Evaluation of Pretransplant Donor-Specific Alloantibodies With Different Crossmatch Techniques.

Donor-recipient crossmatching for kidney transplantation is an obligatory step for the transplant work-up process. Attention is currently put on single bead assay (SBA) that enables virtual crossmatching. In contrast, methods developed for complement binding capacity are still not routinely established. We compared modified, cytolytic flow cytometry crossmatch (cFC-XM) with complement-dependent serological crossmatch (CDC-XM), SBA, and flow cytometry crossmatch (FC-XM) to verify whether newly developed techniques may be beneficial for transplant immunological matching. Also, the cutoff value for donor-specific alloantibodies levels currently used for virtual crossmatch was verified. Serum from 22 sensitized patients was crossmatched with surrogate donors by CDC-XM, FC-XM, and cFC-XM, while anti-HLA antibodies were measured by SBA. In all cases, virtual crossmatch was positive at 5000 mean fluorescence intensity cutoff. Among 22 tested sera with donor-specific alloantibodies above 5000 mean fluorescence intensity, the positive CDC-XM result was noted only in 41% of patients (n = 9), but positive FC-XM was noted in 86% (n = 19), and further lytic antibodies (cFC-XM) were confirmed in 27% of cases (n = 6). Our results suggest that donor-recipient immunological matching for kidney transplantation requires different methods to verify the importance of alloantibodies, and improvement in laboratory investigation is needed. This is especially important for immunized patients that have many types of alloantibodies and virtual crossmatching used as a tool for deceased donor allocation should balance between the likelihood of transplantation and risk of positive crossmatch result.

Paired Analysis of Outcomes After Kidney Transplantation in Peritoneal and Hemodialysis Patients.

BACKGROUND: The impact of dialysis modality before kidney transplantation (hemodialysis or peritoneal dialysis) on outcomes is not clear. In this study we retrospectively analyzed the impact of dialysis modality on posttransplant follow-up. METHODS: To minimize donor bias, a paired kidney analysis was applied. One hundred thirty-three pairs of peritoneal dialysis (PD) and hemodialysis (HD) patients were transplanted at our center between 1994 and 2016. Those who received kidneys from the same donor were included in the study. HD patients were significantly older (44 vs 48 years), but the Charlson Comorbidity Index was similar (3.12 vs 3.46) in both groups. The groups did not differ significantly with respect to immunosuppressive protocols and number of mismatches (2.96 vs 2.95). RESULTS: One-year patient (98% vs 96%) and graft (90% vs 93%) survival was similar in the PD and HD patient groups. The Kaplan-Meier curves of the patients and graft survival did not differ significantly. Delayed graft function (DGF) and acute rejection (AR) occurred significantly more often in the HD recipients. Graft vessel thrombosis resulting in graft loss occurred in 9 PD (6.7%) and 4 HD (3%) patients (P > .05). Serum creatinine concentration and estimated glomerular filtration rate (using the Modification of Diet in Renal Disease guidelines) showed no difference at 1 month, 1 year, and at final visit. On multivariate analysis, factors significantly associated with graft loss were graft vessel thrombosis, DGF, and graft function 1 month after transplantation. On univariate analysis, age, coronary heart disease, and graft loss were associated with death. Among these factors, only coronary heart disease (model 1) and graft loss were significant predictors of death on multivariate analysis. CONCLUSION: The long-term outcome for renal transplantation is similar in patients with PD and HD. These groups differ in some aspects, however, such as susceptibility to vascular thrombosis in PD patients, and to DGF and AR in HD patients.

Association Between HLA Type and Skin Cancer in Kidney Transplant Recipients.

BACKGROUND: Organ transplant recipients (OTRs) are more susceptible to various diseases, among them cancers. Nonmelanoma skin cancers (NMSC) represent the most common malignancies in OTRs in Europe. Due to the significantly higher morbidity, aggressive and rapid progression, and poor prognosis of NMSC in the OTR population, these patients require a special oncological approach. Intensive attention should therefore be paid to factors predisposing OTRs to the development of cancer. The aim of this study was to establish the role of genetic factors in the pathogenesis of skin cancer in kidney transplant recipients (KTRs). METHODS: This single-center study was performed in 39 KTRs with posttransplant NMSC. The frequency of particular types of HLA Class I (HLA-A and HLA-B) and Class II (HLA-DR) in each group were compared to establish the association between the HLA type and risk of skin cancer after renal transplantation. RESULTS: HLA-DR15 were more commonly detected in patients with MNSC than in the control group of KTRs (P = .014) There was also a positive correlation between HLA-B18 and skin squamous cell carcinoma. The antigen was more often recorded in KTRs with squamous cell carcinoma than in KTRs without NMSC (P = .03) and in the general population (P = .002). CONCLUSIONS: Patients who are positive for HLA-BR15 and HLA-B18 should be under special dermatologic surveillance due to the potentially high risk of skin cancer.

Preemptive kidney transplantation: analysis of kidney grafts from the same donor.

BACKGROUND: From November 2003 to December 2012, in the Gdansk Center, 64 patients received preemptive transplantation (PET). PET comprised 8% of 794 kidney transplantations performed during this time. The benefits for individual patients and for the health care system are discussed. METHODS: This study compares the outcomes of these PET patients who had their kidney pairs transplanted after a variable duration of dialysis (PTD), a total of 51 pairs. RESULTS: The mean Charlson comorbidity index was 2.57 vs 3.04 (P > .05) for the PET and PTD groups, respectively. Both groups did not differ significantly with respect to 1-year patient and graft survivals, and incidences of acute rejection. Five (9.8%) PET patients and 20 (39%) PTD patients experienced delayed graft function (P < .05). The graft function (serum creatinine/4p MDRD) 1 year after transplantation was similar in both groups (1.42/53.7 vs 1.43/57.4; mg/dL/mL/min/1.73 m(2)). More PET patients continued normal professional activities or education before and after transplantation (P < .05). CONCLUSIONS: Our single-center results confirmed that for both medical and socioeconomic reasons, PET is an optimal mode of renal replacement therapy.

Modified flow cytometry crossmatch detecting alloantibody-related cytotoxicity as a way to distinguish lytic antibodies from harmless in allosensitised kidney recipients.

The serological complement-dependent cytotoxicity crossmatch (CDC-XM) permits routine identification of anti-donor alloantibodies in the sera of allotransplant recipients. However, in a small group of recipients, antibodies below the threshold of detection may still be responsible for hyperacute rejection. For the same reason, approximately 20% of recipients develop acute rejection episodes. The flow cytometry crossmatch (FCXM) was designed to address these problems, but because of the presence of clinically insignificant antibodies (linked, non-lytic), the FCXM appears to be too sensitive yielding false-positive results. We compared FCXM with its modified version assessing cell viability (cytolytic flow cytometry crossmatch; cFCXM) using sera from previously sensitised kidney recipients. The presence of alloantibodies was detected using the Luminex platform. The cFCXM proved to be of greater sensitivity than CDC-XM, which was additionally confirmed with bead-based Luminex techniques. The cFCXM was also superior to FCXM because it distinguished lytic from non-lytic antibodies. The cFCXM was superior to assess donor specificity, sensitivity, and detection of clinically relevant lytic antibodies.

Sarcoidosis and tuberculosis: a connection to the human leukocyte antigen system.

Infectious, genetic factors, and autoimmunity have been considered as potential causes of sarcoidosis (SA). Pathological similarities between SA and tuberculosis (TB) suggest M. tuberculosis antigen(s) as causative agent(s). Our published comparative analysis of the human leukocyte antigens (HLA) system in patients with SA or TB in the same ethnic group revealed that some antigens were connected with high risk of developing of SA or TB, but other were comparable in both patient populations. Is it possible that the predominating occurrence of HLA antigens characteristic for TB may cause tuberculosis in patients with SA? To answer this question we evaluated the HLA class I and II alleles frequency by PCR amplification with sequence-specific primers in three women with histopathologically proven pulmonary SA, who developed bacteriologically confirmed TB on a corticosteroids (CS) therapy. Analysis of HLA in every case separately revealed a trend for higher occurrence of both alleles predisposing and protecting from TB than SA, in comparison with healthy individuals in our previously mentioned HLA genotyping study. Overall, the number of alleles predisposing to TB was statistically greater than the number of alleles connected with a high risk of developing SA. Also, the frequency of protecting alleles was statistically higher for TB than for SA. Therefore, SA in these patients developed at first, and the presence of additional environmental factors, e.g., age, CS might decrease an immune response and provoked TB. There is a possibility that the occurrence of HLA antigen more associated with high risk of developing TB than SA causes the development of tuberculosis in our patients with sarcoidosis.

Algorithm to manage highly sensitized kidney transplant recipients in Poland.

OBJECTIVE: Programs for immunized transplant recipients are essential to achieve graft survivals comparable to those of non-immunized recipients. The threshold in Poland is a PRA by the complement-dependent cytotoxicity (CDC) method greater than 50%, which includes approximately 3.8% of the patients. At the same time the United Network for Organ Sharing there recipients represent approximately 16% of the waiting list in the United Network for Organ Sharing (UNOS). The underestimation of the immunized group in Poland may be due to differences in laboratory techniques to assess alloantibodies. MATERIALS AND METHODS: This study investigated 55 potential recipients with a PRA by CDC>50%. We used the following algorithm to assess their immunization: Luminex screening test for an HLA antibody; specificity assessed with Luminex Single Antigen, vPRA (evaluation of immunization of the patient); and analysis of acceptable HLA incompatibilities (HLAMatchmaker). RESULTS: All recipients were positive class I anti-HLA antibodies and 94.5% were positive for class II. For the groups of subjects with PRA-CDC from 50% to 79% versus those greater than 80%, the average values of PRA-CDC were 62.2% and 89.5%, respectively. The virtual PRA results for these groups were 95.7% and 97.2%, respectively. In addition, anti-HLA-Cw, anti-DQ and anti-DP antibodies were detected in 77%, 84%, and 51% of recipients, respectively. Immunized recipients reported to the next transplant were characterized by the antibodies against mismatch only in 68%. For all potential recipients, additional acceptable non-compliance was determined with HLAMatchmaker: 152 specificity for locus A and 252 for locus B. CONCLUSIONS: Evaluation of immunization status of recipient candidates should be routinely performed using tests to assess class and specificity as well as level of alloantibodies to enable determination of a safe potential donor. As a routine test, PRA-CDC underestimates the number of highly immunized patients. Exclusion from the list of patients with repeated non-compliance is a simplification, which reduces their chance for transplantation.

11-year single-center experience in living-donor kidney transplantation in Poland.

Living-donor kidney transplantation (LDKT) is a viable option that is characterized by better short- and long-term results than cadaver-donor kidney transplantation (CKT). In Poland number of LDKT is low (2%-3%). We collected data on all 29 LDKTs performed in our center between 1999 and 2010; 82.7% were obtained from living related donors. The mean recipient age was 34.4±12.8 years (range, 15-58) and mean donor age was 48.5±7 years. Most donors were women (n=17; 62%). In 3 cases the LDKT was a second transplantation. The mean numbers of HLA class I and II mismatches were 2.18±0.98 and 0.93±0.6 respectively. The mean total ischemia time was 3.22±1.74 hours. Induction therapy included antithymocyte globulin daclizumab 4; (13.7%) (n=7; 24.1%); 27.6% of recipients were placed on cyclosporine based immunosuppression and the remaining 72.4% on tacrolimus with 69% also receiving mycophenolate mofetil. All recipients were prescribed steroids. Delayed graft function was observed in 3 cases and an acute rejection episode in 4 subjects. One-year patient and graft survivals were both 100% (98% and 83% for CKT). Five-year patient and graft survival were 100% and 89.6%, respectively, compared with 83% and 69% in CKT. The mean serum creatinine levels at 1, 6, 12, and 60 months were 1.59±0.4, 1.51±0.3, 1.51±0.4 and 1.49±0.3 mg/dL respectively. Our results, albeit concerning a small group of patients, confirmed excellent outcomes of LDKT, which should be actively promoted in our country.

Renal transplantation in dialysis patients with the history of coronary artery bypass grafting and cardiac valve replacement.

Death with a functioning kidney is the most frequent cause of graft failure. Cardiovascular disease is the most frequent cause of death after renal transplantation. Therefore, prior to grafting, it is mandatory to diagnose and treat coronary artery disease and heart valve impairment. Transplantation is the best option for renal replacement therapy as far as the quality of life and life expectancy are concerned, although patients with such comorbidities may experience a higher short-term mortality risk. The objective for this study was to analyze both short- and long-term results of patients after coronary artery bypass grafting (CABG) or cardiac valve replacement (CVR). The cardiac surgery recipient group (CSR) included 16 patients (15 men, 1 woman) aged from 44 to 73 (mean 54.9 +/- 7.8) years. CABG was performed in 13/16 patients, and CVR in 3/16. The rest of our patients were treated as a comparative noncardiac surgery recipient (non-CSR) group. It consisted of 422 patients (264 men, 158 women) aged from 14 to 68 years (mean 43.2 +/- 12.9). The comparison revealed that graft function estimated at 1 year after transplantation was not different: serum creatinine concentrations of 1.7 +/- 0.2 and 1.6 +/- 0.5 mg/dL in CSR and non-CSR, respectively. One-year patient survival in the CVR group of 93.8% was slightly worse than that in the non-CSR group (97.9%), but death-censored 1-year graft survivals were comparable in both groups (93.8% vs 92%). Urinary tract and cytomegalovirus infections were the most common complications in the CSR group. One patient lost his graft in month 3(rd) due to many serious infectious complications. One patient died at the end of 12 months as a result of a cardiovascular event (1/16). Our single-center results confirm that transplantation in patients after CABG or CVR is a safe procedure; therefore, such patients should be referred into the waiting list.

A single center experience in preemptive kidney transplantation.

Transplantation is recognized as preemptive if it takes place before the initiation of chronic dialysis. This maneuver has the potential to avoid the morbidity and burden of chronic dialysis. From November 2003 to April 2005, 15 (7 male, 8 female) end-stage renal failure patients of mean age 40 +/- 14.8 years received preemptive grafts from 2 living-related and 13 cadaveric donors, constituting 11.5% of all kidney transplantations performed in our center at that time. The period on the waiting list for preemptive recipients, namely, 2 weeks to 6 months (mean, 2.4 months), was significantly shorter than that of other patients (mean, 13.8 months). The mean creatinine clearance calculated from the Cockroft Gault formula and the mean plasma creatinine level in preemptive recipients before transplantation were 12.7 +/- 3.1 mL/min and 6.6 +/- 0.8 mg/dL, respectively. The donors for preemptive and non-preemptive groups of recipients did not differ significantly in respect to age, gender, and renal function. The mean number of mismatches of 3.73 and 3.25 and the mean total ischemic times of 9.53 +/- 5 and 11.2 +/- 5 hours, in preemptive and non-preemptive groups of recipients, respectively. The incidence of delayed graft function (dialysis in the first week after transplantation) was significantly lower among preemptive recipients (20% versus 42%, respectively). The groups did not differ either in respect to the occurrence of acute rejection episodes or graft and patient survivals. In preemptive patients the mean plasma creatinine levels at 3 and 12 months were 1.37 +/- 0.3 and 1.09 +/- 0.3 mg/dL, and in non-preemptive patients 1.7 +/- 0.5 and 1.4 +/- 0.4 mg/dL. In conclusion, these results are promising, confirming the notion that preemptive kidney transplantation is the optimal treatment for end-stage renal disease patients.

Analysis of DQB1 allele frequencies in pulmonary tuberculosis: preliminary report.

BACKGROUND: The human leucocyte antigen (HLA) system plays an important role in the modulation of the immune response. An association between HLA and pulmonary tuberculosis (TB) has been examined in several populations but the results have been inconsistent. The aim of this study was to evaluate the correlation of DQB1 alleles with TB patients and healthy controls in the same ethnic group in Poland. METHOD: The DQB1 alleles of 38 patients with TB and 58 healthy university staff volunteers were determined by a PCR-SSP low resolution method. RESULTS: The DQB1*05 allele occurred more frequently (p adjusted for multiple comparison=0.002, OR=2.84, 95% CI 1.57 to 5.15) and the DQB1*02 allele occurred less frequently (p=0.01, OR=0.39, 95% CI 0.21 to 0.71) in patients with TB than in controls. The occurrence of DQB1*03,*04,*06 alleles was similar in the two populations. CONCLUSIONS: The occurrence of specific DQB1 alleles may be linked to susceptibility/resistance to tuberculosis.

Nitric oxide synthase type 1 expression in human lung cancer and its relation to p53.

BACKGROUND: The nitric oxide synthases (NOS) have been observed in human tumour cell lines as well as in solid tumours. Neuronal isoform of NOS (NOS1) was particularly abundant in low-differentiated gynaecological, breast and central nerve system tumours, suggesting that it may characterize poorly differentiated tumours. So far, little is known about expression of the neuronal NOS isoform in non-small cell lung cancer. Our aim was to compare NOS1 expression in non-small lung carcinomas with respect to tumor staging and p53 protein expression. MATERIAL AND METHODS: Thirty-two cases of non-small lung carcinomas of all grades of malignancy and ten control lung specimens with neoplastic lesions were examined. Paraffin-embedded tissues were stained with hematoxylin and eosin, or with antibodies to NOS1 and p53, and evaluated under light microscope. RESULTS: No statistical correlation between expression of p53, NOS1 and degree od tumour differentiation was observed. CONCLUSION: Expression of NOS1 can not serve as a marker for highly malignant tumours in the non-small cell lung carcinomas.

Molecular subtypes of the HLA-DR antigens in pulmonary tuberculosis.

OBJECTIVE: The aim of this study was to analyze association between HLA-DRB1 alleles and pulmonary tuberculosis (PTB) in the Polish population. METHODS: The HLA-DRB1 typing was performed using sequence-specific amplification (polymerase chain reaction with sequence specific primer [PCR-SSP] in 31 patients and 58 healthy volunteers. The DRB1 primers were supplied by DYNAL in the standard kit DYNAL DR "low-resolution"-SSP. RESULTS: The study showed that the DRB1*16 alleles frequency was higher in patients with PTB than in the tested group of healthy controls (P < 0.01). When HLA-DR2 alleles were combined (i.e., the DRB1*15 with DRB1*16 alleles), their frequency was comparable with that in the healthy individuals. The highest relative risk (RR) of tuberculosis was associated with DRB1*16 alleles (RR = 9.7). When HLA-DR6 alleles were combined (i.e., the DRB1*13 with DRB1*14 alleles), only a trend for higher frequency in patients with PTB was found. Frequency of DRB1*13 alleles of HLA-DR6 was significantly lower in PTB than in the healthy individuals (P < 0.001; RR = 0.04). CONCLUSIONS: Results suggest that the presence of HLA-DRB1*16 alleles may increase the risk of development of PTB, whereas HLA-DRB1*13 alleles may be resistant to tuberculosis.

Dendritic and cancer cells in the breast tumors--an immunohistochemical study: short communication.

Normal and dysplastic mammary glands express immunocompetent S-100 protein positive dendritic cells (DCs), which are located in a regular pattern, in the suprabasal cell layer of the ducts and alveolar nodules. The epithelial cells, however, are S-100 protein negative. Since some breast cancers also express the S-100 protein, our aim was to check the diagnostic and prognostic value of the S-100 protein distribution combined with the tumor grade and expression of synaptophysin (Syn), chromogranin A (Chg A), c-erbB-2 oncoprotein and p53 protein in infiltrating and metastatic breast tumors. Applying immunohistochemical methods, we show in paraffin- or frozen breast tissue sections that in some cases of the infiltrating breast carcinomas, S-100 protein positive cells do not appear, whereas in other cases, either S-100 protein positive DCs are closely associated with cancer cells, or the cancer cells themselves stain positive to S-100 protein. However, we found no correlation between the S-100 protein expression and other investigated parameters.

HLA-DRB and -DQB1 alleles in Polish patients with hepatitis B associated membranous nephropathy.

We have studied the HLA-DRB and -DQB1 alleles of 42 paediatric patients who have suffered from membranous nephropathy associated with a hepatitis B infection (HBVMN). These patients were all from the Gdansk area of Northern Poland and the disease was diagnosed by light and electron microscopy. The control population consisted of 55 healthy children, approximately age matched, from schools in Gdansk. In addition we have also analysed 40 patients chronically infected with hepatitis B, without any renal involvement, as hepatitis B disease controls. The HLA alleles were defined using PCR/SSP. As idiopathic membranous nephropathy and low responsiveness to hepatitis B vaccine have been found to be associated with DR3 in Caucasoids, our hypothesis was that the HBVMN patients would show an increase in DR3. Our results indicate that, although there is a small increase in the frequency of DRBl*0301 in the HBVMN patients (16/42 38%) when compared to the healthy controls (15/55 31%), this does not approach significance. There is a significant increase in the frequency of DQB1*0303 in the HBVMN patients vs the healthy controls, after correction for the number of antigens detected (Pc)(13/42 vs 2/55, RR=11.6, P=0.0007, Pc=0.02). A similar increase in DQB1*0303 is seen in the HBVMN patients when compared to the hepatitis controls (13/42 vs 4/40) but this is only significant before correction (RR=4.3, P=0.04).

Immunohistochemical study in breast carcinoma. S100-protein positive cells and neuroendocrine differentiation.

In this study S100-protein positive cells were found in all 12 cases of benign dysplastic changes and in all of the investigated 53 cases of breast carcinomas. These cells belong to interdigitating cells (IDC) and were located in a regular pattern in the basal cell layer of the ducts and alveolar nodules in benign lesions. However, in breast carcinomas S100-protein positive IDC were in various pattern of distribution; only in the stroma, in basal cell layer, between cancer cells and S100-protein positivity of IDC and epithelial cancer cells. This phenomenon suggests the antigenic modulation of cancer cells transferring them the property of S100-protein positive cells or it may be the consequence of fusion. Immunoreactivity for chromogranin A (ChgA) and synaptophysin (Syn) was found in epithelial cells in some cases of benign dysplastic changes. However, in some carcinoma cases ChgA and Syn were revealed in carcinoma cells, in myoepithelial cells, in some stromal mesenchymal cells and endothelial cells. Immunoreactivity to smooth muscle actin was shown in the endothelial and smooth muscle cells of the vessel's wall, in myoepithelial cells and was also sporadic in carcinoma cells.

[Development of renal transplantation in the Gdansk center]. / Rozwój transplantacji nerek w osrodku Gdanskim.

The kidney transplantation is one of the renal replacement therapy methods, which prolongs live of the patients with the end stage renal disease for many years. Moreover, this method is well known, safe and not so expensive as dialysotherapy. Our purpose was to present the 15-year activity of the transplantation center in Gdansk. The first renal transplantation took place on the 31st of March 1980 and there have been 137 renal transplantations in Gdansk until now. We can divide the time between the 31st of March 1980 and the end of 1994 into two periods: I from 31.03.80 to 31.12.89 and II from 1991 to 1994. During the first were 46, and during the second were 91 renal transplantations performed. It means that since the second half of 1991 the activity of the center in Gdansk has increased. The graft function was noted in 29 patients (63%) during the first period and in 75 (82%) during the second. The acute graft failure was observed in the most of the cases mentioned above. The 5-year living of the transplanted patients and the dialysed patients is comparable and amounts to 90%. Infections were the main reason of death during the first period, and cardiovascular complications during the second. The 5-year graft's functioning is 60%. Nowadays the results of the kidney transplantation center in Gdansk are good and comparable with the results of other centers in Poland and Europe. Our center, as similar ones in Poland is prepared to extend the kidney transplantation activity. So it is necessary to intensify an effort to gain more organs for transplantations.

Serum protein changes in experimentally induced amyloidosis in hamsters.

After inducing amyloidosis in hamsters by hyperimmunization with Di-Te-Per vaccine, their serum proteins were separated by electrophoresis. Results showed that the level of alpha 1 globulin increased and that of albumin decreased after 6-8 weeks of the hyperimmunization. Gamma-globulins increased by the 10th week of the study, then their level markedly decreased. Electrophoretic separation of the serum proteins refrigerated for 24 h at 4 degrees C showed the presence of a steadily increasing protein fraction induced by the prolonged immunization, which migrated in the prealbumin band. The fraction was observed in all phases of the experiments. This fraction was stained using selective methods for lipids. New bands appearing during electrophoretic separation of hamster serum proteins in amyloidosis may represent monomeric or polimeric forms of amyloid protein.

Some aspects of cell-mediated hypersensitivity in patients with melanoma.

Cell-mediated hypersensitivity in 64 patients with melanoma was assessed by delayed-type skin reactivity to dinitrochlorobenzine (DNCB) and tubercullin as well as by lymphocyte enumerations. The immunological status of the patients was correlated with their clinical stage. The mean absolute and percentage counts of E rosette forming cells were lower in patients than in normal controls. Patients with distant metastases showed lower levels of E rosette forming cells than the patients with localized disease. No differences were found in number of EAC rosette forming cells between melanoma patients and controls. The mean absolute and percentage counts of null cells in melanoma patients in stages II and III were significantly higher than those in controls. Melanoma patients showed reduced skin reactivity to DNCB and normal reactivity to tuberculin. Patients with positive DNCB reactivity showed significantly higher total lymphocyte counts and higher percentages of E rosette forming lymphocytes than patients unreactive to DNCB.