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, Algeriab Department of Hematology and Cell Therapy, Etablissement Hospitalier et Universitaire (EHU) ‘‘1er Novembre 1954’’d’Oran, Algeria

Received 25 August 2012; revised 9 September 2012; accepted 22 September 2012

* Corresponding author at: 26

E-mail address: bmw73dz@yah

2251-7294 ª 2012 T

URL: www.tums.ac

doi:http://dx.doi.or

9, Hai En

oo.fr (M

ehran U

.ir/englis

g/10.1016

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 haematol-

ogy 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) arerarer than diseases related to defects in the soluble clotting

factors [1]. Glanzmann thrombasthenia (GT) and Bernard–Soulier syndrome (BSS) are the best characterised inherited

nakhla, Canastel, 31132 Oran, Al

. Brahimi).

niversity of Medical Sciences. Pub

h/

/j.jmhi.2012.09.003

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 reces-

sive inherited qualitative platelet disorder characterised by

quantitative or qualitative disorder of the platelet glycoproteinGPIIb or GPIIIa [4]. The GPIIb–IIIa complex is the fibrinogenreceptor, which plays a very important role in platelet aggrega-

geria.

lished by Elsevier Ltd. All rights reserved.

12 M. Brahimi et al.

tion. The rarity of this disease and its uneven geographic dis-tribution had been reported by several authors [5–7]. The rea-son for this uneven distribution is the frequent occurrence of

intermarriage in some regions, allowing expression of autoso-mal recessive traits and suggesting a rare occurrence of asymp-tomatic heterozygous subjects [5].

BSS is also an autosomal recessive disorder. The underlyingdefect is a deficiency or a dysfunction of the glycoproteinGPIb-V-IX complex, a platelet-restricted multisubunit recep-

tor, required for normal primary haemostasis [8,9]. BSS is amuch less common bleeding disorder than GT; only about100 cases have been reported in the literature [8,9]. Prevalencehas 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 disor-

ders, they can be almost as frequent as haemophilia and von

Willebrand disease in regions where consanguinity is commonand an accepted custom.

The definitive diagnosis of these congenital defects relies on

aggregometric, flow cytometric and molecular assays. Unfor-tunately, these expensive diagnostic tools are not always avail-able in routine laboratories, especially in developing countries,

leading to misdiagnosis and underestimation of the prevalenceof these defects.

In this paper, the authors suggest a simple and more acces-sible screening method for detection of congenital thrombopa-

thies using only an impedance haematology counter and somereagents.

Concept

It is well known that the platelet count is underestimated, byan 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 asadenosine diphosphate (ADP), thrombin and restocintin can

be easily and cheaply purchased. For these reasons, the pro-posed concept can contribute to develop a more accessiblescreening technique for these hereditary platelet dysfunctions.

As impedance counters are the most widespread systems, thismethod can be generalised in most laboratories.

Evaluation of the method

The blood of normal subjects should be used for the standardi-sation of the method and the definition of the normal ranges.

Blood samples must be drawn into evacuated tubes containing0.1 M buffered citrate (ratio citrate/blood = 1/9). Ethylenediamine tetraacetic acid (EDTA) is not suitable for use in

aggregation testing as it does not leave sufficient Ca2+ avail-able for aggregation to occur [12]. All blood samples mustbe processed within 2 h of collection in order to prevent thechanges in pH [13].

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

¼ðBaselinePlateletCountÞ�ðPost-ActivationPlateletCountÞBaselinePlateletCount

�100

It is well known that impedance instruments are the most

used in routine laboratories. For this purpose, a brief reminderof how an impedance haematology counter works (CoulterPrinciple) is necessary: On most haematology analysers, a

blood sample is diluted in saline, a good conductor of electricalcurrent, and the cells are pulled through an aperture by creat-ing 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 pulsesis proportional to the number of cells counted. The size of the

voltage pulse is also directly proportional to the volume or thesize of the cell. Then, all cells can thus be both counted andsized 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 datafrom counts between two thresholds, for example, 2 and

20 fl. Hence, cells bigger than 20 fl are not recognised as plate-lets 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 pre-cision. As platelets aggregate in response to an activating stim-ulus, they stick together to form ever-increasing sizes ofaggregates until no single platelets remain [15]. Thus, as the de-

gree of aggregation in a sample increases, the platelet countfalls.

An aggregate containing many platelets will be so big so

that it will not be detected by an impedance counter with typ-ical limit settings.

However, an impedance counter can detect microaggre-

gates 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 thecounter, then the platelet count would be theoretically ex-pected to fall by 50% and the mean platelet volume (MPV) willgo up to 200%.

The aim, in the standardisation process, is to determine theamount of normal blood and the agonist dose that leads toplatelet 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 plate-

let count then depends on the time at which the sample is takenafter addition of the agonist [15]. It is therefore essential tospecify this time. Perhaps it might be more interesting to drawa curve, ‘time’ versus ‘percentage of aggregation’, in order to

emphasis the kinetic of the process, that is, aggregation anddisaggregation.

To sum up, in order to standardise this technique, three

parameters must be determined: the amount of whole bloodto be tested, the agonist dose added to blood and finally thetiming of platelet count after agonist addition.

A simple and accessible screening method for congenital thrombopathies using an impedance haematology counter 13

Once the method is standardised, it should be validated onknown GT and BSS patients. The results must be compared tonormal controls.

As it has been mentioned above, there are several models ofimpedance automated counters; therefore the quality controland the calibration of the instrument must be preformed

according to the manufacturer’s instructions. It is well knownthat blood smears have an increasingly important role in thequality control of the platelet count [16]; besides, they repre-

sent a reliable tool for detecting platelet aggregates after theaddition of the agonist.

Discussion

GT and BSS are hereditary autosomal recessive disorders ofplatelet functions. The definitive diagnosis relies essentially

on aggregometric and flow cytometric assays. According tothe literature, these two congenital thrombopathies are veryrare [5,8,9]. This rarity might be due to the misdiagnosis ofthe disease and the lack of reliable screening methods.

In this paper, the authors propose a simple and accessiblescreening method for these disorders using a routine auto-mated blood cell counter and some platelet agonist such as

ADP, ristocetin, thrombin, epinephrine, collagen and arachi-donic acid. Even if this method is simple and intuitive, it hasnever 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 ofaggregates (microthrombi); this might lead to clogging and

damaging the instrument. In a previous article, we describedand validated a platelet counting technique from blood smearson 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 plateletaggregates [11].

Second, platelets must come into contact with each other tobe able to aggregate (the propinquity effect) [13]. If agonistsare added to no stirred platelets, they will become activated

but aggregation will not be sufficient to induce a platelet countdrop. Some cheap semi-automated coagulometers can be usedfor stirring the sample even if they are not dedicated to thispurpose.

Third, BSS are characterised by the presence of macro-thrombocytes and then as their platelets are too large andare usually not detected by the particle counter [3].

Finally, the validation of the technique must be carriedout on a large sample of patients; that is why we are unableto evaluate our hypothesis in our laboratory as GT and BSS

are very rare defects; the number of known patients is notsufficient to validate this method. In contrast, there are someother regions such as Iran where 382 patients with GT hadbeen reported [6].

Conclusion

We can conclude that the proposed technique is simple andaccessible but some problems, cited above, must be solved inorder to validate it. Once done, this method can be set in mostroutine haematology laboratories and must be performed each

time a platelet dysfunction is suspected.

Overview Box

First Question: What do we already know about the

subject?

Congenital thrombopathies are very rare diseases.

The definitive diagnosis of these platelet dysfunctionsrelies on aggregometric, flow cytometric and molecu-lar assays. These expensive diagnostic tools are notalways available in routine laboratories, especially

in developing countries, leading to misdiagnosis andunderestimation 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 screeningmethod 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 ofthe normal ranges. Then, it should be validated onknown patients and the results must be compared

to normal controls. Unfortunately, some unsolvedproblems, cited in this paper, must be worked outbefore the generalisation of the technique.

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