Where are we at with point-of-care testing in haematology?

Point-of-care testing (POCT) in haematology has continued to grow in popularity and uptake throughout the world. The increasing demand to reduce the turnaround time of test results, coupled with rapid improvements in technology, have led to the development of several devices that are designed for use in different clinical settings, with the hope of improving patient care. The most used POCT in haematology is measurement of haemoglobin concentration. Other POCT devices (used primarily in developing countries) for malaria screening and CD4+ T-lymphocytes for quantification of human-immunodeficiency-virus are becoming the cornerstone for the diagnosis and management of these disorders. New devices are also available for red cell indices, white blood cell count and platelets. In this review clinical studies that validate the use of such devices will be discussed, as well as the advantages and disadvantages of POCT in haematology. A disadvantage of POCT is a lack of training, poor standardization in obtaining blood samples and insufficient internal/external quality assessment. As there is every reason to expect that POCT use will increase in all pathology disciplines, including haematology, it is imperative that systems are put in place to oversee these issues.

Guidelines for the laboratory investigation of heritable disorders of platelet function.

The guideline writing group was selected to be representative of UK-based medical experts. MEDLINE was systematically searched for publications in English up to the Summer of 2010 using key words platelet, platelet function testing and platelet aggregometry. Relevant references generated from initial papers and published guidelines/reviews were also examined. Meeting abstracts were not included. The writing group produced the draft guideline, which was subsequently revised and agreed by consensus. Further comment was made by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 40 UK haematologists, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology Committee and comments incorporated where appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 7 of the Procedure for Guidelines Commissioned by the BCSH [http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html]. The objective of this guideline is to provide healthcare professionals with clear guidance on platelet function testing in patients with suspected bleeding disorders. The guidance may not be appropriate to patients receiving antiplatelet therapy and in all cases individual patient circumstances may dictate an alternative approach.

Guidelines for point-of-care testing: haematology.

This guideline provides a framework for the arrangement of point-of-care testing (POCT) services, previously known as near patient testing (patient self-testing not covered). POCT is defined as any analytical test performed outside the laboratory. Primary users are often non-laboratory healthcare workers. The guidance applies to units within hospitals as well as general practioner surgeries, community clinics and pharmacies. The head of the haematology laboratory or a point of care coordinator must take responsibility for all aspects of the POCT service, including quality and training. Depending on the size and nature of the POCT practice, a local POCT manager may also be required. Equipment selected should have received a successful independent performance evaluation. If an independent evaluation has not been performed the purchaser should assess the device according to the protocol in this document. POCT devices should generate results that are comparable to those of the local laboratory. An accredited external quality assessment programme and internal quality control system must be established. Manufacturers promoting POCT devices designed for non-laboratory sites, e.g. pharmacies, should undertake training and annual competency assessment, perhaps using a web-based system. A diagram to illustrate the stages for the implementation of a POCT service is illustrated.

Development of an automated malaria discriminant factor using VCS technology.

Malaria diagnosis presents a challenge to all laboratories. There is a need for rapid, sensitive, and cost-effective screening on all samples, particularly in areas where malaria is endemic. Response to malaria infection involves an increased monocyte count and production of large activated monocytes. These changes can be detected by volume, conductivity, and scatter (VCS) technology on certain automated blood cell counters (Beckman Coulter, Miami, FL). The SD of the volume of lymphocytes and monocytes demonstrates a significant difference from normal when malaria is present. By using a calculation derived from the SD volume of the lymphocytes and monocytes, herein termed the malaria factor, sensitivity of 98% and specificity 94% were demonstrated for the detection of malaria. Based on this derived discriminant, VCS technology should become a useful tool in the detection of malaria. A flag to indicate the potential presence of malaria could then be generated by the instrument if the user or manufacturer chose to do so.

Platelets: the few, the young, and the active.

Many modern automated cell counters in high-volume clinical hematology laboratories use new, improved technologies for routine platelet analysis. The latest progress includes the use of state-of-the art information technology, specific fluorescent dyes, and monoclonal antibodies to obtain more reliable platelet counts. This information allows the accurate and precise enumeration of platelets even in thrombocytopenic patients and the reporting of novel platelet parameters. In the near future, digital image analysis may permit even better platelet analysis.

The value of the white precursor cell channel (WPC) on the Sysmex XN-1000 analyser in a specialist paediatric hospital.

BACKGROUND: Historically, haematology analyser flags for abnormal white blood cells (WBCs) show good sensitivity but lower specificity, causing unnecessary blood film reviews. While the WBC differential channel on Sysmex XE and XN instruments reports a combined flag for blasts/abnormal lymphocytes, the new white precursor cell channel (WPC) on the XN series has been introduced to separate this into a specific flag for either cell type or, if no abnormality, remove the flag entirely. AIMS: To compare the efficiency of abnormal WBC flags from the XN WPC to our existing analyser and determine whether WPC can reduce false positive flags and blood films required. METHODS: Abnormal WBC flags from the Sysmex XE-5000 and XN-1000 were compared to manual differential and blood film morphology on 300 K2EDTA samples from infants and children. RESULTS: The XN WPC flag for blasts was more sensitive and specific than flags indicating blasts on the XE-5000, with a reduction in false positives from 64% (XE) to 36% (XN). Overall efficiency of the WPC flag for abnormal lymphocytes was 94% vs 79% on the XE. WPC reduced false positive flags for blasts and abnormal lymphocytes on neonatal samples by 50%. Automatic reflex analysis by WPC correctly removed a false positive flag from the white cell differential channel on 46% of samples. Total abnormal WBC flags from XN WPC were less (73) than the XE-5000 (92). CONCLUSIONS: XN WPC demonstrated superior efficiency of abnormal WBC flags on paediatric samples, compared to the XE-5000, with greater sensitivity and specificity of flagging, reducing blood films for review.

Performance evaluation of the Sysmex haematology XN modular system.

BACKGROUND: The Sysmex XN haematology instrument performs automatic reflex testing, depending on sample results. A nucleated red blood cell (NRBC) count is provided on all samples. The instrument has a smaller footprint (34%) than previous Sysmex XE analysers. METHODS: An evaluation comparing all results to the Sysmex XE-2100 and manual microscopic differential and morphology (n=390) was performed followed by a workflow study of 1000 samples to compare speed of operation and number of blood films reviews required from both systems. RESULTS: The new features on the instrument are: (1) white cell and NRBC channel, all samples include the NRBC count; (2) white cell precursor channel: false positive flags for blasts, abnormal lymphocytes and atypical lymphocytes are reduced significantly without a statistical increase of false negatives; (3) low white cell count mode: suggested setting of <0.5×10(9)/l. An extended count is more precise and provides an accurate differential. Fluorescent platelet count is performed in a dedicated channel. If the red cell or platelet size histograms are abnormal or if the platelet count is low, then a fluorescent platelet count is automatically performed. Good correlation with the XE-2100 and manual differential was found and the improved results compared to the reference flow cytometric analysis for platelet counts, especially below 30×10(9)/l (XE-2100, R(2)=0.500; XN, R(2)=0.875). CONCLUSION: The XN showed reduced sample turnaround time of 10% and reduced number of blood films for examination, 49% less than the XE-2100 without loss of sensitivity with more precise and accurate results on low cell counts.

The accuracy of platelet counting in thrombocytopenic blood samples distributed by the UK National External Quality Assessment Scheme for General Haematology.

A knowledge of the limitations of automated platelet counting is essential for the effective care of thrombocytopenic patients and management of platelet stocks for transfusion. For this study, 29 external quality assessment specimen pools with platelet counts between 5 and 64 × 10(9)/L were distributed to more than 1,100 users of 23 different hematology analyzer models. The same specimen pools were analyzed by the international reference method (IRM) for platelet counting at 3 reference centers. The IRM values were on average lower than the all-methods median values returned by the automated analyzers. The majority (~67%) of the automated analyzer results overestimated the platelet count compared with the IRM, with significant differences in 16.5% of cases. Performance differed between analyzer models. The observed differences may depend in part on the nature of the survey material and analyzer technology, but the findings have implications for the interpretation of platelet counts at levels of clinical decision making.

Improved flagging rates on the Sysmex XE-5000 compared with the XE-2100 reduce the number of manual film reviews and increase laboratory productivity.

Hematology analyzers generate suspect flags in the presence of abnormal cells. False-positive rates for flags are high on all analyzers. Sysmex, Kobe, Japan, has developed new software for its XE-5000 with improved algorithms for flagging blast cells, abnormal lymphocytes or lymphoblasts, and atypical lymphocytes. This study evaluated the efficiency of these flags in 1,002 samples. The XE-5000 was compared with the XE-2100 (Sysmex) and microscopic examination of cell morphologic features. On the XE-2100, the blast flag demonstrated 90 false-positives, 13 true-positives, and 3 false-negatives. The values on the XE-5000 were 27 false-positives, 14 true-positives, and 2 false-negatives. The abnormal lymphocyte/lymphoblast flag was assessed with the atypical lymphocyte flag. The XE-2100 showed 114 false-positives, 23 true-positives, and 20 false-negatives, and on the XE-5000, there were 45 false-positives, 22 true-positives, and 21 false-negatives. This more specific flagging reduces the number of films that require manual review.

Cold storage of 'cryohydrocytosis' red cells: the osmotic susceptibility of the cold-stored erythrocyte.

'Cryohydrocytosis' is an unusual human haemolytic anaemia of the 'hereditary stomatocytosis' group, in which the red cell membrane is abnormally permeable to Na and K+ at both body and (even more prominently) refrigerator temperatures. If whole cryohydrocytosis blood is anticoagulated in heparin or EDTA and stored on ice overnight, about 50% of the cells will lyse. Citrate phosphate dextrose adenine (CPDa) anticoagulant, empirically verified as an optimal anticoagulant for storage of normal blood before transfusion, very markedly ameliorated this overnight lysis, suggesting that these cells might form an informative model in which cold storage of the red cell could be studied in a short time scale. Accordingly, we conducted studies of ion flux, cell swelling and lysis in different media used historically for blood preservation and compared the experimental data with an 'integrated red cell model', which seeks mathematically to model the osmotic behaviour of red cells under different conditions. Upon experiment, lysis in these cells was reduced by additives that could be regarded as impermeant extracellular solutes (citrate, mannitol) and by low pH, but not by those agents that are regarded as protecting the cell against energy depletion or oxidation (adenine, glucose, nicotinic acid). The protective effects of these extracellular additives were all reproduced by the computer simulation, confirming the validity of this model, although the effect of pH could be simulated only semi-quantitatively, possibly because Na+ permeability itself depends on pH.

Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia.

A new automated method to reliably quantify reticulated platelets, expressed as the immature platelet fraction (IPF), has been developed utilizing the XE-2100 blood cell counter with upgraded software (Sysmex, Kobe, Japan). The IPF is identified by flow cytometry techniques and the use of a nucleic acid specific dye in the reticulocyte/optical platelet channel. The clinical utility of this parameter was established in the laboratory diagnosis of thrombocytopenia due to increased peripheral platelet destruction, particularly autoimmune thrombocytopenic purpura (AITP) and thrombotic thrombocytopenic purpura (TTP). Reproducibility and stability results over 48 h were good. An IPF reference range in healthy individuals was established as 1.1-6.1%, with a mean of 3.4%. Patients in whom platelet destruction might be abnormal, were studied and two of these patients followed serially during the course of treatment. The IPF was raised in several disease states. The most significant increases in IPF values were found in patients with AITP (mean 22.3%, range 9.2-33.1%) and acute TTP (mean 17.2%, range 11.2-30.9%). Following patients during treatment demonstrated that as the platelet count recovered the IPF% fell. These results show that a rapid, inexpensive automated method for measuring the IPF% is feasible and should become a standard parameter in evaluating the thrombocytopenic patient.

Evaluation of immature granulocyte counts by the XE-IG master: upgraded software for the XE-2100 automated hematology analyzer.

We evaluated an automated immature granulocyte (IG) count in the peripheral blood with the XE-IG Master (Sysmex Corporation). The XE-IG Master is the upgraded software package for the XE-2100 automated hematology analyzer. Reproducibility tests demonstrated a mean coefficient of variation of 7.02% for the IG percentage (IG%) and 6.93% for the absolute IG count. Correlations of the IG counts were assessed in two ways. A flow cytometric IG count using CD11b, CD16, and CD45 monoclonal antibodies and a manual differential count were used as reference methods. The regression equation and the correlation coefficient of the IG% for the flow cytometric reference count versus results with the XE-IG Master were: y = 0.91x + 0.10; r = 0.96. For the comparison with the manual differential count of promyelocytes, myelocytes, and metamyelocytes, the regression equation and correlation coefficient were: y = 0.81x + 1.27; r = 0.78. Samples were found to be stable up to 48 hours both at room temperature and when refrigerated. We investigated the clinical significance of the IG count as a new marker of acute inflammation. In this preliminary study, most samples with a high IG count had positive values for C-reactive protein and the erythrocyte sedimentation rate (positive sample rates were 84.0% and 95.0%, respectively) despite neutrophil counts within the normal range. Elevated IG counts correlated most closely with CD64 expression on polymorphonuclear cells and less so with the concentration of interleukin 6. Compared with other available inflammation markers, the IG count is rapidly generated with each full blood count at no extra cost and with no delay in sample analysis.