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Ranjit Chandra
Interest in the use of immunologic assessment in the evaluation of nutrition programmes stems from four sets of observations: (a) the common association of malnutrition, impaired immunity and infection, (b) the similarity between patterns of micro-organisms recovered from malnourished subjects and those seen in primary immunodeficiency states, (c) the marked involution of lymphoid structures in nutritional deficiency, and (d) data on laboratory animals deprived of specific nutrients.
Nutritional deficiency affects cell replication and impairs protein synthesis. the two key elements of optimal immune response. It is to be expected that changes in immunocompetence will precede clinical manifestations of malnutrition and symptomatic infection. Thus, we can use immunologic tests as objective, functional, and early indices of nutritional problems, and therefore, of nutritional improvement due to an intervention.
While immune response in vivo is generally characterized by complex cellular interactions and participation of humoral factors, it is still possible to study and assess individually different components of host defense. Immune response is modulated by a number of inherited and environmental factors; among the latter, dietary intake is an important one. Malnutrition is the most frequent cause of secondary immunodeficiency world wide; it damages almost all layers of the protective umbrella of immunity.
The choice of an individual test will depend on the facilities and funds available and the age of the subjects. Bedside techniques (tonsil size and skin tests) and non-invasive tests (e.g., salivary 1gA concentration) are feasible in all field studies. Other, invasive, methods use capillary blood samples (e.g., lymphocyte count, serum levels of complement C3, and factor B). If larger blood samples are available, the numbers of T lymphocytes and the level of leukocyte terminal deoxynucleotidyl transferase (TdT) can be included in nutritional assessment.
The most important confounding factor in immunocompetence evaluation is the presence of infection. This can be excluded by both clinical examination and by estimation of serum C-reactive protein concentration on capillary blood samples. An elevated level points to a coexisting or recent infection.
Finally, it must be stressed that some of the tests of immunocompetence are highly sensitive to nutritional repletion. In general, appropriate nutritional support will reverse immunologic abnormalities in two to twelve weeks. Ideally, one should have baseline as well as post-intervention evaluation for assessment of changes in the same individual and for intra-group comparison.
What follows is a brief description of the most important tests of immunocompetence which should be considered when evaluating the impact of food and nutrition programmes.
Bedside Procedures
Tonsil Size
The vulnerability of lymphoid tissues to nutritional deficiency is reflected in the small size of the tonsils in malnourished children (1,2).
Method Tonsillar size should be evaluated in a quantitative fashion. The subject should be quiet and not gagging. The following criteria are used:
Grade 0 - vestigial or not visible;
Grade 1 - small tonsil easily visible by pushing the anterior
faucial pillar laterally;
Grade 3 - tonsil easily visible without any additional maneuver;
Grade 4 - large tonsils.
Small tonsils (grades 0 and 1 ) point to the presence of malnutrition.
Delayed Hypersensitivity Skin Tests
The ability of individuals to respond normally to various microbial and chemical agents in a delayed hypersensitivity reaction depends upon the normal integrity of T-cell mediated and inflammatory cell responses.
Delayed hypersensitivity skin reactions are commonly impaired in protein-energy malnutrition (1-4). In many malnourished patients, there is no response to any of the antigens used. Such lack of response, or energy, is a significant finding, but a reduction in the number of antigens to which the individual responds or the size of induration are probably of little consequence. The extent of energy correlates with severity of nutritional deficiency on the one hand and prognosis, sepsis, and death on the other. Many studies have reported similar results.
Skin tests have the merit of inducing in vivo responses. These tests are relatively easy to perform and paramedical individuals can be trained in a short period. The use of a multiple puncture device makes the procedure convenient and less time-consuming. However, the main disadvantage is the need for the patient to return after 48 to 72 hours for a reading of the results. This is feasible for in-patients, but may create a problem for clinic or office patients, particularly if long travel distances and transport difficulties are involved. It is essential to use the standard amounts of antigens recommended by the Scientific Group on Immunodeficiency of the World Health Organization (5).
Method. The following antigens should be employed initially: Tuberculin or Purified Protein Derivative (5 TU), Candida and Trichophyton (1,000 PNU/ml for infants, 100 PNU/ml for older children and adults), streptokinase 5 IU, and streptodornase 25 IU, tetanus toxoid 0.1 Lf unit. Each antigen is administered intradermally in 0.1 ml volume using a small syringe and 1/2" 26 gauge small-bevel needle. Alternatively, a plastic disposable device capable of delivering up to six antigens simultaneously by pressing it against the forearm may be used. The reaction is read after 43 to 72 hours. An induration of at least 5 mm in diameter is considered a positive response.
Most healthy individuals respond to at least one of the above antigens. The number of positive reactions and the diameter of induration should be noted. If there is no reaction to the above recall antigens, sensitization is attempted with the chemical 2, 4 dinitrochlorobenzene (DNCB) in acetone (2 mg/100 ml). The solution is smeared on the volar surface of the forearm within an area of approximately 1 inch diameter, employing a circular metal ring to ensure that no part of the solution touches the outside skin. After three to four weeks, the challenge dose of the same DNCB (10 ug) is applied to the other forearm and the reaction noted after 48 to 72 hours. It is graded as follows: Vesiculation, erythema + induration, erythema alone, no reaction. In patients with varying grades of protein-energy malnutrition, 25 to 100 per cent of subjects show energy to the recall antigens, whereas 0 to 18 per cent do not react to DNCB.
Non-invasive Method: Salivary lgA
Secretory lgA forms an important barrier on mucosal locations. In children with protein-energy malnutrition, the levels of slgA are decreased in the saliva, nasopharyogeal secretions, tears, and other secretions (6, 7).
Method. Samples of saliva can be collected by asking the subject to spit into a clean container. Alternatively, in a small infant, saliva can be obtained by placing a dry cotton ball in the mouth or by gentle suction with a Pasteur pipette. The method for estimation of slgA is similar to that of complement, using specific antiserum against slgA.
Capillary Blood (Finger Prick Samples)
Lymphocyte Count
Blood lymphocyte counts generally parallel the state of integrity of the lymphoid organs. A variable decrease in absolute lymphocyte count is observed in protein-energy malnutrition.
Method. The total leukocyte count is first estimated by drawing venous or capillary blood into a WBC counting pipette and diluting the blood with buffered saline or any other appropriate isotonic solution. With cover slip in place the blood is carefully 'loaded,' ensuring complete filling of the chamber without overflow, and allowed to settle for a minute. The number of leukocytes in the four corner squares is then counted and averaged. Differential count is estimated by examining at least 200 leukocytes on a thin blood smear stained with Giemsa stain. The proportion of lymphocytes (both small and large) in the differential is multiplied by the total leukocyte count and the figure divided by 100 to give the absolute lymphocyte count per mm³.
In healthy subjects, the absolute lymphocyte count should be above 2,500 per mm³. Mild lymphopenia is considered to be between 1500-2000 per mm³, and counts below 1,500 severe lymphopenia. The proportion of malnourished individuals showing lymphopenia varies from 3 to 18 per cent according to the degree of malnutrition (1,2).
Complement Components
The complement system is an efficient amplifying mechanism for immune responses. Complement component C3 and Factor B are measured by the radial immunodiffusion in agar method.
Method. Commercial immunoplates are the most convenient. Alternatively, one can prepare them by the following method: 1 per cent agar solution is prepared by dissolving Agar-Noble in barbitone acetate buffer pH 8.6. Added to this is the specific antiserum to the component being measured. The volume of antiserum required depends on the titer of the preparation (generally 15-50 µl/ml).
The melted agar and antiserum are mixed thoroughly and poured onto glass slides 80 mm x 80 mm. Air bubbles may be removed by rupturing them with a Pasteur pipette, by gentle movement or by flaming. The plate should be placed in a moist chamber in a refrigerator to set.
The sample holes are cut by holding the template over the gel and cutting the gel with a cutter or needle filed to the appropriate size. The gel plugs are removed by suction. The sample wells should be evenly spaced and marked. Serum samples of 3 to 5 µl in three different dilutions are applied to the wells. This should include at least three dilutions of a standard with known quantity of complement. Standard preparations are available commercially and from the World Health Organization. The plate is returned to the moist chamber and diffusion is allowed for 18 to 24 hours. The gels may be placed in 1 per cent tannic acid solution for five minutes to increase the visibility of precipitin rings. The ring diameter is measured to the nearest 0.1 mm and its square calculated. Standard curves are constructed by plotting squared diameter of precipitin rings of standard solutions against known concentrations. The unknown concentration can be read off from this curve. The method has a coefficient of variation of about 7 per cent and the lower limit of sensitivity is about 1 mg per cent. Modifications of this technique include electroimmunodiffusion or rocket immunoelectrophoresis and nephelometry. Their main advantages are, respectively, increased sensitivity and reduced reading time; However, costly equipment is required for the latter. Blood for estimation of complement levels should be collected fresh and allowed to clot at room temperature. The serum should be separated immediately and the samples may be stored at -20° C for several weeks before testing.
The serum concentrations of many of the complement components (particularly C3 and Factor B) are decreased in 21 to 88 per cent of individuals with protein-calorie malnutrition (8,9). This is because of both reduced synthesis and increased consumption in antigen-antibody reactions, the latter as a result of frequently associated infection. The extent of reduction in complement C3 level correlates with morbidity. Infection further lowers C3 concentration. As a direct consequence of these changes, the opsonic activity of plasma (ability of plasma to facilitate ingestion of particles by phagocytes such as neutrophils) is reduced when tests are conducted at low concentrations of 1 to 5 per cent.
Tests Using Venous Blood Samples
Additional immunological tests have been found to be useful in the assessment of nutritional status.
However, these require the processing of cells in the field and/or sophisticated, expensive equipment and reagents. For the present, these methods can be used only in research or in assessment of hospitalized patients.
T-lymphocytes
Small lymphocytes in the peripheral blood appear homogeneous on light microscopic morphology but are comprised of several subsets of cells distinguishable by a variety of methods, including resetting with sheep red blood cells, immunofluorescence, histochemical, cytotoxicity, and cell sorting techniques using specific monoclonal antibodies. Approximately 75 to 80 per cent of the circulating Iymphocytes are T-cells. The functional importance of T-cells in host defense is underscored by the frequency and severity of infections that plague patients with thymic aplasia and reduced number of T-cells.
The assay method most commonly used to identify mature T-lymphocytes is based on their ability to form rosettes with sheep red blood cells. Mononuclear cells are obtained from about 3 to 5 ml of heparinized blood by buoyant density centrifugation. Alternatively, mixing anticoagulated blood and 3 per cent gelatin in a syringe will aid in preparation of leukocyte-rich supernatant. Treatment of sheep cells with neuraminidase beforehand increases the number of rosettes. After washing the cells, equal volumes of suspensions of lymphocytes and sheep red blood cells are incubated in the cold at 4° C for 3 to 15 hours. The gently resuspended cell pellet is mounted on a counting chamber and the proportion of rosetting cells with three or more red cells attached to it is observed. The results are best expressed as the absolute number of T-cells per mm³.
The proportion and absolute number of T-cells is reduced in protein energy malnutrition (3,10,11). This abnormality is rapidly reversed after nutritional supplementation, in some instances in as little as two to eight weeks. Thus, sequential assessment of T-cell number may be a valuable index of nutritional recovery in malnourished patients.
Neutrophil function
The reticuloendothelial system is important for taking up particulate material including micro-organisms, inactivating them and disposing of them. Phagocytes provide a primary line of defense against bacteria and fungi. The ability of neutrophils to inactivate depends upon many lysosomal enzymes, most of which ultimately produce lethal oxygen molecules. Many of these enzymes involved in metabolic activities responsible for intracellular bacterial killing utilize essential nutrients and energy for optimal function.
Polymorphonuclear leukocytes of malnourished children and adults show impaired ability to kill ingested bacteria and fungi (12,13). The abnormality can vary from mild to moderate and is reversed quickly and completely on nutritional recovery.
The main steps of the method for estimation of microbicidal function of neutrophils are: (a) dextran sedimentation of about 3 ml blood, and (b) washing the leukocytes and mixing these with live bacteria or fungi, in a ratio of 5 micro-organisms to 1 phagocyte. Examples of bacteria and fungi include Staphylococcus, aureus. Escherichia coli, or Candida albicans. At intervals, aliquots of cells are sampled, washed, and lysed, to count the number of viable organisms. Normal polymorphonuclear leukocytes can kill more than 95 per cent of ingested mocroorganisms in two hours. An alternative (such as chemiluminescence) to this cumbersome procedure is under investigation.
Leukocyte Terminal Deoxynucleotidyl Transferase (TdT)
The normal ontogenetic development of T-cells involves a series of well-defined stages (14). Stem cells give rise to pre-T lymphocytes that do not show the conventional surface markers of mature T-cells. On the other hand, early thymocytes are very rich in TdT enzyme. Thus, the estimation of leukocyte TdT can be used as an indirect measure of T lymphocyte differentiation.
In protein-energy malnutrition, thymic hormone activity is reduced (15) with resultant maturational effect in thymic lymphocytes. There is an increase in the proportion of "null" cells without the conventional surface markers of mature T or B-lymphocytes (11). The majority of null cells may be immature T cells. TdT is increased in protein-energy malnutrition and there is a direct correlation between the number of null cells and leukocyte TdT (16).
Leukocyte TdT activity can be measured on peripheral venous blood, using a radiolabeled substrate. However, costly scintillation equipment is required. An enzyme-linked immunoassay will be available in the near future.
Recent studies have documented the usefulness of employing immunocompetence as a functional measure of nutrition (17). In growing infants, it may be a sensitive index of imminent failure to thrive and of infectionrelated morbidity.
Obviously, there are many unanswered questions. Many of the immunologic processes act in unison and often interact in a synergistic fashion. Impairment of one facet of immunity may be of little functional consequence for the host if other components are intact. The specificity of tests of immunocompetence is debatable. Other disorders, often clinically apparent, as well as infection, can depress immune responses. The threshold of clinically relevant immuno-deficiency is ill-defined. How much reduction in the number of circulating T cells is biologically significant? Does a 50 per cent decrease in serum complement C3 concentration predispose to more frequent or severe infection? Infection often complicates malnutrition and can suppress immunity.
The answers to these and other crucial questions must await further investigation. Nevertheless, existing knowledge indicates that the assessment of immunocompetence by the methods currently available provides useful prognostic information in relation to nutritional deficiency and in the evaluation of nutrition intervention programmes.
Chandra, R.K., Immunology of Nutritional Disorders (Edward Arnold, London, 1980).
Chandra, R.K., "Immunodeficiency in Undernutrition and Overnutrition," Nutr. Rev. 39: 225-231 (1981).
Chandra, R.K. and P.M. Newberne, Nutrition, Immunity and Infection: Mechanisms of Interactions (Plenum, New York, 1977).
Chandra, R. K., Trace Elements and Immune Responses, (John Wiley, New York, 1982).
Scrimshaw, N.S. C.E. Taylor and J.E. Gordon, "Interactions of Nutrition and Infection," WHO Monograph Series 57 (WHO, Geneva, 1968).
Suskind, R.M., ea., Malnutrition and the Immune Response {Raven Press, New York, 1977).
Thompson, R.A., ea., Techniques in Clinical Immunology (Blackwell, Oxford, 1977).