Roger S. Riley

Reticulocyte analysis by flow cytometry and other techniques

Enumeration of peripheral blood reticulocytes is an essential part of the diagnosis and management of anemic patients, since the number of reticulocytes in the peripheral blood reflects the erythrocytic activity of the bone marrow. Reticulocyte enumeration using flow cytometric methodology is rapidly replacing the inaccurate, imprecise manual counting technique used in the past. This article explores the pathophysiology of the reticulocyte, the various means of counting reticulocytes, and the diverse clinical applications of reticulocyte data.

Clinical utilization of the international normalized ratio (INR)

The prothrombin time (PT) is one of the most important laboratory tests to determine the functionality of the blood coagulation system. It is used in patient care to diagnose diseases of coagulation, assess the risk of bleeding in patients undergoing operative procedures, monitor patients being treated with oral anticoagulant (coumadin) therapy, and evaluate liver function. The PT is performed by measuring the clotting time of platelet‐poor plasma after the addition of calcium and thromboplastin, a combination of tissue factor and phospholipid. Intra‐ and interlaboratory variation in the PT was a significant problem for clinical laboratories in the past, when crude extracts of rabbit brain or human placenta were the only source of thromboplastin. The international normalized ratio (INR), developed by the World Health Organization in the early 1980s, is designed to eliminate problems in oral anticoagulant therapy caused by variability in the sensitivity of different commercial sources and different lots of thromboplastin to blood coagulation factor VII. The INR is used worldwide by most laboratories performing oral anticoagulation monitoring, and is routinely incorporated into dosage planning for patients receiving warfarin. Although the recent availability of sensitive PT reagents prepared from recombinant human tissue factor (rHTF) and synthetic phospholipids eliminated many of the earlier problems associated with the use of crude thromboplastin preparations, local instrument variability in the INR still remains a problem. Presently, the use of plasma calibrants seems the best solution to this problem. Standardizing the point‐of‐care instruments for INR monitoring is another dilemma faced by the industry. Ultimately, new generations of anticoagulant drugs may eliminate the need for laboratory monitoring of anticoagulant therapy. J. Clin. Lab. Anal. 14:101–114, 2000.

Immunophenotypic analysis of acute lymphocytic leukemia

Acute lymphoblastic leukemia (ALL) is one of the most common hematologic malignancies. Flow cytometry is an integral part of ALL diagnosis and also provides significant patient prognostic information. This article is a practical review of the basic principles of the flow cytometric evaluation of acute leukemias, the interpretation of flow cytometric data, and the management of practical problems such as aberrant antigen, hematogones, bone marrow regeneration, and minimal residual disease.

Platelet Satellitism as Presenting Finding in Mantle Cell Lymphoma A Case Report

Platelet satellitism surrounding polymorphonuclear neutrophils has been observed almost exclusively in EDTA-treated blood at room temperature. The mechanism underlying this phenomenon is not understood fully. We report a case of platelet rosetting around atypical lymphocytes in peripheral blood smears made from EDTA-treated and untreated blood. Flow cytometry of the peripheral blood sample and immunohistochemical stains of the subsequent bone marrow biopsy specimen revealed a monoclonal B-cell population positive for CD5, CD20, and cyclin D1 and negative for CD3 and CD23; cytogenetic findings revealed a complex karyotype that included t(11;14). These findings were consistent with mantle cell lymphoma. To our knowledge, the finding of platelet satellitism involving mantle cell lymphoma cells in peripheral blood has not been reported previously.

Bone marrow aspirate and biopsy: a pathologist's perspective. II. interpretation of the bone marrow aspirate and biopsy.

Bone marrow examination has become increasingly important for the diagnosis and treatment of hematologic and other illnesses. Morphologic evaluation of the bone marrow aspirate and biopsy has recently been supplemented by increasingly sophisticated ancillary assays, including immunocytochemistry, cytogenetic analysis, flow cytometry, and molecular assays. With our rapidly expanding knowledge of the clinical and biologic diversity of leukemia and other hematologic neoplasms, and an increasing variety of therapeutic options, the bone marrow examination has became more critical for therapeutic monitoring and planning optimal therapy. Sensitive moleculartechniques, in vitro drug sensitivity testing, and a number of other special assays are available to provide valuable data to assist these endeavors. Fortunately, improvements in bone marrow aspirate and needle technology has made the procurement of adequate specimens more reliable and efficient, while the use of conscious sedation has improved patient comfort. The procurement of bone marrow specimens was reviewed in the first part of this series. This paper specifically addresses the diagnostic interpretation of bone marrow specimens and the use of ancillary techniques. J. Clin. Lab. Anal. 23:259–307, 2009.

The virtual blood film.

The computer and the digital camera offer unprecedented possibilities for improving hematology education, research, and patient service. Peripheral blood smear images of exceptional quality can be acquired rapidly and conveniently from the peripheral blood smear with a modern, high-resolution digital camera and a quality microscope. Digital cameras use CCD or CMOS image sensors to measure light energy and additional circuitry to convert the measured information into a digital signal. Because digital cameras do not use photographic film, images are immediately available for incorporation into web sites or digital publications, printing, transfer to other individuals by e-mail, or other applications. Several excellent consumer digital still cameras are now available for less than

Intragraft cytokine expression and tolerance induction in rat renal allografts.

1000 that capture high-quality images comprised of more than three megapixels. These images are essentially indistinguishable from conventional film images when viewed on a quality color monitor or printed on a quality color or black and white printer at sizes up to 8 x 10 in. Several recent dedicated digital photomicroscopy cameras provide an ultrahigh quality image output of more than 12 megapixels and have low noise circuit designs permitting the direct capture of darkfield and fluorescence images. There are many applications of digital images of peripheral blood smears. Because hematology is a visual science, the inclusion of quality digital images into lectures, teaching handouts, and electronic documents is essential. A few institutions have gone beyond the basic application of digital images to develop large electronic hematology atlases; animated, audio-enhanced learning experiences; multidisciplinary Internet conferences; and other innovative applications. Digital images of single microscopic fields (single-frame images) are the most widely used in hematology education at this time, but single images of many adjacent microscopic fields can be stitched together to prepare zoomable panoramas that encompass a large part of a microscope slide and closely stimulate observation through a real microscope. With further advances in computer speed and Internet streaming technology, the virtual microscope could easily replace the real microscope in pathology education. Interactive, immersive computer experiences may completely revolutionize hematology education and make the conventional lecture and laboratory format obsolete later in this decade. Patient care is enhanced by the transmission of digital images to other individuals for consultation and education, and by the inclusion of these images in patient care documents. In research laboratories, digital cameras are widely used to document experimental results and obtain experimental data.

Widely Used Types and Clinical Applications of D-Dimer Assay

BACKGROUND Intragraft cytokine expression was evaluated in a model of renal transplantation. ACI and Lewis rats were used as donors and recipients, respectively, for heterotopic renal transplantation. METHODS Treated allograft rats (n=10) received a preoperative dose of rapamycin and cyclosporine, followed by 7 days of cyclosporine postoperatively. Isograft rats (n=5) and control allograft rats (n=4) received no immunosuppression. Sacrifice was performed after 120 days. Expression of interleukin (IL)-4, IL-10, and interferon-gamma (IFN-gamma) transcripts was determined with semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS All treated allograft rats had normal function with 50% histologic rejection. All isografts had normal function. IL-4 and IL-10 were in greater density in allografts with normal histology, whereas IFN-gamma was only seen in allografts with cellular rejection. No IL-10 was seen in isografts, but IL-4 was detected in 3/5 isografts. CONCLUSIONS We conclude that the lymphocyte populations elaboration of IL-4 and IL-10 is associated with tolerance, whereas the production of IFN-gamma and absence of IL-4 is associated with histology suggestive of acute cellular rejection.

Immunobiology and long‐term graft function in a transplant heterotopic renal rat model

D-dimers are formed by the breakdown of fibrinogen and fibrin during fibrinolysis. D-dimer analysis is critical for the diagnosis of deep vein thrombosis, pulmonary embolism, and disseminated intravascular coagulation. Modern assays for D-dimer are monoclonal antibody based. The enzyme-linked immunosorbent assay (ELISA) is the reference method for D-dimer analysis in the central clinical laboratory, but is time consuming to perform. Recently, a number of rapid, point-of-care D-dimer assays have been developed for acute care settings that utilize a variety of methodologies. In view of the diversity of D-dimer assays used in central laboratory and point-of-care settings, several caveats must be taken to assure the proper interpretation and clinical application of the results. These include consideration of preanalytical variables and interfering substances, as well as patient drug therapy and underlying disease. D-dimer assays should also be validated in clinical studies, have established cut-off values, and reported according to the reagent manufacturers recommendations.

A pathologist's perspective on bone marrow aspiration and biopsy: I. performing a bone marrow examination

Abstract: Background: The Th1–Th2 paradigm proposes clonal expansion of Th2 lymphocytes as the basis of allograft tolerance. The Th2 cells have been found to be present in recipients with long‐term allograft survival. However, the presence of Th2 cells and tolerance is not a uniform finding. Previously we have shown that pre‐engraftment single dose rapamycin and a 7‐d course of cyclosporin induce transplantation tolerance to 120 d. In the present study, we investigated the immunobiology of grafts in a long‐term follow‐up (>350 d). Methods: Kidney allografts (n = 7), isografts (n = 5) and single nephrectomy (n = 3) groups were followed for 350 ± 87 d. Heterotopic kidney transplant was performed by the same surgeon in the allograft group (ACI‐Lewis) and the isograft group (Lewis‐Lewis). The left kidney was removed in the single nephrectomy group. The allograft group was treated with pre‐engraftment single dose rapamycin and a 7‐d course of cyclosporin. A kidney biopsy was performed at midpoint time for histological study and tissue was frozen for measuring intragraft cytokine expression (IL‐4, IL‐10) in all animals. Prior to biopsy, serum blood urea nitrogen (BUN) and creatinine (Cr) levels were studied. Serum BUN, Cr levels, plus 24‐h urinary protein (PRO) were measured prior to sacrifice. Randomly, four allograft rats received skin grafts (ACI, Lewis and Buffalo skin donors) after kidney biopsy. Skin grafts were studied for a mean of 6 weeks for signs of acceptance or rejection. Analysis of variance (ANOVA) with Tukeys test was used; p < 0.05 was considered statistically significant. Results: The mean follow up was 352 ± 87 d. BUN and Cr levels at biopsy time (mean 214 d) were not statistically different between the three groups (p = 0.19 and p = 0.66). At sacrifice (mean 352 d), BUN, Cr and PRO were statistically different between allograft and isograft groups (p = 0.013), and between allograft and single nephrectomy groups (p = 0.027). Functional and histological signs of graft loss occurred in three of seven (42.8%) of the allografts at 352 d. Using BANFF criteria, three allografts at biopsy time and seven allografts (100%) and four isografts (80%) at sacrifice time developed chronic histologic changes. Intragraft overexpression of IL‐4 and IL‐10 was seen at biopsy and sacrifice time in six of seven allografts and one of five isografts. All donor specific skin grafts (ACI‐Lewis) on allografts were accepted and third party (Buffalo) donor skin grafts were rejected in all animals (>95% skin necrosis). Conclusions: This highly stringent, functional, renal transplant model yields 100% normal renal function as compared with isografts at 120 d follow‐up. With the follow‐up extended to 350 d, 43% of the allografts loose function and develop a chronic allograft histology despite a demonstrated intragraft Th2 cytokine dominance and donor specific skin graft acceptance.