays to test all cancer patients routinely would have no special advantages - especially since the responsiveness of "normal" populations is known to vary widely - assays of immunocompetence can provide needed information about progression of the malignant state during therapy. It is therefore valuable to collect clinical data on the biology of the host-tumor relationship and on possible effects of therapy on tumor progression. Tests such as those described in this article may one day be routine in supporting diagnostic and therapeutic decisions.

General references:

De Vita Jr., V. "Cancer: Principles and Practice of Oncology." Philadelphia, Lippincott, 1981. Di Saia, P.J., and Crossman, W.T. Tumor immunology. In: "Clinical Gynecologic Oncology," 3rd ed., chap. 16. St. Louis, C.V. Mosby, 1989. Fogh, J. "Human Tumor Cells in Vitro." New York, Plenum Press, 1975. Pitot, H.C. "Fundamentals of Oncology," 2nd ed. New York, Marcel Dekker, Inc., 1981. Rose, N.R.; Friedman, H.; and Fahey, J.L. "Manual of Clinical Laboratory Immunology," 3rd ed. Washington, D.C., American Society for Microbiology, 1986. Ultman, J.E., and Golomb, H.M. Neoplasia. In: Petersdorf, R.G., ed. "Harrison's Principles of Internal Medicine," 10th ed., section 9, pp. 777-779. New York, McGraw-Hill, 1983.

PHOTO : A photomicrograph has captured a colony of tumor cells (200X) during the growing process.

PHOTO : Your laboratory's immunology program can be expanded to include tumor vaccines and chemosensitivity assays. Below, a technologist prepares tumor cells for use in one such test.
COPYRIGHT 1990 Nelson Publishing
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Author:Wass, John A.; Patton, Kelly
Publication:Medical Laboratory Observer
Date:Jan 1, 1990
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