Noninvasive monitoring of peak filling rate with acoustic quantification echocardiography accurately detects acute cardiac allograft rejection.

BACKGROUND: Acute cardiac allograft rejection is associated with early diastolic dysfunction. The development of chronic rejection is dependent on the frequency and severity of acute rejection episodes. Therefore, early diagnosis and therapy influence long-term survival significantly. For the first time, acoustic quantification, a new echocardiographic technology for on-line measurement of cardiac volumes and their changes, facilitates quantitative assessment of systolic and diastolic function noninvasively. METHODS: Since May 1996, all consecutive patients after cardiac transplantation (n = 94) underwent 475 endomyocardial biopsies and the same number of echocardiographic studies within 6 hours after biopsy before the histological results were available. RESULTS: Nineteen patients showed 23 episodes of acute rejection (ISHLT > or = 2). There was a significant decrease in left ventricular peak filling rate [PFR: end-diastolic volume (EDV)/ second) as a parameter of diastolic function during rejection (2.9 +/- 0.4, n = 23) as compared to PFR measured under nonrejection status (4.5 +/- 0.8; n = 452; p < 0.0001). Most importantly we found that in these 19 patients showing rejection, the PFR was normal in the last examination before rejection, but was significantly reduced during rejection (2.9 +/- 0.4 vs 4.5 +/- 0.7; n = 23, p < 0.0001). After successful rejection therapy, PFR again normalized in all patients, with the exception of 1 patient with steroid-refractory humoral rejection. We calculated sensitivity and specificity for several cutpoints for the event "first rejection" in 15 patients and plotted them in a receiver operating characteristic curve, showing that a PFR > or = 4.0 EDV/second is never associated with treatable rejection. A decrease of PFR of more than 18% from its prevalue of the last biopsy with no rejection increases the accuracy for the diagnosis of rejection significantly. CONCLUSIONS: We conclude that diastolic dysfunction during acute cardiac allograft rejection can be accurately detected by noninvasive measurement of peak filling rate with acoustic quantification echocardiography. Monitoring of this parameter provides reliable discrimination between treatable and nontreatable rejection.     

Urinary polyamines as markers of cardiac allograft rejection. A clinical evaluation

Histologic evaluation of endomyocardial biopsy specimens is the current method of monitoring rejection after cardiac transplantation. Unfortunately, this technique gives a discontinuous evaluation of the recipient immunologic status. A noninvasive marker of immunologic activation and of allograft rejection that would permit a more continuous monitoring than the biopsy technique would be clinically useful. Urinary polyamine excretion reflects cellular proliferation or degeneration and, as a marker of cellular metabolic activity, may also reflect lymphocyte proliferation and organ rejection. From July 1985 to December 1986, urinary polyamines were studied in 18 patients during hospitalization for heart and heart-lung transplantation. Endomyocardial biopsy was performed twice a week and histologic rejection was characterized by standard criteria. Urinary specimens were collected daily and analyzed for polyamines by high-pressure liquid chromatography. Concentrations of acetylputrescine and total urinary polyamines were significantly higher before the 20 rejection episodes than before the 80 biopsies yielding negative results. So that their clinical usefulness could be evaluated, an elevation of polyamines and a daily level variability of 28% or more was chosen to indicate increased metabolic cellular activity and to predict rejection in the next 8 days. On the basis of these definitions, the sensitivity of polyamine assays to predict rejection was 85%, the specificity 88%, and the positive predictive value 79%. Therefore, serial measurements of urinary polyamines may provide daily information on the recipient's immunologic status after cardiac transplantation.     

Quantification of donor-derived DNA in serum: a new approach of acute rejection diagnosis in a rat kidney transplantation model

Clinical and laboratory findings of acute rejection (AR) are often late and misleading. Core needle biopsy, the most reliable diagnostic method, is usually performed late in the course of AR and is associated with several complications. Therefore noninvasive approaches to monitor the immune system for detection of early AR is one of the major aims in transplant medicine. In a fully MHC-mismatched renal allograft model in the rat, we quantified donor-derived DNA (ddDNA) in the recipient serum using real-time RT-PCR as an alternative screening procedure for the early diagnosis of acute rejection. We also investigated the influence of different immunosuppressive protocols on the levels of ddDNA. Our results show that donor-derived DNA is present in the serum of kidney allograft recipients prior to acute rejection. Animals that received a syngeneic graft and animals that received a mismatched allograft but were treated with immunosuppressive drugs did not show significant elevations of ddDNA. When steroid therapy failed to avoid acute rejection, the animals showed a delayed peak of ddDNA. In summary, the detection of ddDNA in recipient serum offers a noninvasive diagnostic approach to uncover ongoing rejection processes in the graft.     

Soluble CD30 correlates with clinical but not subclinical renal allograft rejection

Soluble CD30 (sCD30) has been proposed as a promising noninvasive biomarker for clinical renal allograft rejection, but its diagnostic characteristics regarding detection of subclinical rejection have not been assessed. We investigated sCD30 in 146 consecutive kidney allograft recipients under tacrolimus–mycophenolate‐based immunosuppression having 250 surveillance biopsies at 3 and 6 months as well as 52 indication biopsies within the first year post‐transplant. Allograft histology results were classified as (i) acute Banff score zero or interstitial infiltrates only, (ii) tubulitis t1, (iii) tubulitis t2‐3 and (iv) isolated vascular compartment inflammation. sCD30 correlated well with the extent of clinical (P < 0.0001), but not subclinical tubulointerstitial rejection (P = 0.06). To determine diagnostic characteristics of sCD30, histological groups were assigned to two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (tubulitis t1‐3 and isolated vascular compartment inflammation). For clinical allograft inflammation, AUC was 0.87 (sensitivity 89%, specificity 79%; P = 0.0006); however, for subclinical inflammation, AUC was only 0.59 (sensitivity 50%, specificity 69%; P = 0.47). In conclusion, sCD30 correlated with clinical, but not subclinical renal allograft rejection limiting its clinical utility as a noninvasive rejection screening biomarker in patients with stable allograft function receiving tacrolimus–mycophenolate‐based immunosuppression.     

彩色多普勒超声诊断早期移植肾急性排斥

目的探讨彩色多普勒超声图像数据分析在诊断早期移植肾急性排斥中的作用及临床价值.方法总结203例肾移植术后6～30d内行彩色多普勒超声检查,结合临床症状、生化检查并参照病理诊断,对二维灰阶图像、彩色血流图、血流频谱及阻力指数(RI)、搏动指数(PI)、收缩期与舒张期血流速度比(S/D)等参数进行回顾性分析.结果以彩色多普勒RI>0.78、PI>1.82、S/D>4.1为标准,结合彩色血流图及血流频谱形态,早期移植肾急性排斥正确诊断率可达85.7%.结论应用彩色多普勒超声诊断移植肾早期急性排斥并监测其功能恢复,具有快速、准确、无创等优点,可提早发现病情变化并指导治疗,提高移植肾存活率.     

Evaluation of posttransplantation soluble CD30 for diagnosis of acute renal allograft rejection

BACKGROUND: Posttransplantation measurement of soluble CD30 (sCD30) may be useful for identifying kidney graft recipients at risk of impending graft rejection in the early posttransplantation period. METHODS: We measured plasma sCD30 levels and evaluated the levels in relation to the diagnosis of rejection. RESULTS: Receiver operating characteristic curves demonstrated that on posttransplantation days 3 to 5, sCD30 allowed a differentiation of recipients who subsequently developed acute allograft rejection (n=25) from recipients with an uncomplicated course (n=20, P<0.0001) (area under the receiver operating characteristic curve 0.96, specificity 100%, sensitivity 88%) and recipients with acute tubular necrosis in the absence of rejection (n=11, P=0.001) (area under the receiver operating characteristic curve 0.85, specificity 91%, sensitivity 72%). CONCLUSIONS: sCD30 measured on posttransplantation days 3 to 5 offers a noninvasive means for differentiating patients with impending acute allograft rejection from patients with an uncomplicated course or with acute tubular necrosis.     

Flow cytometric evaluation of cytotoxic peripheral blood lymphocytes in acute renal graft rejection.

The presence of the S6F1+ epitope on the surface of CD8+ lymphocytes is believed to be uniquely representative of cytotoxic subpopulations. A preliminary study was conducted to evaluate the CD8+ S6F1+ peripheral lymphocytes by flow cytometry in patients undergoing renal allograft biopsy for allograft dysfunction. Lymphocytes, obtained at the time of biopsy, were analyzed by flow cytometry with CD8-FITC/S6F1-RD1 as the test monoclonal antibody and MsIgG-RD1/MsIgG-FITC as internal control. A 100% increase in S6F1+ cells over internal control was considered to be positive result. The results were correlated with the histopathologic findings in 14 instances of allograft dysfunction occurring 26.5 +/- 11.6 days posttransplantation. The histopathologic diagnosis was acute cellular rejection in eight cases, acute tubular necrosis in four, and cyclosporine nephrotoxicity in two. Flow cytometric detection of an increase in S6F1+ cells yielded a sensitivity of 87.5% and a specificity of 83.3% for the diagnosis of acute rejection. It would appear that the use of a monoclonal antibody to detect increases in the number of CD8+ S6F1+ peripheral lymphocytes is a valuable test for the detection of acute allograft rejection in the initial period after transplantation.     

Feasibility of diagnosing renal allograft dysfunction by oligonucleotide array: Gene expression profile correlates with histopathology

BackgroundEffective non-invasive monitoring method to tell histopathology is a big challenge in renal transplantation.MethodsWe used 70-mer long oligonucleotide array with 449 immune related genes to determine gene expression profiles of peripheral blood mononuclear cells (PBMCs) under different immune status including stable renal function (TX), acute tubular necrosis (ATN), biopsy conformed acute rejection (AR), clinical rejection with pathology of borderline changes (BL), clinical rejection without biopsy proven/presumed rejection (PR) and renal dysfunction without rejection (NR).ResultsDistinct molecular expression signatures in each group were found to correlate with histopathology. And we concluded that B cell chemokine CXCL13 and mast cell may play a role in renal allograft rejection through significant difference analysis and functional pathway analysis.ConclusionsIt provides a potential non-invasive method for monitoring renal allograft function and immune status of renal transplant recipients.     

Immunosuppression, diagnosis, and treatment of cardiac allograft rejection

The success of cardiac transplantation has been largely attributable to the development of effective immunosuppressive regimens. The calcineurin inhibitors were pivotal in reducing the frequency of acute rejection and improving early survival. Newer agents, including mycophenolate mofetil and proliferation signal inhibitors, show some promise in further reducing episodes of acute allograft rejection and also having a significant impact on reducing transplant vasculopathy. The role of cytolytic induction therapy remains unclear, although its use may be most appropriate in the high risk presensitized transplant recipient. While the endomyocardial biopsy remains the gold standard for the diagnosis of acute allograft rejection, its invasive nature makes the test suboptimal. The presence of biopsy 'negative' rejection has also led to an appreciation for the role of antibody-mediated rejection in cardiac allograft dysfunction. Effective noninvasive evaluation of allograft rejection remains the ultimate challenge. While imaging technologies have some utility because of their ease of use, more sophisticated techniques such as gene-expession analysis from peripheral blood may show the greatest promise. Management of established acute allograft rejection has significantly improved with our understanding of the immune mechanisms involved. Therapies may be targeted specifically at cellular or humoral components of the immune system.     

Noninvasive immune monitoring assessed by flow cytometry and real time RT-PCR in urine of renal transplantation recipients

BACKGROUND: Monitoring recipient's alloreactivity has shown to be critical for limiting overimmunosuppression besides allowing preemptive treatment of acute rejection (AR). METHODS: Flow cytometry and real time RT-PCR were performed in urine of kidney transplant recipients with AR (n = 13) and compared with pyelonephritis (n = 10), chronic allograft nephropathy (n = 13), acute tubular necrosis (n = 13) and stable graft function (n = 11). Expression of CD3, CD4, CD8, HLA-DR, Fas-L, ICAM-1 and CD25 were assessed using flow cytometry. mRNA of perforin, granzyme B and Fas-L were quantified by real time RT-PCR. RESULTS: Frequencies of CD3+, HLA-DR+, Fas-L+, ICAM-1+ and CD25+ cells were significantly higher in AR group (p < 0.05). ROC curves showed sensitivity from 70% to 91% and specificity from 30% to 100%, whereas the highest sensitivity and specificity was 91% and 100% respectively, for Fas-L+ cells. Levels of mRNA of perforin, granzyme B and Fas-L were significantly augmented in AR, while the sensitivity and specificity ranged from 85% to 88% and from 55% to 100%, respectively. CONCLUSIONS: Analyses of immune activation markers by flow cytometry and real time RT-PCR are equally useful for noninvasive monitoring kidney allografts.     

The influence of primary non-function on the accuracy of ultrasound measurements in the diagnosis of renal allograft rejection

Daily ultrasonographic measurements of transplant cross-sectional area were used to quantify allograft swelling as a diagnostic test for acute rejection in a series of 120 renal transplants. Initial graft function (IF) occurred in 86 patients (72%) and primary non-function (PNF) occurred in the remaining 34 (28%). An increase in allograft cross-sectional area greater than or equal to 10% was defined as a positive ultrasound scan suggesting an acute rejection episode and was investigated by needle core biopsy. During periods of PNF, allografts with consistently negative ultrasound scans were submitted to needle core biopsy on a weekly basis. The diagnosis of rejection was based exclusively on the histological findings. In the IF group, agreement between ultrasound and histological diagnosis was good (k=0.63, sensitivity 81%, specificity 83%, positive predictive value 76%, negative predictive value 86% and overall accuracy 82%). In the PNF group, agreement between ultrasound and histology was only fair (k=0.46, sensitivity 77%, specificity 70%, positive predictive value 69%, negative predictive value 78% and overall accuracy 73%). It it concluded that a degree of allograft swelling is sometimes associated with acute tubular necrosis, and this makes ultrasound measurements of transplant size a less useful technique of monitoring kidneys with PNF.     

Urine Metabolite Profiles Predictive of Human Kidney Allograft Status

Noninvasive diagnosis and prognostication of acute cellular rejection in the kidney allograft may help realize the full benefits of kidney transplantation. To investigate whether urine metabolites predict kidney allograft status, we determined levels of 749 metabolites in 1516 urine samples from 241 kidney graft recipients enrolled in the prospective multicenter Clinical Trials in Organ Transplantation-04 study. A metabolite signature of the ratio of 3-sialyllactose to xanthosine in biopsy specimen-matched urine supernatants best discriminated acute cellular rejection biopsy specimens from specimens without rejection. For clinical application, we developed a high-throughput mass spectrometry-based assay that enabled absolute and rapid quantification of the 3-sialyllactose-to-xanthosine ratio in urine samples. A composite signature of ratios of 3-sialyllactose to xanthosine and quinolinate to X-16397 and our previously reported urinary cell mRNA signature of 18S ribosomal RNA, CD3ε mRNA, and interferon-inducible protein-10 mRNA outperformed the metabolite signatures and the mRNA signature. The area under the receiver operating characteristics curve for the composite metabolite–mRNA signature was 0.93, and the signature was diagnostic of acute cellular rejection with a specificity of 84% and a sensitivity of 90%. The composite signature, developed using solely biopsy specimen-matched urine samples, predicted future acute cellular rejection when applied to pristine samples taken days to weeks before biopsy. We conclude that metabolite profiling of urine offers a noninvasive means of diagnosing and prognosticating acute cellular rejection in the human kidney allograft, and that the combined metabolite and mRNA signature is diagnostic and prognostic of acute cellular rejection with very high accuracy.     

Technetium-99m sulfur colloid accumulation as a predictor of acute renal transplant rejection.

Renal transplantation is the preferred treatment for end-stage renal disease in children. Most transplant failures are due to allograft rejection. To date, only histopathological findings on renal biopsy can establish this diagnosis. Prior to the availability of cyclosporine, technetium-99m sulfur colloid nuclear scans (TSC) were used in a limited number of institutions to detect rejection episodes. The purpose of this study was to determine whether TSC could predict acute rejection in the cyclosporine era. A prospective study involving 41 pediatric renal transplant patients (M = 25, F = 16) was conducted from 6/1/89 to 10/31/91. Patients who received a TSC and biopsy (41 patients, 62 studies) within one week of clinical and laboratory evidence of acute rejection were included in the study. A qualitative method of determining sulfur colloid uptake was used by comparing allograft uptake with that of the fifth lumbar vertebrae (L5) marrow uptake: 3(+)--allograft with greater than L5 marrow uptake, 2(+)--same as, 1(+)--less than, and 0--no allograft uptake. Transplant accumulation of > or = 2+ was considered consistent with acute rejection (P < 0.001). Acute rejection was noted in 53 of 62 renal biopsies. Of those with biopsy-proved acute rejection, SC was positive (> or = 2+) in 46 of 53. SC of > or = 2+ has proved to be a good predictor of acute rejection. This technique has a sensitivity of 98%, specificity of 53%, positive predictive value of 87%, and negative predictive value of 89%.     

Fine-needle aspiration cytology and conventional histology in 200 renal allografts

The diagnoses in 200 parallel fine-needle and core biopsies taken in acute renal allograft dysfunction, reduced function in long-term allografts, or in well-functioning grafts were compared. Fine-needle aspiration biopsy (FNAB) was found to be a reliable diagnostic tool with both a high sensitivity and specificity in acute cellular rejection (81 and 92%, respectively) and in normal kidney grafts (78 and 82%). On the other hand, the method was less valuable in the diagnosis of vascular rejection or interstitial fibrosis. Further evaluation is needed regarding the diagnostic implications of isometric vacuolization of tubular cells in FNAB specimens as a marker for acute cyclosporine nephrotoxicity.     

Immunocytology of urinary sediments as a method of differentiating acute rejection from other causes of declining renal graft function

We have previously reported that during acute rejection of renal allografts T lymphocytosis and increased HLA-DR expression on tubular epithelial cells can be demonstrated in urinary sediments by incubating cytospin preparations with monoclonal antibodies against T cells and HLA-DR antigen in an indirect alkaline phosphatase technique. We now tested whether immunocytological analysis of urinary sediments can be used to differentiate acute rejection from other causes of declining graft function. For this we retrospectively selected, from a series of urinary samples that were taken either at random or as part of a longitudinal study in unselected graft recipients, those specimens that were taken at the time of increasing creatinine levels, and compared the original immunocytological diagnosis, made without knowledge of clinical data, with the final clinical one. In 44 of 74 evaluable cases an immunocytological diagnosis of rejection was made, which in 37 patients was consistent with the eventual clinical diagnosis. In 28 of 30 cases the diagnosis no rejection proved to be correct. This indicates a sensitivity of 95% and a specificity of 80% for the immunocytological diagnosis of rejection. Of 38 patients who underwent a renal core biopsy, the immunocytological diagnosis was consistent with the histological diagnosis in 36 cases (31 rejections, 5 no rejections). In this subgroup the sensitivity of the immunocytology was 97% and the specificity 83%. We conclude that immunocytological examination of urinary sediments in renal allograft recipients can be a valuable new tool in discriminating acute interstitial rejection from other causes of deteriorating graft function.     

The signal-averaged electrocardiogram in cardiac transplantation. A noninvasive marker of acute allograft rejection.

Cardiac allograft rejection represents a major cause of morbidity and mortality in transplanted patients. Noninvasive markers of rejection have been sought, though transvenous endomyocardial biopsy remains the "gold standard" for the diagnosis of rejection. Sixty-one signal-averaged electrocardiograms (five in patients with rejection and 56 in patients without rejection) were obtained on 41 patients and prospectively analyzed in frequency domain via fast Fourier transform (FFT). Patients with acute allograft rejection demonstrate a significant increase in the high-frequency components of the QRS complex upon FFT analysis (QRS area ratio 203 +/- 57 vs. 66 +/- 10, P = 0.0007) compared with patients without rejection. Thus, frequency domain analysis may be a useful noninvasive marker of acute cardiac allograft rejection.     

Prediction of subclinical renal allograft rejection by vascular endothelial growth factor in serum and urine

BACKGROUND: Until now subclinical renal allograft rejection has only been recognized through a protocol biopsy. The aim of this study was to assess whether measurement of vascular endothelial growth factor (VEGF) in serum and urine could be adopted as a new noninvasive tool to predict subclinical rejection. METHODS: Concentration of VEGF in serum and urine was determined by ELISA in 132 recipients of a renal allograft with stable renal transplant function who were to undergo protocol biopsy and 80 healthy controls. A conventional receiver operating characteristic (ROC) curve was used to determine the sensitivities and specificities for patients with subclinical rejection. RESULTS: Levels of VEGF in serum (126.96 +/- 20.13 pg/mL; 95% confidence interval [95% CI], 83.10-170.83) and urine (16.14 +/- 4.09 ng/mmol creatinine; 95% CI, 7.21-25.06) of 13 patients with subclinical rejection significantly differed from those of 119 patients with no allograft rejection (No-AR) and health controls. The areas under the ROC curve were 0.771 (95% CI, 0.0.64-0.901) and 0.819 (95% CI, 0.662-0.976), respectively. Levels of VEGF in serum and urine after antirejection therapy (50.45 +/- 6.58 pg/mL and 2.60 +/- 0.83 ng/mmol creatinine, respectively) were lower than those at the time of protocol biopsy. No difference in urinary and serum VEGF expression was observed between cyclosporine and tacrolimus treatment. CONCLUSION: It is first reported that the monitoring of VEGF in serum and urine might be a new noninvasive approach to supplement a protocol biopsy for detection of subclinical rejection.     

CD103 mRNA levels in urinary cells predict acute rejection of renal allografts

BACKGROUND: CD103 is displayed on the cell surface of alloreactive CD8 cytotoxic T lymphocytes (CTLs) and is a critical component for the intraepithelial homing of T cells. Because intratubular localization of mononuclear cells is a feature of acute cellular rejection of renal allografts, we explored the hypothesis that CD103 messenger (m)RNA levels in urinary cells predict acute rejection. METHODS: We collected 89 urine specimens from 79 recipients of renal allografts. RNA was isolated from the urinary cells, and we measured CD103 mRNA levels and a constitutively expressed 18S ribosomal (r)RNA with the use of real-time quantitative polymerase chain reaction assay. RESULTS: CD103 mRNA levels, but not 18S rRNA levels, were higher in urinary cells from 30 patients with an episode of acute rejection (32 biopsies and 32 urine samples) compared with the levels in 12 patients with other findings on allograft biopsy (12 biopsies and 12 urine samples), 12 patients with biopsy evidence of chronic allograft nephropathy (12 biopsies and 12 urine samples), and 25 patients with stable graft function after renal transplantation (0 biopsies and 33 urine samples) (P = 0.001; one-way analysis of variance). Acute rejection was predicted with a sensitivity of 59% and a specificity of 75% using natural log-transformed value 8.16 CD103 copies per microgram as the cutoff value (P = 0.001). CONCLUSION: CD103 mRNA levels in urinary cells are diagnostic of acute rejection of renal allografts. Because CD103 is a cell surface marker of intratubular CD8 CTLs, a noninvasive assessment of cellular traffic into the allograft may be feasible by the measurement of CD103 mRNA levels in urinary cells.     

Heightened peripheral blood lymphocyte CD69 expression is neither sensitive nor specific as a noninvasive diagnostic test for renal allograft rejection

It has been reported that acute allograft rejection is associated with heightened expression of the peripheral blood lymphocyte (PBL) early activation marker CD69 and that this may serve as a potential biomarker of rejection. This study sought to determine whether PBL CD69 expression correlates with both acute clinical and subclinical renal allograft rejection as well as clinically inapparent cytomegalovirus (CMV) infection. Flow cytometric determination of PBL CD69 expression was performed at the time of clinical and protocol biopsies (n = 131) in 45 renal transplant recipients. Nineteen patients also underwent weekly monitoring of PBL CD69 expression for the initial 15 wk after transplantation. Simultaneous screening for CMV viremia was performed with a semiquantitative PCR assay. No differences were seen in either CD4+ or CD8+ lymphocyte CD69 expression between the biopsy diagnoses. CMV viremia however, independent of rejection, was associated with greater CD69 expression on CD8+ lymphocytes (17.8 +/- 10.4% versus 9.6 +/- 4.8%; P < 0.0001) but not CD4+ lymphocytes. No individuals experienced clinical CMV disease. Weekly monitoring of PBL CD69 expression did not change coincident with the diagnosis of rejection; however, CMV viremia coincided with a substantial rise in the proportion of CD8+69+ lymphocytes in a number of individuals. Thus, PBL CD69 expression is neither sensitive nor specific for the noninvasive diagnosis of renal allograft rejection. Furthermore, clinically inapparent CMV viremia is associated with heightened expression of this activation marker on CD8+ lymphocytes. This latter finding suggests that clinically inapparent CMV viremia may be a potential confounder for biomarkers of rejection that examine peripheral blood lymphocytes.