International Council For Standardization In Haematology Research Articles

Diagnosis in thrombotic microangiopathy

Thrombotic microangiopathies (TMA) are relatively rare multisystem diseases characterised by microvascular endothelial damage with platelet-rich thrombus formation, consumption thrombocytopenia, mechanical haemolytic anaemia with schistocytes and multiorgan failure of variable severity. Thrombotic thrombocytopenic purpura (TTP) and haemolytic uraemic syndrome (HUS) are the two major disorders; other clinical entities that can be associated with TMA, are the HELLP syndrome, (haemolysis, elevated liver enzymes, low platelet count), the catastrophic antiphospholipid syndrome (CAPS) and bone marrow transplant (BMT). On the peripheral blood smears, the detection of schistocytes, that are fragments of red blood cells produced by extrinsic mechanical damage within the circulation, still remains an important morphological clue for the diagnosis of TMA. The International Council for Standardization in Hematology (ICSH) has prepared specific recommendations to standardise schistocyte identification, enumeration, and reporting. Measurement of activity of ADAMTS13 (a zinc-containing metalloprotease enzyme which cleaves and degrades large multimers of von Willebrand factor) remains the most reliable test to distinguish HUS from TTP, presenting with detectable and undetectable ADAMTS13 activity, respectively. Main clues in HELLP are weight gain, arterial hypertension, massive proteinuria, DIC, marked elevation of AST/ALT/LDH and detectable ADAMTS13 activity, while patients with CAPS present with weight loss, recent polyuria–polydipsia, vomiting, hypoglycaemia, jaundice (conjugated bilirubin), prolonged prothrombin time, reduced factor V and antithrombin levels, DIC and renal failure.

Serum Ferritin and Iron Status of Mothers and Newborns in Lubumbashi in the DRC

Introduction: Ferritin is the primary form of intracellular iron storage. The serum concentration generally gives a faithful reflection of the level of iron reserves in the body. Hyperferritinemia is an early and specific marker of iron deficiency. Methodology: A descriptive cross-sectional study with an analytical component was carried out in DR Congo in order to determine the reference values of serum ferritin for mother-newborn couples and to establish the link between these values in mothers and their newborns. Mothers who delivered at term after a singleton pregnancy with no history of breakthrough bleeding or blood transfusion and eutrophic newborns with good APGAR showing no pathology, neither malformation, were included in the study. A blood sample was taken from the mother and the newborn for the determination of ferritin and CRP (C-reactive protein) and certain elements of the hemogram according to the methods recommended by the IFCC (International Federation of Clinical Chemistry) and ICSH (International Council for Standardization in Hematology). We have coupled the ferritin assay to CRP in order to eliminate cases of inflammatory syndrome that can influence ferritinemia. Iron supplementation and consumption of kaolin and tea during pregnancy, and some sociodemographic parameters of mothers and newborns were studied. Results: A total of 103 mother-newborn couples were selected. For mothers: the average age was 27.68 ± 6.42 years and the average parity was 3.80 ± 2.52. About 98.06% lived as a couple, 78.64% had a secondary education, 51.48% engaged in a revenue-generating activity and 20.40% had a poor nutritional status. Regarding the sex of the newborn, 56.3% were female. The mean serum ferritin level of the mothers is 59.21 ± 48.09 μg/l with a variability of 81.22; while it is 290.18 ± 212.69 μg/l with a variability of 73.29 in newborns. Reference values of serum ferritin ranged from 21.10 to 114.00 μg/l in mothers and 60.99 to 749.01 μg/l in newborns. There is a positive and significant correlation in the mothers between the hemoglobin level and the CCMH (0.216 and p = 0.029) and between the reticulocytes and the CCMH (0.270 and p = 0.006). In newborns, there is a positive and significant correlation between hemoglobin and ferritin (r = 0.288; p = 0.003) and between hemoglobin and TCCM (0.191 and p = 0.037). We found a positive and significant correlation between maternal ferritin and the newborn VGM (0.191 and p = 0.037). Iron Supplementation during pregnancy causes significant increase of hemoglobin in the newborn (p = 0.020). While the consumption of tea and Kaolin during pregnancy although statistically insignificant tends to decrease the values of hemoglobin, VGM, CCMH, reticulocytes and ferritin in newborns. Conclusion: The mother-newborn Lubumbashi has a rate of ferritin serum in usual standards. To better understand the origins-deficiency anemia of the newborn within 24 hours of birth in our midst, we propose that we consider as reference values of ferritin in serum, those in the range [60.99 - 749.01 μg/l]. Although small and not statistically significant, there is a relationship of negative correlation between ferritin maternal serum and the newborn.

Standardization of Schistocyte Identification and Quantitation for the Diagnosis of Thrombotic Microangiopathic Anemia at University of Cincinnati Medical Center

Background: Thrombotic microangiopathic anemia (TMA) is a hematological emergency that requires prompt review of a peripheral blood smear for the presence of schistocytes. Within our institution, we had concern for lack of consistency in identifying schistocytes, lack of consistency in reporting methods, possible variation in schistocyte quantitation due to microscope differences, and the threshold for which our laboratory had been reporting increased schistocytes. The objective of our quality improvement project was to implement published practice standards for morphological identification, quantitation, and reporting of schistocytes across different groups at the University of Cincinnati Medical Center (UCMC). Methods: The International Council for Standardization in Haematology (ICSH) recommendations for the identification, diagnostic value, and quantitation of schistocytes (Zini et. al. International Journal of Laboratory Hematology 2012) were reviewed prior to designing this project. We next conducted a survey of groups that were reviewing peripheral blood smears for schistocytes on a routine basis: medical technologists (n=9), pathology residents (n=10) and hematology-oncology fellows (n=13). The survey included questions about schistocytes, including desire for standardization and normal reference values, desire for pathologist instruction, and reporting patterns. One question included 8 images of various red cell morphologies, and participants were asked to select ones they would classify as schistocytes. The microscopes were compared for objective and field diameter measurements. Six (6) peripheral blood smears with previously reported schistocytes were re-reviewed. Schistocytes were identified per ICSH guidelines and counted as a percentage (%) of 1000 total red blood cells using a Miller optical disc and also as an average number per field for a total of 10 high power fields. The results were plotted on a linear X-Y axis graph (Fig 1) and compared to the ICSH guidelines (Fig 2) and the policy for reporting schistocytes in our laboratory. Results: Survey results showed that the majority of each group desired standardization, normal reference values, and pathologist instruction. For reporting of schistocytes, residents answered present/absent (50%) or average per high power field (HPF) (50%), whereas 92% of fellows answered average per HPF. No participants reported a percentage of 1000 red blood cells, the current ICSH recommendation. For the morphological identification, 50% of residents, 62% of fellows and 67% of technologists correctly identified keratocytes as schistocytes. Fifteen (15%) of fellows misidentified bite cells as schistocytes, whereas one fellow (8%) and one technologist (11%) misidentified acanthocytes as schistocytes. Only 70% of technologists correctly identified helmet cells as schistocytes, whereas all residents and fellows chose them correctly. Almost all participants failed to recognize microspherocytes as schistocytes. The microscopes all showed the same objective and field diameter measurements. The schistocyte percentage plotted versus the number of schistocytes per HPF showed a 0.99% correlation co-efficient (R=0.99%) (Fig 1). The results were compared to the ICSH threshold of 1%, above which the ICSH reports is a robust morphologic indicator for the diagnosis of TMA (Fig 2), and to the current reporting policy at UCMC. Two (2) schistocytes per HPF correlated with 1% schistocytes on the linear plot. At UCMC, policy had been to report 2-8 schistocytes per HPF as present and >8 per HPF as increased. These findings indicated that the threshold for reporting increased schistocytes should be lowered from >8 per HPF to >2 per HPF. The above data were reviewed with each survey group. A process was also initiated to change the laboratory schistocyte reporting policy. Conclusion: All survey groups needed and desired education on schistocyte identification and reporting per ICSH guidelines. All microscopes showed the same measurements and were therefore expected to produce the same quantitation results. Schistocyte percentage correlated with the number of schistocytes per HPF. By implementing the ICSH guidelines, we aimed to decrease inter-observer bias and to standardize the quantitation and reporting of schistocytes at UCMC, thereby assisting in timely and accurate diagnosis of thrombotic microangiopathic anemia. Disclosures No relevant conflicts of interest to declare.

International Council for Standardization in Haematology Recommendations for Hemostasis Critical Values, Tests, and Reporting.

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) 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.

PS1478 EVALUATION OF COMBINED MEASUREMENT OF IMMATURE PLATELET FRACTION AND SCHISTOCYTES IN PREGNANCY‐ASSOCIATED THROMBOTIC THROMBOCYTOPENIC PURPURA AND SEVERE PREECLAMPSIA/ HELLP SYNDROME

Background:The occurrence of thrombotic microangiopathy (TMA) in pregnancy is an unfortunate emergency condition. Proper diagnosis is mandatory which requires the consideration of two overlapping diagnoses: severe preeclampsia/ haemolysis, elevated liver enzymes, and low platelet syndrome (SPE/HELLP) and thrombotic thrombocytopenic purpura (TTP). The long turn‐around times of ADAMTS13 testing precludes the timely distinction between the two conditions.Aims:We aimed at evaluating schistocyte counts and immature platelet fraction (IPF%), as both increase in TMAs, to discriminate between TTP and SPE/HELLP of pregnancy.Methods:IPF% was measured using SysmexXE‐2100 automated hematology analyzer, and schistocyte counts were estimated microscopically as per the International Council for Standardization in Hematology (ICSH)‐Schistocyte Working Group guidelines. The study included 30 pregnant patients with SPE/HELLP, 13 pregnant patients with TTP, and 30 women with normal pregnancy. The discrimination between the two patient categories was based on clinical judgment and TTP cases were identified using the PLASMIC score.Results:TTP patients had higher values of IPF% than SPE/HELLP 19.5 (16.9–27.1) vs 13 (9.5 – 23.25); p <0.001); similar results were revealed regarding schistocyte counts 6.5 (3.9–8.6) vs 2.1 (1.6 – 3.5); p <0.001). IPF% and schistocyte counts were able to discriminate between TMA patients and normal pregnant women, and between and SPE/HELLP and TTP patients. Moreover, the discriminatory function of each was improved when the two parameters were used in combination.Summary/Conclusion:IPF% analysis should be used in conjunction with manual schistocyte counting in TMA cases to distinguish TTP pregnant patients from patients having SPE/HELLP.

Quantification of Schistocytes from the Peripheral Blood Smear in Thrombotic Thrombocytopenic Purpura (TTP) Compared to Non-TTP Thrombocytopenic Hospitalized Patients

IntroductionThe diagnosis of TTP requires thrombocytopenia without an underlying cause and microangiopathic hemolytic anemia (MAHA)1. TTP is usually confirmed with a low ADAMTS level. However, the turnaround time for an ADAMTS level is several days and treatment for this rare disease cannot be delayed, since it is uniformly fatal if left untreated but highly responsive to plasma exchange therapy. A rapid diagnosis is required and still must be made on clinical findings and peripheral smear morphology. The degree of MAHA that is seen with TTP, though, has not been well defined, or correlated with ADAMTS results. Automated fragmented red cell analyzers are not yet widely available or accepted, so the determination of MAHA still relies on manual smear reviews. Presenting smears from pts with TTP over a 1 ½ year period were reviewed, with manual counting of schistocytes, and were compared to smears from in-patient non-TTP cases of thrombocytopenia where hematology was consulted to rule out TTP.Methods29 cases were identified and peripheral smears from date of presentation were reviewed, initially by a hematologist and experienced special hematology technician, then by 2 medicine residents after they were given a 1 hour tutorial on smear interpretation. All smear reviewers were instructed on schistocyte identification as defined by the International Council for Standardization in Hematology (ICSH)2. The number of schistocytes per 1000 red cells was counted at 1000 power magnification. The blood smears were coded/de-identified and then read “blindly” by the 4 observers. Degree of agreement between the 1st 2 observers was measured (using Pearson correlation coefficient), as was the their average as compared to the average #schistocytes counted between the 2 med residents. Time taken to review smears was also measured.ResultsAve age 54 (19-83); 59% female, 41% male; 62% Caucasian; 35% AA; 3% Hispanic. 9 had TTP and 20 did not. All cases of TTP had a low ADAMTS level (med <3%, range <3-13%) and all but one had a +inhibitor (one had congenital TTP, confirmed by ADAMTS mutation analysis). The non-TTP cases included sepsis (8/20), DIC (3/20), metastatic cancer (2/20), scleroderma crisis (2/20) and single cases of HIT, EtOH, gestational, MPN/MDS and unknown causes for thrombocytopenia. For TTP cases: med plt count was 22k (4-52k); LDH 760 (309-1175); hgb 9.4 (7.1-10.9); creat 1.59mg/dL (0.6-4.85). For non-TTP cases: med plt count was 44k (3-126k); LDH 300 (155-1111); hgb 8.1 (5.9-14.7); creat 1.36mg/dL (0.9-9.1). 3 non-TTP pts had ADAMTS levels sent (med= 69%, 57-100%).The med #schistocytes in TTP cases was 6% (ave 6.3, 1.85-11.95%) compared to 0.625% (ave 1.52%, 0.25-7.5%) in the non-TTP group (p= 0.00002). The R-coefficient for the 1st 2 observers was 0.93 (95% CI 0.85-0.96) and for their average compared to the 2 medicine residents it was 0.82. The average time to read smears overall was 10.5 minutes.Conclusions:This retrospective study showed that manual quantification of schistocytes is feasible, may be done in a timely fashion and with excellent interobserver reproducibility. All pts had some degree of MAHA but the TTP cases, not surprisingly, had a significantly higher percentage of schistocytes/1000 RBCs than non-TTP thrombocytopenic in-pt consult cases (6% vs 0.625%, respectively, p= 0.00002). All cases of low ADAMTS, positive inhibitor confirmed TTP had >2.5% schistocytes, compared to 4/20 non-TTP cases. A larger cohort of acquired TTP should be studied to determine if there is a cut-off % schistocytes which, in the appropriate clinical setting, should warrant urgent plasma exchange while waiting for ADAMTS levels to return.1George JN and Al-Noun ZL. Diagnostic and therapeutic challenges in the TTP and HUS syndromes. ASH Education Book. 2012, p. 604-609.2Zini G et al. ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes. International J of Lab Hematology. 2011; 34: 107-116. DisclosuresNo relevant conflicts of interest to declare.

Issue Highlights - November 2018.

Issue Highlights - November 2018.

International Council for Standardization in Haematology (ICSH) Recommendations for Laboratory Measurement of Direct Oral Anticoagulants.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH) for providing haemostasis-related guidance documents for clinical laboratories. This inaugural coagulation ICSH document was developed by an ad hoc committee, comprised of international clinical and laboratory direct acting oral anticoagulant (DOAC) experts. The committee developed consensus recommendations for laboratory measurement of DOACs (dabigatran, rivaroxaban, apixaban and edoxaban), which would be germane for laboratories assessing DOAC anticoagulation. This guidance document addresses all phases of laboratory DOAC measurements, including pre-analytical (e.g. preferred time sample collection, preferred sample type, sample stability), analytical (gold standard method, screening and quantifying methods) and post analytical (e.g. reporting units, quality assurance). The committee addressed the use and limitations of screening tests such as prothrombin time, activated partial thromboplastin time as well as viscoelastic measurements of clotting blood and point of care methods. Additionally, the committee provided recommendations for the proper validation or verification of performance of laboratory assays prior to implementation for clinical use, and external quality assurance to provide continuous assessment of testing and reporting method.

The Clinical Significance of Schistocytes: A Prospective Evaluation of the International Council for Standardization in Hematology Schistocyte Guidelines.

Objective:The presence of ≥1% schistocytes on a peripheral blood smear (PBS) is an important criterion for the diagnosis of thrombotic microangiopathy (TMA). The reporting of schistocytes has been standardized by the International Council for Standardization in Hematology (ICSH). Despite the availability of guidelines, however, the assessment of schistocytes remains subjective. More recently, the automated fragmented red cell (FRC) parameter has been evaluated. However, local studies are not available.Materials and Methods:A prospective study was performed at the Charlotte Maxeke Johannesburg Academic Hospital in order to evaluate the ICSH recommendations for schistocyte measurement in 146 PBSs with schistocytes. Schistocytes were evaluated by microscopy and ADVIA 2120 automated hematology analyzers.Results:Schistocytes were frequently observed in patients with TMA (n=76), infection (n=20), hematologic malignancy (n=10), renal failure (n=5), and hemoglobinopathy (n=15), and in neonates (n=11). Schistocytes were ≥1% in all PBSs with TMA (n=76) with a mean of 3.44±1.84. Schistocytes of ≥1% were also observed in cases of renal failure and hemoglobinopathy, and in neonates. In these conditions, schistocytes were mainly observed in conjunction with moderate red blood cell changes. The agreement between two morphologists gave a correlation coefficient of 0.63 [confidence interval (CI): 0.52-0.75], while the correlation coefficient between the average of the morphologists and the FRC percentage was -1.97 (CI: -1.60 to -2.34). The ADVIA 2120 underestimated the schistocyte count in patients with TMA.Conclusion:Observer bias can be decreased by implementing the standardized procedures recommended by the ICSH. However, estimation of schistocytes by the ADVIA 2120 analyzer requires further evaluation as a screening tool. A higher threshold for schistocytes in thrombotic thrombocytopenic purpura is recommended to distinguish this hematological emergency from other conditions associated with ≥1% schistocytes.

Issue highlights - May 2016.

Issue highlights - May 2016.

ICSH: on board for new projects.

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.

ICSH guidelines for the verification and performance of automated cell counters for body fluids

One of the many challenges facing laboratories is the verification of their automated Complete Blood Count cell counters for the enumeration of body fluids. These analyzers offer improved accuracy, precision, and efficiency in performing the enumeration of cells compared with manual methods. A patterns of practice survey was distributed to laboratories that participate in proficiency testing in Ontario, Canada, the United States, the United Kingdom, and Japan to determine the number of laboratories that are testing body fluids on automated analyzers and the performance specifications that were performed. Based on the results of this questionnaire, an International Working Group for the Verification and Performance of Automated Cell Counters for Body Fluids was formed by the International Council for Standardization in Hematology (ICSH) to prepare a set of guidelines to help laboratories plan and execute the verification of their automated cell counters to provide accurate and reliable results for automated body fluid counts. These guidelines were discussed at the ICSH General Assemblies and reviewed by an international panel of experts to achieve further consensus.

Evaluation of ICSH schistocyte measurement guidelines in France

The schistocytes are fragmented red blood cells mainly observed in the setting of hemolytic anemias where they remain an important criterion for the diagnosis. As the identification of these cells is still problematic, the International Council for Standardization in Hematology (ICSH) set up a consensus report in November, 2011. The French Group of Cellular Hematology (GFHC) aimed to collect the opinion of French biologists directly confronted to schistocytes measurements, about these guidelines. Among the 578 professionals, 169 (29%) answered to the 10 questions dealing with the identification and measurements of schistocytes as proposed by the ICSH. A consensus was reached for the urgent need of such guidelines documents, especially in the current background of the European accreditation EN ISO 15189 rules. A traduction in native (French) language was warmly wished in order to facilitate the diffusion of the information. The pathologic threshold for the diagnosis of thrombotic microangiopathic anemia (TMA) (>1%) remained questionable. For half of the biologists, the new fragmented red blood cell (FRC) parameter recently provided by two manufacturers of automated blood cell counters was still doubtful for routine use. This survey assessed the impact of international 'guidelines' on the French biological community. The will to implement validated recommendations was strong, reflecting the awareness of the biologists to standardize the laboratory investigations.

Microscopic schistocyte determination according to International Council for Standardization in Hematology recommendations in various diseases

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. 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. 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. 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.

English

VesMATIC cube80™ is a newly developed automated method for measurement of erythrocyte sedimentation rate (ESR). AIMS: The aim of our study was to compare the ESR values by VesMATIC cube80™ against the modified Westergren method. MATERIALS AND METHODS: This cross-sectional study was conducted at clinical laboratory in Kasturba Hospital, Manipal on 162 patients. The samples taken were as per the recommendations charted out by International Council for Standardization in Hematology (ICSH) for comparing automated and manual Westergren method. STATISTICAL ANALYSIS USED: Bland and Altman statistical analysis was applied for evaluating VesMATIC cube80™ against the conventional Westergren method. RESULTS: The analysis revealed a low degree of agreement between the manual and automated methods especially for higher ESR values (25mm/hr.), i.e., mean difference -0.44 (95% limits of agreement, -41.23 to 40.37). This discrepancy which is of clinical significance was less evident for ESR values in the normal range <25 mm/hour (-1.73 mean of difference; -18.0 to 10 limits of agreement). CONCLUSIONS: The fully automated system VesMATIC cube80™ for ESR measurement tends to underestimate the manual ESR readings. Further studies and validation experiments would be required.

In the realm for standardization in immunophenotyping

STANDARDIZATION and quality control is the keystone of reliable and trustworthy diagnosis. This is particularly true for cellular diagnostics where the preanalytics, instrumentation, and reagents are very diverse and prone to innumerable variations affecting the results (1). Furthermore, as critical are the objects of interest—the cells from biological sources—that are fine-tuned in their composition and behavior in the organism and may be sensitive to various modifications of environmental changes once outside the body. So defining the best and most reproducible assays for their detailed analysis is of utmost relevance for trustworthy diagnosis. Roussel and colleagues from Rennes, France, Bangor, Maine and Seattle, Washington USA (this issue, page 973) scrutinized on behalf of the International Council for Standardization in Hematology (ICSH) three different flow cytometric methods in order to find the reference method for leukocyte differential counts in human blood. In clinical laboratories, nowadays hematology analyzers are routinely used to obtain whole blood differential leukocyte counts. Unfortunately, each of the systems has its own bias as has recently been shown (1) when it comes to rare or unusual cells. In this study, the ICSH study group compared in a multicenter endeavor (three countries), different instruments including automated analyzers, manual counting and in addition two different standard flow cytometers were used. The immunophenotype panels and whole blood, no wash staining and analysis protocols were adapted to each of the flow cytometers from different providers. There are several conclusions from this thorough study. First of all, the bias of hematological automates is that up to 50% of the analysis has to be confirmed by tedious and time consuming manual counting on a microscope. This is not necessary when using flow cytometry analysis. The use of staining of the cell nucleus by DNA specific dyes is useful in order to detect all nucleated cells including premature red blood cells but is difficult to standardize. Finally, the protocols need to be extended to quantify the clinically relevant immature leukocytes and blasts by extending the antibody panel. Also, the characterization of other specific clinically relevant subsets like resident and activated monocytes may be of relevance (2). So in summary, new higher level consensus protocols need evaluation in a clinical setting to approach clinical cytomics in diagnosis. In this respect, various research groups took the effort to increase the depth of analysis by combining a multitude of differentially colorized monoclonal antibodies in a painful and tedious development of optimized multicolor immunofluorescence panels (OMIPs). As examples, recently a 10-color OMIP for the differentiation of hematopoietic cell samples (3) or for memory B-cells (4) were introduced. Now, Mahnke and colleagues from Bethesda, Maryland, USA (this issue, page 935) present a new 13-color 12-antibody panel in order to detail the differentiation of human T-cells. This panel has the advantage of combining phenotypic markers for the unequivocal identification of specific cell types with differentiation and activation markers for their further characterization. Many of the published OMIPs have the potential of giving invaluable input for future standardized clinical panels and many more are to come. Next to the identification of the right instrument and the staining panel, the correct way to isolate and store cells until the

Microscopic examination of bone marrow aspirate in healthy adults – comparison of two techniques of slide preparation

According to the International Council for Standardization in Hematology (ICSH) guidelines for the standardization of bone marrow specimens and reports, smears from bone marrow aspirates for microscopic examination should be prepared using two techniques simultaneously: the wedge-spread and the crush technique. However, the outcomes of these techniques have never been compared. We investigated the bone marrow of 105 adult, haematologically healthy subjects, using bone marrow smears prepared via both techniques simultaneously. Comparison of the two techniques revealed significant differences in terms of the composition of bone marrow cells. Only the percentages of lymphocytes, mature eosinophils and basophils did not differ significantly. The reference ranges for each technique were established. The crush technique seems to be more valuable than the wedge-spread technique because of the lack of a blood dilution effect and better assessment of megakaryopoiesis. We recommend the crush technique for the evaluation of the percentage composition of bone marrow cells. In a very small number of patients with irregular cell localization in the bone marrow particles, the wedge-spread technique may be more beneficial for the assessment of total cellularity. The recommendation to routinely prepare slides using both of these techniques is fully justified.

ICSH recommendations for identification, diagnostic value, and quantitation of schistocytes

Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage within the circulation. The detection of schistocytes is an important morphological clue to the diagnosis of thrombotic microangiopathic anemia (TMA). Reporting criteria between different laboratories, however, are not uniform, owing to variability of shape and nature of fragments, as well as subjectivity and heterogeneity in their morphological assessment. Lack of standardization may lead to inconsistency or misdiagnosis, thereby affecting treatment and clinical outcome. The Schistocyte Working Group of the International Council for Standardization in Haematology (ICSH) has prepared specific recommendations to standardize schistocyte identification, enumeration, and reporting. They deal with the type of smear, method of counting, morphological description based on positive criteria (helmet cells, small, irregular triangular, or crescent-shaped cells, pointed projections, and lack of central pallor). A schistocyte count has a definite clinical value for the diagnosis of TMA in the absence of additional severe red cell shape abnormalities, with a confident threshold value of 1%. Automated counting of RBC fragments is also recommended by the ICSH Working Group as a useful complement to the microscope, according to the high predictive value of negative results, but worthy of further research and with limits in quantitation.

A flow cytometric method for platelet counting in platelet concentrates

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. 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. 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%. 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.

Evaluation of an automated erythrocyte sedimentation rate analyzer as compared to the Westergren manual method in measurement of erythrocyte sedimentation rate

Monitor 100® (Electa Lab, Italy) is a newly developed automated method for measurement of erythrocyte sedimentation rate (ESR). The aim of our study was to compare the ESR values by Monitor 100® against the standard Westergren method. This cross-sectional study was conducted at a Level I trauma care center on 200 patients. The samples taken were as per the recommendations charted out by International Council for Standardization in Hematology (ICSH) for comparing automated and manual Westergrens method. Bland and Altman statistical analysis was applied for evaluating Monitor 100® against the conventional Westergren method. The analysis revealed a low degree of agreement between the manual and automated methods especially for higher ESR values, mean difference -11.2 (95% limits of agreement, -46.3 to 23.9) and mean difference -13.4 (95% limits of agreement-58.9 to 32.1) for 1 and 2 hours, respectively. This discrepancy which is of clinical significance was less evident for ESR values in the normal range <25 mm/hour (-7.7 mean of difference; -18.9 to 3.5 limits of agreement). The fully automated system Monitor 100® for ESR measurement tends to underestimate the manual ESR readings. Hence it is recommended that a correction factor be applied for the range of ESR values while using this equipment. Further studies and validation experiments would be required.