Int J Hematol (2015) 102:157–162 DOI 10.1007/s12185-015-1819-8

ORIGINAL ARTICLE

Use of a microchip flow‑chamber system as a screening test for platelet storage pool disease Hiroaki Minami1 · Keiji Nogami1 · Kenichi Ogiwara1 · Shoko Furukawa1 · Kazuya Hosokawa2 · Midori Shima1 

Received: 1 April 2015 / Revised: 19 May 2015 / Accepted: 2 June 2015 / Published online: 14 June 2015 © The Japanese Society of Hematology 2015

Abstract  Platelet storage pool disease (SPD) is a platelet function disorder characterized by a reduction in the number or content of α-granules, dense granules, or both, and is diagnosed by specialized tests. Patients with SPD often present with prolonged bleeding time (BT), but the sensitivity and reproducibility of this test have limitations, often resulting in false negatives. It has recently been reported that an automated microchip flow-chamber system (T-TAS®) is useful in the assessment of anti-platelet therapy, and could have potential as a screening test for SPD. We examined the utility of T-TAS in three individuals from one family diagnosed with δ-SPD. The propositus had a mildly prolonged BT, and the standard tests for platelet function were close to the normal range. Whole blood samples were anti-coagulated with hirudin and applied to T-TAS microchips coated with collagen (PL-chips) at shear rates of 1000 and 2000 s−1. Platelet thrombus formation (PTF) was monitored with a pressure sensor. Markedly depressed PTF was observed in all cases at both shear rates. These findings indicate that T-TAS is highly sensitive to the defect in these patients with SPD, and may represent a good candidate screening test for a wide range of platelet function disorders. This work was partly supported by grants for MEXT KAKENHI 24591558. An account of this work was presented at the XXIVth Congress of the International Society on Thrombosis and Haemostasis, July 2, 2013, Amsterdam, Netherlands. * Keiji Nogami roc‑noga@naramed‑u.ac.jp 1

Department of Pediatrics, Nara Medical University, 840 Shijo‑cho, Kashihara, Nara 634‑8522, Japan

2

Research Institute, Fujimori Kogyo Co., Ltd., Yokohama, Kanagawa, Japan



Keywords  Storage pool disease · Flow-chamber system · Platelet aggregation

Introduction Platelet storage pool disease (SPD) is a platelet function disorder characterized by a reduction in the number or content of α-granules, dense granules or both. A diagnosis of SPD is initially suspected in the presence of characteristic hemorrhagic symptoms, including nasal bleeding, easy bruising, and menorrhagia, together with a family history, and laboratory results demonstrating a prolonged bleeding time with normal platelet counts and an abnormal pattern of platelet aggregation [1, 2]. Conclusive diagnosis depends on specialized tests including electron microscopy, mepacrine labeling, and quantitative measurement of dense granules content. The bleeding time technique is commonly utilized as a routine screening test in SPD, but the sensitivity and reproducibility of the method are limited, with results often within the normal range. Platelet aggregometry is widely accepted as the gold standard method for assessing platelet function but again, normal responses are often seen in SPD [2]. Moreover, platelet aggregation techniques are labor intensive and usually require the experienced preparation of platelet-rich plasma from a relatively large volume of blood together with multiple agonists at various concentrations. To solve these problems described above, the flowchamber-based systems using the whole blood under the near-physiological condition had been performed as the functional assessments of VWF and platelet in the limited laboratory [3, 4]. Development of these assays provided more significant advances in basic elucidation of VWFand platelet-centered hemostatic mechanisms. Since these

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systems require considerable technical expertise, however, the micro-devices of flow chamber have been improved for similar purposes [5–7]. In particular, a newly automated microchip flow-chamber system (total thrombus-formation analysis system, T-TAS®) has been recently developed, and some groups have already reported on the comprehensive assessment for hemostatic functions of anti-thrombotic or anti-platelet drugs [7–9], hemodilution at cardiopulmonary bypass [10], coagulant bleeding disorder such as hemophilia [11] and von Willebrand disease [12]. This automated device has advantages in evaluating thrombus formation quantitatively in whole blood. From these reports, by utilizing this new system for platelet function disorders, overall platelet function might be possibly assessed by analyzing in vitro thrombus formation. In the present study, we here describe the investigation of platelet thrombus formation at high shear rates using T-TAS as a screening test for diagnosis in patients with δ-SPD.

H. Minami et al.

precision pump. Generated thrombi were appeared over the surface area coated with collagen, and the resulting backpressure was monitored in real time through a pressure transducer located on upstream of the capillary. Thrombus formation was also recorded using a video microscope located under the microchip. For all subjects, 350 µl of hirudin-added whole blood was perfused into the PL-chips at flow rates of 12 and 24 µl min−1, corresponding to high shear rates of 1000 and 2000 s−1, respectively. Flow pressure curves were visualized for 10 min. The platelet thrombus formation on the collagen surface of the PL-chips was quantitatively assessed by the parameters as follows; T10 (time to 10 kPa): time (min) reached to the increase of 10 kPa from baseline of flow pressure, indicating the beginning time of thrombus formation, and T60 (time to 60 kPa): time (min) reached to the increase of 60 kPa from baseline, representing the capillary occlusion, and AUC: the area under the flow pressure curve less than 60 kPa, quantifying the thrombus stability. Platelet aggregometry

Materials and methods Blood samples Written informed consents following ethical guidelines at Nara Medical University were obtained from all subjects before beginning the study. Whole blood samples were collected from 3 patients in one family previously diagnosed with δ-SPD and 20 healthy volunteers (10 men, 10 women). The whole blood samples were taken by venepuncture into plastic tubes containing one-tenth of volume of 3.2 % (w/v) trisodium citrate or into hirudin-containing tubes (final concentration of hirudin; 25 μg/ml). Platelet-rich plasmas or platelet-poor plasmas were prepared by centrifuging whole citrated blood at 800 rpm for 10 min or at 3000 rpm for 10 min, respectively. Individuals had taken any little medication that might affect the platelet or coagulation function in the preceding 1 week of sampling. Blood samples were kept at room temperature for at least 1 h after blood sample collection, followed by performing all platelet function tests. Microchip flow‑chamber system The microchip flow-chamber system, T-TAS® (Fujimori Kogyo, Kanagawa, Japan) was utilized with a minor modification according to the protocol proposed by Hosokawa et al. [7, 9] to analyze the flow-based thrombus formation. Evaluation of platelet thrombus formation was performed with type 1 collagen-coated PL (platelet)-chips (Nitta Gelatin, Osaka, Japan). Briefly, blood samples obtained were perfused into the capillary path of the microchip with a

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Platelet aggregation was evaluated by the light transmission aggregometry using the PRP313M® instrument (IMI, Saitama, Japan) with platelet-rich plasmas. A whole blood impedance aggregometry was also performed using the Multiplate® system (Verum Diagnostica, Munich, Germany). In the PRP313 M, platelet aggregation was monitored by changes in light transmission observed by the addition of individual agonist with a final concentration of 4.0 µM adenosine diphosphate (ADP), 4.0 µg/ml collagen, or 1.5 mg/ml ristocetin. The maximum percentage aggregation achieved within the measurement time was monitored. In the Multiplate technique, the electrical impedance changes induced by the addition of individual agonist with a final concentration of 6.5 µM ADP, 3.2 µg/ml collagen, or 1.2 mg/ml ristocetin were plotted against time, and the AUC was used as a measure of the platelet aggregation response. Data analysis Data are shown as the average and standard deviations (SD). Data analysis was performed using Microsoft Excel software.

Results The initial propositus was a 6-year-old boy who presented with intracranial bleeding after head injury and a postoperative ruptured suture. His mother and elder brother had suffered repeated subcutaneous and nasal hemorrhage since

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Use of a microchip flow-chamber system…

childhood. Laboratory data and bleeding scores in these three patients are summarized (Table 1). Platelet counts and sizes were within the normal range in all cases, and routine coagulation tests showed no abnormality. The bleeding time of the patient was slightly prolonged, but that of his mother and elder brother was normal. Electron microscope findings demonstrated the presence of few dense granules. In addition, quantitative analysis of ADP and ATP released from Table 1  Patients’ laboratory data

Red blood cell count Hemoglobin Platelet count PT APTT Fibrinogen VWF:RCo VWF:Ag Bleeding timea

Reference

Patient Mother Brother

3.76–5.00 (106/µl) 11.3–15.2 (g/dl) 131–362 (103/µl) 10–15 (sec) 25–50 (sec) 200–400 (mg/dl) 50–150 (%) 50–150 (%)

4.88 13.7 204 11.0 30.2 233 74 61.2

4.36 13.1 141 11.5 27.3 236 80 96.2

5.49 14.9 184 11.5 30.8 233 96 94.8

<5′00 (min)

7′00

5′00

4′00

6

6

6

Bleeding scoreb

PT prothrombin time, APTT activated partial thromboplastin time, VWF:RCo von Willebrand factor (VWF) ristocetin cofactor activity, VWF:Ag VWF antigen a

  Bleeding time was measured by the Duke method

b

 The scores were evaluated using the condensed version of the MCMDM-1VWD bleeding assessment tools

Collagen

Ristocetin

Control

Patients

AUC

AUC

AUC

ADP

washed platelets stimulated with convulxin and PAR1P revealed that ADP was 0–1.09 µM at 1.0 × 106/µl platelets (reference 11.7–13.7 µM) and 0–1.55 µM (10.5–12.0 µM), respectively, and that ATP was 0 µM (14.0–16.3 µM) and 0 µM (13.7–14.8 µM), respectively (data not shown). These data were consistent with a diagnosis of δ-SPD. Platelet aggregometry using platelet-rich plasma was examined using the PRP313 M, and demonstrated that the maximum percentage platelet aggregation induced by ADP, collagen and ristocetin was 55–61 %, 26–46 % and 59–76 %, respectively, broadly similar to normal controls (data not shown). With the Multiplate method using whole blood anti-coagulated with hirudin, responses to ADP, collagen and ristocetin were quantified using the AUC, and demonstrated significant statistical differences between the controls and the three δ-SPD cases (p < 0.001) (Fig. 1). However, the measurements of AUC in the patients overlapped those of the healthy controls, and the results appeared to be highly variable between samples and on different days. The results indicated that neither of these tests of platelet aggregation reliably reflected the defect in δ-SPD. In contrast, all measurements using the flow-chamber system (T-TAS) with the collagen-coated microchip were distinctly abnormal in these cases. Platelet thrombus formation was little observed during the measurement period at shear rates of either 1000 or 2000 s−1. In particular, the T10 calculations were markedly defective (Fig. 2a, b;

Control

Fig. 1  Platelet aggregation in whole blood samples from δ-SPD patients using the Multiplate® analyzer. Platelet aggregation induced by ADP, collagen, and ristocetin in whole blood samples from three patients and control individuals (n = 20) was measured as described in “Materials and methods”. Samples obtained on different days were examined in each patient. AUC values are illustrated. The sym-

Patients

Control

Patients

bols used are: open circles; healthy controls, closed circles; propositus, closed squares; elder brother, closed triangles; mother. The mean AUC values in controls and patients were 77.4 ± 16.8 and 47.4 ± 16.0 with ADP (p < 0.001), 95.6 ± 15.6 and 60.7 ± 16.9 with collagen (p < 0.001), and 102.5 ± 25.2 and 52.4 ± 19.8 with ristocetin (p < 0.001), respectively

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Fig. 2  Flow-pressure waveforms observed in whole blood from δ-SPD patients using PL-chips in T-TAS. Thrombus formation on PL-chips was assessed at shear rates of 1000 s−1 (a) and 2000 s−1 (b) using whole blood samples from normal controls (n = 20) and from each of the three patients obtained on several different days as described in “Materials and methods”. Intralumen pressures were monitored and recorded. Vertical axes show the flow pressure after subtracting the baseline values. The horizontal axes show the measurement time

Table 2  Parameters for quantification of platelet thrombus −1

−1

1000 s

2000 s

T10

AUC

T10

AUC

Controls (n = 20)

2.63– 6.83 min

113.2– 468.3

1.45– 5.97 min

123.2– 509.2

Patients (n = 3)

ND

1.9–8.6

ND

0.4–7.0

Values show 95 % confidence interval (CI) AUC area under the curve, ND not detected

Table 2). Furthermore, these measurements were similar in all three patients in samples obtained on several different days. In addition, observations using the video microscope confirmed only slight adhesion of platelets to the capillary surface, and no subsequent growth of platelet aggregates, compared with the extensive platelet thrombi formation within a few minutes in the normal controls (Fig. 3). The findings suggested that the T-TAS technique could be usefully applied as a screening test for δ-SPD.

Discussion The current manuscript reports the sensitive demonstration of platelet dysfunction in individuals with δ-SPD using a

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microchip flow-chamber system. The technique reflects physiological platelet mechanisms more precisely than other static methods, and monitors the processes in which platelets adhere to the surface of a capillary surface and gradually aggregate to form thrombi in the presence of the blood flow. Patients with SPD often illustrate normal platelet responses in commonly used laboratory tests, resulting in difficulties with diagnosis. We have shown that using a PL-chip in T-TAS sensitively detects abnormal platelet aggregation (and little platelet thrombus formation) in δ-SPD. These findings suggested that this method could be applicable as a screening test for the diagnosis of δ-SPD. However, the current investigation involved a small number of patients from only one family (n = 3), and further studies are required to confirm these data. Hosokawa et al. [7] demonstrated that T-TAS could differentiate and quantify the effects of multiple anti-platelet agents (against GPIb or GPIIb/IIIa) using a single type of collagen-coated capillaries, and our results extend those observations. Moreover, in preliminary studies using the same type of PL-chip we have also demonstrated markedly defective platelet thrombus formation in three patients with Bernard–Soulier syndrome (data not shown). It seems likely, therefore, that T-TAS offers the possibility of being used as an initial screening test for a variety of suspected platelet function disorders.

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Use of a microchip flow-chamber system… Fig. 3  Video-microscopy images of platelet thrombi formation on PL-chips at a share rate of 1000 s−1. Platelet thrombus formation of normal control on the microchip was captured at (a) 1 min, (b) 4 min, and (c) 7 min. Patient’s video-microscopy images were captured at (d) 1 min, (e) 4 min, and (f) 10 min. Blood flow direction was from right to left. In normal control, platelet thrombus formation began at 1 min and the capillaries were occluded after 7 min monitor. However, there was any little platelet thrombus formation even at the measurement time of 10 min

A number of earlier reports have suggested that an alternative platelet function analyzer (PFA-100®; Simens USA, Deerfield, IL) may be utilized as a screening test for suspected primary bleeding diatheses. However, this method is not widely used in Japan, and was not available in our laboratory. In addition, Sladky et al. [13] indicated that PFA-100 results were significantly abnormal in fewer than 50 % of patients diagnosed with δ-SPD, and did not correlate with the presence or the severity of the platelet granule deficiency. In addition, previous research indicated that although the PFA-100 analyzer effectively detected severe platelet disorders, including Glanzmann’s thrombasthenia and Bernard–Soulier syndrome, it was less helpful in milder abnormalities [14–17]. In general, therefore, T-TAS seems likely to be more valuable for screening a wider range of platelet defects than PFA-100. Our patients with δ-SPD showed almost normal platelet aggregation responses using platelet-rich plasmas in light transmittance aggregometry (PRP313M). These findings were consistent with those of Nieuwenhuis et al. [18] who reported that 23 % of 106 patients with storage pool deficiency showed completely normal aggregation responses to agonists such as ADP, epinephrine, and collagen. We extended our studies to include the use of different agonists added to whole blood in impedance aggregometry (Multiplate). Quantitative measurements of the mean AUC in these circumstances were significantly lower than in normal individuals, but the results in our patients largely overlapped the normal range in spite of these statistical differences. Furthermore, AUC values varied widely between different samples obtained on different days, even in the same patient. Our data confirmed, therefore, that it is often difficult to reliably assess platelet function by platelet aggregometry alone.

In conclusion, we have investigated three individuals with δ-SPD, and demonstrated that standard platelet function tests were close to normal except for a mildly prolonged bleeding time in one case. In contrast, T-TAS results were markedly abnormal in all cases, and reflected severe platelet dysfunction. The diagnosis of platelet abnormalities can be very complex and require analyses and interpretation from specialists. An appropriate and simple screening test could provide valuable assistance for the selection of patients for further thorough investigation. The T-TAS technique appears to be highly sensitive to physiological platelet mechanisms, and offers considerable potential as a screening test for functional platelet disorders including δ-SPD. Acknowledgments  We would like to thank Dr. Kazuhiro Kashiwagi (Dept. Hematology and Oncology, Osaka University) for measurements of ADP and ATP release in platelets and Dr. Hidenori Suzuki (Dept. Morphological and Biomolecular Research, Nippon Medical School) for the electron microscope analysis of platelets. Conflict of interest  K. Hosokawa is an employee of Fujimori Kogyo Co. The other authors do not have any direct or indirect conflicts of interest.

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