Reduction of the platelet review rate using the two-dimensional platelet method.

Accurate platelet enumeration is critical for optimal treatment of patients with platelet and bleeding disorders, leukemias, and other neoplasias. The majority of automated hematology analyzers count platelets by size differentiation alone, which may result in falsely elevated platelet counts for samples containing interfering particles such as RBC fragments, microcytes, and cell debris. Most analyzers flag questionable platelet counts, necessitating review of results with confirmation by an alternative method, thus increasing the cost of performing platelet counts and delaying results. We studied the effect of a new platelet analysis method, based on measurement of size and refractive index, on the laboratory review rate for platelet counting. We demonstrated that this method yields higher accuracy for platelet counts in samples with interferences, especially for platelet counts less than 50 x 10(3)/microL (< 50 x 10(9)/L). As a result of the 2-dimensional analysis, the review rate for platelet counts was reduced by 65% in our institution, resulting in substantial savings.

Analysis of the diversity of a sheep antibody repertoire as revealed from a bacteriophage display library.

We have applied bacteriophage display technology to construct and analyze the diversity of an IgG library of >1 x 108 clones from an adult sheep immunized against the hapten atrazine. We have identified eight new VH gene families (VH2-VH9) and five new Vkappa gene families (VkappaV-VkappaIX). The heavy and kappa light chain variable region gene loci were found to be far more diverse than previously thought.

Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules.

Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.

Health care practice and the minimisation of patient medical litigation.

This article explores why patients sue their health care providers. Following an extensive literature review, it identifies 'sue' motivators and then examines the legal basis of medical litigation by reviewing contemporary case law. Armed with the 'sue' motivators and having considered the types of claims brought against health care providers, the article focuses upon what health care providers may do to minimise litigation. It recommends specific provocative measures, which are based upon satisfying a myriad of patient needs through consenting practices, and early recognition and resolution of patient issues by providers.

Cell lines of novel type derived from a diabetic secrete tissue-reactive human monoclonal antibodies.

Human monoclonal antibodies have been produced from lymphocytes of an acute-onset insulin-dependent diabetic patient. Peripheral blood lymphocytes were hybridized with a fusion partner HMY-1320. Initial screening of human immunoglobulin secretion was made by a nitrocellulose dot blot assay. Ten stable cell lines of novel type, secreting human immunoglobulin, were obtained. These cell lines have been maintained in continuous culture over 6 months and cryopreserved in liquid nitrogen for 14 months. Human monoclonal antibodies of IgG and IgM class have been produced and are secreted at a rate of 150-650 ng/ml/10(6) cells/day. Monoclonal antibodies were tested for histological staining against a variety of endocrine and non-endocrine tissues. One monoclonal antibody, LT1E12, demonstrates a staining pattern in human, rat, and mouse tissues, similar to that of mitochondrial antibodies. Another antibody, LT3C4, demonstrates weak staining of smooth muscle in rat and mouse kidney sections. Neither specificities were detected in the diabetic patient's serum. The variety of immune tissue specificities obtained in this study demonstrates the potential value of human monoclonal antibodies as probes to analyze the complexity of autoimmunity in diabetes mellitus.

Flow cytochemical patterns of white blood cells in human haematopoietic malignancies. I. Acute leukaemias.

Peripheral blood samples from 118 patients with acute leukaemia (68 untreated; 50 treated) were measured with the Technicon H-6000 automated haematology analyser. This instrument provides, in addition to measurements of the classical haematology parameters (i.e. cell counts, haemoglobin concentration, etc.), a differential count on 10(4) WBC effected by means of flow cytochemistry (peroxidase content) and volume (light scatter) discrimination. Disregarding RBC and platelet counts and their volume distribution profiles, the most important diagnostic parameters for leukaemic disease were the WBC count, the WBC differential count, and the proportions of large unstained cells (LUC) and high peroxidase (HPX) cells obtained by the automated differential count as well as the mean value of the WBC peroxidase content distribution (MPA). Granulocytic leukaemias had lower MPA than normal and lymphocytic leukaemias had MPA values above normal. M1 leukaemias were also characterized by large proportions of LUC and low fractions of HPX, while M2 leukaemias showed low LUC with high HPX. M3 leukaemias had low LUC and very high HPX. M4 leukaemias had large LUC and 'monocytic' components and a modest fraction of HPX. M5 leukaemias had very large numbers of LUC, 'monocytes' and 'lymphocytes' and a normal HPX. For M1 leukaemia, the presence of less than 7% LUC following induction treatment was related to morphological changes of normal cells induced by chemotherapy while LUC above 10% usually indicated unsuccessful induction associated with the presence of residual blasts. If treatment was successful, M2 and M3 leukaemias characteristically decreased their HPX population. All M4 leukaemias studied by us failed to enter remission and continued to display high proportions of HPX and LUC. Similarly, most M5 leukaemias had a poor response to treatment and always showed a very high proportion of LUC. Untreated lymphocytic leukaemias demonstrated high LUC, normal HPX and a high proportion of 'lymphocytes'. Hairy cell leukaemias showed almost equal proportions of 'lymphocytes' and LUC. Successful chemotherapy of all lymphoid leukaemia entities was associated with rapid decreases in LUC, slower decrements of 'lymphocytes' and moderate and transient increments in HPX. Thus, flow cytochemistry can assist not only in the segregation of acute leukaemias along with FAB classification with nonmorphologic criteria, but also in the follow up of patients with these diseases.

Localization of human muscle carbonic anhydrase isozymes using immunofluorescence.

Human muscle sections were examined for the presence of carbonic anhydrase using fluorescent second antibody and antisera specific for the three isozymes, carbonic anhydrase I (CAI), carbonic anhydrase II (CAII), and carbonic anhydrase (CAIII). CAIII was present in all fibers, CAII only in the connective tissue, and CAI showed a weak association with the sarcolemma.

Quantification of bands produced by isoelectric focusing using immunoperoxidase.

An improved, rapid, and sensitive (to autoradiographic levels) method for the quantification of IgG bands from cerebrospinal fluid is presented. The principle of an internal standard is employed using the discrete bands of a paraprotein to correct for any run to run variability in the enzyme detection system which is linked to the immunoassay of immobilised antigen.

Viral immunoblotting: a sensitive method for detecting viral-specific oliogoclonal bands in unconcentrated cerebrospinal fluid.

A new method for detecting viral antibodies in cerebrospinal fluid is described. The technique has many advantages over previously published methods in that it is highly sensitive eliminating the need to concentrate the CSF, takes 5 h to complete, avoids the use of radionucleides, and most importantly circumvents problems associated with prozone effects which occur in immunoprecipitation reaction since the viral antigen is immobilized on nitrocellulose membranes.