Journal of Clinical Laboratory Analysis 4:102-114 (1990)

Natural Killer Cytotoxicity in the Diagnosis of Immune Dysfunction: Criteria for a Reproducible Assay Theresa L. Whiteside,lY2John Bryant, li5 Roger Day, lY4 and Ronald B. Herberman,

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'Pittsburgh Cancer Institute, and Departments of 2Pathology and 3Medicine, University of Pittsburgh School of Medicine and Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania; 5Department of Quantitative Analysis, University of Cincinnati, Cincinnati, Ohio Current evidence indicates that natural killer (NK) cells, which are large granular lymphocytes that mediate non-major histocompatibility complex (MHC)-restricted cytotoxicity and antibody-dependentcytotoxicity and that are involved in multiple regulatory, developmental, and immunologic processes, are important in health. Immunodeficiencystates presentingwith low NK activity are often associated with malignancies, chronic viral infections, or autoimmune diseases. Monitoringof NK function appears to be indicated as an aid to diagnosis, prognosis, and follow-up after therapy. Reliable performance of NK assays in a clinical laboratory requires that uniform

Key words:

Laboratory evaluation of NK function, NK assays, quality control, human N K cells, statistical analysis of NK activity, serial monitoring of NK cells

INTRODUCTION Natural killer (NK) cells appear to play a role in a variety of human diseases. Compromised or absent natural immunity, as measured in vitro by decreased NK activity and/or depressed absolute numbers of circulating NK cells, has been linked to the development and progression of cancer and chronic and acute viral infections, including the acquired immunodeficiency syndrome (AIDS), and to chronic fatigue syndrome, psychologic dysfunction, various immunodeficiencies, and certain autoimmune diseases (1 -4). Recent evidence indicates that NK cells may be involved in multiple effector, regulatory, and developmental activities of the immune system and that deficiencies or abnormalities in NK cell function may contribute to, or be a biologic marker for, disease. For that reason, it is important to reliably detect abnormalitites in NK cell function. Unfortunately, the NK cell assay has not been included in the roster of routine diagnostic immunology tests, and there are no currently established criteria for reliable performance of the assay. It was our objective to demonstrate that NK cell function can be measured and serially monitored in a reliable fashion in human peripheral blood and body fluids. Like all cellmediated cytotoxicity assays, the one for measuring NK cell function is prone to errors, many of which are difficult to 0 1990 Wiley-Liss, Inc.

criteria be established and followed for the acceptability of results. Statistical analysis of daily variability can be of great assistance in identifying and tracking sources of error, but routine statistical adjustments are not generally advisable. The quality control program described here provides a degree of assurance that this cytotoxicity assay can be dependable whether performed at one time point or serially. The successful implementation of this program requires laboratory resouces, biostatistical support, and interpretative skills, all of which are available in a modern clinical laboratory.

recognize and to avoid. In this article we describe and discuss quality control measures that may help to eliminate some of these erron or, at least, to identify them. Because monitoring of NK activity is increasingly utilized in the context of clinical trials with biologic response modifiers, this quality control program may offer a means for decreasing day-to-day variability and improving reproducibility of serial measurements. NK cells represent about 10% (middle 80% range = 5.4-16.3 in our normals in Fig. 1) of circulating blood lymphocytes in humans. Morphologically, NK cells belong to a subset of large granular lymphocytes (LGLs) (5,6). Using monoclonal antibodies, NK cells can be distinguished from other lymphocytes, because they express a characteristic set of surface markers; they are CD3 -, C D 2 + , C D 1 6 + , and CDS6(NKH1)+. They do not productively rearrange T cell receptor genes and do not express the CD3 complex. NK cells are cytotoxic effectors that are capable of spontaneously killing tumor or virus-infected cells (7,8). Their cytolytic activity is not restricted by or dependent on expression of the major histocompatibility complex (MHC) on target cells. Although

Received July 6 , 1989; accepted July 14, 1989. Address reprint requests to Theresa L. Whiteside, Ph.D., Pittsburgh Cancer Institute, Room 7201.2, CBB, 203 DeSoto Dt, Pittsburgh, PA 15213.

Criteria for a Reproducible NK Assay

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in terms of their maturation, differentiation, and activation states as evidenced by the different combinations of surface markers that they express. Cytokines-in particular interferon alpha (IFN-alpha) and interleukin 2 (IL-2)-are efficient activators of NK cells (2,3). NK cells have been demonstrated to express medium affinity receptors (p70-75) for IL-2 (1 1) and can respond directly to IL-2 by proliferation and augmentation of cytotoxic reactivity, without prior activation. The effector cell function of NK cells, involving the ability to eliminate or inhibit the growth of transformed cells, intracellular pathogens, and certain immature normal cells, is one of multiple activities performed by NK cells. By way of their expression of Fc receptor type 111, NK cells can also interact with the Fc portion of IgG and mediate antibodydependent cell-mediated cytotoxicity ( 12). Their involvement in natural immunity to tumors and certain infectious diseases has been well-documented in experimental animals and humans (1 3). More recent data have provided support for the participation of NK cells in immunoregulation, stem-cell development, and interactions with tissue cells [reviewed by Trinchieri (3)1. NK cells can be stimulated to produce tumor necrosis factoralpha (TNF-alpha), interferon-gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3) (14), as well as other growth factors necessary for the development of hematopoietic stem cells (15,16). They also may exert an inhibitory influence on developing and maturing stem cells (17,lS). By interacting with other cells of the immune system and with various tissues, NK cells may be an important and active component of the pathologic processes in human diseases.

NK CELLS IN HUMAN DISEASES

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Fig. 1. A: Distribution of N K activity in a population ot 93 healthy individuals. The arrows indicate the lower boundary and the upper boundary of the 80% middle range of activity. The median activity was 132 LU (2.12 log lytic units). B: Distribution of total Leu-I9 cells (CD56+) in a population of 84 healthy individuals. The median percentage of total CD56 + cells was 10; the 80%range was 5- 16. C: Distribution of CD3 - CD56 + NK cells in a population of 68 normal individuals. The median percentage of CD3 -CD56 cells was 8; the 80%range was 3-14.

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the basis for target cell recognition by NK cells is not clear at this time, they are capable of distinguishing and sparing most normal tissue cells. NK cells arise from precursors in the bone marrow (9,lO). Circulating NK cells may be heterogeneous

We recently reviewed the role of NK cells in human disease (4). Briefly, it has been well-established that patients with a variety of solid malignancies and large tumor burdens have decreased NK activity in the circulation and that this low NK activity may be significantly associated with the development of distant metastases. Furthermore, in patients treated for metastatic disease, the survival time without metastases correlates positively with levels of NK activity (19-21). In patients with hematologic malignancies, there appears to be a correlation between NK activity and the status of disease: the more advanced the disease, the lower the NK activity (22,23). Decreased NK activity may also be an important risk factor for the development of malignancy in humans (24). The prognostic significance of low NK activity in patients with cancer has been recently emphasized; thus, low NK activity may have prognostic value in predicting relapses, poor responses to treatment, and, especially, decreased survival time without metastases (25-27). NK cells are sensitive indicators of activation by biologic response modifiers, and their monitoring has been used to document alterations in the activity of circulating immune cells during therapy with these agents (28,29). In cancer patients treated with radiation or chemo-

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therapy, NK activity becomes depressed as a result of ther- to plastic-and T and B lymphocytes can be removed by apy; given a role for natural immunity in tumor control, it gradient centrifugation or other cell separation procedures may be clinically important to monitor the extent and dura- ( 35,36). Usually, however, NK activity in the blood is measured by tion of the suppression of NK activity in order to minimize it using unfractionated mononuclear cells. These have to be prethrough adjustments in the extent and/or duration of cytopared fresh and maintained in medium supplemented with reductive therapy (3). In human bone marrow transplantation, 5% (v/v) FCS prior to the assay. The assay itself is relatively NK cells may influence the outcome through help in engraftsimple to perform. A constant number (usually 5 X lo3) of ment and control of viral infections and may mediate anti"Cr-labeled K562 target cells is mixed with graded numbers leukemic effects important for the elimination of residual tumor of effector cells in triplicate wells of U-shaped microtiter cells (30,31). On the other hand, there is also evidence for plates. The plates are centrifuged briefly to bring effectors the ability of NK cells to suppress hematopoietic developand targets in contact with each other, and then they are placed ment ( I 7,18). Furthermore, recent evidence indicates that there in a 37°C incubator for 4 h. Killed targets are quantitated by is a relationship between an individual's reaction to emotional measuring the amount of 51Crreleased into the supernatant stress and NK activity, and attempts are being made to define following the 4-h incubation of effectors and targets. After the mechanism responsible for low NK activity in individucentrifugation, the supernatants can be harvested with microals who have difficulties in handling stress and in those sufpipets by hand from each well; however, semiautomated harfering from behavioral disorders (32,33). The role of NK cells in viral disease has been known for a long time. The correla- vesting systems are available and are practical to use if large tion between low NK activity and serious viral infections in numbers of wells need to be harvested at one time. Each plate immunocompromised hosts--e.g., in AIDS, after transplan- must contain wells for determination of the spontaneous chrotation, and in certain congenital immunodeficiencies-has been mium release from target cells (i.e., target cells in medium well-documented (3). Abnormalities in NK function have been only) and of the maximal chromium release from target cells described in a variety of autoimmune diseases, and since these lysed by added acid or detergents. The harvested supernadiseases are frequently associated with serious viral infec- tants from control and experimental wells are counted in a tions and malignancy, low levels of NK activity may be bio- gamma counter. The amount of "Cr released into the superlogically important in individuals with autoimmune disorders natant is directly related to the proportion of target cells killed (34). The above examples indicate that there is a consider- by the effector cells. able amount of evidence available at this time to substantiate the important involvement of the NK cell in the spectrum of CALCULATION OF RESULTS IN NK human diseases. Therefore, reliable measurements of NK activ- CYTOTOXICITY ASSAY ity in health and disease appear to be needed for an adequate Results of the NK cell assay for each effectodtarget (E:T) evaluation of patients. ratio can be expressed as a percentage of specific lysis, which is calculated according to the following formula: PERFORMANCE OF AN NK CYTOTOXICITY ASSAY NK activity is usually measured in a short-term (generally % specific lysis = 4 h) cytotoxicity assay using cultured tumor cells as targets and mononuclear cells isolated from the blood or tissues as effectors. Tumor cells used as targets need to be sensitive to human NK cells, and K562, a cell line derived from a patient with chronic myelogenous leukemia in blast crisis, is most commonly utilized. The targets are first labeled with radioactive chromium (51Cr)and extensively washed to remove any unbound radioisotope. In preparation for cytotoxicity assays, the K562 line is maintained in culture in the presence of 10% (viv) fetal calf serum (FCS) and is provided regularly with fresh medium to assure that cells used as targets are in the log phase of growth; this is important to ensure high viability and uniform and good uptake of the radioisotope. Mononuclear cells prepared by Ficoll-Hypaque centrifugation of human blood, body fluids, bone marrow, or disaggregated lymphoid tissues may be used as effectors. Preparations enriched in NK cells can be obtained by removing monocytes-e.g., by adherence

experimental cpm - spontaneous release cpm maximal release cpm - spontaneous release cpm

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It is important that the spontaneous release be as low as possible. For example, with K562 targets is the log phase of growth, this spontaneous release seldom exceeds 5% of the maximal chromium release. This low a figure may not be achievable with other cell lines, particularly with fresh tumor cell targets; however, spontaneous release exceeding 20% of maximal release is generally not acceptable and indicates that target cells have poor viability or for other reasons are not adequately retaining the label. The NK assay should be performed at least four E:T ratios, e.g., from 50: 1 to 6: I , in order to accurately assess the doseresponse relationships between target lysis and number of effector cells present. The presentation of NK activity as percentage of specific lysis at several E:T ratios is cumbersome and inconvenient, particularly for comparing assays run at different E:T

Criteria for a Reproducible NK Assay

ratios. For that reason, an alternative way of expressing NK activity has been devised and is now widely used (37): NK activity may be expressed in terms of "lytic units" (LUs). LUs are a convenient way to quantitatively compare the relative cytotoxic activities of effector cell populations from different individuals or from the same individual over time. To calculate LUs of NK activity, it is necessary to combine the percentage of specific lysis at all the measured E:T ratios. First, the E:T ratio yielding 20% lysis (E:TZo)is estimated from these measurements. The choice of 20% as a reference level of lysis is arbitrary; however, it is quite common and seems to be a good choice, since experimental E:T ratios can be chosen so that the calibration will rarely require extrapolation beyond the range of the experiment. The estimation of E:T2()is usually accomplished by fitting a curve to the measured points on the graph of percentage of lysis versus E:T ratio and calibrating; however, substantially simpler and more robust methods are available (38). The reciprocal of this estimated E:T ratio increases with increasing cytotoxic activity and seems therefore to be a good summary measure. To complete the calculation of LU, this number is rescaled. A standard number of effectors (ESTD), typically lo7, is chosen, together with a standard number of target cells (TsTD), typically 5 x lo3.Then the lytic activity is reported as

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the physical concentrations of cells at 20% lysis. The calibrated E:TZoencourages us to think about questions such as, How do the E:T ratios in the cytolytic assay compare with the E:T ratios in the bloodstream? Nevertheless, the LU has become entrenched in common practice, and we continue to use the LU calculation routinely. The choices of 20% lysis, experimental E:T ratios and cell densities, incubation time, temperature, and other experimental conditions are not arbitrary. However, they may need to be adjusted depending on the types of effectors and targets being studied. To promote comparability of results among studies, and among laboratories, these conditions should be in conformance as much as possible for each kind of cytolytic assay. One feature of the LU, in contrast with using the percentage of lysis at a single E:T ratio, is that four (or more) values at four (or more) distinct ratios are used, implying greater amounts of information and, therefore, greater precision-just as the mean of four measurements has half the standard deviation that a single measurement has. Also, LUs provide a measure of potency and, therefore, of dosage, when effector cells are infused in a clinical trial or experiment. The total activity of a bolus of cells might be well-represented as the size of the bolus divided by the size of a LU.

DEFINING OPTIMAL CONDITIONS FOR NK CELL CYTOTOXICITY ASSAY There are several requirements for the optimal performance of an NK cell assay. The time between blood collection and The number of LUs is thus derived from the ratio of the expertesting and the viability of effector cells are important. Ideimental T:E ratio to a standard T:E ratio. The standard explanation of LUs is a bit different. LU ally, blood samples should be tested for NK activity immedidenotes a quantity or batch of effector cells needed to kill a ately after their collection; however, this is often not possible specific percentage (e.g., 20%) of a predetermined number because of the time needed for the blood to reach a laboraof target cells (e.g., TsTD = S x lo3).The size of this batch tory. It is important to separate mononuclear cells on Ficolldepends on the cytotoxic potential of effector cells, and the Hypayue gradients prior to storage; in our experience, storage smaller the batch, the more potent, on average, each effector of whole blood at room temperature or 4°C is less satisfaccell is. However, since it is convenient to have a measure of tory, resulting in greater losses in NK activity with time than lytic ability that increases rather than decreases with potency, observed with separated mononuclear cells (MNCs) placed the number of such batches required to make up EsTD = in a complete medium and stored for a comparable period of lo7 effector cells is computed and reported as the number of time. However, it is not recommended to store the separated LUs. Thus, the size of a lytic batch, which is (E:TZO)(TSTD) effectors for longer than 18 h at 4°C; substantial losses in cytoeffector cells, represents 1 LU; therefore, toxicity generally and variably occur during any prolonged storage of human effector cells. In our laboratory, bloods drawn number of LUs per lo7 effector cells = in the course of a morning are separated promptly after reach1o7 ing the lab. MNCs are washed, resuspended in a complete ESTD (i.e., containing 10%FCS) medium, stored overnight at 4"C, ( E : T ~ T s T D ) (E:T~o)(SX 10') and a l w q s tested the following morning. Uniformity in handling the blood samples is advisable. For example, if E:T2" = 10, then cytolytic activity is reported Sometimes, blood specimens for NK activity have to be as 200 LU, or 200 LUI lo7 cells, because lo7/(IO)(S x lo3) shipped from a distance. We recommend that they be col= 200. The choice of EsTD and TyrD seems arbitrary, and the lected early in the morning and hand-carried or air-expressed rescaling has the disadvantage of obscuring visualization of (at room temperature) to reach a laboratory by late afternoon.

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At that point, the specimens should be separated and the recovered MNC processed and tested as described above. Although it has been suggested in the past that cryopreserved effectors may be used in determinations of NK activity (39), it is preferable that fresh rather than cryopreserved cells be used. Cryopreservation, even under optimal conditions, has been shown to significantly decrease NK activity in cells from some individuals (unpublished data). While this decrease does not always occur, it is unpredictable in both occurrence and magnitude. While cells of some individuals can be cryopreserved without a loss of NK activity, cryopreservation generally destroys from 10%to 20% and sometimes more of NK activity in human mononuclear cells. We performed an experimental study of repeatedly measured normal controls over the course of 2 mo, in conjunction with repetaed measurement of frozen samples; we found a rather strong error of a systematic kind that affected the frozen samples but not the fresh samples (see below). It is essential that tumor cell lines used as targets be in the log phase of growth and be mycoplasma-free. The line should be tested for mycoplasma on a regular basis. The presence of mycoplasma contamination may lead to spurious results due to high spontaneous release of "Cr or increased and variable sensitivity of the targets to NK cells. The activity of human effector cells is generally measured in medium containing FCS and not human serum. Human IgG has been shown to inhibit NK activity (40); for that reason, the addition of 5%-10% FCS to the assay appears to be preferable. It could be argued, however, that FCS artifactually increases NK activity above that seen in human serum. An alternative might be to measure NK activity in serum-free media (e.g., AIM-V from Gibco, Grand Island, NY). However, in our hands, AIM-V medium did not support the effector function of human NK cells as well as FCS-containing media (Fig. 2 ) . The rather striking decrease of NK activity in this serum-free medium that we observed could not be explained on the basis of cell viability, because the cells survived well under these conditions. Clearly, further investigations into conditions for optimal NK activity in vitro are necessary.

DIFFERENT ASSAYS FOR HUMAN NK CELLS The NK cell assay based on a specific release of radioactive chromium is both labor-intensive and costly. Therefore, attempts have been made to modify the cytotoxicity assay in order to eliminate radioisotope, increase sensitivity, and reduce labor and reagent costs. Some of the newer assays utilize colorimetric measurements of the number of viable cells remaining after incubation with effector cells (41). We recently introduced an MTT-based colorimetric assay for measurement of tumor cell sensitivity to NK and lymphokine-activated killer (LAK) cell effectors (42). Although much more sensitive and better suited to testing of large numbers of samples than the 5'Cr-release NK cell assay, the MTT-based assay performed

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Fig. 2. Effects of serum-free AIM-V medium (Gibco) on NK activity of human MNCs. A: Fresh MNCs were resuspended either in RPMl 1640 containing 10% (viv) of FCS or in AIM-V medium and used as effectors in 4-hr "Cr-release assays. B: AIM-V medium was supplemented with ITS (insulin, transferin, selenium). K562 targets were cultured in FCS-containing medium in all experiments.

well only with adherent tumor cell targets. Other researchers have developed modifications based on the release of intracellularly trapped fluorescent dye (43). These modifications are useful and practical; however, their acceptance has been slow and their equivalence to the "Cr-release assays would need to be well-documented. Thus, the 4-h "Cr-release NK cell assay remains the one most widely accepted and used. Assays for the measurement of human NK cells in heterogeneous samples of cells in the blood or body fluids fall into two categories: those that measure function and those that

Criteria for a Reproducible NK Assay

quantitate the number or proportion of the effector cells. Measurement of both parameters offers an oppotunity to distinguish between the quantity of NK cells and their level of function, providing insight into the possible basis for decreased NK activity, i.e., whether it is due to a deficiency in the number of effector cells or in their functional ability. It is also possible, but definitely less practical, to first purify NK cells from heterogenous mixtures and then measure the function of such purified NK cells. Both approaches are of interest; while the use of unseparated mononuclear cells or even whole blood cell populations provides a deeper insight into the nature of the cellular interactions on a physiological level, the second approach allows for a measurement of NK activity after these interactions are eliminated. NK cell function can be measured at a single-cell as well as a population level (44). A single-cell NK assay developed by Grimm and Bonavida (44) provides a useful approach to determining the ability of NK cells to bind to a target and kill it. Although the singlecell NK assay has been most useful for measuring several important biologic functions of the NK cell simultaneously, it has remained a research tool because of its complexity in performance and interpretation. As indicated above, NK cells have many different functional characteristics, killing of tumor cells or virus-infected targets being only one of them. Assays are available to look at other functions of NK cells; for example, one can measure release of natural killer cytotoxic factor (NKCF) or the production of cytokines such as TNF-alpha or interferon gamma (14). The degradation of NK cells with the release of the granule-associated enzymes can also serve as a basis for measurements of NK activity (45). However, the release of granules in the presence of suitable targets, or the production of cytokines such as interferon gamma or TNF, are not functions specific to NK cells, as other cytotoxic effectors may be positive. Thus, in mixed populations of mononuclear cells, it would not be possible to distinguish NK cell function from that mediated by other cytotoxic or cytokine-producing cells. While NKCF release appears to be specific to NK cells, this cytokine has not been purified, and only a laborious biologic assay exists for its quantitation (46). The number of NK cells in the blood or cellular suspensions can be determined by staining mononuclear cell populations with monoclonal antibodies against surface antigens on NK cells such as CD56 (NKH1 or Leu-19) or CD16 (antiFc-gamma-RIII). Flow cytometry of the antibody-labeled cells then allows for a precise and specific quantitation of these cells among lymphoid cells. NK cells need to be quantitated by two-color flow cytometry to combine anti-CD3 and anti-CD56 or anti-CD16, since only the CD3- cells expressing these other markers can be considered NK cells (47). Two-color flow cytometry also makes it possible to quantitate activated NK cells, that is, NK cells that simultaneously express CD56 and HLA-DR antigens or CD 16 and HLA-DR antigens as well as the subpopulations of NK cells that may

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be CD56+CD16+, C D 5 6 + C D 1 6 - , and CD56-CD16+ (48). Ideally, these subpopulations should be assessed by three-color flow cytometry to combine anti-CD3 and the other two antibodies. It could be expected that a positive correlation exists between the number of circulating NK cells and levels of NK activity measured in the blood of normal individuals. We have recently shown that, although there is a significant positive correlation for the population of over 50 healthy volunteers examined, this relationship is not particularly robust (4), probably because circulating NK cells may vary considerably in their state of activation. Difficulties also exist in accurate quantitation of activated NK cells (i.e., CD56+HLA-DR+ or CD16+HLA-DR+) in the blood by two-color flow cytometry, because they represent less than 1% of total NK cells in normal individuals. In addition, it remains unclear whether the expression of these “activation” markers accurately reflects an increased functional state of the NK cells. It thus appears that enumeration of NK cells by flow cytometry is not a reliable substitute for the measurements of cytotoxic reactivity. Nevertheless, in certain disease states, e.g., in chroic fatigue immunodeficiency syndrome (CFIS), we have observed both low NK cell numbers and low or absent NK activity (49), whereas in other clinical conditions, low NK activity appears to occur despite normal numbers of the effector cells (50). Based on these observations, we conclude that both phenotypic and functional assays for NK cells are necessary in patients evaluated for natural immunity.

NORMAL DISTRIBUTION OF NK ACTIVITY IN HUMAN BLOOD AND TISSUES

In humans, NK activity and NK cell numbers are usually measured in venous blood or in body fluids in patients from whom such fluids are available. Active NK cells are present in the blood and spleen of all healthy individuals (1). In contrast, human lymph nodes and tonsils contain low, virtually undetectable, levels of NK activity, although the presence of NK cells in human lymph nodes has been documented by immunoperoxidase as well as flow cytometry of lymph node cell suspensions (51,52). Other organs, e.g., liver, contain variable proportions of NK cells among tissue-infiltrating lymphocytes. We have recently determined the NK cell content as well as NK activity among mononuclear cells recovered from liver tissues of patients with various end-stage liver diseases. The process of recovery involved mechanical dissociation of liver tissue, brief enzymatic treatment, and differential Ficoll-Hypaque centrifugation (unpublished data). We found that NK cells represented from 7% to 53% of mononuclear cells recovered from liver tissues of these patients and that NK activity of the liver-resident NK cells was higher than that measured in the circulation of the same patients (unpublished observations). The reasons for decreased NK activity in human lymphoid tissues relative to that measured in blood

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are not clear. Immune complexes may play a role, through the occupancy of the Fc-gamma-R111, in the downregulation of the cytolytic functions of tissue-resident NK cells. Also, there may be cells in the tissues with the ability to suppress NK activity. NK activity as well as the numbers of circulating NK cells detectable in the circulation may vary substantially among healthy individuals. Thus, in our studies of a healthy adult population, NK activity in the blood, detectable in a 4-h chromium-release assay, ranged from a low of 50 LU to a high of 302 LU (80% middle range), with a median of 132 LU (Fig. 1A). In an individual, however, the level of NK activity tends to be rather stable over time, provided the individual does not experience infections or other events (e.g., unusual stress) or take certain drugs (i.e., corticosteroids or other hormones) known to alter NK activity (32,33). Within a population of healthy individuals, measurements of NK activity can define low and high responders, but it is necessary to remember that levels of NK activity may be influenced by age, sex, exercise, and other general health factors (4,53). Furthermore, circadian variations in NK activity have been demonstrated in humans, with the maximum activity occurring in the morning or early afternoon (54). It is apparent that there may be a considerable degree of biologic variability in NK activity measured in the same individuals over time. This fluctuation represents an important consideration in serial monitoring of normal individuals and patients for NK activity.

QUALITY CONTROL IN NK CYTOTOXICITY ASSAY The challenge of distinguishing true biologic variability from assay-related variability is considerable and very important. To make this distinction, it is necessary to establish and perform an assay for NK activity that is reliable, reproducible, and free of errors. Errors that influence the reliability and reproducibility of any biologic assay can be divided into systematic and sporadic. The first type of error occurs when there is a deviation from a true measurement that happens every time the assay is performed, affecting more or less equally each measurement made over a period of time. A systematic error may go undetected because it affects control as well as experimental samples. Systematic errors may be reagent-related, technicianrelated, or procedure-related. As an example of the latter, consider the counting of target cells. This is done once a day following labeling with 5'Cr and washing of cells. The radiolabeled-cell suspension is mixed, and the concentration of cells is determined by tallying cells in a hernacytometer. If a mean count would be 100 t 10 (SD), then the coefficient of variation of the error in this count is around 10%. This error biases all measurements equally for that day, including control measurements, A technician performing the assay may systematically miscount the number of effector cells added to a cytotoxicity assay; the result would be a systematically

higher percentage of specific "Cr-release observed in control and experimental samples alike, and the error might not be identified until another technician took over the performance of the assay. If there are several systematic errors of the same magnitude, the total measurement error is a serious problem, reducing the precision and potentially introducing technical bias. A sporadic error occurs when a variation is introduced into the assay procedure. The variation may be a result of the technical eror, or it may be due to circumstances beyond the technician's control. For example, pipetting of samples may be a source of a sporadic error, if a technician omits cell delivery to one or more microtiter wells; or a semiautomatic micropipettor may be inappropriately set for a volume delivery on a given day; or a semiautomated device for collecting supernatants may not be operating optimally on a given day. Errors of this type are easier to detect when appropriate controls are included in daily cytotoxicity assays. There may be sporadic erros that affect only certain samples in the set, and there may be a tendency to interpret such results as biologic variability. As indicated below, means should be found to prevent such incorrect interpretations of the results. While the distinguishing of sporadic errors from biologic variability may be difficult at times, the identification of systematic errors represents a real challenge in the cytotoxicity assay. To meet the challenge of avoiding a systematic error and identifying sporadic ones, a set of control measures may be introduced as follows. To monitor for daily variability in an NK-cell assay, we recommend the following controls be included daily: 1. Three preparations of cryopreserved mononuclear cells should be obtained from whole blood or leukapheresis, with low, intermediate, and high NK activity. They should be aliquoted and frozen in quantities sufficient for many weeks of daily testing. Such cryopreserved normal control cells are not easy to prepare, because it is necessary to select only individuals wholse cells cryopreserve without a loss of activity, to avoid variability due to cryopreservation artifacts. The laboratory is thus obliged to perform a substantial amount of screening to identify such individuals. Once identified, however, these individuals can be repeatedly used as a source of control cells. 2. At least one fresh normal control from a group of repeatedly tested healthy volunteers should be available to the laboratory. The inclusion of fresh normal cells in the assay accomplishes two things: it allows the laboratory to establish a normal range for an NK cell assay, using the results obtained over time, and with the same normal individual's cells reDeatedly tested over time, it provides a measure of the biologic variability in that individual. Also, measurements of NK activity in a fresh normal control are necessary to ensure that a change in activity of frozen control cells that may be observed on a given day is not due to technical problems affecting only frozen cells. From the statistical point of view, the mainte-

Criteria for a Reproducible NK Assay nance of a fresh normal control series allows, over time, a determination of the degree to which systematic effects affecting fresh and frozen samples are correlated. 3. In addition, if cells from a fresh normal control are included in two different positions in a series of samples each day, the evaluation of intra-assay variability may be obtained. If possible, it may be advisable to similarly split patient cells for testing at two different positios in a series of samples. With these three types of control samples incorporated, implementing a routine NK cell assay becomes feasible in a clinical laboratory setting. To illustrate how these controls allow us to obtain useful information about the NK cell assay, Figures 1 and 3-5 have been provided. In Figure 1 A, the distribution of NK cell activity among 93 healthy individuals tested in our laboratory is presented. Figure 1B shows the distribution among 84 healthy individuals of total CD56+ (NKHl+ or Leu-19+) cells in the circulation, and Figure IC shows the distribution among 68 normal volunteers of CD3 -CD56+ NK cells (shown as the percentage of positive cells). Figure 1 emphasizes that the distribution of biologic activity or percentages of NK cells in the normal population needs to be established by every laboratory performing these determinations on a routine basis. In Figure 3, the performance of three frozen control cells in the NK cell assay over a period of several months is illustrated. Although there appears to be a certain degree of variability from day to day, the high, medium, and low control cells appear to retain their rank over the period of time. In Figure 4, the variability in NK cell assays performed by different technicians over time is illustrated. The data suggest that rotations of technicians have some impact on NK activity measurements. Finally, in Figure 5, NK activity obtained in serial measurements repeated over several months in a healthy volunteer is shown. This activity appears to be quite stable over time.

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Fig. 3. Daily variations in NK cell activity of frozen cells from three healthy individuals. The cells were defrosted and tested daily over a period of several months. Data are presented in base-10 log lytic units (LLU). Solid vertical lines indicate measurements done on Saturdays.

CRITERIA FOR REJECTION OF A CYTOTOXICITY ASSAY Based on the performance of frozen and fresh control samples, criteria for acceptability of the NK cell assay need to be established. In our laboratory, we also consider the perfor-

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mance of target cells used for cytotoxicity. If spontaneous release ofK562 targets is greater than lo%, the assay is rejected. We use frozen control cells to calculate mean NK activity ? 2 SD for each of the three cell preparations tested serially over a period of time. For example, for NK cell assays performed from November 1988 to 1989, the established normal ranges of NK activity for the frozen cell controls were as follows: control cell 1 = 1.4 log LU (LLU) & 0.5 (mean 2 2 SD); control cell 2 = 1.8 LLU ? 0.6; control cell 3 = 1.5 LLU & 0.4. Similarly, fresh normal control cells tested serially enabled us to compute a mean k SD for NK activity of fresh mononuclear cells from each healthy control individual. For example, the mean 2 SD NK activity measured in 10 assays over 1 y for the volunteer in Figure 5 is 1.7 LLU 0.4. This represents a normal established range for this volunteer. If on any given day, all three frozen control cells and a fresh normal control give results that fall outside of their established normal range, the assay is rejected. When the fresh normal control is in range, but the three frozen cells are out of range, the assay is not rejected because possible problems with frozen cells that day. Similarly, if the fresh cell control is out of the established range, but at least two of the three frozen cells are within the established range, the assay is not rejected. The latter situation can be explained by the biologic variation, which may cause shifts in NK activity of a fresh sample. These criteria provide a basis for a control program that should be established and practiced rigorously in every laboratory performing cytotoxicity assays. The introduction of and adherence to this quality control program represents a considerable amount of work and perseverance; nevertheless, where a reliable performance of cytotoxicity assays is important, the above quality control measures need to be established and adhered to.

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STATISTICAL ANALYSIS OF DAILY VARIABILITY IN NK CELL ASSAYS Several authors (29,55) have noted the presence of daily effects in cytolytic assays and have, therefore, recommeded standardization of LU by reference to concurrently run control tests. For example, Maluish and coleagues (29) described an adjustment procedure based on cryopreserved samples from three healthy individuals, each of whom had donated blood sufficient for many determinations. In each assay, all three standards were tested along with the patients' cells. For each patient, LU were standardized by the formula: set standard mean StandardNK = observedNK

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Fig. 6. N K cytotoxicity of cryopreserved and fresh normal controls. Cytotoxicity is expressed in base-I0 logarithmics of lytic units. A: cytotoxicities of cryopreserved cell samples donated by normal individuals A-D B: Cytotoxicities of fresh cell samples donated by normal individuals E-H. C: Frozen ( I ) and fresh (2) data.

This adjustment scheme is appealing. However, the adjusted patient data could actually be considerably less accurate than the unadjusted data, because the correction factor in this equation, especially the denominator, is subject to considerable random error. Experiments performed in our laboratory provided the cytotoxicity results shown in Figure 6, which proved useful in examining the efficacy of the LU adjustment as defined above. The data in Figure 6A were obtained from 162 cryopreserved cell samples obtained from four different healthy individuals (A-D), each of whom gave blood sufficient for at least 30 determinations. Generally, three of these frozen specimens, each from a different donor, were thawed and analyzed on

Criteria for a Reproducible NK Assay

each working day. The data shown in Figure 6B consist of 53 fresh normal samples donated by four additional healthy individuals (E-H). These donors gave blood daily for 3-5 consecutive working days, alternating weeks. Typically, one fresh normal control was assayed each day. The data are plotted as LU/ lo7 effectors on a base-10 logarithmic scale. The presence of systematic daily variability is evident in Figure 6A. In addition to obvious between-donor differences in the level of cytolytic activity, it can be seen that the NK activities of cryopreserved cells assayed on the same day tend to vary. Furthermore, Figure 6C, in which both fresh and frozen test results are graphed over time, gives evidence that the daily effects impacting fresh and frozen data are positively correlated. Additional analyses and formal statistical tests reinforcing these conclusions have been previously described (56). These results led us to attempt a quantitative assessment of the effect of standardizing LUs based on cryopreserved controls. A suitable criterion for this assessment is the withindonor variance of adjusted fresh-sample determinations, where the adjustments are estimates of daily effect based on the cryopreserved data. If standardization is effective in reducing variability caused by daily effects, then the within-donor variance of the adjusted determinations should be substantially less than the within-donor variance of the unadjusted determinations. Since the within-donor distribution of raw lytic units is highly skewed, but that of log lytic units is reasonably symmetric, additive adjustments were made to log lytic units. The adjustment equation used was as follows:

Where Y,id= log LUs of fresh sample j assayed on day d; Kd = average of the log LUs of all frozen controls assayed on day d; and X, = average of the averages over all days of each frozen control assayed on day d. Note that the additive adjustments on the log scale given by eq. 2 are virtually equivalent to the multiplicative adjustments of eq. 1 , the sole difference being that in eq. 1 the set standard mean and the daily standard mean are found by averaging LUs, whereas the averaging in eq. 2 is done on logarithms. Thus, eq. 2 corresponds to the use of geometric means in computing eq. 1. After adjusting the data, the within-donor variance of the adjusted log LUs of all freshly assayed tests was computed and compared with the variance of the unstandardized log LUs. Surprisingly, the two variances were essentially equal. The unadjusted variance was 0.0527 and the adjusted variance was 0.0512, less than a 3% reduction. Therefore, the reduction in variance provided by the controls through the elimination of daily effects was offset by the introduction of noise due to variation of the frozen control data unrelated to fresh determinations.

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This conclusion can be reinforced by estimating variance components based on the fresh and frozen cytotoxicity data. This permits an estimation of the variance of adjusted data, which is a function of the variance components. The results remain identical to those described above. This second approach also pzrmits an estimation of the efficacy of adjustment, if the number of daily frozen controls is to be increased. It turns out that increasing the number beyond three is predicted to have a negligible effect in reducing the variance of adjusted fresh data. The estimated reduction in variance approaches only 8% as the number of controls is increased indefinitely. The primary cause for this disappointing result appears to be the existence of substantial systematic daily error among frozen controls unrelated to fresh determinations. Hence, it may be that the use of a large number of cryopreserved controls would very accurately estimate a daily effect that is relevant to cryopreserved specimens but largely irrelevant to fresh patient specimens. Empirical Bayesian concepts of statistics suggest the use of “shrunken” estimates of daily effect that modify the “ 100% adjustment” (daily frozen control mean less long-term control mean) by multiplying it by a constant between 0 and 1:

(3) The choice of R in eq. 3 represents a way of compromising between full adjustment (R = 100%) and use of unstandardized LUs (R = 0%). For our data, an estimate of the value of R that minimized the within-donor variance of adjusted fresh log LUs turned out be roughly one half (52%). However, even this method of adjustment reduced within-donor variance by a practically inconsequential 13%. To interpret this reduction, we note that the spread of the distribution of donor-adjusted log LUs was reduced only 7%. We conclude that the use of standardized LUs based on cryopreserved controls should not automatically be assumed to lead to a reduced variability of determinations. Apparently, the principal cause for this is the existence of factors that affect frozen controls but are unrelated to fresh data. An example of such a factor might be daily variability in thawing times or temperatures. Care should be taken in extrapolating our conclusions to an environment where conditions may differ substantially. The usefulness of adjustment schemes is strongly dependent on the relative magnitudes of the components of variance affecting patient and control data. These magnitudes may well vary from laboratory to laboratory or, even with the same laboratory. Our conclusions should not be construed to mean that frozen controls have no role to play in data quality control. In fact, they are potentially very useful in detecting assay failure. Likewise, frozen controls might be useful in detecting long-term trends in assay results (i.e., gradual increases or decreases in level). However, their usefulness for purposes of routine adjustment of patient data is limited. On the other

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hand, the practical feasibility of using freshly prepared controls, either alone or in conjunction with frozen controls, shows some promise. The daily effects impacting fresh controls probably also impact patient date, and statistical studies hint at substantial potential for such adjustment schemes (56).

SERIAL MONITORING: DETECTING TREATMENTINDUCED CHANGES IN NK ACTIVITY Treatment-induced changes in NK activity can be reliably detected only by comparing a patient's posttreatment levels with his or her own pretreatment levels. A reasonable program of serial monitoring of cytolytic activity must, therefore, include at least three pretreatment measurements in order to establish each patient's baseline level of NK activity. Since all of the results of the posttreatment samples are compared with the baseline values, multiple baseline measurements are necessary not only to dampen out the effects of biological variability and measurement error, but also to allow for the detection and elimination of the extreme outliers that sometimes occur. Two classes of methods can be used for the detection of treatment effects (a) those that conceptualize treatment-induced response as a dichotomous variable (i.e., a patient either does or does not respond to treatment) and that attempt to identify responders; and (b) those that are based on the notion of a continuous measure of response (i.e., elevation of LUs relative to baseline levels) and that attempt to establish the presence of a non-0 mean response averaged over a conceptual population of treated patients. To illustrate the first method, a time series plot is shown in Figure 7. The plot displays the pre- and posttreatment NK activity of an individual who clearly responded to treatment with a biologic response modifier as evidenced by a sustained increase in NK activity.

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Fig. 7. A time series plot showing changes in NK activity during treatment of a cancer patient with a biologic response modifier (BRM). Arrows indicate days on which BRM was administered. Serial measurements of NK activity were performed in 4-hr "Cr-release assays. Cytotoxicity data are presented in natural logarithms.

In circumstances where it makes sense to conceptualize "response" on a continuum, it is meaningful to test whether the application of a treatment in a hypothetical population of patients would result in a non-0 average response. For example, it may be of interest to test the hypothesis that a specific treatment leads, on average, to an elevation of NK activity over pretreatment levels, as measured by the cytolytic assay. The second class of methods would be applicable in such situations. Generally, various forms of nonparametric repeated measures analyses are used to test such hypotheses. These are recommended in place of classical methods, because the classical methods are too sensitive to isolated extreme observations. As a simple example, an experimental situation my be considered in which a sustained elevation in NK activity over a given posttreatment period may be hypothesized. Then, a test for the detection of a mean increase in NK activity may be constructed by computing, for each patient, a post- versus pretreatment difference in median log LUs and applying the signed rank test to these differences. Similarly, if a protocol provides for multiple dosage levels, a test for differential dosage effects might be carried out by application of the KruskalWallis test to post- versus pretreatment differences. This would be appropriate if one did not wish to assume a monotone relationship between dosage and efficacy.

CONCLUSIONS We have attempted to show that the NK cell assay needs to be a component of a clinical immunodiagnostic and immunomonitoring laboratory. To be useful in diagnosis and in monitoring patients with various immunodeficiency disorders, the NK cell assay must be performed and analyzed reliably. Both the assay and the statistical analysis of the results will require attention and special expertise. The requirements for successful and reproducible performance of the NK cell assay in a clinical laboratory are considerable. It is reasonable, however, to expect that, with an appropriate quality control program such as the one outlined above, and with semiautomatic modifications of the assay, the laboratory performance can be reproducible and dependable. Likewise, statistical approaches that have been developed for handling cytotoxicity data form single measurements as well as serial monitoring facilitate interpretations of changes in NK activity over time, especially when therapeutic intervention is implemented. We have outlined one way of performing a statistical analysis of serial cytotoxicity measurements. Such an approach is particularly valuable in the context of clinical trials, where serial measurements of NK activity that may be influenced by therapy are being accumulated. We believe that the NK cell assay can be performed in a diagnostic or monitoring setting with a great deal of precision and accuracy, provided that a quality control program supported by meaningful data analysis is available to the laboratory performing the assay.

Criteria for a Reproducible NK Assay

ACKNOWLEDGMENTS Supported by NCI Core Grant # 1 P30 CA47904-01 and Biomedical Research Support Grant #BRSG-RR0545 1-26, University of Pittsburgh.

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49. Eby N, Grufferman S, Whiteside TL, Herbennan RB: Natural killer cell activity as a marker of immune dysfunction in the chronic fatigue syndrome (abst.) Narlmmun Cell Growrh Regul7:48, 1988. 50. Roder JC, Haliotis T, Klein M , Korec S , et al: A new immunodeficiency disorder in humans involving NK cells. Nature (London) 284553-555, 1980. 5 1. Si L and Whiteside TL: Tissue distribution of human NK cells studied with anti-Leu 7 monoclonal antibody. Jlmmunol 130:2149-2155, 1983. 52. Antonelli P, Steward W 11, Dupont B: Distribution of natural killer cell activity in peripheral blood, cord blood, thymus lymph nodes and spleen, and the effect of in vitro treatment with interferon preparations. Clin lmmunolIrnmunopatho1 19:168-172, 1981. 53. Kay NE: Natural killer cell. CRC Crir Rev Clin Lab Sci 22:343-359, 1986. 54. Gatti G, DelPonte D, Cavalla R, et al: Circadian changes in human natural killer cell activity. Prog Clin Bio Res 227:399-409, 1987. 55. Pross HF, Rubin P and Baines MG: The assessment of natural killer cell activity in cancer patients. In NK Cells and Other Natural Effector Cells, RB Herberman, ed. Academic Press, New York, 1982, pp 1175-1181. 56. Bryant JL, Day RS: Empirical B q e s Analysis for Systems of Mixed Models With Linked Autocorreluted Random Effects. Technical Reporr. Pittsburgh Cancer Institute, Pittsburgh, PA, 1989.