Air-dried Blood Research Articles

Establishing tools for early diagnosis of congenital toxoplasmosis: Flow cytometric IgG avidity assay as a confirmatory test for neonatal screening

The aim of this study was to evaluate the performance of conventional serology (Q-Preven™ and ELFAVIDAS™) and flow cytometry-based serologic tools for early serologic diagnosis of congenital toxoplasmosis. The study groups included prospectively confirmed cases of congenital toxoplasmosis (TOXO=88) and age-matching non-infected controls (NI=15).The results demonstrated that all samples tested positive/indeterminate for anti-T. gondii IgM screening at birth using air-dried whole blood samples. Serum samples collected at 30–45days after birth tested positive for ELFAVIDAS™ IgG in both groups. While all NI tested negative for ELFAVIDAS™ IgM and IgA, only 78% and 36% of TOXO tested positive for IgM and IgA, respectively. Flow cytometry-based anti-T. gondii IgM, IgA and IgG reactivity displayed moderate performance with low sensitivity (47.6%, 72.6% and 75.0%, respectively). Regardless the remarkable specificity of IgG1, IgG2 and IgG3 subclasses for early diagnosis, weak or moderate specificity was observed (Se=73.9%, 60.2% and 83.0%, respectively). The analysis of IgG avidity indices (AI) demonstrated the highest performance among the flow cytometry-based methods (Se=96.6%; Sp=93.3%), underscoring the low avidity index (AI<60%) within TOXO (97.0%) in contrast with the high avidity index (AI>60%) in NI (93%). Analysis of anti-T. gondii IgG and IgG3 reactivity for mother:infant paired samples may represent a relevant complementary tests for early diagnosis. In conclusion, a feasible high-standard algorithm (Accuracy=97.1%) was proposed consisting of Q-Preven™ IgM screening at birth, followed by ELFAVIDAS™ IgM and flow cytometric IgG avidity analysis at 30–45days after birth as a high performance tool for early serological diagnosis of congenital toxoplasmosis.

Diagnosis of inherited platelet disorders on a blood smear: a tool to facilitate worldwide diagnosis of platelet disorders

Background Many hereditary thrombocytopenias and/or platelet function disorders have been identified, but diagnosis of these conditions remains challenging. Diagnostic laboratory techniques are available only in a few specialized centers and, using fresh blood, often require the patient to travel long distances. For the same reasons, patients living in developing countries usually have limited access to diagnosis. Further, the required amount of blood is often prohibitive for pediatric patients. Objectives By a collaborative international approach of four centers, we aimed to overcome these limitations by developing a method using blood smears prepared from less than 100 μL blood, for a systematic diagnostic approach to characterize the platelet phenotype. Methods We applied immunofluorescence labelling (performed centrally) to standard air-dried peripheral blood smears (prepared locally, shipped by regular mail), using antibodies specific for proteins known to be affected in specific hereditary platelet disorders. Results By immunofluorescence labelling of blood smears we characterized the underlying cause in 877/3217 (27%) patients with suspected hereditary platelet disorders (HPD). Currently about 50 genetic causes for HPD are identified. Among those, the blood smear method was especially helpful to identify MYH9 disorders/MYH9-related disease, biallelic Bernard-Soulier syndrome, Glanzmann thrombasthenia and gray platelet syndrome. Diagnosis could be established for GATA1 macrothrombocytopenia, GFI1B macrothrombocytopenia, ß1-tubulin macrothrombocytopenia, filamin A-related thrombocytopenia and Wiskott-Aldrich syndrome. Conclusion Combining basic and widely available preanalytical methods with the immunomorphological techniques presented here, allows detailed characterization of the platelet phenotype. This supports genetic testing and facilitates diagnosis of hereditary platelet disorders for patients of all ages around the world.

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimens

Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.

BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimen

Introduction Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. Objective To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. Methodology 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. Results V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. Conclusion DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy. Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy.

A Study of Peripheral Blood in Hedgehogs in Turkey

The aim of this study was to determine diameters of blood cells, differential counts of peripheral blood leukocytes, alpha-naphthyl acetate esterase (ANAE), acid phosphatase (ACP-ase) activity of some leukocyte types, and enzymatic positivity percentages of peripheral blood lymphocytes in two hedgehogs species, Hemiechinus auritus, the long-eared hedgehog, and Erinaceus concolor, the southern white-breasted hedgehog. Air-dried peripheral blood smears were stained with May-Grünwald-Giemsa stain. ANAE and ACP-ase were stained in glutaraldehyde-acetone-fixed smears. ANAE-positive lymphocytes displayed a dot-like positivity pattern characterized with 1-5 reddish brown cytoplasmic granules, whereas ACP-ase positive lymphocytes displayed a dot-like positivity pattern characterized with 1-3 pinkish cytoplasmic granules. Monocytes gave a diffuse and strong reaction while neutrophils displayed a weak positive reaction for ANAE and ACP-ase. No difference was observed in mean diameters of peripheral blood cells of these species. It was found that lymphocytes made up the majority (64.3% and 65.5%) of leukocytes, followed by neutrophils (23.9% and 23.3%), eosinophils (9.0% and 7.6%), monocytes (1.8% and 2.3%), and basophils (1.0% and 1.3%) in H. auritus and E. concolor, respectively. Mean ANAE positivity oflymphocytes was 36.6% and 51.3% and ACP-ase positivity was 32.1% and 37.5% for H. auritus and E. concolor, respectively. The ANAE positivity of lymphocytes in E. concolor was significantly (P < 0.05) higher than that of H. auritus.

Exploring the limits for the survival of DNA in blood stains

It is generally recognized that usable DNA may be retained in dry biological stains for years. We have explored the environmental limits for this property. Air-dried blood stains were incubated at different conditions of relative humidity (RH) and temperature. The quality of the extracted DNA was assessed by the ability to amplify 273 bp and 1600 bp DNA fragments by PCR, and by quantitative estimation of a 147 bp DNA fragment using real time PCR. Despite the fact that the availability of water is important for processes that degrade DNA, no significant difference was observed in the stability of DNA at 50%, 80% or 93% RH at room temperature or at 35 °C, and even the 1600 bp fragment was amplifiable after one year. Microbial growth was not observed at these conditions and the number of template molecules did not drop significantly over time. At 100% RH, however, microbial growth was observed after varying amounts of time. This may explain the decreased stability of DNA observed at these conditions. Even so, the 273 bp fragment was amplifiable for at least 3 months, and the 1600 bp fragment for at least two months. Microbial growth was not observed at higher temperatures (45–65 °C) at 100% RH, and the 1600 bp fragment was amplifiable after eight months at 45 °C, but only survived for one month at 55 °C or 65 °C. Thus DNA remains amplifiable in blood stains for many months, even at extreme RH and temperatures up to 45 °C. Even in humid climates the average RH is usually not more than 80% and RH rarely exceeds 93%; therefore we conclude that normal climatic conditions are not critical for the long time survival of DNA in untreated blood stains.

Haematological tests in sheep health management

The evaluation of sheep complete blood cell count (CBC) can be of significant aid to clinical examination. Accurate assessment of haematologic data depends on the proper collection, preparation and transportation of blood samples. Blood should be drawn from animals at rest, calm and well restrained at the time of sampling, in order to avoid artifactual changes, such as a stress leucogram caused by endogenous steroid or epinephrine release or haemolysis from traumatic collection. Proper venipuncture is important to avoid contamination by tissue thromboplastin, which encourages clot formation and invalidates haemostatic function tests. Tubes must be filled to capacity to ensure the proper blood–anticoagulant ratio. When haemostasis tests are indicated, blood should be carefully transferred to vials containing anticoagulant, in order to prevent haemolysis, which precludes the use of evacuated tubes. Blood samples for haematologic studies are best processed as soon as possible after collection, but if a delay is expected they should be refrigerated at 4 °C and for a maximum period of 24 h (with the exception of platelet counts which should be performed within 4–6 h after sample collection), while air-dried blood smears must be prepared immediately after sampling to be stained with Wright or any modified Romanovsky stain. Complete blood counts can be performed either manually or with the aid of sophisticated instrumentation. Automated haematology instruments provide rapid cell counts of blood, haemoglobin calculation, potentially useful erythrocyte indices, but they cannot detect morphologic abnormalities and haemoparasites. Red blood cell counting and morphological changes are useful when evaluating anaemia. The differential white blood cell count is more important than the total white blood cell count, because increases and decreases in individual cell types may occur simultaneously, leaving total count unchanged. Overall, CBC can provide valuable information to the sheep clinician. Moreover, evaluation of a well-prepared blood film is an essential adjunct to interpretation of all cell counts, allowing detection of significant morphologic abnormalities or presence of haemoparasites.

Whole genome microarray analysis, from neonatal blood cards

BackgroundNeonatal blood, obtained from a heel stick and stored dry on paper cards, has been the standard for birth defects screening for 50 years. Such dried blood samples are used, primarily, for analysis of small-molecule analytes. More recently, the DNA complement of such dried blood cards has been used for targeted genetic testing, such as for single nucleotide polymorphism in cystic fibrosis. Expansion of such testing to include polygenic traits, and perhaps whole genome scanning, has been discussed as a formal possibility. However, until now the amount of DNA that might be obtained from such dried blood cards has been limiting, due to inefficient DNA recovery technology.ResultsA new technology is employed for efficient DNA release from a standard neonatal blood card. Using standard Guthrie cards, stored an average of ten years post-collection, about 1/40th of the air-dried neonatal blood specimen (two 3 mm punches) was processed to obtain DNA that was sufficient in mass and quality for direct use in microarray-based whole genome scanning. Using that same DNA release technology, it is also shown that approximately 1/250th of the original purified DNA (about 1 ng) could be subjected to whole genome amplification, thus yielding an additional microgram of amplified DNA product. That amplified DNA product was then used in microarray analysis and yielded statistical concordance of 99% or greater to the primary, unamplified DNA sample.ConclusionTogether, these data suggest that DNA obtained from less than 10% of a standard neonatal blood specimen, stored dry for several years on a Guthrie card, can support a program of genome-wide neonatal genetic testing.

What is your diagnosis? Blood smear from an injured red‐tailed hawk

An injured juvenile red-tailed hawk (Buteo jamaicensis) was evaluated at the Veterinary Medical Teaching Hospital at the University of California, Davis. The hawk was quiet, alert, and emaciated, and had a closed comminuted, mid-diaphyseal ulnar fracture. CBC results included heterophilia with a left shift, monocytosis, and increased plasma fibrinogen concentration. The blood smear included rare heterophils containing small, dark blue inclusions approximately 1-2 mum in diameter that ranged from round to coccobacillary in shape and formed variably shaped aggregates; the morphology of the inclusions was suspicious for Chlamydophila or Ehrlichia spp. pathogens. The hawk died, and histopathologic examination of tissues obtained at necropsy found severe multifocal histiocytic and heterophilic splenitis in addition to chronic hepatitis, myocarditis and epicarditis, meningoencephalitis, and airsacculitis. Using immunohistochemistry the presence of Chlamydia/Chlamydophila spp. antigen within multiple tissues was confirmed. Chlamydophila psittaci DNA was demonstrated in whole blood and fresh splenic tissue via real-time PCR. Direct fluorescent antibody staining of air-dried blood smears was positive in rare leukocytes for Chlamydia/Chlamydophila spp. antigen, and immunocytochemical staining of blood smears for Chlamydia/Chlamydophila spp. antigen was focally positive in rare heterophils. These findings may represent the first reported diagnosis of natural avian C. psittaci infection by visualization of organisms in peripheral blood heterophils. Immunocytochemical evaluation of blood smears was valuable in confirming the diagnosis and may be a useful antemortem test to discriminate between bacteria and other inclusions within heterophils.

A Cheap and Quick Method for DNA-based Sexing of Birds

Abstract Although the collection of air-dried blood samples on filter paper has been used for field work as a routine, it has required the use of special and expensive types of paper. In the present manuscript the use of common filter paper to collect and store air-dried bird blood samples is validated for its use in DNA based sexing techniques with a very simple method avoiding DNA purification for PCR. This method not only simplifies the laboratory work but also does not add any additional cost for DNA-based sexing techniques. The method was tested and successfully employed for sex determination by molecular techniques on blood samples taken on common filter paper during different field trips from four seabird species.

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

Surprising stability of DNA in stains at extreme humidity and temperature

The stability of DNA in air-dried blood and buccal stains at various conditions of relative humidity and temperature was tested by the ability to amplify 1600 and 273 bp DNA fragments by PCR and a 147 bp DNA fragment by real time PCR. Surprisingly it was observed that even the long DNA fragment was amplifiable after incubation for more than 1 month at 100% humidity and temperatures up to 65 °C. The 273 bp fragment was amplifiable after 3 months at 65 °C, 100% humidity. Fungal/microbial growth was observed on some stains after incubation at room temperature or 35 °C at 100% humidity, but never at lower humidity. However, in wet stains DNA degraded rapidly. Thus, even at extreme humid and hot conditions simple air-drying is adequate for the preservation for month of stains and reference samples to be analysed for STRs and SNPs.

FISH techniques.

AbstractThe impression is sometimes given that fluorescence in situ hybridization (FISH) studies are simply a matter of buying a kit with the right DNA probe, following the supplier’s instructions, and reading a simple positive or negative result. In practice, getting a reliable result from a FISH study requires experience, time spent in testing to determine the precise local conditions needed for optimum hybridization, and time spent in assessing and scoring positive and negative controls to determine local baseline levels. The techniques described here will provide some useful guidelines about those aspects of the procedures that can be varied and those that are critical, and advice about the origin and resolution of commonly encountered problems. For simplicity, it will be assumed that the material to be studied is a fixed cell suspension, which is what is usually available in a cytogenetics laboratory. It is possible to study air-dried bone marrow or blood films, fresh tumor touch prints, and wax-embedded sections of solid tumors. The techniques are very similar to those described here (1,2). It is also possible to perform FISH studies on preparations that have already been banded and analyzed as part of a conventional cytogenetics study. However, the simplest and most reliable methods use freshly spread cells, and this is what is described here.KeywordsDextran SulfateSaline Sodium CitrateHybridization EfficiencyCytogenetic LaboratoryUltraviolet Light SourceThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Spectrophotometric Method of Quantifying Adult Cat Flea (Siphonaptera: Pulicidae) Feces

Cat flea, Ctenocephalides felis felis (Bouché), feces are an essential part of larval nutrition under natural conditions. The mass values of adult flea feces can be measured by dissolving samples of flea feces in Drabkin's reagent, filtering, centrifuging, and measuring absorbance spectrophotometrically at 540 nm. Either flea feces or air-dried host blood can be used to generate the standard curve used to convert absorbance (optical density) values into mass values. Debris collected from flea-infested house cats averaged 23.02 mg debris per cat per h with an average of 10.41 mg flea feces per cat per h. Flea feces deposited in the environment serve as potential larval food. Adult flea feces comprised an average of 44.28% of the debris deposited from infested domestic house cats in this study.

Immunohistochemistry in the Community Practice of Pathology: Part II: Detailed Methods, Procedures, and Sources of Reagents

This article details the methods and procedures used in the author’s diagnostic immunohistochemistry laboratory. Included are methods for handling paraffin sections, cryostat sections, and air-dried blood and bone marrow smears. Sources of reagents used in the author’s laboratory are also provided.

A rapid, simple method for leukemia immunophenotyping using air-dried blood and bone marrow smears

This paper describes a modification of the peroxidase technique by which immunophenotyping may be carried out on routinely air-dried blood and bone marrow (BM) smears. The method is simple and quick, requires no special equipment, can be performed on fresh or stored specimens and gives a standard of morphological detail equal to that of routine blood films. With a monoclonal anti-HLA-DR antibody as a prototype, it was possible to demonstrate reliably, the presence of positively and negatively stained cells of appropriate morphological types in the peripheral blood of leukemia patients. Although only about one-third of antibodies tested were effective with the technique, we identified monoclonal antibodies capable of demonstrating myelomonocyte, granulocyte, monocyte, pan-leukocyte, transferrin, platelet, pan-T, ‘cALLA plus B cell’ and other antigens. However, we have not yet found antibodies able to identify T cell subsets, nor to distinguish ‘common’ acute lymphoblastic leukemia from its rare B-cell counterpart. With these limitations the method is suitable for routine use alongside cytochemistry in the differential diagnosis of leukemias and lymphomas.

Microscopic radiation damage to air-dried human blood cells caused by 1.7-MeV 1,2,3H and 4He beams

Monocellular layers of air-dried human blood on glass microscope slides were bombarded by 1.7 MeV 1,2,3H or 4He beams at ambient room temperature at one-third atmosphere pressure of helium gas, with particle fluences up to 3×10 17 cm −2. Air-dried blood cells were studied because such specimens are readily available in hospitals and because air-drying is not expected to alter elemental composition or distribution. Observations of microscopic radiation damage to these specimens indicate approximate upper limits of tolerable fluence prior to cell destruction and therefore indicate where the determination of elemental concentrations in air-dried cells by the technique of charged particle-induced X-ray fluorescence may encounter problems. Characteristic P, S, Cl, and Ca X-ray yields were measured from multicellular layers of air-dried blood during proton bombardment, and neutron emission was measured from triton-bombarded cells fixed in deuterated formaldehye. These measurements are discussed with reference to disruption of cell structure expected from particle irradiation.

Recommended methods for cytological procedures in haematology. International Committee for Standardization in Haematology (ICSH).

Preamble It is well known that histochemical techniques are useful in haematology; especially for the classification of acute leukaemias. However, it is often difficult to compare the results obtained in different laboratories because of the variety of techniques used by workers in various parts of the world. For this reason, the International Committee for Standardization in Haematology (ICSH) convened an expert panel in order to examine the technical problems related to the standardization in cytochemical procedures for blood cells. The panel members met to discuss the problems, review various techniques, compare results and make recommendations for standardization of enzyme chemistry for light microscopy in air-dried peripheral blood films and bone marrow preparations. Five enzymes were identified as requiring methodological standardization: peroxidase, alkaline phosphatase, acid phosphatase, non-specific esterase and chloroacetate esterase. Beta-glucoronidase was excluded because of its minor clinical significance. Each enzymatic reaction was tested by at least two panel members in their laboratories before conclusions were made. The aim was to identify reference methods and also reliable selected techniques suitable for use in routine laboratory practice in order to obtain international comparability of data. For some enzymes the identification of a ‘reference’ method remains to be determined.

Damage to air-dried human blood cells and tissue sections by synchrotron radiation

Damage to unfixed, air-dried human blood cells and tissues caused by synchrotron radiation was studied by scanning electron microscopy (SEM) and by light microscopy (LM). Morphological changes were found in unstained cell specimens after irradiation with photon fluences around 2 × 10 19 photons/cm 2 at an average energy of 15.2 keV. At lower fluences (7 × 10 18 photons/cm 2), morphological changes in irradiated cells are seen by LM after, but not before, staining by a standard hematological technique.

ENERGY DISPERSIVE X-RAY MICROANALYSIS OF PLATELETS AND MEGAKARYOCYTES OF SEVERAL MAMMALS USING AIR-DRIED SPREADS AND FRESH FROZEN DRIED SECTIONS

Semiquantitative energy dispersive X-ray microanalysis of blood platelets on fresh air-dried blood smears and megakaryocytes on fresh frozen dried ultrathin sections of rat bone' marrow and monkey spleen indicates that the dense bodies of blood platelets are derived from the dense granules of megakaryocytes and that their calcium and magnesium content is determined when they are produced in the megakaryocytes. Dense bodies of human blood platelets contain 5-HT, nucleotides (ATP and ADP) and calcium (1). Electron microscopy (EM) of human, rat, mouse, guinea pig and dog platelets on fresh air-dried spreads of blood disclosed dense bodies, alpha granules, bodies with fine dense particles and peripheral vacuoles in the platelets (2,3,4,5). EM of megakaryocytes in mouse spleen and rat bone marrow on fresh frozen dried ultrathin sections (120 nm) revealed a large cytoplasm containing many dense granules and a larger number of less dense granules and a huge irregular nucleus (4,5). Energy dispersive X-ray microanalysis (EDX) of the dense bodies of human blood platelets revealed high peaks for phosphorus and calcium (2,3,6). Semiquantitative EDX of platelet dense bodies of the rat, mouse, guinea pig, rabbit and dog and dense granules of megakaryocytes of the rat and mouse detected high amounts of phosphorus and magnesium and varying small quantities of calcium (4). Examination of blood platelets and spleen megakaryocytes of a crab-eating macaque and cat blood platelets are included in this study. MATERIALS AND METHOD A male monkey (Macaca irus) (1.5 Kg), five humans (three males and two females, two adults and three children), six albino adult Wistar rats (female), six albino adult DDY mice (male, 25 gO, four adult Hartley guinea pigs (female) and five adult and young tabby cats (two males and three females) were used for the study of blood platelets. Drops of blood from finger and tail tips were placed on collodion-film covered copper grids (200 mesh), immediately blotted with filter paper and air-dried. Wedge biopsy specimens of the monkey spleen were obtained by laparotomy, and bone marrow of six rats (female adult) was removed after decapitation. Fresh frozen dried ultrathin sections (120 nm) were prepared by pressing the specimens on the chuck onto the copper disk surface plated with gold (RF-2,Eikow Eng.) precooled with liquid jnitrogen, and cutting them in a cryochamber (FTS, Sorvall) attached to the ultramicrotome MT2-B (Sorvall). The sections were frozen dried in a glass apparatus within the cryochamber with a continuous flow of dry nitrogen gas from a liquid nitrogen dewar (7). They were further dried in a high vacuum at less than 10^ Torr Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphyscol:19842110 C2-486 JOURNAL DE PHYSIQUE f o r more than 48 hr . The specimens were evaporated with carbon about 15 nm thick. A 200 kV e lec t ron microscope (JEM-200CX), equipped with an energy d i spers ive spectrometer (EEDS-11, Ortec) , a scanning transmission attachment (STEM) (JEM-ASID), a low dose system (MDS) and a goniometer s tage was used. The specimens were observed a t an acce le ra t ion vol tage of 200 kV by t h e low dose method and then with the STEM a t an acce le ra t ion vol tage of 80 kV. EDX was performed a t an accelerat ion vol tage of 80 kV, a t a bean current of 95 PA, a t a magnification of 30,000, with condenser aperture of 300 Urn i n diameter with a t i l t i n g of goniometer s tage a t 30 degrees and a t an in tegra ted pulse counting time of 100 sec. The specimen current was about 0.04 nA. Count r a t i o s of t h e elements, Na, Mg, P, S, C 1 , K and Ca were calculated by dividing t h e peak counts of K-alpha X-ray l i n e s of these elements by those of t h e background over t h e energy range of 4.50-5.50 KeV (8) . Dense bodies, alpha granules and regions between t h e granules of t h e p l a t e l e t s of t h e above species and dense granules, l e s s dense granules and regions between t h e granules i n t h e megakaryocyte cytoplasm and regions i n t h e nucleus were examined with a beam spot of about 10 nm i n diameter.