BACKGROUND

To provide target values for the manufacturers' survey of the Japanese Society for Laboratory Hematology (JSLH), accurate standard data from healthy volunteers were needed for the five-part differential leukocyte count. To obtain such data, JSLH required an antibody panel that achieved high specificity (particularly for mononuclear cells) using simple gating procedures. We developed a flow cytometric method for determining the differential leukocyte count (JSLH-Diff) and validated it by comparison with the flow cytometric differential leukocyte count of the International Council for Standardization in Haematology (ICSH-Diff) and the manual differential count obtained by microscopy (Manual-Diff).

METHODS

First, the reference laboratory performed an imprecision study of JSLH-Diff and ICSH-Diff, as well as performing comparison among JSLH-Diff, Manual-Diff, and ICSH-Diff. Then two reference laboratories and seven participating laboratories performed imprecision and accuracy studies of JSLH-Diff, Manual-Diff, and ICSH-Diff. Simultaneously, six manufacturers' laboratories provided their own representative values by using automated hematology analyzers.

RESULTS

The precision of both JSLH-Diff and ICSH-Diff methods was adequate. Comparison by the reference laboratory showed that all correlation coefficients, slopes and intercepts obtained by the JSLH-Diff, ICSH-Diff, and Manual-Diff methods conformed to the criteria. When the imprecision and accuracy of JSLH-Diff were assessed at seven laboratories, the CV% for lymphocytes, neutrophils, monocytes, eosinophils, and basophils was 0.5~0.9%, 0.3~0.7%, 1.7~2.6%, 3.0~7.9%, and 3.8~10.4%, respectively. More than 99% of CD45 positive leukocytes were identified as normal leukocytes by JSLH-Diff.

CONCLUSIONS

When JSLH-Diff method were validated by comparison with Manual-Diff and ICSH-Diff, JSLH-Diff showed good performance as a reference method.

Proteolytic enzyme preparations and techniques used routinely in blood group serology for the detection of atypical patient antibodies prior to transfusion vary widely and are often poorly standardised. Recent advances have been made in the use of biochemical methods to standardise and stabilise the potency of the enzyme preparations used. A joint working party of the International Council for Standardization in Haematology (ICSH) and the International Society of Blood Transfusion (ISBT) has investigated possibilities for the provision of standards for the protease preparations and techniques. The specification for these standards was that the performance of enzyme reference preparation in the reference technique should be of equivalent sensitivity to the ICSH/ISBT LISS spin indirect antiglobulin test using a titration series of a reference weak anti-D, and be free from false-positive reactions. The working party circulated materials for evaluation in inter-laboratory trials, followed by a laboratory workshop meeting to achieve agreement on the specification for reference materials and methods. Reference freeze-dried papain at 0.6 azoalbumin units and weak anti-D preparations (91/562) have been prepared and validated to meet these specifications. The performance of a test enzyme preparation in the technique for which it is recommended for use should be at least equal to that of the reference papain preparation, by the reference two-stage technique in terms of sensitivity, using a titration series of the reference anti-D, and freedom from false-positive reactions, using six fresh inert sera. The reference papain and weak anti-D can also be used to calibrate the level of proteolytic activity required in other procedures in blood group serology, such as new technology methods for antibody detection, and automated and microplate cell grouping procedures. These preparations and an agreed method for their use are now available from listed centres as ICSH/ISBT and Food and Drug Administration reference materials.

Background: Isolated chronic subdural hematoma (ICSH), as a special rare species, has great controversy over its treatment. A retrospective analysis was performed to compare craniotomy with endoscopic-assisted trepanation drainage (EATD) of ICSH.

Methods: The data of ICSH patients for craniotomy or EATD from January 2011 to April 2019 were retrospectively collected and analysed. Of 106 patients, 49 and 57 patients received craniotomy and EATD treatment respectively. Recurrence rate, morbidity and mortality rate were the main outcome.

Result: There was no recurrence in both groups. The morbidity rate of the EATD group (2/57, 3.5%) was significantly lower than that of the craniotomy group (17/49, 34.7%, p = 0.0033). There was no death in the EATD group, but 3 cases died of operative produce in the craniotomy group. The average operation time of the craniotomy group (95.3min) was significantly longer than that of the EATD group (66.5min, P = 0.0032). Craniotomy group had more intraoperative blood loss (213.2ml) than EATD group (34.5ml, P = 0.0044). EATD patients had shorter hospital stay and recovered faster.

Conclusions: Compared with craniotomy, EATD is a more effective and safer method for the treatment of ICSH.

Keywords: Chronic subdural haematoma; Craniotomy; Endoscope; ICSH; Trepanation drainage.

Using recently established ICSH recommended methods, red cell pyruvate kinases (PK) of 20 patients with PK deficiency were characterized and 7 new PK variants were found. Analysis of partially purified red cell pyrimidine 5'-nucleotidase (P5N) from a patient with P5N deficiency provided the evidence for a structural alteration of the enzyme protein. Red cell adenosine deaminase (ADA) from a patient with 40-fold increase in ADA activity associated with hemolytic anemia was purified and compared with that from normal subjects. It is most conceivable that the increased ADA activity represents increased amount of structurally normal enzyme.

Before a new method is used for clinical testing, it is essential that it is evaluated for suitability for its intended purpose. This document gives guidance for the performance, verification and implementation processes required by regulatory and accreditation bodies. It covers the planning and verification of specialist haemostatic tests, including factor assays, D-dimers, direct anticoagulants and thrombophilia testing.

Keywords: assays; haemostasis; laboratory automation; precision.

The fourth ISBT/ICSH platelet serology workshop took place in 1988. It consisted of a wet workshop carried out by the 17 participating laboratories during March 1988, followed by a discussion of the results at a meeting of the ISBT/ICSH Expert Panel on Platelet Serology at the ISBT/BBTS Congress in London in July 1988. Sixteen samples, including 1 negative control serum, were analysed by the participants using methods of their choice. There was general agreement on 10/16 samples. The results indicated progress in the following areas: (i) antibody identification; (ii) differentiation of platelet-specific and HLA antibodies; (iii) glycoprotein localisation of platelet specific antigens; (iv) identification of new platelet antigen systems.

The erythrocyte sedimentation rate (sedimentation rate, sed rate, and ESR for short) is a common hematology test that may indicate and monitor an increase in inflammatory activity within the body caused by one or more conditions such as autoimmune disease, infections or tumors. The ESR is not specific for any one disease but is used in combination with other tests to determine the presence of increased inflammatory activity. The ESR has long been used as a "sickness indicator" due to its reproducibility and low cost. Over many decades, several methods have evolved to perform the test. However, the reference method for measuring the ESR proposed by the International Committee for Standardization in Haematology (ICSH) is based on the findings described by Westergren a century ago. Newer automated systems using closed blood collection tubes and automatic readers have been introduced into laboratories to decrease the biohazardous risk to operators and to decrease the time that it takes to perform the ESR. The Westergren method measures the distance (in millimeters) at which red blood cells in anticoagulated whole blood fall to the bottom of a standardized, upright, elongated tube over one hour due to the influence of gravity. The tube used for the test is called the Westergren tube. Today, these tubes are made of either glass or plastic, with an internal diameter of 2.5 mm and lengths of 190 to 300 mm long. Perhaps the first to notice a change in the sedimentation of blood due to illness was a British surgeon John Hunter (1728–93) in his posthumous publication A Treatise on the Blood, Inflammation, and Gun-Shot Wounds. A Polish physician, Edmund Faustyn Biernacki (1866–1911), later refined the clinical use of the ESR near the end of the 19 century. Biernacki detailed his findings in 2 articles in 1897 (the Gazeta Lekarska in Poland and the Deutsche Medizinische Wochenschrift in Germany), and he developed his test for measurements. These findings were not widely propagated in the English speaking medical communities. Because of his work, the ESR is occasionally referred to as the Biernacki Reaction world-wide. The applied use of ESR in clinical diagnostics by Biernacki was furthered refined by Dr. Robert Fahraeus in 1918 and by Dr. Alf Vilhelm Albertsson Westergren in 1921. Dr. Westergren defined the standard measurement of the ESR that is still in use today. Together, Robert Fahraeus and Alf Vilhelm Albertsson Westergren are often remembered for the test, historically called the Fahraeus-Westergren test (FW test or Westergren test), which uses a standardized tube and sodium citrate anticoagulated blood. The Westergren method for measuring the ESR proposed by the International Committee for Standardization in Haematology (ICSH) has allowed reproducibility for almost a century. Over time, the use of this same method has established comparable reference values within the same laboratory and even between different facilities across the globe. The Westergren method was adopted as the gold standard for ESR measurement in 1973 by the ICSH. Even after the advent of automated machines used for the analysis of the ESR, the Westergren method was still confirmed as the gold standard in 2011 by both the ICSH and by the Clinical and Laboratory Standards Institute (CLSI).

Pyruvate kinase (PK) from four patients with moderate to severe congenital non-spherocytic haemolytic anaemia was characterized by methods recommended by the ICSH. The possibility that two of the patients are true homozygotes cannot be ruled out, while the other two apparently represent double heterozygotes. All but one had levels of PK activity between 44 and 65% of normal. The variant enzymes were designed 'PK Pontos', 'PK Macedonia', 'PK Athens' and 'PK Larisa'. Multiple physicochemical as well as kinetic aberrations were detected in the above variants. Their altered kinetic behaviour is discussed in terms of the concerted transition model for allosteric enzymes and their abnormal properties are compared with other known variants, while it is also attempted to correlate them with possible mechanisms resulting in chronic haemolytic anaemia.

Seven new pyruvate kinase (PK) variants were characterized by the recently established recommended methods by the International Committee for Standardization in Haematology (ICSH). The seven cases were all true homozygotes as evidenced by consanguineous marriages of the parents. All of them were Japanese. These variants were designated PK Tokyo, Nagasaki, Sapporo, Maebashi, Itabashi, Fukushima, and Aizu. Low substrate affinity for phosphoenolpyruvate and thermal instability seem to play a major role in causing clinical manifestation of chronic hemolysis. Product inhibition of PK by ATP may also play an additional role in some cases.

A red-cell pyruvate kinase (PK) variant from an individual with congenital non-spherocytic haemolytic anaemia was characterized according to the procedure recommended by the International Committee for Standardization in Haematology (ICSH 1979). Activity of the mutant enzyme in haemolysates was one fifth of that found in normal control subjects. The electrophoretic mobility and thermostability of the mutant enzyme were lower than those of the normal enzyme and its apparent affinity for phosphoenolpyruvate was higher.

METHODS

The clinical and biochemical aspects of two cases of PK deficiency associated with chronic hemolytic anemia in two unrelated patients are reported. The residual erythrocyte PK enzymes in both patients were characterized by the recommended methods of the International Committee for Standardization in Hematology (ICSH).

CONCLUSIONS

Patient (W.Q.) had 60% residual PK activity and may be considered homozygous on the basis of consanguinity in the family. This patient suffered from moderate hemolytic anemia that improved after splenectomy. The enzymatic properties were: low activity, moderate thermal stability, reduced affinity for phosphoenol-pyruvate (PEP), and normal electrophoretic mobility. Patient (E.O.) had 42% residual PK activity and may be considered compound heterozygous since his parents are not related. He suffered from moderate hemolytic anemia. The enzymatic properties were: low activity, moderate thermal stability, reduced affinity for PEP and minimal retardation in electrophoretic migration. Theses two cases of PK deficiency are the first to be discovered in Jordan and probably the first in any Arab country.

The glycyl glycine dipeptidase activity under the in vitro influence of two follicle-stimulating hormone preparations of different concentration was investigated in 36 ovaries. A significant impediment was observed under Folistiman, whereas under Anthrogon the impediment could only be detected with higher concentrations. The FSH/ICSH relation evidently plays an important role in this respect. The results are in support of the statement that the glycyl glycine activity in vivo also depends on the gonadotrophin production. At the same time they affirm the fact that not only endopeptidases play a role in the hormone metabolism of the ovary.

The automated Sysmex M-2000 was evaluated according to the ICSH (International Committee for Standardization in Haematology) protocol. After dilution of packed cells with cell-free plasma, blood cell counts were linear. The overall precision of the measured parameters was good; the CV's ranged between 0.64% and 2.06%. The carry-over was negligible; platelets showed the biggest carry-over with 0.25% in the Whole Blood Mode, while red blood cells (RBC) showed a carry-over of 0.55% in the Prediluted Mode. 300 clinical samples were measured on the Sysmex M-2000 and the Sysmex CC-700 with PL-100, and the results were compared. The coefficients of correlation for white blood cells (WBC), red blood cells (RBC), haemoglobin and haematocrit were greater than 0.99; platelets showed an r of 0.982. Comparison of the results from the Sysmex M-2000 trimodal leukocyte distribution with a manual 100 cell differentiation showed a close correlation for lymphocytes (r = 0.948), and neutrophils (r = 0.931). The middle cell fraction corresponding to monocytes, eosinophils and basophils showed a correlation with r = 0.703. Pathological samples showed no interference with the blood count. Leukocyte counts less than 1000 x 10(9)/l did not effect the measurement of haemoglobin. During the period of evaluation, no instrument malfunctions occurred. Because of its precision and reliability, the Sysmex M-2000 is well suited for routine work and stat analysis in medium-sized laboratories.

We report a new potentiometric method for determining pyruvate kinase (PK). Enzymatic activity is measured by monitoring the change in pH produced in the reaction buffer under International Committee for Standardization in Haematology (ICSH) standardized assay conditions, and the lactate dehydrogenase reaction is automatically subtracted in each measuring cycle. The analysis, performed at 37 degrees C, requires a 10-microL sample (isolated erythrocytes or whole blood) and is completed in 2.5 min. The intra-assay CV is < 4% (PK between 3 and 35 U/g Hb); the interassay CV is 4.0% (PK 15 U/g Hb); results are linear from 3 to 30 U/g Hb. A good correlation with the ICSH reference method (x) was found: y = 1.011x - 0.05; n = 32; r = 0.9939; Sylx = 0.75 (units: U/g Hb). The reference intervals of the PK activity in isolated erythrocytes (RBC-PK) were estimated in 89 normal subjects. We found that women possess a higher RBC-PK than do men (P < 0.0001) and that the biological variability (CVb) of RBC-PK is 13.5%. Applications of the proposed method to the hematological routine are reported.

An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti-IgG and anti-C3 (anti-C3c and anti-C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2-ml freeze-dried aliquots from seven assigned distribution centres.

BACKGROUND

The analyzed values in the ICSH reference method for serum iron analysis are affected by non-transferrin(Tf)-bound iron such as ferritin. Also, non-Tf-bound plasma iron (iron citrate) is present in iron-overloaded specimens from patients with hemochromatosis, which was measured as serum iron in previous methods. We developed a specific determination method for serum transferrin-bound iron (serum t Fe) by high-performance liquid immunoaffinity chromatography (HPLAC), and compared it with the ICSH method and a fully automated (FA) method.

METHODS

Tf and t Fe were isolated from interferents in serum by HPLC using an immunoaffinity column. The concentration of t Fe isolated was determined by a colorimetric reaction using a highly sensitive chromogen.

RESULTS

Interferents, except iron saccharate (detected at 5%), do not affect t Fe determination. Within-run and between-run imprecisions were in the ranges of 0.2-0.4% and 0.4-1.0% CV. The results of the HPLAC method correlated well with those of the ICSH method (r=0.9993) and FA method (r=0.9984).

CONCLUSIONS

In contrast to the ICSH and FA methods for determining serum iron, the HPLAC method is simple, highly precise and specific for serum t Fe, which can contribute to the measurement of iron status.

The most common method for determination of haemoglobin is the cyanmet-haemoglobin method which is recommended by the International Committee for Standardization in Haematology (ICSH). Determination of haemoglobin is one of the most used methods in hospital laboratories. One disadvantage with the method is the use of cyanide in the reagent, which demands cautious preparation and use. The intention of our investigation was to replace the cyanide reagent for Hemalog-8 with a reagent not containing cyanide. It is shown that the change of reagent did not influence the results of the analysis.

Androgen biosynthesis in the male gonads may be analysed in some detail by means of in vitro incubation of minor testicular biopsy specimens with various radiolabelled steroid precursors. We have investigated nine adult human male voluteers without apparent gonadal dysfunction with regard to their in vitro metabolism of 3H-progesterone. The following metabolic compounds were recovered: 20a-dihydroprogesterone, 17a-hydroxyprogesterone, 20a, 17a-dihydroxyprogesterone, androstenedione and testosterone. No significant amounts of 5a-reduced delta4-3-oxo-steroids or oestrogens were found. The major metabolites formed were 20a-dihydroprogesterone and 17a-hydroxyprogesterone, which accounted for 13-49 per cent and 17-47 per cent, respectively, of all newly synthesized steroid compounds. Radiolabelled delta4-3-oxo-C19-metabolites were recovered in minor amounts only, possibly due to metabolic interference with the endogenous pool of cold mass compounds. The individual steroid metabolic patterns were found to be poorly related to the individual levels of FSH, ICSH (LH) and testosterone in the peripheral circulation. Adequate knowledge of the steroid metabolic pathways generally utilized in ordinary testicular tissue in vitro is a prerequisite for the evaluation of steroid metabolism in males with various types of gonadal dysfunction.