Immune monitoring of anti-thymocyte globulin (ATG) treatment in transplant patients

Clinical and Applied Immunology Reviews 1 (2001) 347–371

Immune monitoring of anti-thymocyte globulin (ATG) treatment in transplant patients B.K. Shentona,*, D. Manasb, J.H. Darkc, D. Talbotb, K.R. Clarkd, Alison Bella a

Transplantation Laboratory, Department of Surgery, Medical School The University, Newcastle Upon Tyne, NE3 1UB. UK. b Renal Transplant Unit, Freeman Hospital, Freeman Road, Newcastle Upon Tyne, UK c Cardiothoracic Transplant Unit, Freeman Hospital, Freeman Road, Newcastle Upon Tyne, UK d Department of Surgery, Dryburn Hospital, Durham, UK Received 15 April 2001; received in revised form 6 August, 2001; accepted 9 August, 2001

Abstract Over the past few years there has been an increasing array of monoclonal antibodies directed against immunocompetent lymphocytes available for use in the prevention and treatment of rejection in solid organ transplantation. Despite this there has been a continued and expanding use of polyclonal anti-T cell globulins in the management of cardiac, lung and renal transplants in many centres. As with all immunosuppressive drugs over and under-immunosuppression remains a major problem. A number of different methods have been described for monitoring anti-thymocyte globulin (ATG) therapy in transplant patients. These include: monitoring serum IgG levels, the E- rosette assay; flow cytometry and monoclonal antibodies. The measurement of serum IgG levels of the host animal (rabbit or horse) has been shown not to correlate with the presence of acute rejection episodes. The E-rosette assay has been found to be extremely time consuming and unreliable. With flow cytometric techniques, monoclonal antibodies directed against the T lymphocytes have been used to monitor ATG therapy and this has allowed the adjustment of the ATG administered to each individual patient. In patients receiving, renal, cardiac, lung or heart lung transplants we have developed flow cytometric assays to monitor ATG therapy on a daily basis with adjustment of the dosage of ATG administered according to this result. We have progressed from a dual platform assay where the T-cell percent from flow cytometry is combined with the lymphocyte count determined from a Haematology Cell Analy-

Abbreviations: ATG, anti-thymocyte globulin; ALG, anti-lymphocyte globulin; ALS, anti-lymphocyte serum; ATS, anti-thymocyte serum; CMV, cytomegalovirus; ECD, electrocoupled dye; HIV, human immunodeficiency virus; PERCP, peridin chlorophyll; RIT, rosette inhibition test; SRBC, sheep red blood cells. * Corresponding author. Tel.: 44-0191-222-8441; fax: 44-0191-222-8514. E-mail address: [email protected] (B.K. Shenton). 1529-1049/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S1 5 2 9 - 1 0 4 9 ( 0 1 ) 0 0 0 3 7 -X

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ser to a single platform flow cytometric assay using commercially available polyfluorescent beads of two different types. An increasing problem with transplant patients is that ATG once given may induce antibodies against the heterologous rabbit or horse IgG. With time, a number of renal, cardiac and lung transplant patients have lost their grafts and are retransplanted. A similar situation is seen in patients who are given regular ATG therapy for the treatment of aplastic anemia. We have developed a flow cytometric duplex assay for the detection of such antibodies. This has allowed us to screen the sera of transplant patients for the presence of such antibodies before therapy is given and to suggest for that patient the most appropriate ATG to be used. The cellular and humoral assays we have developed are rapid and reproducible and are now used as routine in the clinical management of patients who are receiving or about to receive ATG therapy in transplantation. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Transplantation; ATG; ALG; Immune Monitoring

1. Introduction 1.1. History of ATG therapy The first description of an antilymphoid preparation was by Metchnikoff in 1899 [1] and Chew and Lawrence [2] were the first to test antilymphocyte serum in vivo. Injection of rabbit anti-guinea pig serum into guinea pigs resulted in a reduction of circulating lymphocytes. However, inability to remove all lymphocytes was confirmed by Cruikshank in 1941 [3] using a rabbit anti-rat serum. The ability of such antiserum to block delayed hypersensitivity reactions was shown by Inderbitzen [4] and Waksman and associates [5]. It was not however until 1963 [6] and 1964 [7] that Woodruff and Anderson showed that administration of a rabbit anti-rat antilymphocyte serum produced a lymphopenia that was associated with prolonged skin graft survival. In many subsequent studies the ability of anti-lymphocyte serum (ALS) or anti-thymocyte serum (ATG) to prolong skin graft survival was confirmed (Nagaya & Sieker [8], Levey & Medawar [9], Anderson et al. [10], James & Medawar [11]). Encouraged by these results other groups studied renal allografts in dogs using ALS and ATS as adjunctive therapy to azathioprine and prednisolone or as the sole immunosuppressive agent. All of these studies showed beyond doubt that ALS and ATS were potent immunosuppressive agents (Mitchell et al. [12], Monaco et al. [13,14], Lawson et al. [15], Weil & Simmons [16]). In addition, it was found that the ALS was not only a potent immunosuppressive agent but that it was extremely well tolerated by the dogs, with no increase in infections (Monaco et al. [13]). In 1967, Monaco et al. [14] were the first to administer rabbit ALS to normal human volunteers. However, the results were not as good as they had observed in small mammals and dogs. A more potent ALS with reduced toxicity was prepared by purifying and separating the IgG fraction (Iwasaki et al. [17]). Following initial experience in canine renal transplants, Starzl’s group from Denver were the first to use horse ALG in human renal transplants (Starzl et al. [18]). Starzl et al. [18] had shown incomplete control of rejection with ALG and found that it was best used as an adjunctive agent with azathioprine allowing the effect of allowing the

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sparing of steroids. Following these studies many reports in the 1970s and 1980s showed the beneficial effects of ATG on graft survival in renal transplant patients (Davis et al. [19], Taylor [20], Kountz et al. [21], Launois et al. [22], Novick et al. [23]). In the 1990s, further clinical studies with ATG therapy showed the value of short vs. long treatment (Olausson [24]), the treatment of highly sensitized patients (Thibaudin et al. [25]), the favorable comparison of ATG with OKT3 treatment (Mariat et al. [26]) and the value of ATG therapy in patients showing delayed renal graft function (Lange et al. [27]). 1.2. Development of Monitoring of ATG therapy Many of the variable results obtained from the early clinical studies using ALG or ATG as an adjunctive therapy were due to the variety of preparations used and the lack of standardisation of products. There was an obvious need for an in vitro test that would confirm whether or not a particular preparation of ATG would be a potent immunosuppressive agent in vivo. Of the various tests proposed to assay ATG potency the following have been used the most frequently: lymphocyte agglutination, lymphocytotoxicity, lymphocyte opsonisation and skin allograft survival in lower primates, e.g. chimpanzees or rhesus monkeys and the rosette inhibition titre. Jeejeebhoy [28] and Greaves et al. [29] showed no correlation between the lymphocyte agglutination or the lymphocytotoxicity titres and the immunosuppressive effect of the ATG product. Greaves et al. [29] proposed that only the opsonisation titre, measuring the ability of ALS to bind to lymphocytes and cause attachment of lymphocytes to macrophage monolayers, correlated with the in vivo immunosuppressive effect. The ability of ALS to enhance phagocytosis of T lymphocytes by the liver following opsonization was measured by Martin and Miller [30] and was proposed as an in vitro assay of the immunosuppressive potency of ALS. During the early 1970s, a primate skin allograft survival assay was accepted as the gold standard for measuring the immunosuppressive potency of batches of ATG or ALG (Balner et al. [31], Cosimi et al. [32]). This assay was not only expensive but was difficult to standardise and failed to identify batches of less potent ATG. When batches of ATG, shown to be potent by the primate skin allograft assay, were used in human renal transplantation varying results were still obtained. Bach et al. [33] noted that human lymphocytes were able to form rosettes when mixed with sheep red blood cells (SRBC) and the addition of ATG to the lymphocytes prior to mixing with SRBC inhibited this reaction. Bach et al. [33] developed an assay measuring the ability of ATG to inhibit rosetting of lymphocytes mixed with SRBC and it was called the rosette inhibition test (RIT). A good correlation was shown between the RIT and the primate skin allograft assay for measurement of the immunosuppressive potency of ATG batches. However no correlation between the two tests was found by Kobayashi et al. in 1976 [34]. Currently, for the purposes of internal standardisation and control, in vitro tests of product quality are performed in house by the product manufacturers. The variable results obtained by different groups using the same ATG preparations illustrated that there was no standard way in which to administer the immunosuppressive agent. The majority of centres administer ATG to patients in an empirical fashion according to a fixed dose regime. Due to the idiosyncratic reaction of a transplant patient to ATG within a fixed dose regimen some patients may be underimmunosuppressed and some patients may

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be overimmunosuppressed. This in turn may lead to acute renal allograft rejection or increased incidence of opportunistic infections such as cytomegalovirus (CMV). In fact, following clinical experience with a particular ATG product the manufacturers reduced the recommended doseage by 50%. The problems of oversuppression and undersuppression with fixed dose administration of ATG, due to batch to batch variation and the idiosyncratic reaction of patients, have caused many groups to monitor the effect of ATG therapy and adjust the dosage of ATG accordingly. Three main methods of monitoring the in vivo effect of ATG by in vitro methods have been used. 1.2.1. Serum IgG levels ATG present within the serum may be assayed by measuring the level of the host animal IgG, e.g., rabbit or horse. In 1976, Bieber et al. [35] measured rabbit IgG levels in the serum of cardiac transplants who had received ATG. They showed that those patients with a slower rate of clearance had less rejection episodes and an improved one-year graft survival. Other studies, however, in which groups have looked at IgG levels and acute renal allograft rejection have found no correlation between the two (McAlack et al. [36], Hoitsma et al. [37]). In view of this no studies have been performed where IgG antibody to ATG has been monitored and the ATG dose adjusted based on the titre. 1.2.2. E-rosette assay While the rosette inhibition titre was used initially to assay the immunosuppressive potency of ATG in vitro, Bishop et al. [38] developed the E-rosette assay from this to measure the number of circulating lymphocytes during ATG therapy. To perform the E-rosette assay peripheral blood was taken from patients receiving ATG and the lymphocytes separated. Sensitized SRBC were incubated with the patients’ lymphocytes and then treated with 1% eosin. Rosettes formed between the lymphocytes, and the SRBC were counted using a haemocytometer. In 1976, Cosimi et al. [39] found that monitoring the circulating T cell levels in patients receiving ATG was the best means of defining the immunosuppressive potency and studying the variation in response to ATG amongst identically treated individuals. They concluded that ATG should be administered to reduce the level of circulating T cells to less than 10% of the patient’s pretransplant T lymphocyte count. This study was confirmed by Thomas et al. [40] who found that administration of ATG to keep the circulating T lymphocyte levels to less than 20% of the pretransplant value reduced the incidence of rejection. In 1979, Thomas et al. [41] suggested that the performance of the E-rosette assay allowed tailoring of ATG immunosuppression to the individual. They found that the administration of ATG to keep the total circulating T-cell count to less than 200 cells/mm3 reduced the incidence of acute renal allograft rejection and improved the one-year survival rate. However, other groups reported that administration of ATG according to a dose by rosette monitoring resulted in an increased incidence of acute renal allograft rejection (Geis et al. [42], Uittenbogaart et al. [43]). The reproducibility of the SRBC rosette assay was also questioned when the flow cytometric measurement of monoclonal antibody labeled T cells was introduced (Cosimi et al. [44], Thomas et al. [45]). Flow cytometric analysis of monoclonal antibody labeled lymphocytes proved to be more accurate, more sensitive, more reproducible and easier to perform (Ganghoff [46]) than the E-rosette assay. This difference was most noticeable when there was a low total lymphocyte count.

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1.2.3. Flow cytometry Flow cytometric analysis of the T cells in peripheral blood may be performed using many different monoclonal antibodies directed to different epitopes on the T-cell surface, e.g., antiCD3, antiCD2 and antiCD5. Some workers including us have studied the effect of ATG treatment on many other subsets of lymphocytes (Bourdage & Hamlin [47]). To count T lymphocytes in the peripheral blood the CD3 marker is most commonly used. Thomas et al. [48] reported an increased incidence of acute rejection episodes when at least three CD3 cell levels were greater than 100 cells/l in the week before the rejection episode occurred. In this study, T-cell counts were only performed three times per week and ATG was administered for 90 days using sequential rabbit and horse ATG. These findings were confirmed by Williams et al. in 1985 [49]. Delmonico et al. in 1984 [50] found that administration of ATG to keep CD3 cells to less than 10% of pretransplant values resulted in improved allograft function and reduced the incidence of rejection. In 1987, Thomas et al. [51] reported a prospective study in which dosage of ATG was adjusted to keep the total circulating T-cell count to less than 150 cells/mm3. This resulted in an increase in one-year patient and graft survival and a reduction in acute rejection episodes. Data from our center in 1992 and 1993, (Clark et al. [52,53]) showed that the administration of ATG to maintain an absolute T-cell count of less than 50 T cells /mm 3 as defined by CD3 monoclonal antibody staining provided a threshold count for dosing that allowed reversal of steroid rejection without the problem of overt infection. This represented a 90–95% depletion of the pretransplant T-cell population in the peripheral blood. Studies by Bourdage and Hamlin [47] and Olausson et al. [54] showed the effect of ATG on different cell subsets. We have studied 20 different commercially available ATG/ALG products and found that their lymphocytototoxic, lymphocyte binding and specificity of epitope blocking were extremely variable. Indeed some were directed mainly at B lymphocytes. Of the three T-cell markers available for use, we found that antiCD5 and antiCD2 were reduced to extremely low levels when compared to antiCD3 stained cells. Rosettes in most cases were zero when positive CD3 values of up to 60% of the lymphocyte pool were recorded. This suggested that monoclonal antibody staining was much more sensitive for T-cell enumeration than the use of the E-rosette assay, possibly due to steric hindrance by ATG bound to the cell surface preventing SRBC attachment while permitting penetration by the much smaller labeled monoclonal antibodies. It was also interesting that the blocking of binding for antiCD3, antiCD5 and antiCD2 sites on the lymphocyte cell surface by different ATG products varied with the product being tested. Indeed, we have found that the blocking potency of batches correlated with the biological effect of the product and suggest that pretesting of batches may prevent oversuppression when potency of product is increased and yet a fixed dose regimen is used. In a financially driven testing laboratory, the cost of the testing for ATG is important. This has been addressed by many workers (Abouna et al. [55 ], Gorrie et al. [56], Djamali et al. [57], [58–61]). In one study reported by Abouna et al. [55], a saving in drug costs of $2398 per patient by using T-cell monitoring was reported. It is the aim of the present paper to review both the current state of immunosuppressive therapy used in transplant patients and the various methods used for determining the efficacy of anti-T cell therapy given. We have discussed the development of flow cytometric methodology for the determination of T-cell numbers in ATG-suppressed patients culminating in the

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availability of a single platform assay that is rapid, accurate and allows treatment to be prescribed on an individual patient basis thus minimising the clinical problems of both undersuppression and overimmunosuppression of patients. 2. Materials and methods 2.1. Clinical background to immunosuppressive therapy used in renal transplantation In this study, the clinical course of 396 renal transplants performed between 1991 and 1997 on different immunosuppressive regimes was examined. All patients had received immunosuppression as a matter of routine and had provided informed consent for the blood monitoring tests to be performed as part of their clinical management. The patients could be divided into nine groups based upon the type of immunosuppression given (See Table 1). The first five groups did not receive ATG and comprised: Group 1. Cyclosporin monotherapy, (114 patients); Group 2. Cyclosporin and prednisone (54 patients); Group 3. Cyclosporin, prednisone and imuran (106 patients); Group 4. FK506 (Tacrolimus), prednisolone and imuran (16 patients); and Group 5 Anti-CD45 (22 patients). Results summarizing Table 1 Results for patients receiving different immunosuppressive regimens in Newcastle 1991–1997 Graft Failure

Number with no rejection

ATG Treatment

Infection Minor

Major

2 (1.7%) 1 (2%) 3 (2.8%) 1 (6.3%) 0

12 (10.5%) 3 (5.5%) 8 (7.5%) 2 (12.5%) 1 (4.5%)

18 (15.8%) 15 (27.8%) 42 (40%) 9 (56.3%) 8 (36.8%)

12 (10.5%) 9 (16.7%) 20 (18.9%) 1 (6.3%) 4 (18.2%)

11 (9.6%) 11 (20.4%) 15 (14.2%) 3 (18.8%) 2 (9.1%)

7 (6.1%) 2 (3.7%) 9 (8.5%) 4 (25%) 0

2 (20%) 1 (5.8%)

0

100%

17

2 (20%) 0

0

100%

6 (60%) 11 (64.7%)

3 (30%) 0

23

0

3 (13%)

1 (47.8%)

100%

1 (4.3%)

2 (8.7%)

34

4 (11.8%)

3 (8.8%)

15 (44%)

100%

8 (17.6%)

2 (5.9%)

Therapy

Number of patients

Non ATG Therapy Groups 1. CyA monotherapy

114

2. CyA/Predisolone 3. CyA/ Prednisolone / Azathioprine 4. FK506/Prednisolone/ Azathioprine 5. CD45 ATG Therapy Groups 6. ATG - 3mg/kg/day patients with SRR 7. ATG - 3mg/kg/day patients with SRRT cell monitored 8. ATG /Predisolone/ CYA. T cell monitored 9. ATG/Prednisolone/ CYA/ Azathioprine T cell monitored

54 106 16 22

10

Deaths

Groups 1–5 non-ATG induction groups; groups 6 and 7, ATG administered at 3mg/kg/day; groups 8 and 9, ATG administered at 2 mg/kg/day.

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the clinical course of the patients are shown in Table 1. The greatest graft failure was found in Groups 1 and 4 and Groups 3 and 4 also showed the highest degree of infection–43.8%. Patients showing the highest infection rate also showed the absence of acute rejection. The most potent immunosuppression in Group 4 was associated with a 25% incidence of major infections. Conversely Group 5 with the lowest infection rate showed 63.2% incidence of rejection. In summary, Groups 1–5 show that when any of these therapies are used failure to control rejection is found in between 6.3–18.2% of the patients studied. It is clear that with these therapies reduced rejection is accompanied by increased infections in some cases to exceptionally high levels. Groups 6–9 comprised 84 renal transplant patients who received ATG either for steroid-resistant rejection following treatment for Groups 1–5, were from a high risk group (second transplants, poor HLA match or a high panel reactive antibody status) or showed poor postoperative renal function. In Group 6, ATG was given to all patients showing steroid-resistant rejection, the dosage was stopped in the presence of thrombocytopenia and/or leucopenia. In Group 7, ATG was given only if the T-cell count was above 50 T cells /l. As can be seen in Group 7, no infections were found and graft failure was reduced from 20% in Group 6 to 5.8% in Group 7. In Groups 8 and 9, ATG was given as these patients were either of high risk (second or third transplants, high panel reactivity, poor recipient/donor match). The ATG was given prophylactically or to patients who had not responded to three courses of bolus steroids (steroid-resistant rejection). Both groups were monitored for T cells and as can be seen few showed major problems with infection and half were free of rejection. These results have made the monitoring of T cells in patients mandatory in our center, allowing therapy to be tailored to each individual patient, causing few major infections with low graft failure, and halving the cost of ATG used. 2.2. History of development of T Cell monitoring on ATG-treated patients Since 1989 with the development of monoclonal antibody reagents and flow cytometry we have used both single and dual platform enumeration of peripheral blood T cells on transplant patients. Recently, the enumeration of absolute levels of cells and their subsets has been shown to be of clinical value in human immunodeficiency virus (HIV)  individuals (CD4 T-lymphocyte enumeration during disease progression), in patients who are candidates for autotransplant (CD34 haemopoetic progenitor cells) and in the quality control of leukodepleted blood products (residual white blood cells). For the first 7 years of T-cell monitoring, we performed T% determinations on density gradient separated blood of transplant (renal, cardiac, lung and liver) patients. At this time, the use of the third (FL3) channel for immunophenotyping was not available due to the absence of commercially available directly conjugated monoclonal antibodies with peridin chlorophyll (PERCP) or electrocoupled dye (ECD). Dual monoclonal antibody staining was performed with two color Leucogate (Becton Dickinson, Oxford, UK, (BD); CD45 FITC and CD14 PE) using a dual platform system of analysis. In the dual platform method flow cytometry, immunophenotyping was used to provide the percentage of CD3 positive cells within the lymphocyte population and a Haematology analyser was used to provide both the white blood cell count and the lymphocyte % on the blood sample. From the lymphocyte count and the % T-cell value from flow cytometry the total absolute lymphocyte count was obtained.

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2.3. Dual color with leucogate on separated blood: Dual platform In this method, 5–10ml of peripheral whole blood collected in heparin was diluted 1:1 with Earles balanced salt solution (Sigma, Poole, UK) and layered (either under or over) by pipette over a sodium metriozoate density gradient (Lymphoprep) and centrifuged at 400 g for 20 min at 20 C. The resultant interfacial mononuclear cell layer was recovered by pipette and washed once at 600 G for 10 min in Isoton (Coulter Electronics, Crawley, UK). The cell pellet was then suspended in 50 l of isoton. Three tubes were set up for staining. 1. .10l cells  2l PE isotype control (BD)  2l FITC isotype control (BD) 2. .10l cells  2l CD14-PE (BD)  2l CD45-HLE-FITC (BD) 3. .10l cells  2l CD3-FITC (BD) The cells were incubated at 4 C for 20 min and then washed in a programmable cell washer. The pellets were then resuspended in 200 l of isoton and run through the flow cytometer. By back gating on CD45  CD14 cells (Mandy et al. [62]) a FSC/SSC lymphocyte region was identified (Loken et al. [63]). Using this scatter gate, the % of T cells within the defined lymphocyte gate (usually 99–100% CD45 CD14 cells) was calculated. In order to determine the absolute T-cell count, a lymphocyte count was performed independently on a Haematology Cell Counter. From this lymphocyte count, the T-cell count was determined. Since the development of three-color fluorescent reagents for immunophenotyping, it has been reported that the use of SSC and FSC gating is not now considered acceptable (Mercolino et al. [64] and Gelman & Wilkening [65]). Indeed laboratories not using CD45 gating and side scatter gating have been shown to have 2–3 times more chance of inaccurate results [65].

2.4. Three color with leucogate on whole blood: Dual platform One of the major problems with the two-color method initially used was the separation of the lymphocytes before staining. The separation technique may well have produced abnormal cell populations not truly reflective of the original T-cell composition. As described above, the use of a back-gating technique in analyzing the blood sample for testing is also not now encouraged [66]. We therefore developed a three-color whole blood method about 6 years ago. Whole blood was collected in EDTA and two tubes were prepared for each sample. 4. .5l CD45PERCP (BD)  5l CD14PE (BD)  5l IgG FITC isotype control (BD) 5. .5l CD45PERCP (BD)  5l CD14PE (BD)  5l CD3FITC (DAKO) In addition the commercially available TRITEST reagent comprising 20 l of a CD45PERCP/ CD14CD4/CD3FITC mixture was used in Tube 3. Tube 1 was used as the TRITEST control. To each of the two tubes, 100 l of whole blood was added (either 1 and 2 or 1 and 3) and vortexed at low speed for 3 s. The tubes were incubated for 15 min at room temperature in the dark. In addition, 0.5 ml of 1x FACSlyse (Becton Dickinson, Oxford,UK) solution was added to each tube. Each tube was immediately vortexed gently. The tubes were incubated for 10 min in the dark, and then run on the flow cytometer. No wash methods have been used for over 20 years [67] and a stain no wash procedure is used routinely in the FACS count

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Fig. 1. The figure illustrates the three-color leucogating for dual platform analysis of CD3 %. 1A shows the live side scatter (SSC) / CD45PERCP gate. 1B identifies region R1, i.e., only lymphocytes within the live gate and no monocyte (CD14PE) stain. 1C shows the blood of three patients stained with CD3FITC.

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[68]. Specimen plots illustrating the analysis procedure are shown in Fig. 1. To maximise data acquisition, data for 1–5,000 lymphocytes were collected using a live gate (CD45PERCP/ SSC), (Fig. 1A). A region was defined on a CD45PERCP/CD14PE dot plot and any contaminating monocytes were excluded (Fig. 1B). A CD3FITC histogram was constructed with a 3% marker being set on Tube 1 (the isotype control for CD3). Tube 2 was the analyzed with same gating and histogram marker. Data shown in Fig. 1C show a heart transplant patient (3 days post ATG) with 34.72% CD3lymphocytes, a lung transplant patient (1 day post transplant) with 47.5% CD3 lymphocytes and a renal patient (7 days post ATG) with 12.7% CD3 lymphocytes. The inaccuracy of haematology analysers as the basis for determining the lymphocyte count has been reported as a problem with the dual platform system [69–71] and single stage platforms using only flow cytometric methods have been developed. 2.5. Absolute T-cell counting on ATG blood samples: Single platform In order to obtain accurate absolute T-cell counts on whole blood specimens, two different procedures were used. This method has become known as the single platform analysis and directly provides the absolute number of T cells by counting the identified cells in either a precise known cell volume or by reference to a known number of calibrated fluorescent particles used as an internal ‘spike’. The addition of beads to the samples is used and reverse pipetting is essential for accurate bead pipetting [69–71]. Such methods do not require the use of a Haematology Cell Analyser. 2.5.1. Flowcount beads The first method used was the three-color leucogate described above and in this 100 l of precounted Flowcount beads (Coulter) were added (997 beads/l) after staining and no wash lysis had been performed. The beads are fluorescent 10 Um microsphere standards that are obtained in suspension with a defined concentration (e.g., 1000/l [72–74]). The beads can be used in a lyse no wash procedure and are quite large and so may be gated on FSC/SSC or fluorescence 3 (Fig. 2). Polygonal gates are needed to define the bead and cell populations ([70], [73]; see Fig. 3A). Care in storage is important with special attention to resuspenson, aliquoting and clumping. The beads float naturally and they should not be used after 30 days to avoid clumping. After vortexing, the mixtures were run on the flow cytometer. After initial problems with bead intensity, clear populations were found, which were more clearly identified by using SSC log amplification (Fig. 2). The lymphocytes and beads were identified and gated on a CD45PERCP/SSC dot plot (Fig. 3A) and exclusion of monocytes was confirmed by checking the lymphocyte gate with a CD45PERCP/CD14PE dot plot with quadrant analysis (Fig. 3B). The gated lymphocyte population was then displayed as a FL1–CD3 FITC histogram and a marker was set at a 3% level with the isotype control (Tube 1). CD3 cells were then identified from the histogram with Tube 2 (Fig. 3C). To calculate the T-cell count the following calculation was used: Cell number ----------------------------- × 997 = Number of T cells ⁄ µl blood Bead count

(1)

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Fig. 2. The three bead populations used for single platform flow cytometric absolute count determinations are shown above.

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Fig. 3. The use of Flowcount and Trucount beads is shown above. 3A shows the CD54/SSC dot plots where R1 identifies the lymphocytes, R2 identifies the Trucount beads and R3 identifies the Flowcount beads. 3B shows the low monocyte contamination of the R1 population and 3C shows the CD3 (FITC) histograms.

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Alternatively other software may be used to determine the absolute T-cell count directly as with FCS Express (DeNovo Software, Ontario, Canada) (Fig. 4). 2.5.2. Trucount beads In this method, beads are already in the test tubes as a lyophilised pellet of 4.2 m fluorescent labeled beads. The beads are retained at the bottom of a 12  75 mm polystirene tube by a small metal grid ([75–77]. The beads are too small to be recognized in the scatter channels and may be recorded by using their inherent fluorescence properties (See Fig. 2). The pellet of beads is retained in the bottom of the tube by a stainless steel retainer. The number of beads is recorded. Two test TRITEST reagents were used (BD). 6. . CD45PERCP, CD4PE and CD3FITC. This reagent is already prepared as a pretitred monoclonal antibody mix. 7. . CD45PERCP, CD14PE and CD3FITC (BD). This reagent that comprises the Leucogate and CD3 FITC was supplied pretitered by Becton Dickinson (San Jose, CA, USA).

Fig. 4. This illustrates the direct calculation of CD3 count from flow cytometric analysis.

Fig. 5. The use of the three bead populations is shown above. R1 and R5  lymphocytes. R2  Trucount beads, R3  Flowcount beads and R4  Accucount beads.

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Table 2 Results of linear regression correlation between the assays used Groups Correlated % of T cells 2 Colour Separated / 3 Colour Leucogate Flowcount Leucogate / Trucount Tritest Flowcount Leucogate / Trucount Leucogate Trucount Tritest / Trucount Leucogate Absolute number of T cells Haematology (DP) v Flowcount Leucogate (SP) Haematology (DP) v Trucount Leucogate (SP) Haematology (DP) v Truecount Tritest (SP) Flowcount Leucogate (SP) v Trucount Leucogate (SP) Trucount Tritest (SP) / Trucount Leucogate (SP)

P value

r value

P 107 P 105 P 104 P 105

r  0.996 r  0.977 r  0.991 r  0.983

P  1012 P  1021 P  1015 P  1012 P  1029

r  0.971 r  0.869 r  0.809 r  0.921 r  0.950

(DP) dual platform — % from flow cytometry x haematology absolute lymphocyte count (SP) single platform — absolute lymphocyte count from flow cytometry only

To the Trucount tube, 20 l of Tritest reagent was added directly. By reverse pipetting, 50 l of blood was added. The mixture was vortexed and incubated at room temperature for 15 min. In addition, 450 l of FACSlyse was added and the mixture was incubated at room temperature in the dark for 15 min. The samples were then run on the FACScan. Specimen plots are shown in Figs. 3 and 4. A third commercially available population of fluorescently labeled beads have been recently become available—ACCUCOUNT(Spherotech, Libertyville, USA). They appear very similar in their size to Flowcount beads and they may be gated on SSC and FL3. Plots comparing these beads to Flowcount and Trucount are shown in Fig. 2 and their use with cells is shown in Fig. 5. 3. Comparisons between assays 3.1. Results Linear regression analyses were performed between the different tests comparing the % CD3  values on blood samples from a total of 65 patients (See Table 2). As can be seen excellent correlation was seen between the various assays used. This is to be expected particuTable 3 Comparison between absolute T-cell counts Groups

Agreement

Differences

1. Comparison between Single Platform Assays Flowcount Leucogate v Trucount Tritest Flowcount Leucogate v Trucount Leucogate Trucount Tritest V Trucount Leucogate

51/54 (94.4%) 28/30 (93.3%) 39/40 (97.5%)

22 2 Flowcount  1 CD4 

2. Comparison with Haematology counts Haematology v Flowcount Leucogate Haematology v Trucount Tritest Haematology v Trucount Leucogate

61/65 (94%) 59/63 (94%) 36/38 (95%)

4  Haematology 22 2  Haematology

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larly for the Leucogate pairs. No differences were seen for either the separated (two color) or unseparated (three color) analyses. The Leucogate and Tritest gave a high correlation although in the Tritest reagent the CD14 stain was omitted. The degree of correlation found between the absolute counts determined by the various methods (see Table 2) with extremely high correlation between the Trucount Tritest and the Trucount Leucogate samples; the latter perhaps due to the products being provided by the same manufacturer. A summary of the agreement between the different assays in terms of a 50 T cell/l cut off for treatment is shown in Table 3. Between the tests, 94.4–97.5% agreement was seen. The Flowcount Leucogate method showed two samples with higher values than that for the Trucount Tritest and Trucount Leucogate, which may have been due to pipetting errors. Excellent agreement was found between the Trucount tests. In comparison with the haematology counts, agreement was high although there was a trend for the haematology count to be higher than that found for either the Trucount or Flowcount tests. This may be as already discussed errors in the Haematology Counters where low numbers are often expressed to one decimal place. 3.2. Comparison between ATG dosage based on peripheral blood T-cell count or absolute lymphocyte count In our studies based on a threshold T-cell count of 50 T cells/l of peripheral blood on average 2.3 doses of 2 mg/kg/day of ATG were given to renal transplant patients over the 10day course of therapy. It has been suggested that the peripheral blood lymphocyte count could be used for monitoring the administration of ATG A comparison was made between the use of a threshold of treatment of either 100 lymphocytes/l or 50 T cells/l. The results of 76 samples were studied. If the absolute lymphocyte count was used then 52 of 76 doses of ATG would have not been given. In the ATGtreated group using an absolute lymphocyte count of 100/l, six doses would not have been given. Only 24 of the 76 doses would have been subscribed on agreement of the test systems. If an absolute level of 200 lymphocytes/l was used for ATG prescription of the 24 doses given, 14 would have been omitted in the nontreated group and ATG would been given 56 of the 76 times that it was not used. This data does not support the hypothesis that absolute lymphocyte count mirrors absolute T-cell count. The main source of error in trying to correlate the two absolute counts is that the absolute lymphocyte count makes no allowance for the proportion of B cells in the blood. Our clinical data confirm that by using a 50 T cell/l of blood monitoring index greater efficacy of prescription of ATG is possible. This has the advantages of reducing cost, reducing infection and yet producing a low graft loss in the treated and monitored patients. It is however important that monitoring be performed daily during the administration of ATG. 4. Detection of anti-ATG antibodies 4.1. Introduction While the graft function in patients who have received ATG prophylactically or as therapy for rejection is good, a proportion of patients reject their transplants. When such patients

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Fig. 6. In this figure, R1 identifies the ATG-coated beads, and R2 identifies the ALG-coated beads.

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present for a second or a third transplant the question of whether ATG therapy can be used must be addressed [78]. Such antibodies may also affect ATG therapy in patients with aplastic anemia [79], [80]. It is therefore important to determine whether such patients have developed antibodies against the foreign ATG given. 4.2. Methods In the first part of this study, we have compared the ELISA assay against a bead method we have developed. The principal of the method involves the coating of avidin coated polystirene particles with biotinylated ATG or ALG. Three different bead sizes were used: 5 m, 10 m and 30 m (Spherotech, Libertyville,USA). The antibody preparation was dialysed against 0.1M NaHCO3 and the antibody concentration was adjusted to 2 mg/ml with 0.1 NaHCO3. As biotin spacer, n-Biotinyl-E-aminocaproic acid–hydroxy succinamide ester (BNHS-spacer), was made up of 0.05M in dimethylsulphoxide (DMSO) (0.05M BNHSspacer with 22,72 mg/ml DMSO. One mg of BNHS was added dropwise immediately to 4 mg of antibody. This was stirred for 2 h at room temperature. This was dialysed with 0.01 M PBS at a pH of 7.2 at 4 C as preservative 15 mM NaN3 was added. To couple the biotinylated anti-lymphocyte preparation to the beads, 2 ml of biotinylated protien (100 g/ml of protein) in 0.1M sodium phosphate buffer (pH 7.4) was added to 0.2 ml of avidin coated polystirene particles (5 g/100 ml). The mixture was incubated for 1 h at room temperature. The mixture was centrifuged at 3000G for 10 min. The supernatant was removed and the pellet was resuspended in 4 ml of 0.1 M phosphate buffered saline (PBS). Two further washes of the beads were performed with PBS and the pellet was finally resuspended in 4 ml to obtain a 0.25% particle suspension. To differentiate between the three bead preparations, three different bead sizes were used: 5 M for ALG, 10 M for ATG and 30 m for OKT3. Although outside the scope of the present review we have observed rises in antibodies to OKT3 in OKT3-treated transplant patients, indicating possible escape from therapy.

Table 4 Methodology for ELISA and flow Beads The ELISA methoda

The ATG bead methodb

1. Coating : Overnight at 4 C. 1/2500 ATG diluted in PBS (2ul/ml) 100ul/well. Three washing steps with PBS Tween. 2. Saturation — 90 min at 37 C. 3. Add serum at 1/500 dilution in PBS/ Tween/BSA—90 min. 4. Add Goat anti-human IgG labelled with HRP—90 min at 37°C. 5. Develop with substrate 15 min, stop with sulphuric acid. 6. Read at OD 405 nm

1. 20 ul serum added to 20 ul of ATG coated beads, mixed gently. Room temperature 30 min.

a b

Time for assay: 17 hours Time for assay: 1 hour

2. Wash x 2 with PBS 15 min. 3. Add 50 ul of 1/625 monoclonal antihuman IgG. Incubate on ice for 15 min. 4. Wash x 2 with PBS — 15 min. 5. Make up to 250 Ul and run on the flow cytometer

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Table 5 Comparison of the ELISA test and the Flow Bead test End point titration ELISA 1. 2. 3. 4. 5. 6. 7.

-8–10

10 10-8–10 10-8–10 10-5–6 10-4 –5 10-2 –3 10-4 –5

FACS bead 14

10 1014 1014 107 107 106 105

Improvement of bead 104 104 104 10 102 103 10

In performing the test, 1/40 dilution of the beads was made in PBS. This was centrifuged for 5 min at 200 g and the supernatant was discarded. The beads were reconstituted with PBS at a 1/40 dilution. As controls for the test, six negative AB sera were used at a 1/10 dilution. Positive sera from ATG-treated patients were used as positive controls every time the test was run. All test sera were diluted 1/10 before use. Twenty l of serum (test or control) and an antiIgM and antiIgG control were used. The sera and beads were incubated at room temperature for 30 min. The beads were then washed in a programmed cell washer. Fifty l of 1/ 625 antihuman IgG (Dako F0056-FITC conjugated) or 50 l of 1/625 antihuman IgM (DAKO FOO58-FITC conjugated) were added. The tests were mixed, incubated in an ice/ water bath in the fridge for 30 min. The beads were washed in a programmed cell washer. To prevent nonspecific binding, all washes were performed in PBS containing 1% FCS. Specimen data are shown in Fig. 6. Gates R1 and R2 were used to identify the beads of interest and fluorescent 1 (FITC) histograms were generated. The median fluorescence channel of each test and control were calculated. A mean plus 2 S.D.s was calculated for the six AB control tests and any test value above this was reported as positive. 4.3. Results A summary of the bead and a comparable ELISA assay is shown in Table 4. As can be seen the flow cytometric assay can be completed in 1 h whereas the ELISA requires 17 h for completion. Determination of negative cut off is extremely difficult by the ELISA method where a system of scoring is used. There are also considerable problems with nonspecific adhesion of test sera to the ELISA plates. We have compared the ELISA assay with the Flow Bead test on 31 ATG treated patients who had received ATG between 2 and 2.5 years after the therapy had stopped. Results are shown in Table 5. Twenty-three samples were negative by both methods and seven were positive by both methods. As can be seen the bead method produced from 10–10000 increased sensitivity over the ELISA method. Figure 7 illustrates the results of examining the antibody levels in a renal patient who received a second transplant having been given ATG for rejection of the first transplant. As can be seen despite ATG (raised in the rabbit) therapy, the T-cell count rose dramatically on Day 8 of therapy. This was accompanied by a rise in antibodies to ATG, from a pretransplant level of 1/78,000 to a level of 1/400,000. As there were minimal detectable antibodies to

Fig. 7. The data shows lymphocyte count, therapy and antibody titres on an ATG-treated renal transplant patient.

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ALG (raised in the horse, 1/5) the patient was given ALG. There was an immediate fall in the T-cell count and little increase in titre of antibodies to ALG (1/125). This was accompanied by an improved graft function. For any renal or heart/lung transplant patient who has received ATG or ALG on a previous occasion and requires further ATG therapy we now monitor the antibody titre before administration of the globulin. 5. Summary The results of many studies including our own have shown that the dose administered and efficacy of potent immunosuppressive polyclonal anti-T cell reagents like ATG and ALG must be regulated by CD3 lymphocyte monitoring on patients receiving the therapy on a daily basis. In our experience with lymphocyte monitoring using a threshold of 50 T cells/l in patients, the dose given may be reduced by 20–70% if the incidence of thrombocytopenia, lymphopenia or neutropenia are used as the only indicators of dose prescription. Over the last 12 years, we have used a number of flow cytometric methods and progressed from a dual to a single platform of analysis. A clear description of the precautions of using all bead assays in flow cytometry particularly for absolute counting is laid down by Brando et al. [80]. We have found the single platform analysis for absolute T-cell counting to be rapid, reproducible and consistent for the enumeration of T-cell number on patients requiring T-cell therapy. This has induced a cost saving of 60% on the costs of drugs. In our experience use of the Trucount or Flowcount systems provide equally good results although care and precision are needed with either of these products. In our transplant center, monitoring of T-cell number is used on all patients receiving ATG. These patients comprise all renal transplants who are high risk (previously transplanted, high panel reactive antibodies and poor donor recipient match) or have shown steroid resistant rejection. A 10-day course of ATG is normally given. All lung transplants are given a 4-day course of ALG to spare the use of steroids that impairs bronchial healing. Any heart transplant patients who have possible renal insufficiency are given ATG for 4–5 days to spare the possible nephrotoxic effect of cyclosporin A. In addition to using T-cell monitoring on post transplant ATG-treated patients, we also determine the level of ATG/ALG antibodies using a duplex assay. This determines the correct therapy for patients who have previously received ATG. By the use of these two tests as routine we are able to tailor therapy to each individual patient.

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