The algorithm in Figure 7 summarizes the overall approach to fitting a single reference distribution. If no statistically significant difference is found, the investigation should assess whether the power of the chi-squared test was insufficient to detect differences of practical significance. If a statistical difference is found, Appendices B and C and Figure 6 may be used to determine whether the sample size and the power have led to the detection of an effect size too small to be of importance.

The classification of acute leukaemias is now widely based on a combined morphological, cytochemical and immunophenotyping approach. Difficulties are frequently encountered however in reaching an acceptable degree of diagnostic concordance between different laboratories because of variations in the techniques used (in terms of methodologies, reagents and equipment) and diagnostic interpretation. The International Council for Standardization in Haematology (ICSH) convened an expert panel to consider currently available diagnostic techniques with the aim of defining a minimum cytochemical and immunological diagnostic panel that could be used as core components for the classification of acute leukemia. The proposed ICSH scheme, which attempts to balance the basic requirement for providing precise and informative diagnostic information without limiting its use to only those laboratories with sophisticated facilities, is based on three sequential levels of investigation; primary cytochemistry, intracellular phenotyping and membrane immunophenotyping. The minimum ICSH recommended cytochemistries comprise myeloperoxidase (MPO), chloroacetate esterase (ChlorE) and alpha-naphthyl acetate esterase (ANAE), and standardised methods for these cytochemistries are detailed in this communication. For cases of acute leukaemia that remain unclassified by primary cytochemistry, subsequent immunological analyses for cytoplasmic CD3, CD22, MPO and nuclear TdT are recommended. The ICSH panel considers that the use of these minimum primary cytochemical and intracellular phenotyping procedures will lead to the consistent classification of most acute leukaemias, and that the third level of investigation (membrane immunophenotyping) should be used for the purposes of confirmation, diagnostic clarification of atypical leukaemias, and the subtyping of acute lymphoblastic leukaemias (ALL). The ICSH panel also recognised that there are a number of additional technologies which can provide definitive diagnostic information, such as cytogenetics and DNA genotyping, but these were excluded from the minimum panel because of their restricted availability. While many specialised laboratories, particularly in the areas of diagnostic research, will continue to use individual investigatory protocols, it is considered that the inclusion of the ICSH scheme as core components would lead to greater consistency when comparing independent studies of acute leukemia.

The classification of acute leukaemias is now widely based on a combined morphological, cytochemical and immunophenotyping approach. Difficulties are frequently encountered however in reaching an acceptable degree of diagnostic concordance between different laboratories because of variations in the techniques used (in terms of methodologies, reagents and equipment) and diagnostic interpretation. The International Council for Standardization in Haematology (ICSH) convened an expert panel to consider currently available diagnostic techniques with the aim of defining a minimum cytochemical and immunological diagnostic panel that could be used as core components for the classification of acute leukaemia. The proposed ICSH scheme, which attempts to balance the basic requirement for providing precise and informative diagnostic information without limiting its use to only those laboratories with sophisticated facilities, is based on three sequential levels of investigation; primary cytochemistry, intracellular phenotyping and membrane immunophenotyping. The minimum ICSH recommended cytochemistries comprise myeloperoxidase (MPO), chloroacetate esterase (ChlorE) and alpha-naphthyl acetate esterase (ANAE), and standardised methods for these cytochemistries are detailed in this communication. For cases of acute leukaemia that remain unclassified by primary cytochemistry, subsequent immunological analyses for cytoplasmic CD3, CD22, MPO and nuclear TdT are recommended. The ICSH panel considers that the use of these minimum primary cytochemical and intracellular phenotyping procedures will lead to the consistent classification of most acute leukaemias, and that the third level of investigation (membrane immunophenotyping) should be used for the purposes of confirmation, diagnostic clarification of atypical leukaemias, and the subtyping of acute lymphoblastic leukaemias (ALL). The ICSH panel also recognised that there are a number of additional technologies which can provide definitive diagnostic information, such as cytogenetics and DNA genotyping, but these were excluded from the minimum panel because of their restricted availability. While many specialised laboratories, particularly in the areas of diagnostic research, will continue to use individual investigatory protocols, it is considered that the inclusion of the ICSH scheme as core components would lead to greater consistency when comparing independent studies of acute leukaemia.

The microscopic enumeration of reticulocytes is a tedious assay with poor reproducibility. Its results provide only fragmentory information on the kinetics of erythropoiesis. Thus, attempts have been made for its automation. This study presents results of an evaluation of the first machine exclusively devoted to reticulocyte analysis. The Sysmex R-1 000 uses flow cytofluorometry with argon laser, after precipitation of nucleic acids by auramine-O. The whole study was performed according to ICSH recommendations. At least 2,500 erythrocytes were examined in the microscopic reference technique, after 1 p. cent brilliant cresyl-blue staining. Within and between-batch precision studies gave CV not exceeding 5.46 and 8.72 p. cent respectively for reticulocyte numbers. In dilution study it was shown that no measured result was over 4 p. cent of the expected value, in the range 4-400 x 10(9)/l. There was no significant carry-over in the range tested (35-510 x 10(9)/l). Accuracy testing proved that the significant difference observed when microscopic enumeration was performed in routine conditions disappeared when at least 5,000 erythrocytes were examined. Keeping the samples at 4 degrees C limited the decrease of the reticulocyte number to less than 10 p. cent after 24 hours. Analysis of some particular clinical situations demonstrated the risk of spurious results in case of massive Plasmodium infestation, and the usefulness of obtaining the percentage of newly emerged reticulocytes, i.e. the most fluorescent, for a better estimate of the kinetics of erythrocyte production. This evaluation testifies the quality of the Sysmex R-1 000, and underlines the potential benefit of such analyzers in the equipment of the modern hematology laboratory.

Measurement of the Haemoglobin F in red cell haemolysates is important in the diagnosis of δβ thalassaemia, hereditary persistence of fetal haemoglobin (HPFH) and in the diagnosis and management of sickle cell disease. The distribution of Hb F in red cells is useful in the diagnosis of HPFH and in the assessment of feto-maternal haemorrhage. The methods of quantifying Hb F are described together with pitfalls in undertaking these laboratory tests with particular emphasis on automated high-performance liquid chromatography and capillary electrophoresis.

The International Council for Standardization in Haematology (ICSH), an international organization promoting international agreement on hematological testing, is now restructuring to strengthen its activities. In Asia, a diversity of testing methods exists and the resulting testing levels make it difficult to compare test results internationally among Asian countries. Fortunately, the ICSH is considering regionalizing its organization to 5 sub-societies to increase its activity, and we have been able to establish a new society, ICSH-Asia, under the ICSH umbrella.

BACKGROUND

Laboratory reference intervals are important for both clinical orientations and therapeutic decisions. In Morocco, no reference ranges are available for local population. The ranges commonly used in clinical laboratories and by physicians are those of Caucasian population. We have decided that it is relevant to undertake an epidemiological investigation on local adult healthy population, with the aim of establishing hematology reference intervals in Moroccan population.

METHODS

Blood samples were taken from healthy adult volunteers of the regional transfusion center and measured on a Sysmex XE-2100 analyzer. We have grouped our data samples with regard to gender and retained donors aged between 18 and 45 years old according to ICSH guidelines 1982. Leucocyte, erythrocyte, and platelet parameters were analyzed. For any sample flagged by the automate or thrombopenia with platelets < 100000/microL, a systematic smear was done and checked.

RESULTS

A significant difference between male and female was found with regard to the values for leucocyte, erythrocyte, and platelet parameters as well as for hemoglobin and hematocrit. These data were compared to normal values reported for Arabic, Caucasian, and African population.

CONCLUSIONS

As part of this study, we have given a descriptive approach of normal blood cell count and its peculiarities in North African Arabian and Berber population not explored until now. We have established similarities and differences between our population and other African, Arab, and Caucasian populations.

The International Council for Standardization in Hematology (ICSH) is a not-for-profit organization aimed at improving global quality and harmonization of analytical methods, and achieving reliable and reproducible results in diagnostic hematology. ICSH co-ordinates Working Groups of experts to examine laboratory methods and instruments for hematological analyses, and co-operates with different international organizations which have similar scientific goals. Among seven ongoing approved projects, three ICSH projects have been selected and will be presented in the ICSH session at the XXVIIth ISLH International Symposium on Technological Innovations in Laboratory Hematology in The Hague, on May 2014. The project on 'Guideline for flow cytometric evaluation of patients with suspected acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS)' covers different aspects of the application of immunophenotyping by multiparameter flow cytometry (MFC) in the diagnosis of AML and MDS including integration into multimodal diagnostic workflow, quality control, antibody selection, interpretation of findings, reporting, and personnel. Data from the pilot study of the project for 'International Standardization of Hematology Reporting Units' suggest that there is a wide variation in reporting units for the routine blood cell count and highlights the areas of nomenclature and units of measurement where standardization is necessary and feasible, such as units for cell counts, white cell differentials, and hemoglobin concentration. The project on 'Standardization of HbA2 measurement and its implications for clinical practice' starts from the observation that different instruments give different results for hemoglobin A2; it is aimed at producing recommendations as to how instrument manufacturers and laboratories should assess their equipment before using it to analyze patient samples. These projects are examples of how the ICSH represents a great opportunity for scientists involved in hematology laboratory to participate in a process of expert collaboration and discussion all around the world.

The defective enzymes of 54 patients with pyruvate kinase (PK) deficiency were characterized according to the recommendations of the International Committee for Standardization in Haematology (ICSH). The erythrocyte PK activity in whole blood was calculated considering the 16-fold higher activity of the reticulocyte enzyme (AR) compared to the erythrocyte enzyme (AE). The following parameters turned out to give a good correlation to the degree of haemolytic anaemia and can therefore serve as a prognostic tool: All patients with a severe course of the disease had residual erythrocyte PK activities less than 33% of the normal enzymes (percentage activity), and patients with mild haemolytic anaemia exhibited residual activity values below and above this threshold value. Studies of enzyme cooperative showed that positive cooperative or mixed cooperative phosphoenolpyruvate (PEP) binding with a predominant positive cooperative part appeared in all cases with a mild clinical course, and about one-third of the severe ones. Negative cooperativity or mixed cooperativity with predominant negative cooperative part was observed only with severe haemolytic anaemia. Furthermore, the determination of glucose-6-phosphate (G-6-P) turned out to be a good prognostic criterion, i.e. all patients with mild clinical course exhibited G-6-P-concentrations lower than 0.11 mumol/l red blood cells. In the case of patients with severe haemolytic anaemia, about 80% showed values higher than 0.11 mumol/l RBC.

Automated high performance liquid chromatography and Capillary electrophoresis are used to quantitate the proportion of Hemoglobin A2 (HbA2 ) in blood samples order to enable screening and diagnosis of carriers of β-thalassemia. Since there is only a very small difference in HbA2 levels between people who are carriers and people who are not carriers such analyses need to be both precise and accurate. This paper examines the different parameters of such equipment and discusses how they should be assessed.

The present work was carried out to study the effect of zeranol on the pars distalis of 39 male fat-tailed lambs about 8 months old. The examined animals were classified into 3 groups. The group I was considered as control, group II and III were implanted subcutaneously in the back side of ear with 3 doses each of 12 mg zeranol at 40 - days intervals. Groups I and II were fed on concentrate mixture containing 70.45% starch and 14.42% digestible protein, while that of the group III containing 70.44% starch value and 7.92% digestible protein. Zeranol implants have been shown to increase body weight of animals group II and III. A significant difference was found in the number of the pars distalis cells as well as the histochemical changes in their properties have been observed in zeranol implanted lambs. The STH-cell showed a significant increase in number and signs of cellular hyperactivity. The ICSH-cells, FSH-cells and TSH-cells were significantly decreased in number and exhibited signs of less activity compared with the propriate cells of the nonimplanted animals. It is concluded that Zeranol improve gain, stimulate STH-cells and inhibit the GTH-cells as well as TSH-cells in the pars distalis of zeranol implanted animals.

There was no loss of biological activity in vitro when the 1st International Reference Preparation (IRP) for human pituitary gonadotrophins [FSH and LH/interstitial cell stimulating hormone (ICSH) for bioassay] code no. 69/104 and the 1st IRP for human pituitary LH/ICSH [for immunoassay] code no. 68/40 were stored for 1 year at -70 degrees C in a buffered 0.8% saline solution containing 1% bovine plasma albumin (BPA). However, storage of the 69/104 preparation at -20 degrees C in either 0.1 or 1% BPA, or at -70 degrees C in the presence of 0.1% BPA showed a small but significant decrease (approximately 10%) in activity over the same period. It is, therefore, advantageous to store these reference preparations at -70 degrees C in the presence of 1% BPA.

The prostate of 4 mature pure bred Beagles 12 months old was studied 3 weeks following a single i.m. injection of 1 mg estradiol/kg body weight by means of histochemistry (acid and alcaline phosphatase) and electron microscopy. Four 11 months old Beagles served as controls. Estradiol leads to a variable reaction of glandular epithelium. There is an atrophy of active secretory cells, probably due to an inhibition of the release of ICSH by the anterior pituitary lobe, that in turn leads to a deficiency of androgens. The residual secretory function is not sufficient for normal synthesis of secretory granules, recognizable through the decrease in electron density of secretory granules and the extensive loss of activity of acid phosphatase. Under physiologic conditions it corresponds in its localization to the amount of secretory granules lying in the apical portion of the cytoplasm. The basal reserve cells show an ambivalence. Normally under the predominant influence of androgen they do not show any metaplasia, but they differentiate into the secretorely active epithelial cell. Without stimulation by androgens, estradiol leads to a basal cell proliferation with squamous metaplasia particularly in the dorso-lateral lobes close to the urethra. The activity of alcaline phosphatase shows a minor decrease in the capillary endothelium under estradiol. With increasing maturation of the metaplastic squamous epithelium the activity of alcaline phosphatase increases in the upper cell layer.

Improved caudatum sorghum cultivars have spread since their introduction in the late nineteen-eighties in the Kolokani area, Mali, being cultivated at the turn of the millennium by many farmers over hundred villages, under the name "Gadiabani". Being compact-panicled, these varieties are prone to damage by the panicle-feeding bug Eurystylus oldi. On-farm trials were conducted in 1998 and 1999 in respectively five and 16 villages of this area, to establish the status of castor bean (Ricinus communis) as a source of sorghum infestation by E. oldi, and evaluate the management of this alternate host plant, in combination with sowing date manipulation and use of host plant resistance, as potential control options for this pest. Experimental designs were split-plots with three factors. Both years, eight sorghum cultivars were planted in two dates (DOS). The third factor studied was vicinity with castor in 1998, and castor management in 1999. There were five replicates in 1998, and three in 1999. Head-bug numbers were recorded on maturing grains, and head-bug damage was visually rated at grain maturity. In 1998, infestation and damage by head-bugs on sorghum panicles in trials located close to castor plants were significantly higher than on those with no castor in their vicinity. In 1999, castor management either by insecticidal treatment or physical removal of flowering castor spikes on plants neighboring test plots, immediately before sorghum flowering, significantly reduced E. oldi populations on sorghum panicles. In both years, the effect of sowing date was not significant on head-bug population, while in 1998, head-bug damage scores were significantly higher on DOS1 than on DOS2. In 1998, the genotypic effect was significant for both parameters, the hybrid ICSH 89002 being the most infested and damaged genotype, while Malisor 84-7 and 94-EPRS-GII-1122 were respectively the less infested and the less damaged. In 1999, the genotypic effect was also significant, local guinea and head-bug resistant controls Bibalawili and Malisor 84-7 being the less infested cultivars, compared to ICSH 89002, ICSV 1063, Gadiabani and ICSV 1079, which suffered severe infestation, while 87W810 and 94-EPRS-GII-1122 had an intermediate ranking. These results definitely demonstrate the role played by castor as a significant source of sorghum infestation by head-bugs, and the prospect for reducing both infestation and damage, by management of this alternate host, in conjunction with use of host plant resistance and to a lesser extent planting date manipulation, in IPM programs.

A number of commercial rabbit tissue thromboplastins used in oral anticoagulant control have been calibrated against the first International Reference Preparation for thromboplastins. This was done in a three-stage procedure by one laboratory, each stage representing a different level of thromboplastin comparability. The calibration model recently recommended by ICTH and ICSH was tested. This model proved to be suitable, although a statistically significant aberration was observed for some of the thromboplastins. The bias introduced by using the model in these non-ideal cases was small compared to the overall variation of the International Normalized Ratio, being the universal scale for reporting the prothrombin time during oral anticoagulant control. Batch-to-batch calibration using lyophilized pooled plasmas could be reliably performed for several commercial thromboplastins.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), the aim of which is to provide hemostasis-related guidance documents for clinical laboratories. The current ICSH document was developed by an ad hoc committee, comprising an international collection of both clinical and laboratory experts. The purpose of this ICSH document is to provide laboratory guidance for (1) identifying hemostasis (coagulation) tests that have potential patient risk based on analysis, test result, and patient presentations, (2) critical result thresholds, (3) acceptable reporting and documenting mechanisms, and (4) developing laboratory policies. The basis for these recommendations was derived from published data, expert opinion, and good laboratory practice. The committee realizes that regional and local regulations, institutional stakeholders (e.g., physicians, laboratory personnel, hospital managers), and patient types (e.g., adults, pediatric, surgical, etc.) will be additional confounders for a given laboratory in generating a critical test list, critical value thresholds, and policy. Nevertheless, we expect this guidance document will be helpful as a framework for local practice.

A lot of attempts have been made to standardise both activated partial thromboplastin time (APTT) and prothrombin time (PT). Only the standardization of PT has been successfully implemented while the standardization of APTT is still underway. The PT test is a common method for monitoring oral anticoagulant therapy. Owing to the variable response of the thromboplastins and the different ways of reporting, PT results obtained from patients treated with oral anticoagulants have not been interchangeable between laboratories. In 1977, the World Health Organization (WHO) designed a batch of human brain thromboplastin as the first international reference preparation (IRP) for thromboplastin and a calibration system was proposed in 1982, based on the assumption that a linear relationship exists between the logarithm of the PT obtained with the IRP and test thromboplastins. This calibration model is used to standardize the reporting of the PT by converting the PT ratio observed with the local thromboplastin into an International Normalized Ratio (INR). The INR system is being adopted by an increasing number of hospitals in many countries. With the increasing use of the INR system, a number of problems have been identified with the INR system. The most serious one is that the ISI of a thromboplastin depends on the coagulometer used. Besides, a number of investigators have noted that the ISI value provided by the manufacturer for each new batch of thromboplastin reagent may be incorrect and the use of inappropriate control plasma can lead to erroneous INR calculations. Four solutions have been proposed to solve the problems of the INR system as follows: (a) the local system calibration with lyophilized plasma calibrants with assigned manual PT determined in terms of the relevant IRP for thromboplastin; (b) the use of a mean normal prothrombin time (MNPT) obtained with the coagulometer to derive the prothrombin ratio: (c) PT standardization by means of the procedure using plasma calibrants: and (d) selection of sensitive thromboplastin with low ISI values. The INR system has been adopted in Thailand since 1984. There are 3 steps in the implementation as follows: (a) preparation of National Reference Thromboplastin; (b) selection of high sensitive thromboplastin; and (c) optimal therapeutic range for Thai patients. The anticoagulant effect of heparin is usually monitored by the APTT, a test that is sensitive to the inhibitory effects of heparin on thrombin, factor Xa. and factor IXa. However, the type of clot detection system, the contact activator; and the phospholipid composition of the reagent affect the APTT response. In 1995. ISTH/ICSH proposed a calibration model for APTT standardization. As the problem showed a great similarity to PT standardization, the same model of calibration was applied but no international reference preparation for the APTT is yet available. In 1998. van den Besselaar et al proposed a lyophilized APTT reagent comprising synthetic phospholipids and colloidal silica as a candidate IRP for the APTT.

Background: Malaria is a life-threatening, multisystem disease caused by the plasmodial parasite with a global incidence of approximately 229 million annually. The parasites are known to have unique and crucial interactions with various body tissues during its life cycle, notably the liver, spleen, and recent work has shown the bone marrow to be a reservoir of infection.

Methods: This study is a case series of patients in whom examination of bone marrow revealed malarial parasites. A retrospective record review of 35 parasite-positive bone marrow specimens examined at Aga Khan University Hospital (AKUH), Karachi, Pakistan, over the years 2007 to 2015 was conducted. Bone marrow aspirates were collected as per International Council for Standardization in Haematology (ICSH) guidelines.

Results: The median age of patients was 22 years (range 1-75), and 60 % (n = 21) were male. 22 patients had evidence of Plasmodium falciparum, 12 had evidence of Plasmodium vivax and 1 patient had a mixed infection. Gametocytes and trophozoites were the most common stages identified on both peripheral blood and bone marrow examinations. Indications for bone marrow examination included fever of unknown origin and the workup of cytopenias and malignancies.

Conclusions: The incidental finding of Plasmodium in samples of bone marrow suggests the reticuloendothelial system may be regularly harbour these parasites, be the infection acute or chronic in character.

Keywords: Bone marrow; Malaria; Pakistan; Plasmodium; Reticuloendothelial.

BACKGROUND

The Sysmex DI-60 system (DI-60, Sysmex, Kobe, Japan) is a new automated digital cell imaging analyzer. We explored the performance of DI-60 in comparison with Sysmex XN analyzer (XN, Sysmex) and manual count.

METHODS

In a total of 276 samples (176 abnormal and 100 normal samples), white blood cell (WBC) differentials, red blood cell (RBC) classification and platelet (PLT) estimation by DI-60 were compared with the results by XN and/or manual count. RBC morphology between pre-classification and verification was compared according to the ICSH grading criteria. The manual count was performed according to the Clinical and Laboratory Standards Institute guidelines (H20-A2).

RESULTS

The overall concordance between DI-60 and manual count for WBCs was 86.0%. The agreement between DI-60 pre-classification and verification was excellent (weighted κ=0.963) for WBC five-part differentials. The correlation with manual count was very strong for neutrophils (r=0.955), lymphocytes (r=0.871), immature granulocytes (r=0.820), and blasts (r=0.879). RBC grading showed notable differences between DI-60 and manual counting on the basis of the ICSH grading criteria. Platelet count by DI-60 highly correlated with that by XN (r=0.945). However, DI-60 underestimated platelet counts in samples with marked thrombocytosis.

CONCLUSIONS

The performance of DI-60 for WBC differential, RBC classification, and platelet estimation seems to be acceptable even in abnormal samples with improvement after verification. DI-60 would help optimize the workflow in hematology laboratory with reduced manual workload.