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GB2175691A - Bleeding time measurement - Google Patents
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GB2175691A - Bleeding time measurement - Google Patents

Bleeding time measurement Download PDF

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Publication number
GB2175691A
GB2175691A GB08510629A GB8510629A GB2175691A GB 2175691 A GB2175691 A GB 2175691A GB 08510629 A GB08510629 A GB 08510629A GB 8510629 A GB8510629 A GB 8510629A GB 2175691 A GB2175691 A GB 2175691A
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Prior art keywords
filter
blood
fibres
passing
diameter
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GB08510629A
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GB8510629D0 (en
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John Richard O'brien
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BRIEN JOHN RICHARD O
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BRIEN JOHN RICHARD O
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Priority to GB08510629A priority Critical patent/GB2175691A/en
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Publication of GB2175691A publication Critical patent/GB2175691A/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • G01N33/487Physical analysis of biological material of liquid biological material
    • G01N33/49Blood
    • G01N33/4905Determining clotting time of blood

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Ecology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

Blood is tested by passing it under pressure through a filter 1 and measuring the amount of blood passing through as a function of time. The filter 1 is made of fibres passing in all directions and bonded together to define tortuous channels of different sizes permitting the passage through the filter of particles up to 10 mu m in diameter. <IMAGE>

Description

SPECIFICATION Bleeding time measurement This invention relates to a method and apparatus for testing blood in order to obtain a measurement of bleeding time.
The conventional method of measuring bleeding time consists in making, usually, two or three controlled superficial cuts on the forearm after applying a sphygmomanometer to the upper arm and inflating it to 40 mm Hg.
This test is only moderately reliable; it hurts a little and often leaves small scars. It is not usually possible to make many repeated observations, e.g. to monitor treatment.
The conventional test gives an "in vivo" bleeding time which is dependent on the global interaction between the cut vessel wall and the blood, in which the platelets play a major part.
For some time there have been tentative studies involving pushing blood through small holes and observing whether the platelets are "activated" and block the hole. "In vitro" bleeding time tests have been developed in which blood is passed through a filter. In particular, such a test is described in S. Uchiyama et al., "Clinical Evaluation of a New Test of Hemostasis: The Filter Bleeding Time", Thrombosis Research 34, pp. 397-405, 1984. The method is based on the progressive decrease in drop rate of 5 ml of citrated whole venous blood through a filter of low porosity woven "Dacron" (Trade Mark for polyester) ("Meadox"-a Trade Mark for a graft material) under a pressure of 150 mm Hg as platelet aggregates occlude the filter.
The filter bleeding time (FBT) is defined as the time until the drop interval exceeds 1 minute.
If an in vitro bleeding time test is abnormal in all of most people with a haemorrhagic diathese, it should be of great general use. It could be used as a screening procedure before operations; as part of the routine investigation of people with a haemorrhagic diathesis; and to monitor the effect of treatment, for example when a patient is bleeding and a specific clotting factor is being transfused.
However, the test according to Uchiyama et al has some drawbacks. The blood used is citrated, thereby removing calcium and decreasing the value of the test. The pressure of 150 mm Hg tends to cause fragmentation of the red cells and the platelets.
The present invention is based on research into the behaviour of blood when passed through various filters under pressure. As a result of trials with many different filters, the inventor has determined that the structure of the filter is critical to the value of the in vitro bleeding time test.
The invention provides a method of testing blood comprising passing blood under pressure through a filter and measuring the amount of blood passing through as a function of time, in which the filter is made of fibres passing in all directions and bonded together to define tortuous channels of different sizes through the filter, permitting the passage of particles of up to 10 ,um diameter through the filter.
The fibres are preferably glass fibres and are preferably resin-bonded. The fibres constituting the filter preferably are of different diameters; the diameter may be up to 5 ,um, more preferably up to 4 ,us., and may be as small as 0.1 ,um.
The use of such a filter does not require the high pressure of 150 mm Hg in the Uchiyama et al. test.
If the pressure is too low (e.g. 5 mm Hg) there may be no progressive slowing of the amount of blood passing through the filter.
Preferably, the pressure is at least 20 mm Hg.
The preferred pressure range is 40 to 100 mm Hg.
Clotting of the blood may be inhibited by the addition of an anticoagulant such as heparin.
The invention also provides apparatus for testing blood, comprising a filter, means for passing blood through the filter under pressure, and means for continuously measuring the amount of blood passing through the filter, in which the filter is made of fibres passing in all directions and bonded together to define tortuous channels of different sizes through the filter, permitting the passage of particles of up to 10 Crm diameter through the filter.
The apparatus preferably includes a filter holder, and a syringe connectable to the filter holder and having an inlet for air under pressure.
The invention will be described further, by way of example, with reference to the accompanying drawings, in which: Figure 1 schematically shows apparatus for measuring bleeding time; Figures 2 to 4 are graphs of cumulative drop counts versus time, in use of the apparatus; Figure 5 is a graph of the decrease in drop rate versus the percentage of platelets retained by the filter in the apparatus; Figure 6 is a graph of cumulative drop count versus time, comparing normal blood with blood from patients having von Willebrand's disease; and Figures 7 and 8 are electron micrographs of the filter structure (magnification: 180 and 1800 respectively).
In the apparatus illustrated the blood to be tested is passed through a filter 1 fitted in a filter holder 2 (e.g. a commercially available filter holder such as one supplied by Nuclepore Ltd.). The filter 1 is cut with an annular knife from a "Pall Filter U/100" (Trade Mark) sheet (supplied by Pall Biomedical Ltd.). The filter is made of resin-bonded glass fibres of different diameters (ranging from about 3.4 ,um down to about 0.11 Am) passing in all directions, substantially randomly (see Figs. 7 and 8); it permits the passage of particles up to 10 ,um in diameter through tortuous passages of different sizes defined between the fibres.
A syringe 3 already containing 0.5 ml air is filled with 2.5 ml of blood 4 and inserted into the filter holder 2, which has already been primed with blood. This unit is then fitted into an air-tight syringe holder (not shown) and air under pressure is delivered from a reservoir 6 via a tap 7 through a hole 8 in the syringe barrel. The air reservoir 6 is supplied by a pump 9 via a tap 11. A the selected air pressure is recorded by a pressure transducer 12.
To start the test, the tap 7 is opened and the air pressure forces blood through the filter 1. immediately below the filter holder 2, drops of blood fall across the path of a light beam emitted by an emitter 13 and received by a sensor 14 connected to a drop counter 16 and a timer 17. The first drop crossing the light beam triggers both the counter 16 and the timer 17. Thereafter, a cumulative drop count is recorded at 1, 5, 10, 15, etc. seconds. The test is taken to be finished when the drop rate falls below 1 drop per 5 seconds or all the blood has passed through the filter.
The blood coming through the filter in a given period, usually between 10 and 30 seconds after the start, is collected in a receiver 18 containing EDTA (ethylene diamine tetraacetic acid) so that the platelet count of the blood collected can be determined. The loss of platelets relative to the unfiltered platelet count can be expressed as a percentage of the unfiltered count (platelet loss %).
The results can be quantitated in a number of ways. For example, the time to blockage can be determined, blockage being defined as occurring when the flow is reduced to 1 drop or less per 5 seconds. The drop count between 5 and 30 seconds can be divided by the drop count between 0 and 5 seconds (which reflects the maximal rate); this drop ratio gives a measure of the slowing, which is usually greatest between 15 and 30 seconds.
Results obtained by the above-described procedure are explained below, with reference to Figs. 2 to 6.
Normal heparinised blood collected within the last 15 min was loaded into the syringe (Fig. 2) 1) At a pressure of 5 mm Hg. There was a slow initial rate of passage through the filter (0.2-1 drop/sec) and the drop rate continued at an almost constant rate for 1-2 min until all the blood had passed through the filter.
Thus there was almost no progressive slowing in the rate.
2) At 40 mm Hg. Initially (0-5 sec) the blood passed rapidly (e.g. 3 drops/sec) through the filter. Thereafter, normally, there was a rapid progressive slowing in the rate of passage, resulting in the complete blocking of the filter by 30-40 sec. This time for complete blocking may be called the "in vitro bleeding time".
3) At 100 mm Hg. Normal blood behaved similarly at 40 and 100 mm Hg. Abnormal blood that showed little slowing at 40 mm Hg was sometimes found to behave more normally at 100 mm Hg, i.e. to show more marked progressive slowing.
Native blood usually produced slightly more rapid blocking than did heparinised blood. Fig.
3 shows graphs of cumulative drop count versus time, for native blood and for blood containing heparin, citrate, and EDTA respectively.
Heparinised platelet rich plasma (PRP) passed through the filter at 40 or 100 mm Hg initially flowed through at a greater rate than whole blood. Thereafter the slowing was very fast, often with complete blocking of the filter by 15-20 sec (Fig. 4).
Platelet retention. The percentage of platelets normally retained in the filter initially was about 50% irrespective of the pressure.
Thereafter: at 5 mm Hg the loss of platelets remained approximately the same; at 40 and 100 mm Hg as blocking proceeded, i.e. between 10 and 30 sec, more platelets were retained-from 70 to 90%. There is a good correlation (r=0.76; n=30) between percentage platelets retained and the rate of blocking (Fig. 5).
The number of platelets retained in the filter.
At 40 mm Hg with blocking occuring at, e.g., 30 sec, about 1 ml of blood had passed through the filter. If the original platelet count was 200 X 109/1 and 80% were retained then the total number of platelets retained in the filter 160 X 106 platelets.
At 5 mm Hg, 2 ml of blood runs through the filter, which does not get blocked. The retention is about 50% and 200 X 106 platelets are retained.
Time and temperature. The passage of heparinised blood through the filter was not affected within the temperature range 20-37"C.
If blood was kept at room temperature for 1 hour, the rate of blockage was either unaffected or slightly shorter.
Reproducibility. Within 1 subject: (heparin blood 40 mm Hg) cumulative drops 5-30 secs=17+3 (n=9).
The effect of the initial platelet count. Provided the initial platelet count was from 70,000 to 5000,000 X 109/1 the rate of blockage was unaffected.
Idiopathic thrombocytopenia (n=2) and blood rendered artifically thrombocytopenic (platelet count less than 70,000 X 109/1) by passage through cotton wool, always ran through the filter with minimal slowing.
Calcium dependence. The progressive slowing and blocking of the filter at 40 or 100 mm Hg, occurred most rapidly in native blood.
Heparinised blood blocked a little more slowly.
Citrated blood shows little progressive slowing and EDTA blood almost none (Fig. 3).
Transit time. Because the structure of the filter is uneven the transit time through different channels must differ. However, the total volume of the channels was calculated from the difference in weight of a dry filter and one filled with water. If 1 ml of blood passes through the filter in 30 sec it can be calculated that the transmit time is approximately 1/1000 sec.
von Willebrand's disease. 12 patients with von Willebrand's disease had been diagnosed in the past by a low Factor Vllle and a low Factor VIIIR:Ag. All patients had a long bleeding time and poor platelet retention in a glass bead column. Twelve of these bloods were grossly abnormal and did not block the filter.
The 13th showed moderate reduction in the rate of drops emerging and her Factor VIII was higher than the others (Fig. 6).
Renal disease. 12 patients with end stage renal failure on continuous ambulatory peritoneal dialysis (CAPD) have been studied. It is known that this type of patient is at risk of bleeding. Six/12 were repeatedly found not to block the filter, i.e. had long "in vitro bleeding times". Factor VIIIR:AG is high in such patients.
It can be concluded that blocking is due to platelets progressively piling up in the filter channels. No blocking occurs in thrombocytopenia. In pathological bloods in which the rate of blocking is slow, this rate is linearly related to the number of platelets retained in the filter.
It appears that blocking requires stress activation of platelets. Since there was minimal progressive slowing of normal blood passing through the filter at 5 mm Hg, the filter surface itself is not markedly active (but note that some 50% of platelets are retained).
Blocking, however, regularly occurred at 40 mm Hg. This higher pressure might be expected to push blood through the filter faster (which initially it does) and to make it more difficult for platelets to block the channels. In fact the high pressure caused blocking. The only difference from the 5 mm Hg experiment seems to be that at 40 mm Hg there is increased shearing forces exerted on the platelets.
At 5 mm Hg, by 2 minutes 200,000 X 106 platelets were retained but blood still passed freely through the filter. At 40 mm Hg, although the same number of platelets were retained, blocking occurred at 30 sec. Thus it appears that the organisation of the platelets within the filter must differ.
Both the physical effect of red cells in laminar flow and the release of ADP from red cells may influence platelet adhesion in some models. In this model neither mechanism can occur, since heparinised PRP blocks the filter more rapidly than whole blood.

Claims (15)

1. A method of testing blood comprising passing blood under pressure through a filter and measuring the amount of blood passing through as a function of time, in which the filter is made of fibres passing in all directions and bonded together to define tortuous channels of different sizes through the filter, permitting the passage of particles of up to 10 ;im diameter through the filter.
2. A method as claimed in claim 1, in which the pressure is at least 20 mm Hg.
3. A method as claimed in claim 2, in which the pressure is 40 to 100 mm Hg.
4. A method as claimed in any of claims 1 to 3, in which an anticoagulant is added to the blood so as to prevent clotting.
5. Apparatus for testing blood comprising a filter, means for passing blood through the filter under pressure, and means for continuously measuring the amount of blood passing through the filter, in which the filter is made of fibres passing in all directions and bonded together to define tortuous channels of different sizes through the filter, permitting the passage of particles of up to 10 Hm diameter through the filter.
6. Apparatus as claimed in claim 5, in which the fibres are glass fibres.
7. Apparatus as claimed in claim 5 or 6, in which the fibres are resin-bonded.
8. Apparatus as claimed in any of claims 5 to 7, in which the fibres are of different diameters.
9. Apparatus as claimed in any of claims 5 to 8, in which the fibres are up to 5 Xlm in diameter.
10. Apparatus as claimed in claim 9, in which the fibres are up to 4 Am in diameter.
11. Apparatus as claimed in any of claims 5 to 10, in which the fibres are at least 0.1 Am in diameter.
12. Apparatus as claimed in any of claims 5 to 11, including a filter holder, and a syringe coniectable to the filter holder and having an inlet for air under pressure.
13. A method as claimed in any of claims 1 to 4 carried out in apparatus according to any of claims 5 to 12.
14. A method as claimed in claim 1, substantially as described with reference to the accompanying drawings.
15. Apparatus for testing blood, substantially as described with reference to Figs. 1, 7 and 8 of the accompanying drawings.
GB08510629A 1985-04-26 1985-04-26 Bleeding time measurement Withdrawn GB2175691A (en)

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GB2175691A true GB2175691A (en) 1986-12-03

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3802221A1 (en) * 1988-01-26 1989-07-27 Stephan Prof Dr Rer Nat Nees Apparatus for measuring the activation of cells in a cell suspension, of whole blood cells or of components of blood plasma
DE10003093A1 (en) * 2000-01-25 2001-08-02 Goltz Volker Freiherr Von Device and method for recording coagulation functions of global, especially primary, hemostasis
EP1072879A3 (en) * 1999-07-26 2003-03-05 Alvise Cortinovis Method and equipment for the determination of general and capillary viscosity of blood
EP2500096A1 (en) 2011-03-15 2012-09-19 Siemens Healthcare Diagnostics Products GmbH Method and devices for determining the platelet function in a centrifugal analyser

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1263894A (en) * 1969-02-14 1972-02-16 Alfred Fleisch Improvements in fluid agglomeration testing methods and equipment
GB1469151A (en) * 1973-03-13 1977-03-30 Unilever Ltd Apparatus for determining the degree of platelet aggregation in blood
GB2018151A (en) * 1978-03-06 1979-10-17 Asahi Chemical Ind Seperation of leukocytes from leukocyte-containing suspension by filtration
GB2018149A (en) * 1978-03-06 1979-10-17 Asahi Chemical Ind Separation of lymphocytes from lymphocyte-containing suspension by filtration

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1263894A (en) * 1969-02-14 1972-02-16 Alfred Fleisch Improvements in fluid agglomeration testing methods and equipment
GB1469151A (en) * 1973-03-13 1977-03-30 Unilever Ltd Apparatus for determining the degree of platelet aggregation in blood
GB2018151A (en) * 1978-03-06 1979-10-17 Asahi Chemical Ind Seperation of leukocytes from leukocyte-containing suspension by filtration
GB2018149A (en) * 1978-03-06 1979-10-17 Asahi Chemical Ind Separation of lymphocytes from lymphocyte-containing suspension by filtration

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3802221A1 (en) * 1988-01-26 1989-07-27 Stephan Prof Dr Rer Nat Nees Apparatus for measuring the activation of cells in a cell suspension, of whole blood cells or of components of blood plasma
EP1072879A3 (en) * 1999-07-26 2003-03-05 Alvise Cortinovis Method and equipment for the determination of general and capillary viscosity of blood
DE10003093A1 (en) * 2000-01-25 2001-08-02 Goltz Volker Freiherr Von Device and method for recording coagulation functions of global, especially primary, hemostasis
DE10003093C2 (en) * 2000-01-25 2002-05-16 Goltz Volker Freiherr Von Device for investigating properties of the global hemostasis function in whole blood or platelet-rich plasma
EP2500096A1 (en) 2011-03-15 2012-09-19 Siemens Healthcare Diagnostics Products GmbH Method and devices for determining the platelet function in a centrifugal analyser
EP2500095A1 (en) 2011-03-15 2012-09-19 Siemens Healthcare Diagnostics Products GmbH Method and devices for determining the platelet function in a centrifugal analyser
JP2012194181A (en) * 2011-03-15 2012-10-11 Siemens Healthcare Diagnostics Products Gmbh Devices and methods for determining platelet function
US9205424B2 (en) 2011-03-15 2015-12-08 Siemens Healthcare Diagnostics Products Gmbh Devices for determining the platelet function in a centrifugal analyzer

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Publication number Publication date
GB8510629D0 (en) 1985-06-05

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