Mitochondrial growth and division during the cell cycle in HeLa cells.

The growth and division of mitochondria during the cell cycle was investigated by a morphometric analysis of electron micrographs of synchronized HeLa cells. The ratio of total outer membrane contour length to cytoplasmic area did not vary significantly during the cell cycle, implying a continuous growth of the mitochondrial outer membrane. The mean fraction of cytoplasmic area occupied by mitochondrial profiles was likewise found to remain constant, indicating that the increase in total mitochondrial volume per cell occurs continuously during interphase, in such a way that the mitochondrial complement occupies a constant fraction( approximately 10-11(percent)) of the volume of the cytoplasm. The mean area, outer membrane contour length, and axis ratio of the mitochondrial profiles also did not vary appreciably during the cell cycle; furthermore, the close similarity of the frequency distributions of these parameters for the six experimental time-points suggested a stable mitochondrial shape distribution. The constancy of both the mean mitochondrial profile area and the number of mitochondrial profiles per unit of cytoplasmic area was interpreted to indicate the continuous division of mitochondria at the level of the cell population. Furthermore, no evidence was found for the occurrence of synchronous mitochondrial growth and division within individual cells. Thus, it appears that, in HeLa cells, there is no fixed temporal relationship between the growth and division of mitochondria and the events of the cell cycle. A number of statistical methods were developed for the purpose of making numerical estimates of certain three-dimensional cellular and mitochondrial parameters. Mean cellular and cytoplasmic volumes were calculated for the six time-points; both exhibited a nonlinear, approx. twofold increase. A comparison of the axis ratio distributions of the mitochondrial profiles with theoretical distributions expected from random sectioning of bodies of various three-dimensional shapes allowed the derivation of an "average" mitochondrial shape. This, in turn, permitted calculations to be made which expressed the two-dimensional results in three-dimensional terms. Thus, the estimated values for the number of mitochondria per unit of cytoplasmic volume and for the mean mitochondrial volume were found to remain constant during the cell cycle, while the estimated number of mitochondria per cell increase approx. twofold in an essentially continuous manner.

Viral antigen in endocrine cells of the pancreatic islets and adrenal cortex of Pekin ducks infected with duck hepatitis B virus.

Endocrine cells in the pancreas and adrenal glands of duck hepatitis B virus-infected ducks were examined for the presence of viral antigen. Analysis of pancreas tissue was based on double immunofluorescence assays in which anti-duck hepatitis B virus serum was used to detect viral antigen, and anti-glucagon and anti-insulin serum were used, respectively, to identify endocrine alpha and beta cells. Assays of pancreas from infected ducks ranging in age from 3 to 20 weeks indicated that subpopulations of both alpha and beta cells expressed viral antigen. A higher percentage of viral antigen-positive cells was observed in alpha-islets than in beta-islets. As assayed by immunofluorescence and immunoperoxidase staining with anti-viral serum, small clusters of viral antigen-positive cells were detected in the adrenal glands of young infected ducks. These cells were identified as cortical cells on the basis of histologic criteria.

Estimation of lymphocyte percentage and number on the Coulter Counter, Model S Plus Phase II.

The Coulter Counter Model S Plus Phase II provided precise measurements of lymphocyte percentage and count and carry-over was negligible. Lymphocyte percentage values agreed well with those from the stained blood film except when the percentage was high and in these circumstances the instrument gave underestimates. When making artificial mixtures with increasing lymphocyte count a progressive underestimation of the lymphocyte percentage was also noted. The display of leucocyte volume distribution was found to be a useful attribute particularly when the instrument alerted the operator to "rejected" profiles. Rejection by the instrument proved to be a helpful function occurring in the myeloid leukaemias, erythroblastosis, and in some cases when the lymphocytes differed from normal--for example, glandular fever and chronic lymphocytic leukaemia.

Tumor cells induced by the v-src oncogene are heterogeneous for expression of markers of mesenchyme differentiation.

The observation that v-src-induced tumors contain tumor cells of differing morphology, notably fibroblastoid or polygonal, raised the question as to whether the tumor cells are also heterogeneous with respect to expression of markers of cellular differentiation. Of the markers tested here, consistent reactivity for tumor tissue was noted only for antibody probes reactive to muscle actin (HHF35, alpha sm-1) or to procollagen type I (SP1. D8); for any given tumor, whether induced by v-src DNA or by Rous sarcoma virus, each of these markers was found only in a subpopulation of tumor cells. The observation of marker heterogeneity in the one v-src DNA-induced tumor examined here that typed as monoclonal suggests that v-src-induced transformation is consonant with a degree of plasticity in the phenotypes of the clonal progeny of a single transformant.

Differential white cell counts by frequency distribution analysis of cell volumes.

Absolute neutrophil and lymphocyte counts on peripheral blood can be made by analysis of the output from a Coulter particle counter, utilizing the difference in the relative cell volume between these two types of cell. A comparison has been made between the results obtained by volume analysis and those obtained by standard microscopical techniques in 10 normal people and 45 patients. The absolute neutrophil count obtained by volume analysis agreed well with values obtained by microscopy; the lymphocyte count did not give such good agreement, since the smaller number of cells counted gave rise to larger sampling errors. The method of volume analysis is suitable for the assessment of absolute neutrophil counts for clinical use.

Guidelines for organisation and management of external quality assessment using proficiency testing. Expert Panel on Cytometry of the International Council for Standardization in Haematology.

This document is intended to assist towards the WHO objective that external quality assessment (EQA) schemes be established at national and/or regional levels world-wide. Quality assurance is defined as all steps taken by the director of a laboratory to ensure reliability of laboratory results and to increase accuracy, reproducibility and between-laboratory comparability. This includes the use of internal quality control procedures and participation in external quality assessment. Internal quality control provides the means for evaluation of analytic test results at the time of testing in order to decide whether they are reliable enough to be released to the requesting clinicians. EQA, on the other hand, refers to a system of retrospective and objective comparison of results from different laboratories by means of proficiency testing (PT) organised by an external agency. The main purpose is to establish between-laboratory and between-method (including between-instrument) comparability, and agreement with a reference standard where one exists. Internal quality control and EQA complement each other and must never be considered as alternatives.

Coincidence correction in red blood cell counting.

A method has been developed for accurate correction of red blood cell count for coincidence in aperture-impedance electronic blood cell counters. It is based on extrapolation of the slope of regression of the counts which are obtained with sequential dilutions of the samples.

Future needs and expected trends in peripheral blood cell analysis: erythrocyte histograms.

Erythrocyte histograms should be analysed in accordance with the ICSH recommended protocol to determine: How many red cell populations are present and the proportion of the total red cells which are included in each population. The central tendency (mode, median & mean) and dispersion (size ratio, geometric standard deviation & coefficient of variation) for each population. The proportion of microcytic or macrocytic cells in each population. Examples are given to show how the patient's diagnosis is assisted by careful analysis of the histograms and the effect of treatment monitored by sequential testing. Histograms can be generated from blood films or from aperture-impedance and light-scatter volume measurements. Volume measurements on these systems are influenced by the red cell shape and internal refractive index. Mean cell hemoglobin concentration affects both shape in the aperture-impedance orifice and internal refractive index in light-scatter systems. There is poor agreement between volume histograms obtained on the two measuring systems and the histograms available on current automated instruments provide no more useful information than can be obtained from the blood film. Automated instruments need to produce histograms with fewer artefacts and the histograms should then be examined in accordance with the ICSH protocol. This approach should maximise the diagnostic value of the complete blood count.

Recommended methods for the assignment of assay values to stabilized cell suspensions.

Stabilized blood suspensions are required for the transfer of data from accurately calibrated reference instruments to service laboratory instruments. However, the more the cells are stabilized to increase shelf life, the less like fresh blood they become. Fixation is quite satisfactory for producing red count standards but it affects the flexibility, shape factor and the assigned MCV of the erythrocytes. Because of these problems values must be assigned indirectly by assigning values to fresh blood by reference methods and subsequently comparing fresh bloods with stabilized suspensions on a range of user instruments. The details for these methods are given and the reasons for these requirements are discussed.

Discriminant functions.

Discriminant Functions (DFs), first described by Fisher in 1936, have been applied to the classification of microcytic disorders such as iron deficiency and heterozygous thalassemia. Mathematically DFs are weighted linear combinations of variables. If the underlying assumption of multivariate normality is valid DFs provide the best possible classification. Variables may need to be transformed before the DF is derived. When two groups have to be classified it is easy to visualise the DF. With one variable the DF is represented by the point which provides the best separation. In the bivariate situation the two groups form ellipses and the DF is the best line of separation whilst in the trivariate case the two groups are ellipsoids and a plane forms the best separation. Ratios and power functions are equivalent to DFs but they are less efficient and less rigorously derived. To apply DFs in hematological practice it is necessary to carefully select the measurements to be included and to define the case selection criteria. Once the DF has been derived it should be tested on a new data set and its transferability assessed. Like any single test the DF will have sensitivity and specificity which may need to be adjusted by changing the "cut-off" if the DF is used for screening rather than for differential diagnosis.

Total and differential leucocyte count.

PIP: The methods of measurement of the total leucocyte count and the differential leucocyte count, automation of the leucocyte count, and normal values are discussed. Variations between different subjects and variations within each subject should be considered when measuring normal values. Age, sex, race, pregnancy, smoking, and oral contraceptives influence hematological values. In women after menopause the WBC and neutrophil counts fall. Pregnancy can cause leucocytosis, and myelocytes or metamyelocytes may appear in the blood. A consistent fall in neutrophil count at menstruation has been demonstrated; 2 neutrophil peaks in each cycle are most likely related to changes in circulating estrogen levels. Oral contraceptives have been reported to raise the WBC, but this has been absent with the low estrogen preparations.^ieng

Discrimination between iron-deficiency and heterozygous-thalassaemia syndromes in differential diagnosis of microcytosis.

Three formulae have been advocated for interpreting the blood-count when differentiating between iron deficiency and heterozygous thalassaemia: the discriminant function. M.C.V.-R.B.C.-(5 X Hb)-k, the M.C.V/R.B.C. ratio, and the (M.C.V.)2 X M.C.H. product. In a comparison of their diagnostic accuracy in microcytic adults from several countries, the discriminant function was the most satisfactory. 417 of 455 uncomplicated cases (91.6%) of iron deficiency, heterozygous alpha1- and beta- thalassaemia were correctly identified. The function was not able to distinguish heterozygous alpha2-thalassaemia from iron deficiency. A flow chart illustrates how the discriminant function can be used in haematological practice.

A comparison of methods for analysing red cell and platelet volume-distribution curves.

Cell volume-distribution curves may be analysed using log-probability paper or by considering volumes corresponding to frequencies of 50 or 60% of that at the mode. All methods give similar results; the log-probability paper method is better because it allows the researcher to check that the underlying distribution is lognormal; methods based on 50 or 60% modal frequency are slightly less reliable but are simpler for routine use. For platelets it may be convenient to estimate the curve from the modal volume and the volume above the mode at which the frequency is reduced to 60% of the modal frequency.