Clinical cytology and primary health care of children and adults.

Clinical cytology is a diagnostic branch of medicine, best known by the Papa test in gynaecology. But, cytology can be applied in almost all fields of clinical medicine. Its advantages--high accuracy, simplicity, with little or no aggressiveness and low cost--are not used as widely as they could be. Medical practice, as well as medical research, and also medical education, are nowadays often directed at profitable use and not at the real benefit of the patient. Primary practitioners do not have enough chance to get acquainted with clinical cytology as a whole although they need true information, based on the cost-effectiveness and patient-benefit. A panel discussion on this subject was organised at the 4th Croatian Congress of Clinical Cytology, in Split, October 11-14, 2009 by the Croatian Society for Clinical Cytology-Croatian Medical Association, to inform primary practitioners about the possibilities of cytodiagnostics in the health care of children and adults. Indications for cytodiagnostics in infectious diseases (T. Jeren and A. Vince), haematology (I. Kardum-Skelin), pulmonology (S. Smojver-Jezek), thyroid diseases (A. Knezevic-Obad), breast diseases (I. Kardum-Skelin), gastroenterology and urology (G. Kaic) were discussed, as well as technical procedures and the interpretation of the cytological findings. Moderator (Z. Znidarcic) opened the panel with presentation about the role of clinical cytology, particularly in the primary health care. The discussion finally pointed at the necessity of better communication between primary practitioners and cytologists. This review article presents contents of the panel discussion.

Naive and memory CD4+ T-cells in the cerebrospinal fluid of children with aseptic meningitis following measles-mumps-rubella vaccination and enteroviral meningitis.

We investigated the distribution of memory (CD45RO+) and naive (CD45RA+CD62L+) CD4+ T-cells as well as CD8+ T-cells and total T-cells in the CSF of children with aseptic meningitis following measles-mumps-rubella (MMW) vaccination and those with enteroviral meningitis. Flow cytometric analysis of CSF cells was performed in 12 children with MMR vaccine-associated meningitis and 11 children with enteroviral meningitis. Percentages of total T-cells, CD4+ and CD8+ T-cells and monocytes in CSF of patients from the two groups were not significantly different. The majority of CD4+ T-cells in the CSF of both patient groups were of memory phenotype. Percentages of CSF naive CD4+ T-cells were increased in children with aseptic meningitis following MMR vaccination. Further studies focused on the more detailed immunophenotyping of CSF cells are needed to fully establish the usefulness of flow cytometry in the diagnostic workup of inflammatory CNS diseases in children.

Fabry disease--a diagnostic and therapeutic problem.

The authors present a patient with Fabry syndrome that remained undiagnosed for several years. Fabry syndrome is a genetic disease related to changes on the X chromosome. Its complex clinical presentation and diverse symptomatology is caused by deficient activity of lysosomal hydrolase alpha-galactosidase enzyme. Defect in the basic alpha-galactosidase molecule implies genetic change, which can be a predisposing factor for the development of atypical and typical forms of this genetic disease. In the presented case, clinical manifestation and hemizygous symptomatology were the evidence of metabolic and genetic irregularity, typical clinical presentation of Fabry disease. Many authors report generalized vasculopathy as a basic characteristic of Fabry disease and a causative factor of multiorgan changes. Some authors indicate that persons with diagnosed asymmetric hypertrophy of the left ventricle have decreased alpha-galactosidase. Cardiac complications, coronary disease, and acute myocardial ischemia are often present in cases of Fabry disease, frequently causing death in such patients. Characteristic central nervous system symptoms with skin-burning sensation and paresthesia were also present in our case. Cerebrovascular complications were caused by changes on small blood vessels. Clinical signs of renal failure were nonspecific, and the diagnosis was based on extrarenal symptoms. Initial renal manifestations were insignificant as asymptomatic proteinuria and microhematuria, due to which our patient was referred to further examination. The level of alpha-galactosidase was significantly decreased. The severity and progression of this disease depends on the level of alpha-galactosidase enzyme in serum and its catabolic effect. More recent studies have showed that deficient enzyme can be synthetic zed and, accordingly our patient has been successfully enrolled in the replacement therapy program.

Increased expression of CXCR3 and CCR5 on memory CD4+ T-cells migrating into the cerebrospinal fluid of patients with neuroborreliosis: the role of CXCL10 and CXCL11.

The aim of this study was to evaluate the contribution of chemokine receptor CXCR3 and the corresponding ligands CXCL10 and CXCL11 to the recruitment of peripheral blood (PB) memory CD4+ T-cells into the cerebrospinal fluid (CSF) of patients with acute neuroborreliosis. Percentages of memory CD45RO+CD4+ T-cells expressing CXCR3 and CCR5 were significantly enriched in the CSF compared to the PB. Concentrations of CXCL10 and CXCL11 in the CSF of neuroborreliosis patients were significantly higher compared with the corresponding serum samples. Our results suggest that CXCL10 and CXCL11 create a chemokine gradient between the CSF and serum and recruite CXCR3-expressing memory CD4+ T-cells into the CSF of neuroborreliosis patients and that CCR5 also plays a role in this process.

[Guidelines for antimicrobial treatment and prophylaxis of urinary tract infections]. / Smjernice antimikrobnog lijecenja i profilakse infekcija mokracnog sustava.

Recommendations for antimicrobial treatment and prophylaxis of urinary tract infections (UTI) have been made according to the results of investigation of resistance of the most frequent causative agents of UTI to antimicrobial drugs. This investigation has been conducted for the past seven years by the Committee for monitoring bacterial resistance to antibiotics in the Republic of Croatia, with consensus of eight professional societies of the Croatian Medical Association. Uncomplicated cystitis is treated 1, 3, or 7 days, complicated 7 days, pyelonephritis 10-14 days, and complicated UTI 7 to 14 days, rarely longer. For the treatment of cystitis fluorokinolons, nitrofurantoin, betalactam antibiotics, and in the fields of lower resistance trimethoprim/sulfamethoxazol are being used. Single treatment with fluorokinolons is administered to otherwise healthy young women with normal urinary tract in whom cystitis symptoms have been present for less than 7 days. Empiric antimicrobial treatment of pyelonephritis, recurrent and all complicated UTI must be reviewed after urine culture finding is obtained. In the treatment of bacterial prostatitis and febrile UTI in males, the drug of first choice is ciprofloxacin. Asymptomatic bacteriuria (AB) is treated in pregnant women, newborns, preschool children with urinary tract abnormalities, before invasive urologic and gynecologic procedures, in kidney transplant recipients, and in the first days of short term urinary bladder catheterization. Recommendations for the treatment of AB in patients with diabetes mellitus have been controversial in the past two years. Antimicrobial prophylaxis is administered mostly one hour prior to the diagnostic or therapeutic invasive urological procedure, using selected antimicrobial agents.

[MHC tetramers: tracking specific immunity]. / MHC-tetrameri: na tragu specificnoj imunosti.

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.

Center for Disease Control (CDC) flow cytometry panel for human immunodeficiency virus infection allows recognition of infectious mononucleosis caused by Epstein-Barr virus or cytomegalovirus.

AIM: To analyze the distribution of lymphocyte subsets in the peripheral blood of patients with infectious mononucleosis caused by Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to investigate the possible diagnostic usefulness of flow cytometry panel recommended by the Center for Disease Control and Prevention (CDC) for HIV-1 infection. METHODS: The study included 130 immunocompetent adults with infectious mononucleosis caused by EBV (n=103) and CMV (n=27) and 50 controls. EBV-infected patients were divided into two groups based on typical (n=92) or atypical (n=11) clinical presentation of the disease. Lymphocyte subpopulations were determined by flow cytometry and a panel of monoclonal antibodies recommended by the CDC for the immunophenotyping of patients infected with human immunodeficiency virus (HIV). RESULTS: Patients with typical and atypical presentation of EBV-induced infectious mononucleosis showed increased percentages of total T-cells, cytotoxic-suppressor CD8(+) T cells and activated HLA-DR(+) T cells compared to healthy controls. Percentages of CD4(+) T cells, as well as CD4/CD8 ratio, were significantly decreased. Absolute counts of CD4(+) T cells and percentages of B cells did not differ from healthy controls. Pattern of changes in CMV-infected patients was completely identical to that in healthy controls, although less pronounced. CONCLUSION: Lymphocyte subpopulations represented in the CDC panel for HIV are sufficient for the recognition of patients with infectious mononucleosis caused by EBV and CMV. Flow cytometry can be useful support for reaching diagnosis in patients with atypical clinical presentation of EBV-induced infectious mononucleosis.

[Role of the clinician in cytologic diagnosis]. / Uloga klinicara u donosenju citoloske dijagnoze.

In the course of continuing education for physicians on nongynaecological cytology, together with the topics about development and actual situation in clinical cytology, especially in Croatia, as well as those about the technical procedures in cytology, the clinicians' participation in the interpretation of cytological diagnosis was discussed. The term "clinicians" is used here for those physicians who need a rapid, accurate and nonaggressive morphological diagnostic method for the treatment of their patients. The central part of the course was a round-table discussion about the role of clinicians in making cytological diagnosis, with participation of cytologists and clinicians from various branches of medicine, who are either cytologists, or have a good contact with cytologists. They answered questions about the clinicians' information on cytology, about the contact and collaboration between cytologists and clinicians, and about their own experience in everyday cytological practice. The aim of this discussion was to provide sufficient information to clinicians on cytological diagnostics, and the emphasis on team-work was made.