Standardization of the thromboplastin most commonly used for anticoagulant control in The Netherlands has been achieved by the Dutch Reference Laboratory for Anticoagulant Control. The system established for control indicates the performance characteristics of each batch of thromboplastin in patients relative to a national calibration batch. Calibration constants were assessed according to an ICTH/ICSH proposal and with a reproducibility of approximately 2% (CV). With the use of a given calibration constant of the International Reference Preparation of Thromboplastin (1.0) and after assessment of the calibration constant of the National Reference Preparation in international terms (0.93), the accuracy of calibration with the various modifications of the calibration procedure is of the order of magnitude of 3% (CV). On the basis of the calibration protocol, physicians will be provided with correlation tables containing, for their convenience, prothrombin (thromboplastin) times in terms of the reference thromboplastin (uniform, i.e., batch-independent, prothrombin times), conventional prothrombin activity (percentages), and in due course with International Calibrated Ratios. With this approach current practice in the prescription of oral anticoagulants will be maintained. Opportunity is given, however, to aim at internationally proposed therapeutically optimal target values.

Mithun (Bos frontalis) is a unique domestic free-range bovine species available in North Eastern hilly (NEH) regions of India and is reared for its protein rich meat. Mithun suffers severe non-cyclical population fluctuations; however, it is not yet declared as endangered species. Mithun follows some sort of seasonality based on the calving trend and semen production, although it is a perennial breeder. However, there is need to study the rhythmic changes of endocrine profiles to understand the hormone flow pattern in mithun to select the suitable time for blood collection to assess the exact endocrine profiles and to select the suitable time for natural breeding or semen collection by artificial methods for further research, conservation and propagation of its germplasm. Therefore, the present study was designed to evaluate the reproductive as well as metabolic endocrinological profiles in 24:00 h in intact adult mithun bulls during different seasons (winter, spring, summer and autumn) to know the rhythmic changes and flow pattern of the endocrinological profiles to improve its reproductive efficiency. Experimental mithun bulls (n = 6; age: 5-6 years; body condition score: 5-6 out of 10, classified as good) were selected for the study. Endocrinological profiles, follicle stimulating hormone (FSH), luteinizing hormone/interstitial cell stimulating hormone (LH/ICSH), testosterone, cortisol, thyroxine (T4), insulin like growth factor-1 (IGF-1), prolactin and melatonin (MT) were estimated at 04:00 h interval for one whole day in four seasons. The analysis was completed in two different ways as different times of collection (08:00, 12:00, 16:00, 20:00, 24:00 and 04:00 h) and day (08:00 to 16:00 h) & night time (20:00 to 04:00 h) collections. Repeated measures ANOVA analysis revealed that the bulls in winter and spring had significantly (p < 0.05) higher FSH, LH, testosterone, T4, IGF-1 and MT than those in summer whereas the bulls in summer had significantly higher cortisol and prolactin than those in winter and spring seasons. Similarly FSH, LH, testosterone, T4, IGF-1 and MT were significantly (p < 0.05) higher in night than in day time collections whereas cortisol and prolactin were significantly (p < 0.05) higher in day than in night time collections in different seasons. Correlation analysis revealed that FSH, LH, testosterone, T4, IGF-1 and MT had significant (p < 0.05) positive correlation with each other whereas these had significant (p < 0.05) negative correlation with cortisol and prolactin. The study concludes that season and time of blood collection had significant effect on the endocrinological profiles in mithun bulls. Estimation of FSH, LH, testosterone, T4, IGF-1 and MT during night time and cortisol and prolactin during day time was more appropriate to get correct value of the endocrinological profiles. Spring and winter have significantly greater beneficial effects than summer on reproduction and artificial breeding programs in mithun species in the semi-intensive management in the present location.

Keywords: 24-H profiles; Day and night; Endocrinological profiles; Mithun; Seasonality.

An automated method without dialysis for determining serum iron is described. It is an adaptation of the Lauber procedure to the Technicon Auto Analyser I: serum iron is split from its protein combination by exposure to an acetate pH 5.8 buffer, in the presence of a detergent (Teepol 710) which prevents any precipitation of proteins. Iron is maintained in ferrous state by addition of sodium dithionite and is quantitated with bathophenanthrolin colorimetry. Serum samples are analysed at a rate of 60/H. It is necessary to run a blank for each serum sample. Between 20 and 500 mug iron/100 ml the color intensity agrees with Beer's law. The reproducibility is good, and the variation coefficient less than 1.2 per cent. Our results are not significatively different of those obtained with the ICSH standard method. Bilirubin does not interfere. Iron added is satisfactorily recovered. Our method can also be used for determining the transferrin iron binding capacity.

Mithun (Bos frontalis) is a unique domestic free-range bovine species available in North Eastern hilly (NEH) regions of India and is reared for its protein rich meat. Although, mithun suffers from severe non-cyclical population fluctuations, it is not yet declared as endangered. Therefore, present study was conducted to assess the effect of slow release subcutaneous exogenous melatonin (MT) implant on sexual behaviour, scrotal circumference (SC) and testicular parameters, endocrinological profiles and antioxidant and oxidative stress profiles in mithun during different seasons (winter, spring, summer and autumn) to improve its reproductive efficiency. Experimental mithun bulls (5-6 years of age) were selected and divided randomly into two groups, Gr I: Control (n = 6) and Gr II: Treatment (n = 6; melatonin implant @ 18mg/50 kg B. Wt). Scrotal circumference and testicular parameters [total testicular volume and weight], endocrinological profiles [follicle stimulating hormone (FSH), luteinizing hormone/interstitial cell stimulating hormone (LH/ICSH), testosterone, thyroxine (T4), cortisol, prolactin and melatonin (MT)], sex behaviour profiles [libido score, mating ability score and sexual behaviour score], antioxidant profiles [total antioxidant capacity (TAC), catalase (CAT), glutathione (GSH), glutathione reductase (GSHRx) and superoxide dismutase (SOD)] and oxidative stress profile [malondialdehyde (MDA)] were estimated. Statistical analysis revealed that these experimental profiles differed between treatment and control groups within the seasons and among the seasons within the experimental groups. FSH, LH, testosterone, T4 and MT were higher and cortisol and prolactin were lower in spring and winter than in summer season. Concentration of FSH, LH, testosterone and MT were higher and concentration of T4, cortisol and prolactin were lower in MT treated than in untreated control group. Similarly, sexual behavioural scores, SC, testicular parameters and antioxidant profiles were higher and oxidative stress profile was lower in MT treated than in untreated control group and in winter and spring than in summer season. The study concluded that exogenous slow-release melatonin implantation and spring and winter seasons had significant beneficial effects in improvement of the antioxidant profiles, minimization of oxidative stress with cascading beneficial effects on endocrinological profiles, SC, testicular parameters and libido, which will improve the semen production, higher sperm cryo-survivability and fertility rate in mithun species.

Keywords: Antioxidant; Endocrinological profiles; Melatonin; Mithun; Scrotal circumference; Sexual behaviour; Testicular parameters.

Following a short introduction to the physiology and function of gonads in men, the authors present their material and exploration methods. the study covered 39 patients with azoospermia of obstructive origin. The obstruction of v. deferens developed in 29 patients after a non-specific inflammatory reaction, in 7 patients after a trauma, in 2 patients after vasectomy, and one patient was a case of congenital anomaly. Concentrations of FSH and ICSH in the plasma of the patients studied did not show any major deviations. Histological analysis showed normal morphological spermatids and primary spermatocytes with degenerative changes in later stages in 29 patients. In the remaining 10 patients, most probably due to the inflammatory reaction and as the result of congenital anomaly, the destruction of the germinal epithelium was evident. Spermagglutinins in the serum of the patients were detected in 56.8%. Such a high percentage of antispermatic agglutinins is meant to be the result of obstruction and the presence of the inflammatory agent.

OBJECTIVE

To define the prevalence of acquired red cell enzymopathy in leukemia and MDS patients and explore its clinical significance.

METHODS

Red cell enzymes (G6PD, 6PGD, PK, ENOL, ADA, PNP,ALD) activities and PK,ADA isoenzymes were assayed by the methods recommended by ICSH and PAGE electrophoresis.

RESULTS

The prevalence rate of G6PD deficiency was 43.1%, and of PK deficiency was 27.8%, in leukemia and MDS patients. The prevalence rates of PK, G6PD and PNP deficiency in MDS patients were 50%, 48% and 39.1%, respectively, while of ADA, ENOL and ALD over production in MDS patients were 69. 6%, 40% and 30. 4%, respectively. PK activity was increased in CML, while the PK isoenzyme pattern was normal. Administration of antitumor antibiotics to AL decreased G6PD and 6PGD activities (P < 0.05). ADA2 was expressed in MNC of AML (M4 and M5) and in RBC of CMML, but not expressed in ALL (P < 0.001).

CONCLUSIONS

Determination of enzymes may be used as prognostic parameters in CML,a predictor of antitumor drug resistance in acute leukemia, and a differential parameter between MDS-RA and aplastic anemia and between CML and CMML.

Measurement of the erythrocyte sedimentation rate (ESR) using a closed tube system reduces the biohazard risk to laboratory staff. The Diesse Ves-matic system offers manual or vacuum collection of blood into plastic tubes, automated mixing of the sample, and automated reading of the end point after 20 minutes of sedimentation. This system was compared with the 1977 Westergren ESR method of the International Council for Standardization in Haematology (ICSH) and with the 1988 ICSH undiluted ESR method. Manually collected Ves-matic samples showed good agreement with ICSH values, although there was a tendency to false low results at low ESR values which may represent dilution of plasma protein with excess citrate. Vacuum collected Ves-matic samples also showed good agreement with ICSH values, although there was a tendency to false high results which may reflect a change in the blood: citrate ratio caused by loss of anticoagulant diluent or vacuum from plastic tubes during storage. The Diesse Ves-matic system incorporates several improvements over previous technology and offers a safer, quicker, and more standardised ESR.

This guidance document has been prepared on behalf of the International Council for Standardisation in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for collection of blood samples for coagulation tests in clinical laboratories throughout the world. The following processes will be covered: ordering tests, sample collection tube and anticoagulant, patient preparation, sample collection device, venous stasis before sample collection, order of draw when different sample types need to be collected, sample labelling, blood-to-anticoagulant ratio (tube filling) and influence of haematocrit. The following areas are excluded from this document, but are included in an associated ICSH document addressing processing of samples for coagulation tests in clinical laboratories: sample transport and primary tube sample stability; centrifugation; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport and storage; and preanalytical variables for platelet function testing. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Keywords: ICSH; coagulation; sample collection.

The ICTH/ICSH collaborative study failed to achieve its aims because each combination of plasma, thromboplastin and method was tested by too few laboratories in relation to intra and inter laboratory variation, and because the stability of the reference thromboplastin 70/178 is suspect. The evidence of the study does not support the superiority of reference thromboplastins over reference plasmas, but the concept of reference plasma was not adequately explored.

Background: Manual microscopic examination is the gold standard of humoral cell count test. However, it has some limitations and cannot fully meet clinical needs. Compared with the manual method, the automatic blood cell analyzer has the advantages of a high degree of automation, minimal error, high speed, high precision, and easy standardization. This study intends to verify the detection performance of the body fluid model of the Mindray BC-6000PLUS automatic hematology analyzer.

Methods: This study was performed in accordance with the International Committee for Standardization in Haematology (ICSH) Hematology Analyzer Evaluation Guide (version 2014) and the requirements of WS/T662-2020 "Clinical humoral examination technique". The humoral white blood cell-body fluid (WBC-BF), humoral red blood cell-body fluid (RBC-BF), monocyte (MN), polymorphonuclear (PMN) were measured to verify the performance indicators of the instrument, including background counting, intrabatch precision, accuracy, carrying contamination rate, and linear range. Referring to the WS/T514-2017 (Establishment and verification of detection capability for clinical laboratory measurement procedures), the limit of blank (LoB) and limit of detection (LoD) values of WBC-BF and RBC-BF in the humoral mode of the instrument were established.

Results: The blank count of WBC-BF and RBC-BF, and contamination of Mindray BC-6000PLUS analyzer were zero; the coefficient of variation (CV) of intra-batch precision at different levels of each item was less than 10%. There was a high correlation between instrument test results and manual microscopic examination results (r>0.95). The linear range of the instrument was wide, and the linear verification parameters was good (R²>0.999). The LoB value and LoD value of WBC-BF established by the instrument were 0×109 /L and 0.004×109 /L, respectively. The LoB value and LoD value of the RBC-BF established by the instrument were 0×1012/L and 0.004×1012/L, respectively. The lower detection limits of WBC-BF and RBC-BF were set as 0.004×109 /L and 0.004×1012/L, respectively.

Conclusions: All performance indicators of the Mindray BC-6000PLUS automatic blood analyzer met the requirements of the manufacturer's criteria. This instrument can fulfill the requirement of body fluid sample routine test in clinical practice.

Keywords: WS/T662-2020; humoral mode; performance evaluation (PE).

Introduction: The aims of study were to assess: 1) performance specifications of Atellica 1500, 2) comparability of Atellica 1500 and Iris, 3) the accuracy of both analysers in their ability to detect bacteria.

Materials and methods: Carryover, linearity, precision, reproducibility, and limit of blank (LoB) verification were evaluated for erythrocyte and leukocyte counts. ICSH 2014 protocol was used for estimation of carryover, CLSI EP15-A3 for precision, and CLSI EP17 for LoB verification. Comparison for quantitative parameters was evaluated by Bland-Altman plot and Passing-Bablok regression. Qualitative parameters were evaluated by Weighted kappa analysis. Sixty-five urine samples were randomly selected and sent for urine culture which was used as reference method to determine the accuracy of bacteria detection by analysers.

Results: Analytical specifications of Atellica 1500 were successfully verified. Total of 393 samples were used for qualitative comparison, while 269 for sediment urinalysis. Bland-Altman analysis showed statistically significant proportional bias for erythrocytes and leukocytes. Passing-Bablok analysis for leukocytes pointed to significant constant and minor proportional difference, while it was not performed for erythrocytes due to significant data deviation from linearity. Kappa analysis resulted in the strongest agreements for pH, ketones, glucose concentrations and leukocytes, while the poorest agreement for bacteria. The sensitivity and specificity of bacteria detection were: 91 (59-100)% and 76 (66-87)% for Atellica 1500 and 46 (17-77)% and 96 (87-100)% for Iris.

Conclusion: There are large differences between Atellica 1500 and Iris analysers, due to which they are not comparable and can not be used interchangeably. While there was no difference in specificity of bacteria detection, Iris analyser had greater sensitivity.

Keywords: Atellica 1500 analyser; Iris analyser; urine; verification.

In 2018, the International Council for Standardization in Hematology (ICSH) published a consensus document providing guidance for laboratories on measuring direct oral anticoagulants (DOACs). Since that publication, several significant changes related to DOACs have occurred, including the approval of a new DOAC by the Food and Drug Administration, betrixaban, and a specific DOAC reversal agent intended for use when the reversal of anticoagulation with apixaban or rivaroxaban is needed due to life-threatening or uncontrolled bleeding, andexanet alpha. In addition, this ICSH Working Party recognized areas where additional information was warranted, including patient population considerations and updates in point-of-care testing. The information in this manuscript supplements our previous ICSH DOAC laboratory guidance document. The recommendations provided are based on (1) information from peer-reviewed publications about laboratory measurement of DOACs, (2) contributing author's personal experience/expert opinion and (3) good laboratory practice.

In 2012, the International Council for Standardization in Hematology (ICSH) published recommendations for the identification, quantitation, and diagnostic value of schistocytes. In the present review, the impact of these recommendations is evaluated. This work is based on citations in peer-reviewed papers published since 2012. The first 2012 ICSH Recommendations have also been revised to incorporate newly published data in the literature and current best laboratory practice. Recommended reference ranges have been proposed for healthy adults and full-term neonates of 1% or less schistocytes. More than 1% of morphologically identified schistocytes on the blood film are considered suspicious for thrombotic microangiopathy. For preterm infants, a normal level of 5% or less is recommended. The fragment red cell count (FRC) generated by some automated hematological analyzers provides a valuable screening tool for the presence of schistocytes. Specifically, the absence of FRCs can be used as a valuable parameter to exclude the presence of schistocytes on the blood film. The validity and usefulness of microscope schistocytes and automated FRCs, respectively, are discussed in the context of the laboratory diagnostic tests used for thrombotic microangiopathies.

Keywords: cytomorphology; peripheral blood film; red blood cell fragments; schistocytes; thrombotic microangiopathy.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Keywords: ICSH; coagulation; sample processing.

Efficiency and accuracy in automated hematology analyzers are very important for clinical laboratories. The purpose was to evaluate the flags and results reported by the newest Beckman Coulter analyzer DxH 900 compared to the Sysmex XN20 system. Samples were analyzed on the XN20 (Sysmex, Kobe, Japan) and on the Beckman Coulter DxH 900 (Beckman Coulter, Miami, Florida, USA). Slide reviews were performed microscopically. Morphologic criteria were used to identify abnormal cells as recommended by International Consensus Group for Hematology (ICSH): blasts, immature granulocytes (IG%), abnormal lymphocytes (ALs) and plasma cells. Results: there was a strong correlation between the analyzers in almost all clinical parameters tested. Both DxH 900 and XN20 showed an excellent degree of association for the leukocyte differential compared to the reference method (manual microscopy). When it comes to IG%, XN20 showed a positive bias for higher results. Related to platelets, there are no differences between the two methods for PLT count. For mean platelet volume (MPV), DxH 900 provided 100% results of the samples analyzed while XN20 while in the XN20 analyzer, 16% of the results were missing. From our results we came to the conclusion that both analyzers, DxH 900 and XN20 were clinically accurate and efficient. Abnormal Lymphocyte detection highlighted the differences between the two technologies as only minimal agreement was obtained. DxH 900 demonstrated higher sensitivity in detecting IG with good correlation with microscopic review. The DxH 900 for platelet clumps identification provides an excellent flag (PLT Clumps) with the highest sensitivity observed in our evaluation.

Keywords: Beckman Coulter DxH 900; Sysmex XN20; cell blood count (CBC); leukocyte differential.