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Method and kit for diagnosing bleeding disorders. It is an object of the present invention a method for diagnosing bleeding disorders, by determining deficiencies in the dense granules of platelets. Platelets are blood components that play a critical role in hemostasis and thrombosis processes. Human platelets as well as other mammals, have a type of organelles called “dense granules” or “granules delta”.

Similar to other secretory organelles platelet manner, such as lysosomes and alpha granules, dense granule contents are released by activated platelets where they play an essential role in blood clotting. In this regard, the dense granules accumulate molecules which, when secreted, contribute to the activation of additional circulating platelets to clot formation. In some individuals, the dense granules of platelets are deficient resulting in a bleeding disorder called “Delta storage disease” or “delta-storage pool disease” abbreviated as dSPD.

Clinically, dSPD manifests as a moderate bleeding tendency, although some individuals may present severe bleeding. Typical manifestations include epistaxis, menorrhagia, postpartum hemorrhage and subsequent to surgical procedures McNicol, A.

Platelets12, DSPD usually due to a congenital defect dSPD congenital may occur as a separate entity, in which the defect in platelets is the only anomaly White, Crit Rev Oncol Hematol JG4,or as part of some complex hereditary syndromes such as Hermansky-Pudlak syndrome and syndrome where Chediak-Higashi various additional diseases Rendu, F. Platelets12, observed. Blood70, While congenital dSPD infrequently diagnosed, it has been proposed that the actual incidence of the disease is unknown to a diagnosis performed not in individuals with mild symptoms Nieuwenhuis HK et al.

A variant of dSPD interpretcion “dSPD acquired” which occurs as a result of some clinical conditions such as liver and kidney disease, myeloid leukemias, myelodysplasia or intterpretacion syndromes McNicol, A.

The dense granules of platelets are acidic organelles accumulating adenine nucleotides, serotonin and calcium. Am J Clin Pathol, The analysis of the contents of the dense granules of platelets, is included among the tests performed for diagnosing xe, and requires the use of techniques such as aggregometry, flow cytometry, liquid chromatography, high pressure electron microscopy and fluorescence microscopy Lorez, HP et al.

J Clin Pathol Am, Most of these techniques require expensive equipment and long and laborious procedures. Therefore, the generation of new methods to study dSPD that can be used to analyze a large number of samples in tight times, which are cheaper and using common equipment in biomedical laboratories is necessary. The present invention addresses these needs among others. J Biol Chem, It has recently been found that dense granules of platelets accumulate high levels of inorganic polyphosphate polyP Ruiz, FA et al.

J Bacteriol, Figura 1. The polyP is a polymer of various sizes formed by phosphate residues linked together by fosfoanhidro high energy bonds Kornberg, A. J Bacteriol, Figure 1. Besides the dense granules of platelets, polyP accumulates in similar organelles found in other cell types. En este sentido, estos organelos que acumulan polyP se conocen como “acidocalcisomas” y han sido descritos en bacterias Seufferheld, M.

Nat Rev Microbiol3, In this sense, these organelles that accumulate polyP known as “acidocalcisomas” and have been described in bacteria Seufferheld, M. J Biol Chem, and in various unicellular eukaryotes, such as apicomplexa the tripanosomatidios and parasites, alga Chlamydomonas reinhardtii histogrramas and “slime mold” Dictyostelium discoideum Docampo, R.

J Bacteriol, Polyp industry has been widely used in various applications including food processing, use as a food additive and reducing water hardness, among others Kornberg, A. In medicine, polyP has been used for the treatment of gum disease Yamaoka, M. Gerodontologyin pressto stimulate bone regeneration Hacchou, Y. El polyP se ha encontrado en todos los seres vivos donde se ha estudiado Kornberg, A.


Polyp found in all living beings where it was studied Kornberg, A. J Bacteriol, and recently has been linked to the modulation of blood clotting and dissolution of blood clots fibrinolysis Smith, SA et al.

ESA1 – Method and kit for the diagnosis of clotting disorders. – Google Patents

The invention presented here, relates to the correlation between levels of polyP in extracts hematolgia human platelets and the presence or absence of dSPD, which had not been demonstrated so far. It is a first object of the present invention a method for diagnosing bleeding disorders including histogramaa following steps: Specifically, when the bleeding disorder is diagnose disease reservoirs delta “delta storage pool disease” in Englishthe compound is determined in the biological sample is inorganic polyphosphate.

Thromb Haemost74, For the present invention examples in which the biological sample is taken from humans, but in addition to humans, other mammals such as dogs, cats, pigs, cattle, and mice, reported are also susceptible to developing dSPD MeyersKM et al. Therefore, the method of the invention is also useful to study the disease in mammals in een or to validate animal models of dSPD.

The biological sample includes blood platelets specifically.

Cuando la muestra incluye plaquetas, el procedimiento comprende las siguientes etapas: When the sample includes platelets, the method comprises the following steps: The isolation step of platelets is accomplished by a process of simple, or centrifugation by gel filtration or by centrifugation in density gradient and determining the number of platelets is made by a hematological analyzer, or by a Neubauer hemacytometer and microscope.

The step of acid attack on platelets is effected by resuspending the platelets, previously washed with an isotonic buffer, in a strong acid such as perchloric acid or trichloroacetic acid and separation of solubilised polyphosphate is carried out by centrifugation.

Quantitation of solubilized and separated polyphosphate in the previous stages is carried out after neutralization with alkaline solution directly by colorimetry or fluorimetry, colorimetry preferably. Alternatively, the quantitative determination of solubilised and separated polyphosphate includes after neutralization with alkaline solution: Another object of the present invention a kit for diagnosing bleeding disorders by that procedure.

ADENOGRAMA – Definition and synonyms of adenograma in the Portuguese dictionary

Additionally, the kit may include specific processing enzymes polyphosphate detectable products by colorimetry or by luminescence.

In preferred embodiments of the kit, the means for producing acidic extracts platelets are acids selected from perchloric acid or trichloroacetic acid, the means for the detection and quantification of the polyphosphate are reagents for colorimetric assays, fluorescence or luminescence, preferably reagents colorimetric which are selected from basic dyes such as blue toulidina or fluorophores as Fura-2 or DAPI.

For kits that include enzymes, these are selected from the exopolifosafatasas, the polyphosphate kinase, or glucokinasas polyphosphate. Figure 1 shows a schematic of a molecule polyP. Each molecule contains at least three polyP phosphate residues.

The symbol designated as “n” represents an integer greater than 1 and can be up to several hundred. Figure 2 shows a histogram of the measurement of polyP in platelets, following the methodology explained in Example 1. It has identified a correlation between a low content of polyP in platelets and the presence of dSPD.

Therefore, when the contents of polyP in a platelet sample from an individual are significantly smaller than a standard, obtained from healthy individuals, this is an indication that the individual may be suffering from dSPD. Using the methodology described in more detail in Example 1, it has been determined a correlation between low levels of polyP in platelets and dSPD. Briefly, the method began with the collection of blood from patients who have been previously diagnosed with dSPD.

Confirmed healthy donors who did not have clotting disorders and a week before blood donation had not taken medications that could affect platelets. To avoid platelet activation during blood collection and during the initial handling of samples, tubes containing citrate, although other anticoagulants may also be used during this stage is used. Subsequently, platelets were isolated from the blood sample by simple centrifugation and number of platelets was determined with a hematology analyzer platelet counter.


Clin Lab Haematol27, Isolation of platelets can also be performed by other methods such as gel filtration Lages, B.

Also, the total number of platelets in the sample can be determined by other methods, for example by using a hemacytometer or “neubauer chamber” and a microscope Dacie, JV and Lewis, Practical Hematology SM Then the precipitate isolated platelets, and washed with an isotonic buffer, resuspended in a strong acid such as perchloric acid to produce acidic extracts in which the molecules of medium size polyP are soluble, but proteins and lipids platelets form an insoluble mixture is separated by centrifugation.

Then, the acidic extracts containing polyP, were neutralized with an alkaline solution to proceed with the determination of the contents of polyP as described below. Several methods can be used for detection of polyP, which include, but are not limited to, metachromatic reactions with basic dyes such as Blue toulidina Lorenz, B.

J fluorescentin pressor colorimetric determination of the resulting products after incubation of the extracts with specific processing enzymes polyp. J Bacteriol,o la polifosfato glucokinasa Clark, JE y col. J Bacteriol,or polyphosphate glucokinase Clark, JE et al. The products of enzymatic reactions are labeled with chromophores and are detected and quantified with a spectrophotometer or colorimeter or a luminometer.

In Example 1, the polyP been quantified by the use of recombinant and purified form of yeast inorganic exopolyphosphatase Ruiz, FA et al.

Likewise, the phosphate produced by the enzymatic reaction was assessed colorimetrically by the use of molybdenum blue and malachite green Ruiz, FA et al.

Finalmente, se historamas los niveles de polyP obtenidos a partir de las muestras de plaquetas y se compararon los valores obtenidos entre los pacientes con dSPD y los individuos sanos.

En contraste, el polyP de las plaquetas fue significativamente menor en el grupo de pacientes con dSPD, al compararlo con el grupo de individuos sanos.

ES2325520A1 – Method and kit for the diagnosis of clotting disorders. – Google Patents

In contrast, the platelet polyP was significantly lower interpretafion the group of patients with dSPD, when compared to the healthy group. This example describes the methodology used to identify the polyP as dSPD marker. Los experimentos fueron realizados utilizando plaquetas frescas de individuos voluntarios y de cinco pacientes con dSPD. Experiments were performed using fresh platelets individual volunteers and five patients with dSPD.

Leucocitos o Serie Blanca. Alteraciones y Causas.

Donors control group confirmed that according to his knowledge, had clotting disorders. Also, all individuals in the study confirmed that they had not taken any medication that would affect platelets in the week before donating blood. Diagnosing dSPD in five patients used was confirmed by the analysis of the incorporation of mepacrine iterpretacion flow cytometry and by the study of secretion ATD using luminescence.

Las muestras de sangre fueron obtenidas en tubos con citrato de sodio al 3. Blood samples were obtained in tubes containing sodium citrate 3.

Clin Lab Haematol22, The platelet count of the samples was performed using a hematology analyzer Sysmex XE Hiztogramas, C. The resulting supernatants were centrifuged at 1, xg for 15 min, and the resulting precipitates were washed once with the following buffer: The precipitates were then extracted with 0. After incubation on ice for 30 min, the acidic extracts were centrifuged at 13, xg for 7 min.

The KClO4 precipitate was separated by centrifugation at 13, xg for 5 min, and the resulting supernatant was used for determination of polyP.

PolyP levels were determined in extracts neutralized by measuring inorganic phosphate Pi resulting after treatment with an excess of purified enzyme exopolyphosphatase recombinant yeast Saccharomyces cerevisiae Enzyme ScrPPX1. J Bacteriol,en la cepa de bacteria E. J Bacteriol, in the strain E.