Certain modifications to the previously published ICSH recommendations (1978) have been approved by the Iron Panel of the International Committee for Standardization in Haematology (ICSH). They include substitution of the chromogen bathophenanthroline sulphonate with ferrozine or ferene, which are more sensitive and cheaper, and a reduction in the volume of the test sample from 2 ml to 0.5 ml.

The following method has been approved by the International Committee for Standardization in Haematology (ICSH) Board for publication as an ICSH standard (EP7/1978).

These guidelines provide information on how to develop and manage a point-of-care (POCT) service so that reliable haematology results are produced regardless of where the test is performed. Many of the issues addressed here are relevant to POCT within hospitals or health centres; however, the principles are equally applicable to care in the community and doctors' offices. Other aspects discussed in this guideline are the initiation of the service (including indications for and limitations of a POCT service), staff training, type of haematology equipment selected, the blood results, monitoring of quality, accreditation, safety and cost. Equipment selected should generate results that are comparable to those of the local reference laboratory. If a complete independent evaluation of the POCT device has not been performed, the purchaser should perform a local assessment according to the protocol in this document. A literature search should also be undertaken to find independent peer reviewed evaluations on POCT equipment. Often the ideals discussed here may not be achievable in some developing countries but long-term training and education of POCT workers needs to be supported and constantly kept on government agendas to reach the recommendations advised here. Users should interpret these recommendations for their particular POCT needs and setting.

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called "home brew" assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part II - Preanalytical issues. © 2013 International Clinical Cytometry Society.

Normal blood smears were stained by the standardised azure B-eosin Y Romanowsky procedure recently introduced by the ICSH, and the classical picture resulted. The effects of varying the times and temperature of staining, the composition of the solvent (buffer concentration, methanol content, & pH), the concentration of the dyes, and the mode of fixation were studied. The results are best understood in terms of the following staining mechanism. Initial colouration involves simple acid and basic dyeing. Eosin yields red erythrocytes and eosinophil granules. Azure B very rapidly gives rise to blue stained chromatin, neutrophil specific granules, platelets and ribosome-rich cytoplasms; also to violet basophil granules. Subsequently the azure B in certain structures combines with eosin to give purple azure B-eosin complexes, leaving other structures with their initial colours. The selectivity of complex formation is controlled by rate of entry of eosin into azure B stained structures. Only faster staining structures (i.e. chromatin, neutrophil specific granules, and platelets) permit formation of the purple complex in the standard method. This staining mechanism illuminates scientific problems (e.g. the nature of 'toxic' granules) and assists technical trouble-shooting (e.g. why nuclei sometimes stain blue, not purple).

Regulation of testosterone secretion is presumably mediated by interstitial cell-stimulating hormone (ICSH). However, there is little information on the actions of other chemical messengers in regulating testosterone secretion. We have shown that follicle-stimulating hormone augments testosterone secretion stimuated by ICSH in rabbit testes perfused in vitro with an artificial medium.

This revision is intended to update the 1994 ICSH guidelines. It is based on those guidelines but is updated to include new methods, such as digital image analysis for blood cells, a flow cytometric method intended to replace the reference manual 400 cell differential, and numerous new cell indices not identified morphologically are introduced. Haematology analysers are becoming increasingly complex and with technological advancements in instrumentation with more and more quantitative parameters are being reported in the complete blood count. It is imperative therefore that before an instrument is used for testing patient samples, it must undergo an evaluation by an organization or laboratory independent of the manufacturer. The evaluation should demonstrate the performance, advantages and limitations of instruments and methods. These evaluations may be performed by an accredited haematology laboratory where the results are published in a peer-reviewed journal and compared with the validations performed by the manufacturer. A less extensive validation/transference of the equipment or method should be performed by the local laboratory on instruments prior to reporting of results.

Clinical diagnostic assays, may be classified as quantitative, quasi-quantitative or qualitative. The assay's description should state what the assay needs to accomplish (intended use or purpose) and what it is not intended to achieve. The type(s) of samples (whole blood, peripheral blood mononuclear cells (PBMC), bone marrow, bone marrow mononuclear cells (BMMC), tissue, fine needle aspirate, fluid, etc.), instrument platform for use and anticoagulant restrictions should be fully validated for stability requirements and specified. When applicable, assay sensitivity and specificity should be fully validated and reported; these performance criteria will dictate the number and complexity of specimen samples required for validation. Assay processing and staining conditions (lyse/wash/fix/perm, stain pre or post, time and temperature, sample stability, etc.) should be described in detail and fully validated.

These guidelines provide information on how to reliably and consistently report abnormal red blood cells, white blood cells and platelets using manual microscopy. Grading of abnormal cells, nomenclature and a brief description of the cells are provided. It is important that all countries in the world use consistent reporting of blood cells. An international group of morphology experts have decided on these guidelines using consensus opinion. For some red blood cell abnormalities, it was decided that parameters produced by the automated haematology analyser might be more accurate and less subjective than grading using microscopy or automated image analysis and laboratories might like to investigate this further. A link is provided to show examples of many of the cells discussed in this guideline.

Scientific symposia on haemoglobinometry were held at the 9th Congress of the European Society of Haematology, Lisbon, 1963 (ESH 1964) and the 10th Congress of the International Society of Haematology (ISH), Stockholm, 1964 (ISH 1965). The International Committee for Standardization in Haematology (ICSH) made recommendations endorsed by the General Assembly of ICSH in Sydney on 23 August 1966 (ICSH 1967), for a reference method for haemoglobinometry and for the manufacture and distribution of an international reference preparation. Further symposia were held at the 12th Congress of the ISH, New York, 1968 (Astaldi, Sirtori & Vanzetti 1979) and at the 13th Congress of ISH, Munich, 1970 (Izak & Lewis 1972). The recommendations were reissued in 1978 (ISH 1978). On the basis of continuing experimental studies, the reference method and the specifications for the international reference preparation have been modified. The revised recommendations are described in this document.

An in vitro bioassay method for hFSH is presented. The method is based on the principles previously described by Dorrington et al. (1976b) and involves the assay of oestradiol produced from 19-hydroxyandrostenedione by dispersed Sertoli cells of 10-day old rats when cultured in the presence of graded doses of FSH. Using the 1st International Reference Preparation for human pituitary gonadotrophins (FSH and LH/ICSH) for bioassay (code no. 69/104) as standard, the useful range of the method is from 0.5 to 32 mIU/chamber (2 to 128 mIU/ml). The sensitivity of the method is 0.5 mIU/chamger. The mean index of precision (lambda) obtained from 16 multiple assays over 2 or 3 dose levels was 0.084. Parallelism was obtained between the 69/104 preparation and all preparations under study. The practicability of the proposed assay method is such that 15 preparations at 3 dose levels can be assayed by one person in 3 days. The specificity of the assay was investigated by determining the FSH activity in the following preparations: hFSH alpha- and beta-subunits, hLH, hCG, hTSH, ACTH, human growth hormone (hGH) human prolactin (hPRL) and luteinizing hormone-releasing hormone (LH-RH). The ACTH, hGH, hPRL and LH-RH preparations studied showed no detectable FSH activity in the assay. In the remaining preparations very low levels of FSH activity were found, corresponding to 0.004 to 0.6% of the weight of these preparations when compared with a highly purified hFSH preparation, suggesting that the method is specific for FSH. The possible synergistic or antagonistic influence of the above preparations when assayed in the presence of the 69/104 preparation was also assessed. No evidence of a synergistic or antogonistic effect was found. The assay of the hFSH potencies of a limited number of hFSH preparations of varying purity by the proposed in vitro bioassay, an hFSH radioreceptor method and an hFSH specific radioimmunoassay technique revealed that - although the relationship of the various potencies obtained with each method showed a close agreement - the bioassays yielded the highest potency estimates, and the radioimmunoassays the lowest ones. Since the proposed bioassay method is sensitive and considered to be specific for hFSH activity, it provides a suitable basis for the assessment of the specificity of other in vitro methods (radioreceptor and radioimmunoassay) currently used for detecting low levels of FSH activity.

Seven new red-cell pyruvate kinase (PK) variants were characterized by the methods recently recommended by the International Committee for Stanardization in Haematology. The cases were all true homozygote as evidenced by consanguineous marriages of the parents; all are Japanese. These variants were designated as PK Tokyo, PK Nagasaki, PK Sapporo, PK Maebashi, PK Itabashi, PK Fukushima and PK Aizu, respectively. Low substrate affinity (high K0.5S for phosphoenolpyruvate) and thermal instability appear to play major roles in causing defective enzyme function, resulting in chronic haemolytic anaemia. Product inhibition of PK by ATP may also play an additional role in causing haemolysis in more than half the cases.

Although the length of sedimentation reaction in blood (LSRB) (commonly, but improperly called erythrocyte sedimentation rate, ESR) has long been used in clinical laboratories because it is simple and low-cost, its sensitivity and specificity are unsatisfactory. Usually, the values are reported using the Westergren method with sodium citrate-anticoagulated specimens. We used a new procedure, the TEST1, which measures the length of sedimentation reaction in undiluted K3EDTA anticoagulated blood samples following ICSH (International Committee for Standardization in Haematology) recommendations. Samples obtained from 840 reference individuals (430 females and 410 males, mean age 44 and 46.5 years respectively, range 1 to 90 years) were utilised to estimate the reference limits. The subjects, classified by sex, were subdivided into four statistically different age groups to determine the reference limits (2.5th and 97.5th percentiles). Sex difference was statistically significant in two age groups, from 14 to 50 (p < 0.0001) and from 50 to 70 years (p < 0.01). We did not observe significant sex difference within the age bracket from 1 to 14 years and from 70 to 90 years. In both sexes LSRB values increased with age, in significant correlation with fibrinogen concentration (p < 0.0001), and became significantly higher in subjects older than 70 compared to all the younger subjects (p<0.01 in females and p < 0.02 in males). Thus, we defined adequate reference ranges in elderly.

OBJECTIVE

In thrombocytopenia, high accuracy and precision of low platelet count is essential for appropriate decisions. The recently introduced Sysmex XN2000 analyzer (Sysmex, Kobe, Japan) offers 3 methods for platelet counting: impedance (PLT-I), optical (PLT-O), and a new fluorescence method (PLT-F). The precision of the PLT-F method in blood samples with platelet counts less than 50 × 10(3)/μL (50 × 10(9)/L) was investigated and compared with the ICSH CD61-ImmunoPLT reference method. For comparison, PLT-I and PLT-O were determined on the Sysmex XN2000 and Sysmex XE2100 analyzer.

METHODS

Blood samples with platelet counts less than 50 × 10(3)/μL (50 × 10(9)/L) (n = 37) were analyzed on the Sysmex XN2000 and XE2100 analyzers. The CD61-ImmunoPLT method was performed on a Beckman Coulter FC-500 flow cytometer (Miami, FL).

RESULTS

At a platelet count of 20 × 10(3)/μL (20 × 10(9)/L), reproducibility for PLT-I, PLT-O, and PLT-F on the XN2000 demonstrated coefficients of variation of 9.3%, 8.5%, and 3.0%, respectively. Correlation between PLT-O on the XN2000 and XE2100 yielded an r value of more than 0.977. Linear regression analysis between the PLT-F and CD61-ImmunoPLT methods resulted in a PLT-F of 0.71*CD61 - 0.8 (r = 0.988). Linear regression between PLT-F and PLT-O on the XN2000 resulted in a PLT-F of 1.05*PLT-O - 2 (r = 0.975), and using the transfusion threshold of 20 × 10(9)/L platelets resulted in a PLT-F of 0.90*PLT-O - 0.4 (r = 0.956).

CONCLUSIONS

The new PLT-F method demonstrated excellent results for reproducibility in samples with platelet counts less than 50 × 10(9)/L. PLT-F could be helpful in making better decisions for platelet transfusions.

OBJECTIVE

The presence of ≥1% schistocytes on a peripheral blood smear (PBS) is an important criterion for the diagnosis of thrombotic microangiopathy (TMA). The reporting of schistocytes has been standardized by the International Council for Standardization in Hematology (ICSH). Despite the availability of guidelines, however, the assessment of schistocytes remains subjective. More recently, the automated fragmented red cell (FRC) parameter has been evaluated. However, local studies are not available.

METHODS

A prospective study was performed at the Charlotte Maxeke Johannesburg Academic Hospital in order to evaluate the ICSH recommendations for schistocyte measurement in 146 PBSs with schistocytes. Schistocytes were evaluated by microscopy and ADVIA 2120 automated hematology analyzers.

RESULTS

Schistocytes were frequently observed in patients with TMA (n=76), infection (n=20), hematologic malignancy (n=10), renal failure (n=5), and hemoglobinopathy (n=15), and in neonates (n=11). Schistocytes were ≥1% in all PBSs with TMA (n=76) with a mean of 3.44±1.84. Schistocytes of ≥1% were also observed in cases of renal failure and hemoglobinopathy, and in neonates. In these conditions, schistocytes were mainly observed in conjunction with moderate red blood cell changes. The agreement between two morphologists gave a correlation coefficient of 0.63 [confidence interval (CI): 0.52-0.75], while the correlation coefficient between the average of the morphologists and the FRC percentage was -1.97 (CI: -1.60 to -2.34). The ADVIA 2120 underestimated the schistocyte count in patients with TMA.

CONCLUSIONS

Observer bias can be decreased by implementing the standardized procedures recommended by the ICSH. However, estimation of schistocytes by the ADVIA 2120 analyzer requires further evaluation as a screening tool. A higher threshold for schistocytes in thrombotic thrombocytopenic purpura is recommended to distinguish this hematological emergency from other conditions associated with ≥1% schistocytes.

This protocol is proposed for the evaluation of automated blood cell counters to assess the performance, advantages and limitations of such instruments. It is based on the International Committee for Standardization in Haematology (ICSH) 'Protocol for type testing equipment and apparatus used for haematological analysis' (1978a) and the British Committee for Standardization in Haematology 'Guidelines for the evaluation of instruments used in haematology' (Shinton, England & Kennedy, 1982). The document has been prepared by the ICSH Panel on Cytometry after discussion with colleagues. This tentative protocol will be reviewed 1 year after publication, in accordance with the ICSH rules, before it is adopted as a definitive standard.