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A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter
Journal of Medical Hypotheses and Ideas (2013) 7, 11–14
Available online at www.sciencedirect.com
Journal of Medical Hypotheses and Ideas journal homepage: www.elsevier.com/locate/jmhi
REGULAR ARTICLES
A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter Mohamed Brahimi a,b,*, Mohamed Nazim Bennaoum a, Hassiba Lazreg a, Affaf Adda a, Hadjer Beliali a, Amel Mihoubi a, Abdessamad Arabi b, Mohamed Amine Bekadja b a
Department of Hemobiology, Etablissement Hospitalier et Universitaire (EHU) ‘‘1er Novembre 1954’’ d’Oran, Algeria Department of Hematology and Cell Therapy, Etablissement Hospitalier et Universitaire (EHU) ‘‘1er Novembre 1954’’ d’Oran, Algeria
b
Received 25 August 2012; revised 9 September 2012; accepted 22 September 2012
KEYWORDS Congenital thrombopathies; Glanzmann thrombasthenia; Bernard–Soulier syndrome; Screening method; Haematology counter
Abstract Glanzmann thrombasthenia (GT) and Bernard–Soulier syndrome (BSS) are hereditary autosomal recessive disorders of platelet functions. These two congenital thrombopathies are very rare. This rarity might be due to the misdiagnosis of the disease and the lack of reliable screening methods. Usually, the definitive diagnosis of these congenital defects relies on aggregometric, flow cytometric and molecular assays. Unfortunately, these expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects. In this paper, the authors suggest a simple and accessible screening method for detection of congenital thrombopathies using only a haematology counter and some reagents. ª 2012 Tehran University of Medical Sciences. Published by Elsevier Ltd. All rights reserved.
Introduction Hereditary defects of platelet functions (thrombopathy) are rarer than diseases related to defects in the soluble clotting factors [1]. Glanzmann thrombasthenia (GT) and Bernard– Soulier syndrome (BSS) are the best characterised inherited
disorders of platelets [2]. A number of other congenital thrombopathies are exceptional and difficult to diagnose [3]. GT is the most common defect [3]. It is an autosomal recessive inherited qualitative platelet disorder characterised by quantitative or qualitative disorder of the platelet glycoprotein GPIIb or GPIIIa [4]. The GPIIb–IIIa complex is the fibrinogen receptor, which plays a very important role in platelet aggrega-
* Corresponding author at: 269, Hai Ennakhla, Canastel, 31132 Oran, Algeria. E-mail address: [email protected] (M. Brahimi). 2251-7294 ª 2012 Tehran University of Medical Sciences. Published by Elsevier Ltd. All rights reserved. URL: www.tums.ac.ir/english/ doi:http://dx.doi.org/10.1016/j.jmhi.2012.09.003
12 tion. The rarity of this disease and its uneven geographic distribution had been reported by several authors [5–7]. The reason for this uneven distribution is the frequent occurrence of intermarriage in some regions, allowing expression of autosomal recessive traits and suggesting a rare occurrence of asymptomatic heterozygous subjects [5]. BSS is also an autosomal recessive disorder. The underlying defect is a deficiency or a dysfunction of the glycoprotein GPIb-V-IX complex, a platelet-restricted multisubunit receptor, required for normal primary haemostasis [8,9]. BSS is a much less common bleeding disorder than GT; only about 100 cases have been reported in the literature [8,9]. Prevalence has been estimated at less than 1/1 000 000 cases/Hab/year but is probably higher due to misdiagnosis and underreporting [9]. Therefore, although BSS and GT are typically rare disorders, they can be almost as frequent as haemophilia and von Willebrand disease in regions where consanguinity is common and an accepted custom. The definitive diagnosis of these congenital defects relies on aggregometric, flow cytometric and molecular assays. Unfortunately, these expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects. In this paper, the authors suggest a simple and more accessible screening method for detection of congenital thrombopathies using only an impedance haematology counter and some reagents. Concept It is well known that the platelet count is underestimated, by an automated cell counter, each time platelet aggregates are present in the sample tube [10–12]. The adjunction of a platelet agonist to a normal sample tube will lead to a formation of aggregates and therefore to a drop of the platelet count. In the case of a hereditary platelet dysfunction, upon the adjunction of a platelet agonist, aggregates cannot be formed, and then the platelet count will remain the same. Nowadays, haematology cell counters are available in the majority of routine laboratories and platelet agonists such as adenosine diphosphate (ADP), thrombin and restocintin can be easily and cheaply purchased. For these reasons, the proposed concept can contribute to develop a more accessible screening technique for these hereditary platelet dysfunctions. As impedance counters are the most widespread systems, this method can be generalised in most laboratories. Evaluation of the method The blood of normal subjects should be used for the standardisation of the method and the definition of the normal ranges. Blood samples must be drawn into evacuated tubes containing 0.1 M buffered citrate (ratio citrate/blood = 1/9). Ethylene diamine tetraacetic acid (EDTA) is not suitable for use in aggregation testing as it does not leave sufficient Ca2+ available for aggregation to occur [12]. All blood samples must be processed within 2 h of collection in order to prevent the changes in pH [13].
M. Brahimi et al. Platelet count must be determined before (baseline count) and after (post-activation count) the addition of the agonist. The percentage of platelet aggregation can be calculated using the following formula: PercentageofAggregation ðBaselinePlateletCountÞðPost-ActivationPlateletCountÞ 100 ¼ BaselinePlateletCount
It is well known that impedance instruments are the most used in routine laboratories. For this purpose, a brief reminder of how an impedance haematology counter works (Coulter Principle) is necessary: On most haematology analysers, a blood sample is diluted in saline, a good conductor of electrical current, and the cells are pulled through an aperture by creating a vacuum [14]. Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage. This change in voltage generates a pulse. The number of pulses is proportional to the number of cells counted. The size of the voltage pulse is also directly proportional to the volume or the size of the cell. Then, all cells can thus be both counted and sized from the electrical impulses that they generate [10]. Depending on the model of instrument, platelets and red cells may be separated from each other by a fixed threshold, for example, at 20 fl, or by a moving threshold or the data from counts between two thresholds, for example, 2 and 20 fl. Hence, cells bigger than 20 fl are not recognised as platelets but as red cells [10]. In order to standardise this technique, the amount of whole blood and the dose of the agonist must be determined with precision. As platelets aggregate in response to an activating stimulus, they stick together to form ever-increasing sizes of aggregates until no single platelets remain [15]. Thus, as the degree of aggregation in a sample increases, the platelet count falls. An aggregate containing many platelets will be so big so that it will not be detected by an impedance counter with typical limit settings. However, an impedance counter can detect microaggregates that contain two (or even three or four) platelets (although it will count this as one particle) [15]. Therefore, if all the platelets in a sample formed aggregates of two platelets per aggregate, which were measured by the counter, then the platelet count would be theoretically expected to fall by 50% and the mean platelet volume (MPV) will go up to 200%. The aim, in the standardisation process, is to determine the amount of normal blood and the agonist dose that leads to platelet counts fall of 33–50% and an MVP rise of 200– 300%, that is, aggregates containing two to three platelets. Low levels of activation of platelets frequently result in transient and reversible aggregation responses. The final platelet count then depends on the time at which the sample is taken after addition of the agonist [15]. It is therefore essential to specify this time. Perhaps it might be more interesting to draw a curve, ‘time’ versus ‘percentage of aggregation’, in order to emphasis the kinetic of the process, that is, aggregation and disaggregation. To sum up, in order to standardise this technique, three parameters must be determined: the amount of whole blood to be tested, the agonist dose added to blood and finally the timing of platelet count after agonist addition.
A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter Once the method is standardised, it should be validated on known GT and BSS patients. The results must be compared to normal controls. As it has been mentioned above, there are several models of impedance automated counters; therefore the quality control and the calibration of the instrument must be preformed according to the manufacturer’s instructions. It is well known that blood smears have an increasingly important role in the quality control of the platelet count [16]; besides, they represent a reliable tool for detecting platelet aggregates after the addition of the agonist. Discussion GT and BSS are hereditary autosomal recessive disorders of platelet functions. The definitive diagnosis relies essentially on aggregometric and flow cytometric assays. According to the literature, these two congenital thrombopathies are very rare [5,8,9]. This rarity might be due to the misdiagnosis of the disease and the lack of reliable screening methods. In this paper, the authors propose a simple and accessible screening method for these disorders using a routine automated blood cell counter and some platelet agonist such as ADP, ristocetin, thrombin, epinephrine, collagen and arachidonic acid. Even if this method is simple and intuitive, it has never been used for screening of congenital thrombopathies. Some reasons come to mind: First, when platelets aggregate in response to an activating stimulus, they stick together to form ever-increasing sizes of aggregates (microthrombi); this might lead to clogging and damaging the instrument. In a previous article, we described and validated a platelet counting technique from blood smears on the basis of platelet/red cell ratio [16]. This latter method can be used as an alternative to automated counters. Besides, blood smears are suitable tools for detecting platelet aggregates [11]. Second, platelets must come into contact with each other to be able to aggregate (the propinquity effect) [13]. If agonists are added to no stirred platelets, they will become activated but aggregation will not be sufficient to induce a platelet count drop. Some cheap semi-automated coagulometers can be used for stirring the sample even if they are not dedicated to this purpose. Third, BSS are characterised by the presence of macrothrombocytes and then as their platelets are too large and are usually not detected by the particle counter [3]. Finally, the validation of the technique must be carried out on a large sample of patients; that is why we are unable to evaluate our hypothesis in our laboratory as GT and BSS are very rare defects; the number of known patients is not sufficient to validate this method. In contrast, there are some other regions such as Iran where 382 patients with GT had been reported [6]. Conclusion We can conclude that the proposed technique is simple and accessible but some problems, cited above, must be solved in order to validate it. Once done, this method can be set in most routine haematology laboratories and must be performed each time a platelet dysfunction is suspected.
13
Overview Box First Question: What do we already know about the subject? Congenital thrombopathies are very rare diseases. The definitive diagnosis of these platelet dysfunctions relies on aggregometric, flow cytometric and molecular assays. These expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects.
Second Question: What does your proposed theory add to the current knowledge available, and what benefits does it have? The authors suggest a simple and accessible screening method for detection of congenital thrombopathies using only a haematology counter and some reagents.
Third question: Among the numerous available studies, what special further study is proposed for testing the idea? The blood of normal subjects should be used for the standardisation of the method and the definition of the normal ranges. Then, it should be validated on known patients and the results must be compared to normal controls. Unfortunately, some unsolved problems, cited in this paper, must be worked out before the generalisation of the technique.
References [1] Roberts HR, Ma AD. Overview of inherited hemorrhagic disorders. In: Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, George JN, editors. Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. [2] Nurden AT, George JN. Inherited abnormalities of the platelet membrane: Glanzmann thrombasthenia, Bernard–Soulier syndrome, and other disorders. In: Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, George JN, editors. Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. [3] Cox D. Platelet function studies. In: Quinn M, Fitzgerald D, editors. Platelet function: assessment, diagnosis, and treatment. Totowa, NJ: Humana Press Inc.; 2005. [4] Kannan M, Ahmed RPH, Jain P, Kumar R, Choudhry R, Saxena R. Type I Glanzmann thrombasthenia: most common subtypes in North Indians. Am J Hematol 2003;74:139–41. [5] George JN, Caen JP, Nurden AT. Glanzmann’s thrombasthenia: the spectrum of clinical disease. Blood 1990;75(7):1383–95. [6] Toogeh G, Sharifian R, Lak M, Safaee R, Artoni A, Peyvandi F. Presentation and pattern of symptoms in 382 patients with Glanzmann thrombasthenia in Iran. Am J Hematol 2004;77:198–9.
14 [7] Awidi AS. Increased incidence of Glanzmann’s thrombasthenia in Jordan compared with Scandinavia. Scand J Haematol 1983;30:218. [8] Lanza F. Bernard–Soulier syndrome (Hemorrhagiparous thrombocytic dystrophy). Orphanet J Rare Dis 2006;1:46–52. [9] Arabi A, Brahimi M, Bekadja MA. Is flow cytometry (alone) sufficient for diagnosis of Bernard–Soulier syndrome: a case report. Acta Med Mart 2010;10(3):19–21. [10] Bain BJ. Blood cells (a practical guide). 4th ed. London, England: Blackwell Publishing; 2006. [11] Bahadır A, Erduran E, Dilber B, U¨c¸u¨ncu¨ N. EDTA-dependent pseudothrombocytopenia. Turk J Hematol 2012;29:311–2. [12] Bain BJ. Diagnosis from the blood smear. N Engl J Med 2005;353:498–507.
M. Brahimi et al. [13] White MM, Jennings LK. Platelet protocols (research and clinical laboratory procedures). San Diego, CA: Academic Press; 1990. [14] Lentowski L, Ciesla B. Basic procedures in a hematology laboratory. In: Ciesla B, editor. Hematology in practice. Philadelphia: F.A. Davis Company; 2007. p. 297–330. [15] Jarvis GE. Platelet aggregation in whole blood (Impedance and Particle Counting Methods). In: Gibbins JM, Mahaut-Smith MP editors. Methods in molecular biology, vol. 272, Platelets and megakaryocytes, functional assays, vol. 1. Totowa, NJ: Humana Press Inc.; 2004. p. 77–87. [16] Brahimi M, Osmani S, Arabi A, Enta-Soltan B, Taghezout Z, Smain Elkahili B, et al.. The estimation of platelet count from a blood smear on the basis of the red cell platelet ratio. Turk J Hematol 2009;26:21–4.
Available online at www.sciencedirect.com
Journal of Medical Hypotheses and Ideas journal homepage: www.elsevier.com/locate/jmhi
REGULAR ARTICLES
A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter Mohamed Brahimi a,b,*, Mohamed Nazim Bennaoum a, Hassiba Lazreg a, Affaf Adda a, Hadjer Beliali a, Amel Mihoubi a, Abdessamad Arabi b, Mohamed Amine Bekadja b a
Department of Hemobiology, Etablissement Hospitalier et Universitaire (EHU) ‘‘1er Novembre 1954’’ d’Oran, Algeria Department of Hematology and Cell Therapy, Etablissement Hospitalier et Universitaire (EHU) ‘‘1er Novembre 1954’’ d’Oran, Algeria
b
Received 25 August 2012; revised 9 September 2012; accepted 22 September 2012
KEYWORDS Congenital thrombopathies; Glanzmann thrombasthenia; Bernard–Soulier syndrome; Screening method; Haematology counter
Abstract Glanzmann thrombasthenia (GT) and Bernard–Soulier syndrome (BSS) are hereditary autosomal recessive disorders of platelet functions. These two congenital thrombopathies are very rare. This rarity might be due to the misdiagnosis of the disease and the lack of reliable screening methods. Usually, the definitive diagnosis of these congenital defects relies on aggregometric, flow cytometric and molecular assays. Unfortunately, these expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects. In this paper, the authors suggest a simple and accessible screening method for detection of congenital thrombopathies using only a haematology counter and some reagents. ª 2012 Tehran University of Medical Sciences. Published by Elsevier Ltd. All rights reserved.
Introduction Hereditary defects of platelet functions (thrombopathy) are rarer than diseases related to defects in the soluble clotting factors [1]. Glanzmann thrombasthenia (GT) and Bernard– Soulier syndrome (BSS) are the best characterised inherited
disorders of platelets [2]. A number of other congenital thrombopathies are exceptional and difficult to diagnose [3]. GT is the most common defect [3]. It is an autosomal recessive inherited qualitative platelet disorder characterised by quantitative or qualitative disorder of the platelet glycoprotein GPIIb or GPIIIa [4]. The GPIIb–IIIa complex is the fibrinogen receptor, which plays a very important role in platelet aggrega-
* Corresponding author at: 269, Hai Ennakhla, Canastel, 31132 Oran, Algeria. E-mail address: [email protected] (M. Brahimi). 2251-7294 ª 2012 Tehran University of Medical Sciences. Published by Elsevier Ltd. All rights reserved. URL: www.tums.ac.ir/english/ doi:http://dx.doi.org/10.1016/j.jmhi.2012.09.003
12 tion. The rarity of this disease and its uneven geographic distribution had been reported by several authors [5–7]. The reason for this uneven distribution is the frequent occurrence of intermarriage in some regions, allowing expression of autosomal recessive traits and suggesting a rare occurrence of asymptomatic heterozygous subjects [5]. BSS is also an autosomal recessive disorder. The underlying defect is a deficiency or a dysfunction of the glycoprotein GPIb-V-IX complex, a platelet-restricted multisubunit receptor, required for normal primary haemostasis [8,9]. BSS is a much less common bleeding disorder than GT; only about 100 cases have been reported in the literature [8,9]. Prevalence has been estimated at less than 1/1 000 000 cases/Hab/year but is probably higher due to misdiagnosis and underreporting [9]. Therefore, although BSS and GT are typically rare disorders, they can be almost as frequent as haemophilia and von Willebrand disease in regions where consanguinity is common and an accepted custom. The definitive diagnosis of these congenital defects relies on aggregometric, flow cytometric and molecular assays. Unfortunately, these expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects. In this paper, the authors suggest a simple and more accessible screening method for detection of congenital thrombopathies using only an impedance haematology counter and some reagents. Concept It is well known that the platelet count is underestimated, by an automated cell counter, each time platelet aggregates are present in the sample tube [10–12]. The adjunction of a platelet agonist to a normal sample tube will lead to a formation of aggregates and therefore to a drop of the platelet count. In the case of a hereditary platelet dysfunction, upon the adjunction of a platelet agonist, aggregates cannot be formed, and then the platelet count will remain the same. Nowadays, haematology cell counters are available in the majority of routine laboratories and platelet agonists such as adenosine diphosphate (ADP), thrombin and restocintin can be easily and cheaply purchased. For these reasons, the proposed concept can contribute to develop a more accessible screening technique for these hereditary platelet dysfunctions. As impedance counters are the most widespread systems, this method can be generalised in most laboratories. Evaluation of the method The blood of normal subjects should be used for the standardisation of the method and the definition of the normal ranges. Blood samples must be drawn into evacuated tubes containing 0.1 M buffered citrate (ratio citrate/blood = 1/9). Ethylene diamine tetraacetic acid (EDTA) is not suitable for use in aggregation testing as it does not leave sufficient Ca2+ available for aggregation to occur [12]. All blood samples must be processed within 2 h of collection in order to prevent the changes in pH [13].
M. Brahimi et al. Platelet count must be determined before (baseline count) and after (post-activation count) the addition of the agonist. The percentage of platelet aggregation can be calculated using the following formula: PercentageofAggregation ðBaselinePlateletCountÞðPost-ActivationPlateletCountÞ 100 ¼ BaselinePlateletCount
It is well known that impedance instruments are the most used in routine laboratories. For this purpose, a brief reminder of how an impedance haematology counter works (Coulter Principle) is necessary: On most haematology analysers, a blood sample is diluted in saline, a good conductor of electrical current, and the cells are pulled through an aperture by creating a vacuum [14]. Electrical resistance or impedance occurs as the cells pass through the aperture causing a change in voltage. This change in voltage generates a pulse. The number of pulses is proportional to the number of cells counted. The size of the voltage pulse is also directly proportional to the volume or the size of the cell. Then, all cells can thus be both counted and sized from the electrical impulses that they generate [10]. Depending on the model of instrument, platelets and red cells may be separated from each other by a fixed threshold, for example, at 20 fl, or by a moving threshold or the data from counts between two thresholds, for example, 2 and 20 fl. Hence, cells bigger than 20 fl are not recognised as platelets but as red cells [10]. In order to standardise this technique, the amount of whole blood and the dose of the agonist must be determined with precision. As platelets aggregate in response to an activating stimulus, they stick together to form ever-increasing sizes of aggregates until no single platelets remain [15]. Thus, as the degree of aggregation in a sample increases, the platelet count falls. An aggregate containing many platelets will be so big so that it will not be detected by an impedance counter with typical limit settings. However, an impedance counter can detect microaggregates that contain two (or even three or four) platelets (although it will count this as one particle) [15]. Therefore, if all the platelets in a sample formed aggregates of two platelets per aggregate, which were measured by the counter, then the platelet count would be theoretically expected to fall by 50% and the mean platelet volume (MPV) will go up to 200%. The aim, in the standardisation process, is to determine the amount of normal blood and the agonist dose that leads to platelet counts fall of 33–50% and an MVP rise of 200– 300%, that is, aggregates containing two to three platelets. Low levels of activation of platelets frequently result in transient and reversible aggregation responses. The final platelet count then depends on the time at which the sample is taken after addition of the agonist [15]. It is therefore essential to specify this time. Perhaps it might be more interesting to draw a curve, ‘time’ versus ‘percentage of aggregation’, in order to emphasis the kinetic of the process, that is, aggregation and disaggregation. To sum up, in order to standardise this technique, three parameters must be determined: the amount of whole blood to be tested, the agonist dose added to blood and finally the timing of platelet count after agonist addition.
A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter Once the method is standardised, it should be validated on known GT and BSS patients. The results must be compared to normal controls. As it has been mentioned above, there are several models of impedance automated counters; therefore the quality control and the calibration of the instrument must be preformed according to the manufacturer’s instructions. It is well known that blood smears have an increasingly important role in the quality control of the platelet count [16]; besides, they represent a reliable tool for detecting platelet aggregates after the addition of the agonist. Discussion GT and BSS are hereditary autosomal recessive disorders of platelet functions. The definitive diagnosis relies essentially on aggregometric and flow cytometric assays. According to the literature, these two congenital thrombopathies are very rare [5,8,9]. This rarity might be due to the misdiagnosis of the disease and the lack of reliable screening methods. In this paper, the authors propose a simple and accessible screening method for these disorders using a routine automated blood cell counter and some platelet agonist such as ADP, ristocetin, thrombin, epinephrine, collagen and arachidonic acid. Even if this method is simple and intuitive, it has never been used for screening of congenital thrombopathies. Some reasons come to mind: First, when platelets aggregate in response to an activating stimulus, they stick together to form ever-increasing sizes of aggregates (microthrombi); this might lead to clogging and damaging the instrument. In a previous article, we described and validated a platelet counting technique from blood smears on the basis of platelet/red cell ratio [16]. This latter method can be used as an alternative to automated counters. Besides, blood smears are suitable tools for detecting platelet aggregates [11]. Second, platelets must come into contact with each other to be able to aggregate (the propinquity effect) [13]. If agonists are added to no stirred platelets, they will become activated but aggregation will not be sufficient to induce a platelet count drop. Some cheap semi-automated coagulometers can be used for stirring the sample even if they are not dedicated to this purpose. Third, BSS are characterised by the presence of macrothrombocytes and then as their platelets are too large and are usually not detected by the particle counter [3]. Finally, the validation of the technique must be carried out on a large sample of patients; that is why we are unable to evaluate our hypothesis in our laboratory as GT and BSS are very rare defects; the number of known patients is not sufficient to validate this method. In contrast, there are some other regions such as Iran where 382 patients with GT had been reported [6]. Conclusion We can conclude that the proposed technique is simple and accessible but some problems, cited above, must be solved in order to validate it. Once done, this method can be set in most routine haematology laboratories and must be performed each time a platelet dysfunction is suspected.
13
Overview Box First Question: What do we already know about the subject? Congenital thrombopathies are very rare diseases. The definitive diagnosis of these platelet dysfunctions relies on aggregometric, flow cytometric and molecular assays. These expensive diagnostic tools are not always available in routine laboratories, especially in developing countries, leading to misdiagnosis and underestimation of the prevalence of these defects.
Second Question: What does your proposed theory add to the current knowledge available, and what benefits does it have? The authors suggest a simple and accessible screening method for detection of congenital thrombopathies using only a haematology counter and some reagents.
Third question: Among the numerous available studies, what special further study is proposed for testing the idea? The blood of normal subjects should be used for the standardisation of the method and the definition of the normal ranges. Then, it should be validated on known patients and the results must be compared to normal controls. Unfortunately, some unsolved problems, cited in this paper, must be worked out before the generalisation of the technique.
References [1] Roberts HR, Ma AD. Overview of inherited hemorrhagic disorders. In: Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, George JN, editors. Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. [2] Nurden AT, George JN. Inherited abnormalities of the platelet membrane: Glanzmann thrombasthenia, Bernard–Soulier syndrome, and other disorders. In: Colman RW, Clowes AW, Goldhaber SZ, Marder VJ, George JN, editors. Hemostasis and thrombosis: basic principles and clinical practice. Philadelphia, PA: Lippincott Williams & Wilkins; 2006. [3] Cox D. Platelet function studies. In: Quinn M, Fitzgerald D, editors. Platelet function: assessment, diagnosis, and treatment. Totowa, NJ: Humana Press Inc.; 2005. [4] Kannan M, Ahmed RPH, Jain P, Kumar R, Choudhry R, Saxena R. Type I Glanzmann thrombasthenia: most common subtypes in North Indians. Am J Hematol 2003;74:139–41. [5] George JN, Caen JP, Nurden AT. Glanzmann’s thrombasthenia: the spectrum of clinical disease. Blood 1990;75(7):1383–95. [6] Toogeh G, Sharifian R, Lak M, Safaee R, Artoni A, Peyvandi F. Presentation and pattern of symptoms in 382 patients with Glanzmann thrombasthenia in Iran. Am J Hematol 2004;77:198–9.
14 [7] Awidi AS. Increased incidence of Glanzmann’s thrombasthenia in Jordan compared with Scandinavia. Scand J Haematol 1983;30:218. [8] Lanza F. Bernard–Soulier syndrome (Hemorrhagiparous thrombocytic dystrophy). Orphanet J Rare Dis 2006;1:46–52. [9] Arabi A, Brahimi M, Bekadja MA. Is flow cytometry (alone) sufficient for diagnosis of Bernard–Soulier syndrome: a case report. Acta Med Mart 2010;10(3):19–21. [10] Bain BJ. Blood cells (a practical guide). 4th ed. London, England: Blackwell Publishing; 2006. [11] Bahadır A, Erduran E, Dilber B, U¨c¸u¨ncu¨ N. EDTA-dependent pseudothrombocytopenia. Turk J Hematol 2012;29:311–2. [12] Bain BJ. Diagnosis from the blood smear. N Engl J Med 2005;353:498–507.
M. Brahimi et al. [13] White MM, Jennings LK. Platelet protocols (research and clinical laboratory procedures). San Diego, CA: Academic Press; 1990. [14] Lentowski L, Ciesla B. Basic procedures in a hematology laboratory. In: Ciesla B, editor. Hematology in practice. Philadelphia: F.A. Davis Company; 2007. p. 297–330. [15] Jarvis GE. Platelet aggregation in whole blood (Impedance and Particle Counting Methods). In: Gibbins JM, Mahaut-Smith MP editors. Methods in molecular biology, vol. 272, Platelets and megakaryocytes, functional assays, vol. 1. Totowa, NJ: Humana Press Inc.; 2004. p. 77–87. [16] Brahimi M, Osmani S, Arabi A, Enta-Soltan B, Taghezout Z, Smain Elkahili B, et al.. The estimation of platelet count from a blood smear on the basis of the red cell platelet ratio. Turk J Hematol 2009;26:21–4.