Twenty-five monoclonal antibodies (MABS) to complement components were evaluated according to the ISBT/ICSH protocol by twelve laboratories. Seven detected some form of C3c, but one of them, 174, did not react with EiC3b, although it was positive with 'EC3b' (Fruitstone). 174 may detect some form of enzyme sensitive C3b antigen, but C3a was not evaluated (present on 'EC3b' Fruitstone). Twelve of the antibodies were anti-C3d, one was an anti-C3g and five were anti-C4c.

The leukocyte count, the differential leukocyte count and the erythrocyte sedimentation rate (ESR) are the more commonly used tests for diagnosing or managing an inflammatory process. Measurements of acute-phase proteins has an advantage over that of the leukocyte count and ESR. Especially microscopic examination of peripheral blood smear can be time consuming, but the simple and inexpensive technique is still clinically useful when a high grade bacteremia is likely to be present. Although the results are examiner dependent, it should be reliable in the proper clinical setting. In the guidelines for the selection of laboratory tests for monitoring the acute phase response, published in 1988, the International Committee for Standardization in hematology (ICSH) considered the biohazzard of ESR. Therefore the ESR should not routinely be performed on blood samples from patients who show a positive test for hepatitis virus or human immunodeficiency virus. The subcommittee for laboratory tests in daily care situations in Japan Society of Clinical Pathology published the "Essential Laboratory Tests" in 1989. We conclude that the differential leukocyte counts and the ESR should be used to follow the activity and response to treatment of certain inflammatory disorders when other objective indicators are not available.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune-mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.

Keywords: ADAMTS13; VWF; haemostasis; thrombosis.

METHODS

The operating performance of the Coulter Counter S Plus STKR was evaluated in two hospital laboratories in Rome and in Florence. Experimental design conformed to both the ICSH and NCCLS Standards for the evaluation of hematologic analyzers, and to the ECCLS guidelines for the multicenter evaluation of analyzers in clinical chemistry.

CONCLUSIONS

Cell counts in K3 EDTA were unchanged over 6 hours at room temperature and 72 hours at 4 degrees C, while MCV, MPV and leukocyte differentials were far less stable. Carry over, precision and linearity met the manufacturer's specifications, while a satisfactory relative accuracy was demonstrated by determining reference values on an adult reference group and by comparing the instrument with the previous model S Plus IV D. The accuracy of the leukocyte differentials was evaluated by the microscope reference method, and our results seemed to validate the hypotheses that the STKR model counts: i) eosinophils, basophils and banded neutrophils among GR; ii) variant lymphocytes among LY, and iii) various abnormal cells among mononuclear cells. However, in spite of this statistical significance, some difficulties in correctly classifying the mononuclear population were demonstrated.

BACKGROUND

The aim of this study was to perform a verification of the hematology analyzer Sysmex XN-2000 by comparing with the previous XE-5000. This study assessed the precision and carryover on the XN-2000 and the systematic error between the both counters according to desirable biological variability criterion and a flag comparison study.

METHODS

Within-run precision and between-batch precision were measured according to the ICSH guidelines. A comparative study was performed analyzing two hundred and six samples of peripheral blood from patients. The statistical study was conducted using the Passing-Bablok and Bland-Altman analyses. The leucocyte flag comparison was made by measuring the efficiency rate.

RESULTS

Between-batch precision was lower than that recommended by the biological variability criterion and manufacturer specifications. The comparison gave nonagreement results for neutrophil and basophil counts according to the criterion of biological variability. Erythroblasts and immature granulocytes showed nonagreement, but there is no available biological variation database for these parameters to compare with. Nevertheless, excellent absolute agreement was found for red blood cell parameters, and for platelet, lymphocyte, monocyte, and eosinophil counts.

CONCLUSIONS

The global results obtained for the precision, comparability, and efficiency provide a satisfactory integration of the XN-2000 in the core laboratory routine and accomplish an optimal reliability.

This paper describes the preparation and nature of the First International Standard for Human Urinary Follicle Stimulating Hormone and for Human Urinary Luteinizing Hormone, for Bioassay, and of two batches of working standard which were prepared from the same material. A collaborative study of these materials was carried out by six laboratories in six different countries. The FSH and LH activities of the Standard were assayed in terms of those of the Second International Reference Preparation of Human Menopausal Gonadotrophins, Urinary, for Bioassay, which it replaces. The results from 20 valid FSH assays and 30 valid LH assays (using four different methods) obtained in this way gave a weighted combined potency estimate for FSH of 53.7 IU, with 95 % fiducial limits of 47.2-61.1 IU, and for LH of 46.2 IU, with 95 % fiducial limits of 43.3-49.3 IU. Accelerated degradation studies of the Standard stored at elevated temperatures suggested that the stability of both FSH and LH activities under normal storage conditions would be satisfactory. The FSH and LH activities of the two batches of working standard (WS-A and WS-B) were compared with those of the Standard and were not found to differ significantly, except for the LH activity of WS-B which appeared to be slightly higher than that of the Standard. Accelerated degradation studies did not show any significant differences in stability between the Standard and batches of working standards. On the basis of these results the Standard has been established by WHO and allocated a potency for FSH of 54 IU per ampoule and for LH 46 IU per ampoule. The International Units for FSH, Human Urinary, for Bioassay and for LH (ICSH), Human Urinary, for Bioassay are thus defined as the activities contained in 0.1138 mg and 0.13369 mg of the International Standard, respectively.

According to the International Committee for Standardization in Haematology (ICSH), we determined the reference values for reticulocytes using an automated blood cell analyzer Technicon H*3 RTC in 200 healthy adult blood donors, aged between 17 and 60 years, 100 of whom were male and 100 female. The parameters included reticulocyte count, and its corpuscular indices; mean reticulocyte corpuscular volume (MCVr), mean reticulocyte corpuscular hemoglobin concentration (CHCMr), mean reticulocyte hemoglobin content (CHr), reticulocyte distribution width (RDWr), reticulocyte hemoglobin distribution width (HDWr) and reticulocyte corpuscular hemoglobin concentration distribution width (CHDWr). The reference ranges were established by setting the reference limits at two standard deviations from the arithmetic reference mean.

We estimated the measurement uncertainty (MU) of platelet concentration measured using the Sysmex XN system with two reference platelet counting methods described by DIN 58932-5 (PTB method) and the International Council for Standardization in Haematology (ICSH method). Ten blood samples were used to estimate and compare the MU of the XN system, and 30 samples were used to compare the methods. The standard uncertainty of the reference method was significantly higher for the ICSH method; the PTB method showed higher platelet concentrations than the ICSH method. When applying different methods with the XN system, optic counting showed higher MU compared to the other methods. There was good correlation among the two reference methods and three automated platelet-counting methods. We evaluated the MU in platelet concentrations measured using an automated hematology analyzer. Our results suggest that using the PTB method for calculating MU of the automated hematology analyzer is superior to the ICSH method because of its lower standard uncertainty.

BACKGROUND

We evaluated the measurement of length of sedimentation reaction in blood (LSRB) by TEST 1 and compared the results with those for the Westergren and Sed Rate Screener 100 (SRS 100) methods.

METHODS

LSRB was measured in 113 paired blood samples.

RESULTS

TEST 1 correlated significantly with the Westergren (r=0.94) and SRS 100 (r=0.90) methods with low bias (-0.29 and -1.92 mm/h, respectively) and limits of agreement (-14.5 to 13.9, and -23.4 to 19.6 mm/h, respectively). Hematocrit (Htc) correlated negatively with LSRB in TEST 1 (r=-0.54) and SRS 100 (r=-0.53) only in samples with high Htc >>/=35%). The bias and limits of agreement between TEST 1 and Westergren in samples with low (-1.46 and -22.3 to 19.3 mm/h) and high (0.43 and -7.29 to 8.14 mm/h) Htc were comparable to those between SRS 100 and Westergren (1.83 and -27.2 to 30.9 mm/h for low, 0.71 and -7.27 to 8.70 mm/h for high Htc samples). Total protein and fibrinogen correlated similarly with LSRB in both TEST 1 (r=0.23 and 0.48, respectively) and SRS 100 (r=0.30 and 0.51, respectively).

CONCLUSIONS

The findings suggested that TEST 1 is a reliable, precise and accurate system for measurement of LSRB in clinical laboratories with high workload.

BACKGROUND

The aim of this study was to present and compare the results of proposed methods for optimal red cell mass and plasma volume (RCM&PV) estimation, and their influence on the interpretation of obtained results.

METHODS

In 120/280 patients with polycythaemia rubra vera, subjected to RCM&PV determination with autologous erythrocytes in vitro labelled with 51Cr-sodium chromate, optimal volumes were determined using: 1. traditional ml/kg of: --the real body weight method (ml/kg RBW); --the optimal body weight method (ml/kg OBW). 2. the body weight, height, and sex based method (Retzlaff's tables), 3. the method recommended by the International Council for Standardization in Haematology (ICSH), based on body surface area.

RESULTS

Different interpretation of the same results of 120 RCM&PV measurements was registered in 48/120 patients (40%). The greatest disagreement existed between ml/kg RBW and ml/kg OBW methods (in 39/120 subjects, 32.5%). In underweight patients the ml/kg RBW method, and in overweight patients the ml/kg OBW method, offered better agreement with ICSH&Retzlaff's methods. The ml/kg RBW method disagreed with ICSH&Retzlaff's methods and ml/kg OBW in 25% and 19.2% of patients respectively. ICSH and Retzlaff's methods disagreed in 10/120 patients (8.3%). The ICSH method yielded significantly lower optimal volumes than Retzlaff's.

CONCLUSIONS

Three methods for optimal RCM&PV estimation lead to different interpretations of the same results of RCM&PV measurements with 51Cr-erythrocytes in 40% of patients. Two ml/kg body weight methods show greater disagreement in comparison with ICSH and Retzlaff's methods, which differ significantly. The ICSH method yields lower optimal values compared to Retzlaff's.

The Cell-Dyn 3500 is an automated haematology analyzer which quantitatively measures and computes haematological quantities including a full "five-part" white cell differential. It measures 22 parameters for erythrocytes, white blood cells and platelets, also giving the respective histograms. Evaluation of the Cell-Dyn 3500 was performed according to the International Committee for Standardization in Haematology (ICSH) norms, for a period of 5 weeks. A total of 1,235 samples were studied by comparison with the Coulter MaxM. The five-part white cell differential and the flagging system were estimated and compared with the smear examination of 506 samples, by four clinical pathologists trained in cytology. Good correlation was obtained within, between batches, and day-to-day, for the following parameters: red blood cells (RBC), haemoglobin (HGB), mean cell volume (MCV), white blood cells (WBC) and platelets (PLT). The accuracy was estimated (RBC, HGB, VGM, WBC and PLT) each day with three different levels of titrated controls with good results. The linearity was established for RBC, HGB, WBC and PLT. The results obtained were good. Carry-over studies were performed according to the Broughton method for the same parameters and the results were also good. Stability studies for the automatic parameters including the differential white blood cell count showed that these parameters were stable at 4 degrees C for 48 hours. At room temperature the stability was reduced to 7 hours. Agreement was good between the Cell-Dyn 3500 and the Coulter MaxM, for the automatic haemocytometric values. The comparative studies between the five-part white cell differential of the haematologic analyzer and the manual differential showed excellent results for neutrophils and lymphocytes, very good for monocytes and eosinophils. For the flag estimation two criteria were established, one based on the clinical significance and the other based on the alarm detection described in the analyzer manual. The specificity was good for both criteria. In general the sensibility was better for the second criteria. The Cell-Dyn 3500 has thus shown to be a good haematology analyser which greatly reduces the morphological examination of smears.

Two new erythrocyte pyruvate kinase (PK) variants with severe nonspherocytic hemolytic anemia are presented. These cases are both considered to be homozygous because of the consanguineous marriages in their parents. Their erythrocyte PK's were characterized by the recommended methods of the International Committee for Standardization in Haematology (ICSH). These two variants have been named PK Sendai and PK Shinshu. PK Sendai showed a high K0.5S (phosphoenolpyruvate), was remarkably inhibited by ATP, and was thermolabile, while PK Shinshu demonstrated remarkably low enzyme activity and required a high level of fructose 1,6-diphosphate for activation.

Testicular atrophy in myotonic dystrophy is caused by a primary and progressive atrophy of the seminiferous tubules. Endocrinological studies with synthetic LH(ICSH)-RH in two patients demonstrated the complete absence of FSH feed back mechanism. Glycogen content was markedly diminished or completely absent, as demonstrated histochemically, both in the morphologically intact and in damaged tubules. The endocrinological and morphological results both demonstrate that insufficiency of the Sertoli cells is the primary damage in the pathogenesis of testicular atrophy in myotonic dystrophy.

Results obtained with the original calibration procedure of Biggs and Denson obtained by one expert laboratory compare well with those obtained from the ICTH/ICSH collaborative study. The simplified calibration procedure described in 1975 should only be used for the assessment of inter-batch variability of a given brand of thromboplastin; for the calibration of unlike thromboplastins, the simplified procedure should be revised by using more than two abnormal plasmas, e.g. different plasmas representing seven levels of anticoagulation between international calibrated ratios (ICRs) from 1.5 to 4.5. The formula for the calculation for the proposed ICRs based on the calibration constant should be modified to allow for instances where the calibration line for dissimilar thromboplastins, fitted in the therapeutic range, does not pass through the origin of ratio 1,1.

BACKGROUND

The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study.

METHODS

Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate-labeled anti-CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers.

RESULTS

The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%.

CONCLUSIONS

The BEST flow cytometric method has a smaller intercenter CV and a smaller center-to-center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers.

Red cells from 5 healthy adults and an unstable mutant pyruvate kinase (PK), PK Maebashi, were stored for 12 months in a -80 degrees C freezer. With an exception of triosephosphate isomerase and phosphoglycerate kinase activities, normal red-cell enzyme activities remained essentially unchanged. Mutant PK was characterized by the methods recommended by the International Committee for Standardization in Haematology (ICSH). Normal control and PK Maebashi remained essentially unchanged in their characteristics after storage in a -80 degrees C freezer for 12 months. These results indicate that the red cells stored over a long term in a -80 degrees C freezer can be used for the study of unstable mutant PK.

Anion exchange microcolumn chromatography is the ICSH (1978) recommended procedure for the quantitation of Hb A2 and Hb S. The use of isoelectric focusing (IEF) followed by laser densitometry of the separated haemoglobin bands is evaluated as a quick and reliable method for quantitation for Hb A2 and Hb S. A paired comparison of 35 specimens, 25 normal and 10 with beta thalassaemia trait, were quantitated for Hb A2. The two groups were well separated by both procedures and Hb A2 levels were similar (r = +0.93, P less than 0.001). In addition, paired analyses of 30 specimens, 20 sickle cell trait and 10 with sickle cell trait combined with alpha thalassaemia, were quantitated for Hb S. The two groups were also well separated by both procedures and Hb S levels were similar (r = +0.94, P less than 0.001). In conclusion, IEF followed by laser densitometry appears to be a reliable, quick procedure for the screening of populations at risk from beta thalassaemia and populations at risk from sickle cell trait, with or without alpha thalassaemia interaction.