A simple, sensitive, precise and reproducible micromethod for the determination of plasma iron in a 50-microliter sample has been devised by modification of the ICSH reference procedure, which requires a 2-ml sample. The micromethod uses microcuvettes and substitutes ferrozine for the bathophenanthroline chromogen of the ICSH. Comparison of plasma iron values on 47 paired specimens obtained by fingerpuncture and venipuncture determined by the micro- and ICSH-methods respectively gave identical means of 15.8 mumol/l (88 microgram/dl); the correlation of the two methods was r = 0.92 (p less than 0.0001). The micromethod has been successfully used to measure plasma iron levels in fingerpuncture specimens collected in a field study of 429 children.

We evaluated performance of the TEST 1 (SIRE Analytical Systems, Udine, Italy), a fully automated analyzer for the measurement of the erythrocyte sedimentation rate (ESR). Intra-assay reproducibility was satisfactory for a wide range of ESR values, whereas there was a significant decrease in ESR data when the samples were stored at 4 degrees C for up to 24 hours. We compared TEST 1 with the Westergren ESR method approved by the International Council for Standardization in Haematology (ICSH) and with the Diesse Ves-Matic analyzer Linear regression analysis comparing the TEST 1 and the ICSH reference method yielded satisfactory correlations for K3 EDTA- and sodium citrate-anticoagulated samples. Bland-Altman analysis showed no evidence of a systematic bias between the TEST 1 and the reference method. A close correlation was found between the TEST 1 system and the Diesse Ves-Matic analyzer despite a significant positive systematic bias. Reference values for men and women were analyzed according to nonparametric statistics. The TEST 1 was easy to use, had a satisfactory operative practicability required minimal maintenance, and reduced contact with potential biohazards. This system enables the determination of ESR with any common standard-sized tube; the use of samples anticoagulated with K3 EDTA can widely reduce the workload in clinical laboratories.

An in vitro bioassay method for measuring LH activity was applied to male plasma. This method is based on the specific testosterone response to LH activity by interstitial cells from mouse testes. In contrast to assays conducted on female plasma, non-parallel response lines were obtained between serial dilutions of untreated male plasma and the International Reference Preparation for Human Pituitary Gonadotrophins FSH and LH/ICSH) for bioassay (code no. 69/104). In an attempt to eliminate this source of error, which would invalidate the assays, plasma was subjected to either ether extraction or charcoal adsorption prior to assay. While ether extraction was ineffective, charcoal treatment eliminated the source of non-parallelism. Evidence is presented indicating that the inclusion of a charcoal pre-treatment step provides an assay method for LH which fulfils the recognized criteria of reliability when applied to male plasma. An investigation of the likely causes of non-parallelism was undertaken by incubating mouse interstitial cells with various steroids and steroid sulphates at concentrations likely to be present in plasma. While most of the presumed precursors of testosterone were converted to testosterone, steroid sulphates (dehydroepiandrosterone sulphate and pregnenolone sulphate) at high concentrations as present in male plasma were the most active compounds in forming testosterone. However, the amount of testosterone produced from these precursors under controlled conditions was insufficient to account entirely for the deviation from parallelism observed with male plasma. Hence, the non-parallelism observed with untreated plasma samples cannot be entirely explained by the presence of steroidal testosterone precursors in male plasma.

Thirteen cases of pyruvate kinase (PK) deficiency, considered to be heterozygous for different PK mutants because of no consanguinities in their parents, were characterized by the International Committee for Standardization in Haematology (ICSH) recommended methods. These deficiency cases are named PK "Kagoshima," PK "Kyoto," PK "Takamatsu," PK "Abeno," PK "Kobe," PK "Marugame," PK "Hoenzaka," PK "Osaka," PK "Motomachi," PK "Gifu," PK "Hiroshima" PK "Matsumoto," and PK "Tama." The characteristics of mutant PK enzymes suggest that the cause of chronic hemolysis depends mainly on decreased affinity for phosphoenolpyruvate, thermolability, increased inhibition by adenosine triphosphate, and low activation by fructose-1, 6-diphosphate.

BACKGROUND

We investigated the effect of iron deficiency anemia (IDA) on levels of glycated hemoglobin (HbA1c) and to compare its levels before and after iron supplementations.

METHODS

Age and sex matched subjects were enrolled and clustered in 2 groups: IDA (n=62) and healthy controls (HC; n=60). HbA1c levels were estimated by HPLC. Hemogram were estimated by hematology analyser. Serum ferritin (ELISA) and other parameters of iron profile were measured by standard guidelines of ICSH. HbA1c values and iron studies were repeated after 3months of iron supplementation to determine the effect of iron therapy on HbA1c levels.

RESULTS

Significantly higher HbA1c levels were observed in IDA subjects compared to HC (5.51±0.696 v/s 4.85±0.461%, p<0.001). A significant negative correlation was observed between HbA1c and hemoglobin, hematocrit, RBC count, MCH, MCHC and serum ferritin in IDA subjects (r=-0.632, -0.652, -0.384, -0.236, -0.192 and -0.441). Significant decline was noticed in HbA1c levels in IDA subjects after iron supplementation (5.51±0.696 before treatment v/s 5.044±0.603 post-treatment; p<0.001). Post treatment, 70% subjects (14/20) with HbA1c in pre-diabetes range normalised to normal glucose tolerance (NGT) range and out of 6 patients with pre-treatment HbA1c in diabetes range, 5 reverted to pre-diabetes range while 1 of them reverted to the NGT range.

CONCLUSIONS

Caution must be exercised in interpreting the results of HbA1c in patients of IDA and iron deficiency must be corrected before diagnosing diabetes and pre-diabetes solely on the basis of HbA1c criteria.

Bone marrow (BM) tissue biopsy evaluation, including trephine biopsy and clot section, is an integral part of BM investigation and is often followed by ancillary studies, in particular immunohistochemistry (IHC). IHC provides in situ coupling of morphological assessment and immunophenotype. The number of different IHC tests that can be applied to BM trephine biopsies and the number of indications for IHC testing is increasing concurrently with the development of flow cytometry and molecular diagnostic methods. An international Working Party for the Standardization of Bone Marrow IHC was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines for the standardization of BM IHC based on currently available published evidence and modern understanding of quality assurance principles as applied to IHC in general. The guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus and represent further development of the previously published ICSH guidelines for the standardization of BM specimens handling and reports.

Idiopathic transdural spinal cord herniation (ICSH), a rare condition which may cause progressive myelopathy, can be diagnosed preoperatively by MRI. Surgical treatment usually results in the resolution of symptoms and, thus, familiarity with the imaging findings in this condition impacts patient management. We present the case of a 36-year-old woman in whom the initial MRI findings were thought to be consistent with only an arachnoid cyst compressing the spinal cord. After surgery, her symptoms remained unchanged, and a repeat MRI study was interpreted as being compatible with a transdural spinal cord herniation. Repeated surgery with reduction of the herniation resulted in significant clinical improvement.

BACKGROUND

Recently, a consensus report for microscopic schistocyte determination was prepared by International Council for Standardization in Hematology (ICSH). ICSH focused on diagnosis of thrombocytopenic purpura (TTP)/hemolytic uremic syndrome (HUS). We aimed to reanalyze schistocytes according to ICSH recommendations, to study diseases other than TTP/HUS related to the schistocytes, and to compare the percentage of schistocytes among the various diseases.

METHODS

We retrieved all reported cases of peripheral blood (PB) smear in a single institution during 6 years. Schistocytes on 282 PB smears showing previous peripheral schistocytes and hemoglobin ≤10 g/dL were recounted according to ICSH recommendations.

RESULTS

The schistocytes were frequently observed in patients with microangiopathic hemolytic anemia (MAHA), metastatic carcinoma, sepsis, chronic renal failure, preterm infant, and infection. Only two among 34 patients categorized as MAHA were diagnosed as TTP/HUS. Schistocytes were observed with other morphological changes in 169 of 170 cases with schistocyte ≤1% and in 102 of 112 with schistocyte >1%. The median schistocyte percentages of patients with hematologic malignancy, megaloblastic anemia, acute renal failure, and preterm infant were 1.20%, 1.30%, 1.35%, and 1.70%, respectively.

CONCLUSIONS

Schistocytes were observed above 1% in many diseases other than TTP /HUS. Therefore, it is important to understand that schistocytes could be seen in various diseases, and in these cases, schistocytes were usually detected together with other red blood cell morphologic changes. These data support ICSH recommendation that a schistocyte count should be considered clinically meaningful if schistocytes represent the main morphological abnormality in the PB smear.

The International Council for Standardization in Haematology (ICSH) and the International Society of Laboratory Hematology (ISLH) recommend the counting of specifically labeled platelets relative to the RBCs with a fluorescence flow cytometer, together with an accurate RBC count determined with a semiautomated, single-channel aperture-impedance counter as a reference method for the enumeration of platelets. Fresh EDTA-anticoagulated venous blood specimens are measured within 4 hours of the draw. The specimen is prediluted (1:20) and the platelets labeled with two monoclonal antibodies specific to a cluster of differentiation common to all platelets. A final 1:1,000 dilution is made and at least 50,000 events with a minimum of 1,000 platelet events are counted with a flow cytometer to determine the RBC/platelet ratio. The platelet count is then calculated from this ratio and the RBC concentration of the original blood specimen.

As little as 5 micrograms of interstitial cell stimulating hormone (ICSH) or 20 micrograms of ICSH-beta is effective for the induction of ovulation in 100 percent of hamsters treated at 0500 hours on day 4 after lordosis, whereas as much as 800 micrograms of ICSH-alpha is ineffective. Both ICSH and ICSH-beta are also effective for induction of ovulation in hypophysectomized animals. Thus, the ovulation-inducing activity of the ICSH molecule resides in its beta subunit.

In an international collaborative study three candidate reference and the local APTT reagent have been tested with lyophilized plasmas containing heparin added in vitro and fresh plasmas from heparinized patients to determine whether it was possible to standardize APTT heparin monitoring. The APTT responses of patients with thrombosis were much less to the same concentrations of heparin so only these plasmas were used to calibrate the reagents. A calibration constant (b) was determined for each local APTT system from the orthogonal regression slope of the patients' and normals' plasmas. Although the calibration was based on relatively limited numbers of patients' plasmas the results gave no consistent indication that this method of analysis with the reference preparations was incorrect. Two of the three candidate reference reagents performed almost equally well and one of these reagents is recommended as the APTT reference preparation. Use of a single b value for a brand of reagent for all laboratories is not advised. Each laboratory must perform its own local calibration. The adoption of a reference APTT reagent together with the use of orthogonal regression analysis would be a useful step to initiate APTT heparin monitoring standardization and to develop safe and effective local therapeutic ranges.

The use of molecular genetic technology for blood group typing is becoming routine procedure in many reference laboratories worldwide. A First International Workshop was organized on behalf of the International Society of Blood Transfusion (ISBT) and the International Council for Standardization in Haematology (ICSH). Thirty laboratories that provide a molecular diagnostic service participated in the workshop. Six samples were distributed: two represented DNA from transfusion-dependent patients for testing for multiple polymorphisms; two represented fetal DNA prepared from amniotic fluid for RhD, Rhc and K-testing; and two represented plasma from RhD-negative pregnant women for fetal RhD testing. Error rates varied from 0 to 11% for different polymorphisms. A consensus arising from discussion on the workshop results between participants at a feedback meeting and by e-mail has resulted in seven recommendations for molecular blood group genotyping. Further international workshops will take place every 2 years, with a more limited exercise being organized in the intervening years.

Histopathological and biochemical effects of gossypol acetate (GA) on pituitary-gonadal axis were investigated. 10 and 25 mg GA/kg were administered orally to sexually mature adult male Wistar rats for 4 and 5 weeks, respectively. STH and LTH/PRL cells showed no significant changes as compared to those of controls while TSH cells showed hypertrophy, hyperplasia and degranulation in both experimental groups. Pituitary FSH, LH/ICSH cells showed progressive regression. Gonosomatic indices of sex accessory glands at 25 mg showed significant reduction in the experimental animals as compared to those of controls. The diameter of seminiferous tubules reduced and azoospermia developed. Sertoli and Leydig cells also regressed. At 10 and 25 mg GA treatment, spermatogenesis ceased at secondary spermatocytes and spermatogonia stages, respectively. Epididymis and prostate regressed. Seminal vesicle showed no significant histological variations as compared to that of control except reduction in secretion. Biochemical observations revealed increased levels of acid phosphatase, fructose and citric acid and significant reduction in glycerylphosphoryl choline in reproductive glands of both experimental groups as compared to those of controls. Possible mechanism of antifertility action of GA is discussed.

An international collaborative exercise has been undertaken to calibrate a secondary international reference preparation (IRP) of thromboplastin on behalf of the International Committee for Standardization in Haematology (ICSH). This preparation of British Comparative Thromboplastin (BCT/441) is required because supplies of the WHO primary IRP (BCT/253) are necessarily limited. The calibration was performed at seven centres with only a small degree of interlaboratory variation. As a result of this study an ISI value of 1.04 has been assigned to the preparation. Opportunity was also taken to assess the reliability of a simplified calibration based on lyophilised plasmas. The results of the latter appeared reliable. BCT/441 will be available to officially designated National Control Laboratories for calibration of local thromboplastins to promote prothrombin time standardization in oral anticoagulant control.

BACKGROUND

The gold standard for the determination of the erythrocyte sedimentation rate (ESR) is the Westergren method. Other methods to measure the ESR have become available. They range from modest modifications of the Westergren method to very different methodologies. The ICSH therefore established a Working Group to investigate these new approaches and compile recommendations for their validation and verification.

METHODS

A panel of six experts in laboratory hematology examined the peer-reviewed literature and EQA surveys from over 6000 laboratories on four continents performing ESR testing. This information was used to create lists of ESR instrument manufacturers and their methods.

RESULTS

Only 28% of laboratories surveyed used the unmodified Westergren method, while 72% of sites used modified or alternate methods. Results obtained with the new instruments could differ from results obtained with the Westergren method by up to 142%. Different non-Westergren methods showed differences from each other of up to 42%. The new methods were often significantly faster, safer, and less labor-intensive. They reduced costs and often used standard EDTA tubes, eliminating the need for a dedicated ESR tube.

CONCLUSIONS

Based on the consensus of the Working Group, recommendations for manufacturers for the validation of new ESR methods were developed. In addition, a list of recommendations for laboratories that are moving to modified or alternate methods was compiled, addressing instrument performance verification and communications of results to clinical users.

BACKGROUND

various modifications of the Erythrocyte Sedimentation Rate (ESR) determination have been suggested since the original Westergren procedure that has been adopted as the gold standard by the International Council for Standardization in Haematology (ICSH). Recently, an automated method, (Alifax Test 1), based on a technique completely different from Westergren, has been introduced.

METHODS

In this comparative study, ESR of blood from 680 patients with various rheumatic diseases was determined on both Test 1 and the StaRRsed automated ESR analyser which performs measurements in accordance with ICSH specifications. Furthermore the robustness of the new technique was evaluated.

RESULTS

Direct correlation of Test 1 and StaRRsed measurements confirmed the results of previous studies: an overall correlation coefficient of R = 0.90. However, further statistical analysis showed that, depending on the instrument that was used, in 78 samples (i.e. 11.5%) the results could lead to different treatment suggestions. Furthermore it appeared that several procedural factors could influence the final Test 1 outcome.

CONCLUSIONS

Due to its sensitivity for procedural variations, Test 1 measurements should be carried out under strictly standardized conditions. Especially at the higher ESR levels the Test 1 technique is, however, not a reliable alternative for the ICSH approved 'Westergren' method.

The Sysmex XS-1000i is a compact new, fully automated haematology analyser, designed to generate complete blood counts with five-part leucocyte differential. In our study, a Sysmex XS-1000i instrument was evaluated according to Clinical Laboratory Standards Institute (CLSI) and International Council for Standardization in Haematology (ICSH) guidelines. Precision, carry-over and linearity were determined. Using a total of 700 patient samples, results from the Sysmex XS-1000i were compared with those from a Sysmex XE-2100, an Abbott Cell Dyn 4000 and the manual reference leucocyte differential. Using quality control material, total and within-run imprecision was less than 3% except for platelets. The system demonstrated good linearity over the entire reporting range and no carry-over (<0.5%). The Sysmex XS-1000i showed good correlation with XE-2100, CD-4000 and the manual reference leucocyte differential. Overall flagging sensitivity and specificity were 91% and 48%, respectively. In conclusion, the Sysmex XS-1000i demonstrated good analytical performance, is able to generate a complete blood count with five-part differential on low blood volumes and has considerable back-up capacity.

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

Although serum iron determinations play an important role in the diagnostic process, the accuracy of routine methods is suspect. The poor performance of currently used methods is well documented in the quarterly College of American Pathologists survey reports. Compared with the 1990 method of the International Committee for Standardization in Haematology (ICSH), the 1978 ICSH Reference Method shows a +3.4% bias and the standard method of the National Committee for Clinical Laboratory Standards shows a -17.8% bias for iron results. All routine methods checked--DuPont aca, Abbott TDx, Kodak Ektachem (except one lot), BM/Hitachi 717, and Synermed--show a negative bias over the entire analytical range, a significant negative intercept, and extremely poor correlation with the 1990 ICSH method for iron values < 750 micrograms/L. Individual discrepancies of several hundred percent were observed. Imprecision of the methods is not the reason for these discrepancies. We question whether most routine methods measuring low iron concentrations provide results sufficiently reliable for confirmation of iron deficiency.

The International Council for Standardization in Haematology (ICSH) in conjunction with Eurotrol, B.V. has released a new lot of the haemiglobincyanide or haemoglobin standard. This technical report describes the purpose, methodology in manufacturing, summary of value assignment data and availability of this standard material used for the standardization and calibration of whole blood haemoglobin measurements on most haemoglobinometers and automated blood cell counters throughout the world.

In connection with systematic studies of steroid and peptide hormone interactions during follicular growth, we have measured ovarian weight responses to graded doses of highly purified human chorionic gonadotropin (hCG), human interstitial cell stimulating hormone (hICSH)in hypophysectomized immature female rats (HIFR) treated with diethylstilbestrol in silastic capules (desc) implanted subcutaneously. Our results are consistent with earlier reports of enhancement of ovarian weight responses to hCG and FSH. Contrary to results of similar experiments reported by others, we have found that estrogen treatment of HIFR enhanced ovarian weight response to ICSH. In addition, we report for the first time that small doses of hCG and hICSH inhibit ovarian weight responses to estrogen in HIFR. Our observations on effects of small doses of hCG and hICSH and the long-known fact that ovarian interstitial cells are stimulated in HIFR given similar doses of these hormones lead us to hypothesize that ovarian interstitial cell stimulation is involved in the control of follicular maturation.

Relationships between the spermiogram and sex hormones in 104 andrologic patients were studied. Endocrine connections in normal and in pathologic spermiogenesis have been studied. A rather close correlation was found between FSH and ICSH levels. ICSH increases parallel with the increase of FSH level. There is also a close relationship between sperm concentration and FSH level. Between ICSH and sperm count the correlation is not significant. Between steroid hormone level and sperm concentration no correlation ws found. It is only the plasma FSH level which show a close relationship with the disorder of spermiogenesis and it has diagnostic significance.