- Email: [email protected]
VON WILLEBRAND'S DISEASE ASSOCIATED WITH INTERMITTENT THROMBOCYTOPENIA
966
The
density of the nephrogram, on the other hand, improves with increasing dosage of contrast medium. There is good evidence that the nephrogram is the result of glomerular filtration only (Sherwood, Dollery, and Breckenridge 1967). The density of the nephrogram must therefore be directly related to the plasma-level of the contrast
medium. This has been shown
to
be determined
by the dose of contrast medium administered and to be independent of renal function (Sherwood, Breckenridge, et al. 1967). Therefore at all levels of renal function, the nephrogram can be improved by increasing the dose of contrast medium. In patients with severely impaired renal function (G.F.R. < 25 ml. per min.), calyceal detail is rarely satisfactory whatever the dose of contrast medium. In this group two aims are paramount-adequate delineation of kidney size and shape, and sufficiently clear demonstration of ureters to exclude ureteric obstruction without resort to retrograde pyelography. In patients with isosthenuria it can be an advantage to increase the dose of urografin 60% beyond 1 ml. per kg. In our experience 4 ml. contrast medium per kg. is excessive and can produce unpleasant side-effects. We suggest that, for patients with severely impaired renal function, a dose of contrast medium equivalent to 2 ml. per kg. of urografin 60% should be used. Tomography should be available as a routine in this group, to deal with the problem of overlying gut shadows.
of detailed disorder.
coagulation
studies in
cases
of
hxmorrhagic
Introduction
THE pathogenesis of the abnormal bleeding tendency in Willebrand’s disease has been a matter for great controversy. von Willebrand and Jurgens (1933) considered the disease to be a thrombocytopathy, Macfarlane (1941) described it as a capillary defect, while Nilsson et al. (1959) demonstrated that a plasma factor present in normal and haemophilic plasma could correct both the prolonged bleeding-time and the antihxmophilic globulin (A.H.G., factor vm) deficiency generally observed in these patients. The role of the platelets in this disorder has lately attracted renewed attention since decreased platelet adhesiveness in vivo (Borchgrevink 1961) and in vitro (Zucker 1963, O’Brien and Heywood 1967) has been demonstrated. We describe here, in three generations of a family, a haemorrhagic diathesis with the characteristics of von Willebrand’s disease except for the additional finding of
von
thrombocytopenia. Case-reports pedigree of the family is shown in the figure. The husband, parents, and seventeen siblings of case 4 had no known bleeding tendency. There was no consanguinity. The The
We are grateful to Mrs. P. Hopgood for skilled technical assistance, and to Schering Chemicals Ltd. and Schering A.G. (Berlin) for financial support. Requests for reprints should be addressed to T. S. REFERENCES
Breckenridge, A. (1967) Unpublished. Metcalfe-Gibson, A. (1965) Lancet, ii, 265. Cattell, W. R., Kelsey Fry, I., Spencer, A. G., Purkiss, P. (1967) Br. J. Radiol. 40, 561. Dure-Smith, P. (1966) ibid. 39, 655. Foley, T. H., Jones, N. F., Clapham, W. F. (1966) Lancet, ii, 86. Friedenberg, M. J., Carlin, M. R. (1964) Radiology, 83, 405. Glanville, J. N., Herlinger, H. (1966) Clin. Radiol. 17, 230. Hartley, W. (1966) ibid. p. 237. Powell, T., Lentle, B. C., Dew, B., ApSimon, H. T., Pitman, R. G. (1967) Br. J. Radiol. 40, 30. Schencker, B. (1964) Radiology, 83, 12. Sherwood, T., Breckenridge, A., Dollery, C. T., Doyle, F. H., Steiner, R. E. (1967) Clin. Radiol. (in the press). — Dollery, C. T., Breckenridge, A. (1967) Lancet, i, 784. Whitesel, J. A., Heller, E. (1964) J. Urol. 92, 224. —
younger sister of case 2 and 3 was premature and died immediately after birth without having shown evidence of
bleeding tendency. Case1 A 14-month-old girl. The pregnancy and birth had been normal. Birthweight, 3000 g. (6 lb. 10 oz.); length, 50 cm. No
VON WILLEBRAND’S DISEASE ASSOCIATED WITH INTERMITTENT THROMBOCYTOPENIA E. GRAVEN NIELSEN Cand. Med. Odense OF THE DEPARTMENT OF PEDIATRICS, COUNTY AND CITY HOSPITAL OF ODENSE, DENMARK
A. SVEJGAARD Cand. Med. Århus OF THE BLOOD BANK AND BLOOD GROUPING LABORATORY, MUNICIPAL HOSPITAL OF ÅRHUS, DENMARK
Summary Summary
Four
cases
of
von
Willebrand’s disease
accompanied by intermittent thrombocytopenia in three generations of a family are described. Possible explanations for the coincidence of these two conditions are that the thrombocytopenia is inherited in close linkage with von Willebrand’s disease or that it is part of the disease. It is tentatively suggested that decreased platelet adhesiveness caused by lack of a plasma factor leads to increased loss of platelets during each bleeding episode. These reports stress the importance
bleeding episodes neonatally. Psychomotor development during normal. The parents first observed bleeding 2 months, after a superficial scratch. At ages tendency 10 and 13 months there were episodes of gingival bleeding, and, when she had learned to walk, she bruised easily. Epistaxis or articular, gastrointestinal, or urogenital bleedings have not been observed. On admission, the patient was small for her age and delicately built. Weight, 9020 g. (19 lb. 13 oz.). Many ecchymoses, about 1 cm. in diameter, were observed in the regio frontalis and on the legs. Petecchiae were not seen. The spleen was not palpable and the peripheral glands were not enlarged. Laboratory findings: Hb 14-8 g. per 100 ml.; erythrocytes, 4,760,000 per c.mm.; leucocytes, 13,200 per c.mm. with a normal differential count; platelets, 107,000-33,000 per c.mm. There
the first year
was
at age
agglutinating or complement-fixing platelet antibodies. serum-proteins were 7-3 g. per 100 ml. The electrophoretic pattern was normal. were no
Total
Case 2 The 27-year-old father of case 1. He has always bruised easily and has a tendency to epistaxis and prolonged bleeding from injuries. Aged 5 years, he had been admitted to hospital for prolonged epistaxis. Laboratory findings were: Hb, 8-1 g. per 100 ml.; leucocytes, 6320 per c.mm. with a normal differen-
967 TABLE I-COAGULATION STUDIES
*
On
a
previous occasion the
Not investigated. Duke bleeding-time exceeded 30 minutes.
tial count; platelets, 70,500-50,000 per c.mm.; Duke bleedingtime more than 15 minutes; recalcification-time 21/2 minutes; prothrombin-proconvertin time, 24 seconds (normal); capillary fragility (Bexelius) normal. At age 7 brisk bleeding after tonsillectomy, and adenoidectomy; was stopped after tamponade. At age 12 years, platelets were 69,300-82,390 per c.mm. Splenectomy was considered, but was refused by the parents. Since then there has been no serious hsemorrhagic
episodes. Case3 The 22-year-old paternal uncle of case 1. He has always bruised easily, and during childhood had frequent epistaxes often lasting several days. At age 13 years he had been admitted for epistaxis, which ceased after tamponade and transfusion of 1 litre of blood. Laboratory findings were: Hb, 5-5 g. per 100 ml.; platelets, 22,950 per c.mm.; Duke bleeding-time more than 10 minutes. Recalcification-time and capillary fragility were normal. During the next 8 months, platelet-counts varied between 73,240 and 238,510 with a mean of 135,740 per c.mm. The bleeding-time was always prolonged and unrelated to platelet-count. Splenectomy was considered, but instead cortisone therapy was tried during a few weeks without affecting the thrombocytopenia or the bleeding-time. At age 20 he was readmitted, for extraction of wisdom teeth. Before and after the extractions plasma infusions were given and no serious bleeding occurred. Laboratory findings were: platelet-count, 262,000, 97,000, 252,000, and 223,000 per c.mm.; Duke bleeding-time more than 30 minutes; A.H.G. concentration, 12, 24, 15, and 20%. Case 4 55-year-old paternal grandmother of case 1. She has bruised easily since childhood and had prolonged bleeding after small skin lesions and extraction of teeth. The menstruations were prolonged and profuse. She had four children. The first delivery was accompanied by excessive bleeding, the others were uncomplicated. At age 37, carcinoma of the neck of the uterus was diagnosed, and radiation therapy was instituted; the
platelet-count
was
74,400-53,110 per
c.mm.
Aged 40, profuse
hasmaturia necessitated transfusion of 2 litres of blood. At age 53, uneventful tooth-extractions were carried out under cover of plasma infusions. Laboratory findings were: Hb, 11-0 g. per 100 ml.; leucocytes, 2000-5200 per c.mm. with a normal differential count. Erythrocyte-sedimentation rate, 90-110 mm. in lst hour (Westergren); Duke bleeding-time 18 minutes; platelet-counts, 47,000, 61,000, 66,000, 140,000, 36,000, and 75,000 per c.mm.; recalcification and prothrombinproconvertin times were normal; plasma-fibrinogen, 0-23 g. per 100 ml.; A.H.G. 38, 50, 50, 56%. Serum-proteins were 8-23 g. per 100 ml. with slight hypergammaglobulinxmia; flocculation test for syphilis was positive, but Wassermann’s reaction was negative.
Methods The Duke bleeding-time was used. Platelet-counts were performed on platelet-rich plasma from sequestrene/heparinblood (1 drop of 15% sequestrene, 1 drop of heparin /500 units
mi./and 2 ml. of blood) using a Thoma counting chamber. Capillary fragility was estimated by the tourniquet test described by Biggs and Macfarlane (1962). The partial thromboplastin (kaolin) time and the assay for A.H.G. concentration have previously been described (Laursen et al. 1967). The methods for determining clot retraction, recalcificationtime, thromboplastin generation, Quick’s time, plasmathrombin time, fibrinogen concentration, and euglobulin fibrinolysis time were those described by Dybkjaer et al. (1964). In addition, the thromboplastin-generation test of Biggs and Macfarlane (1962) and the adenosine-diphosphate platelet aggregation test described by Hutton (1966) were used. Fibrinolytic activity was also estimated with the fibrin-plate methods described by Astrup and Mullertz (1952) and Lassen (1952). Plasminogen was measured by the method of Biggs and Macfarlane (1962). per
Results
The family members with a history of bleeding tendency investigated in greater detail in March, 1967 (table i). The abnormalities observed were’ a prolonged bleedingwere
time, prolonged partial-thromboplastin time, delayed generation of thromboplastin, and decreased A.H.G. levels. Cases 1 and 4 also had low platelet-counts, which easily explains the decreased prothrombin consumption, diminished clot retraction, and decreased response to A.D.P. of the platelet-rich plasma. Consistent with the low A.H.G. levels, thromboplastin generation was delayed when the aluminium-hydroxide treated plasma came from the patients. In contrast, the patients’ sera responded normally in this test, as did the platelets from case 4 (after concentration of the platelet-rich plasma to normal platelet-counts) and from case 3. There was not enough platelet-rich plasma from the other two patients for this test. No morphological platelet abnormalities could be observed in peripheral blood-smears, and the leucocytes contained no May-Hegglin inclusions. Fibrinolytic activity and plasminogen activity were normal. The coincidence of prolonged bleeding-time and low A.H.G. levels suggested von Willebrand’s disease, and so we tested the effect of fraction i-o on the bleeding-time of case 2. Before the infusion, bleeding had not ceased within 13 minutes; infusion of 300 ml. fraction 1-0 (obtained from the Danish State Serum Institute) with an A.H.G. concentration of 345% was started, and 7 minutes TABLE II-CASE
2:
EFFECT OF FRACTION 1-0 INFUSION
968 later the bleeding stopped. After the infusion the bleedingtime was 4 minutes. The results are shown in table n. Unfortunately, it was not possible to follow the A.H.G. values over a longer period. The serum of case 4 contained the irregular red-cell antibody, anti-S, and leucoagglutinins, lymphocytotoxic antibodies, and platelet-agglutinins. The direct Coombs’ test on her red cells was weakly positive. These tests were all negative with blood from the other patients. The Rose-Waaler test and Hyland’s rheumatoid arthritis and lupus erythamatosus latex tests were negative throughout. Discussion
According Nilsson and Blomback (1963) a diagnosis of von Willebrand’s disease requires demonstration of A.H.G. deficiency, prolonged bleeding-time, and an autosomal dominant hereditary pattern. Our patients fulfil these criteria, except that the pedigree does not (yet) exclude the possibility of an X-linked dominant inheritance. Moreover, the correcting effect of fraction 1-0 on the bleeding-time seems definitely to confirm the diagnosis. We could exclude thrombocytopenia as the cause of the prolonged bleeding-time, at least in cases 2 and 3. The possibility of a thrombasthenia or a thrombopathy seems to be ruled out by the observation that the platelets had normal morphology, exhibited normal clot retraction, responded adequately to A.D.P., and showed normal to
thromboplastin generation. We think it reasonable to classify the hxmorrhagic disorder in this family as von Willebrand’s disease accompanied by intermittent thrombocytopenia. The thrombo-
cytopenia may be independently inherited, inherited in close linkage with von Willebrand’s disease, or be part of the von Willebrand’s disease. The first possibility cannot be completely excluded, but seems rather unlikely since the two disorders coincided in all four patients. The second proposal cannot be discounted since the defects might have arisen by a structural, gametic mutation affecting closely linked genes in one of the grandmother’s chromosomes. The third possibility, that the thrombocytopenia is a result of the von Willebrand disorder, is challenged by numerous published case-reports with normal platelet-counts. Furthermore, Nilsson (1967) has found normal platelet-counts in about two hundred Swedish patients with this disease. At least two cases of transient thrombocytopenia in patients most probably having von Willebrand’s disease have been published (Sharp and Ellis 1960, Karaca and Stefanini 1963), and rather low platelet-counts have been reported (Raccuglia and Neel 1960, Cornu et al. 1963). We do not think it impossible that the thrombocytopenia observed intermittently in our patients is a consequence of the von Willebrand disorder. If so, a tentative explanation could be that, owing to decreased platelet adhesiveness resulting from lack of a plasma factor (Meyer et al. 1967), every bleeding episode tends to cause an unusual loss of platelets. During prolonged haemorrhage this loss might exceed the capacity of the bone-marrow to produce new platelets, and thrombocytopenia would result. Accordingly, one should expect the periods with low platelet-counts to be preceded by bleeding episodes, and this was usually so in our patients and in the patient described by Sharp and Ellis (1960). Our case 4 presented signs of " autoimmune " disease, and it may be of interest that Karaca and Stefanini (1963) suggested that the thrombocytopenia in their patient was due
to
"
autoimmune " mechanisms. However,
none
of
patients in our family presented evidence of autoimmunity ". Whatever the explanation of the intermittent thrombocytopenia in this family, its mere presence will clearly tend to aggravate the bleeding tendency caused by the von Willebrand disorder. Furthermore, the case-reports stress the importance of performing detailed coagulation studies whenever hsemorrhagic disorders are encountered. These patients were initially assumed to be instances of thrombocytopenia alone. However, treatment with platelet concentrates will not correct the abnormality in von Willebrand’s disease, which should be treated with A.H.G. preparations (Nilsson and Blomback 1963).
the other "
We thank Miss Grethe Krogh for skilled technical assistance. Requests for reprints should be addressed to A. S. REFERENCES
Astrup, T., Mullertz, S. (1952) Archs Biochem. 40, 346. Biggs, R., Macfarlane, R. G. (1962) Human Blood Coagulation and its Disorders. Oxford. Borchgrevink, C. F. (1961) Acta med. scand. 170, 231. Cornu, P., Larrieu, M. J., Caen, J., Bernard, J. (1963) Br. J. Hœmat. 9, 189. Dybkjær, E., Berg, E., Kissmeyer-Nielsen, F. (1964) Acta chir. scand. 128, 350. Hutton, R. A. (1966) J. med. Lab. Technol. 23, 161. Karaca, M., Stefanini, M. (1963) Acta hœmat. 29, 102. Lassen, M. (1952) Acta physiol. scand. 27, 37. Laursen, N. P. R., Glavind-Kristensen, S., Kissmeyer-Nielsen, F. (1967) Dan. med. Bull. 14, 49. Macfarlane, R. G. (1941) Q. Jl Med. 1941, 10, 1. Meyer, D., Larrieu, M. J., Maroteaux, P., Caen, J. P. (1967) J. clin. Path. 20, 190. Nilsson, I. M. (1967) Personal communication. Blombäck, M. (1963) in Heparin and Thromboplastin (edited by E. Koller); p. 103. Stuttgart. Blombäck, B. (1959) Acta med. scand. 164, 263. O’Brien, J. R., Heywood, J. B. (1967) J. clin. Path. 20, 56. Raccuglia, G., Neel, J. V. (1960) Blood, 15, 807. Sharp, A. A., Ellis, H. (1960) Br. med. J. ii, 356. Willebrand, E. A. von, Jurgens, R. (1933) Dt. Arch. klin. Med. 175, 453. Zucker, M. B. (1963) Nature, Lond. 197, 601. —
—
—
A TEST FOR THE BACTERICIDAL ACTIVITY OF EYE-DROPS M.D. SENIOR
KEVIN ANDERSON Lond., M.R.A.C.P., M.C.P.A., M.C.Path.
MEDICAL
BACTERIOLOGIST, INSTITUTE OF MEDICAL SCIENCE, ADELAIDE, SOUTH AUSTRALIA
AND
VETERINARY
M.B.
DAVID CROMPTON Adelaide, D.O. Melb., F.R.A.C.S.
HONORARY EYE
SURGEON, ROYAL ADELAIDE HOSPITAL, ADELAIDE, SOUTH AUSTRALIA
The bactericidal efficacy of eye-drops in which sterility has been preserved by adding chlorbutol, benzalkonium chloride, or mercurial or combined preservatives or drops with no stated preservative can be checked by a simple test in which viable cells of Pseudomonas aeruginosa are added. After filtration, the membrane filters are transferred on to neutralising medium and incubated for ten days. Growth on the membrane is recorded: a figure of not less than 99.99% destruction is accepted as evidence of bactericidal activity. The ability to destroy an experimental inoculum of Ps. aeruginosa varied with the active ingredient, even when identical preservatives had been incorporated. Summary
Introduction
THE preservation of ophthalmic solutions after terminal sterilisation is an accepted principle, and many agents are used for this purpose. Ideally, the chosen substance
The
density of the nephrogram, on the other hand, improves with increasing dosage of contrast medium. There is good evidence that the nephrogram is the result of glomerular filtration only (Sherwood, Dollery, and Breckenridge 1967). The density of the nephrogram must therefore be directly related to the plasma-level of the contrast
medium. This has been shown
to
be determined
by the dose of contrast medium administered and to be independent of renal function (Sherwood, Breckenridge, et al. 1967). Therefore at all levels of renal function, the nephrogram can be improved by increasing the dose of contrast medium. In patients with severely impaired renal function (G.F.R. < 25 ml. per min.), calyceal detail is rarely satisfactory whatever the dose of contrast medium. In this group two aims are paramount-adequate delineation of kidney size and shape, and sufficiently clear demonstration of ureters to exclude ureteric obstruction without resort to retrograde pyelography. In patients with isosthenuria it can be an advantage to increase the dose of urografin 60% beyond 1 ml. per kg. In our experience 4 ml. contrast medium per kg. is excessive and can produce unpleasant side-effects. We suggest that, for patients with severely impaired renal function, a dose of contrast medium equivalent to 2 ml. per kg. of urografin 60% should be used. Tomography should be available as a routine in this group, to deal with the problem of overlying gut shadows.
of detailed disorder.
coagulation
studies in
cases
of
hxmorrhagic
Introduction
THE pathogenesis of the abnormal bleeding tendency in Willebrand’s disease has been a matter for great controversy. von Willebrand and Jurgens (1933) considered the disease to be a thrombocytopathy, Macfarlane (1941) described it as a capillary defect, while Nilsson et al. (1959) demonstrated that a plasma factor present in normal and haemophilic plasma could correct both the prolonged bleeding-time and the antihxmophilic globulin (A.H.G., factor vm) deficiency generally observed in these patients. The role of the platelets in this disorder has lately attracted renewed attention since decreased platelet adhesiveness in vivo (Borchgrevink 1961) and in vitro (Zucker 1963, O’Brien and Heywood 1967) has been demonstrated. We describe here, in three generations of a family, a haemorrhagic diathesis with the characteristics of von Willebrand’s disease except for the additional finding of
von
thrombocytopenia. Case-reports pedigree of the family is shown in the figure. The husband, parents, and seventeen siblings of case 4 had no known bleeding tendency. There was no consanguinity. The The
We are grateful to Mrs. P. Hopgood for skilled technical assistance, and to Schering Chemicals Ltd. and Schering A.G. (Berlin) for financial support. Requests for reprints should be addressed to T. S. REFERENCES
Breckenridge, A. (1967) Unpublished. Metcalfe-Gibson, A. (1965) Lancet, ii, 265. Cattell, W. R., Kelsey Fry, I., Spencer, A. G., Purkiss, P. (1967) Br. J. Radiol. 40, 561. Dure-Smith, P. (1966) ibid. 39, 655. Foley, T. H., Jones, N. F., Clapham, W. F. (1966) Lancet, ii, 86. Friedenberg, M. J., Carlin, M. R. (1964) Radiology, 83, 405. Glanville, J. N., Herlinger, H. (1966) Clin. Radiol. 17, 230. Hartley, W. (1966) ibid. p. 237. Powell, T., Lentle, B. C., Dew, B., ApSimon, H. T., Pitman, R. G. (1967) Br. J. Radiol. 40, 30. Schencker, B. (1964) Radiology, 83, 12. Sherwood, T., Breckenridge, A., Dollery, C. T., Doyle, F. H., Steiner, R. E. (1967) Clin. Radiol. (in the press). — Dollery, C. T., Breckenridge, A. (1967) Lancet, i, 784. Whitesel, J. A., Heller, E. (1964) J. Urol. 92, 224. —
younger sister of case 2 and 3 was premature and died immediately after birth without having shown evidence of
bleeding tendency. Case1 A 14-month-old girl. The pregnancy and birth had been normal. Birthweight, 3000 g. (6 lb. 10 oz.); length, 50 cm. No
VON WILLEBRAND’S DISEASE ASSOCIATED WITH INTERMITTENT THROMBOCYTOPENIA E. GRAVEN NIELSEN Cand. Med. Odense OF THE DEPARTMENT OF PEDIATRICS, COUNTY AND CITY HOSPITAL OF ODENSE, DENMARK
A. SVEJGAARD Cand. Med. Århus OF THE BLOOD BANK AND BLOOD GROUPING LABORATORY, MUNICIPAL HOSPITAL OF ÅRHUS, DENMARK
Summary Summary
Four
cases
of
von
Willebrand’s disease
accompanied by intermittent thrombocytopenia in three generations of a family are described. Possible explanations for the coincidence of these two conditions are that the thrombocytopenia is inherited in close linkage with von Willebrand’s disease or that it is part of the disease. It is tentatively suggested that decreased platelet adhesiveness caused by lack of a plasma factor leads to increased loss of platelets during each bleeding episode. These reports stress the importance
bleeding episodes neonatally. Psychomotor development during normal. The parents first observed bleeding 2 months, after a superficial scratch. At ages tendency 10 and 13 months there were episodes of gingival bleeding, and, when she had learned to walk, she bruised easily. Epistaxis or articular, gastrointestinal, or urogenital bleedings have not been observed. On admission, the patient was small for her age and delicately built. Weight, 9020 g. (19 lb. 13 oz.). Many ecchymoses, about 1 cm. in diameter, were observed in the regio frontalis and on the legs. Petecchiae were not seen. The spleen was not palpable and the peripheral glands were not enlarged. Laboratory findings: Hb 14-8 g. per 100 ml.; erythrocytes, 4,760,000 per c.mm.; leucocytes, 13,200 per c.mm. with a normal differential count; platelets, 107,000-33,000 per c.mm. There
the first year
was
at age
agglutinating or complement-fixing platelet antibodies. serum-proteins were 7-3 g. per 100 ml. The electrophoretic pattern was normal. were no
Total
Case 2 The 27-year-old father of case 1. He has always bruised easily and has a tendency to epistaxis and prolonged bleeding from injuries. Aged 5 years, he had been admitted to hospital for prolonged epistaxis. Laboratory findings were: Hb, 8-1 g. per 100 ml.; leucocytes, 6320 per c.mm. with a normal differen-
967 TABLE I-COAGULATION STUDIES
*
On
a
previous occasion the
Not investigated. Duke bleeding-time exceeded 30 minutes.
tial count; platelets, 70,500-50,000 per c.mm.; Duke bleedingtime more than 15 minutes; recalcification-time 21/2 minutes; prothrombin-proconvertin time, 24 seconds (normal); capillary fragility (Bexelius) normal. At age 7 brisk bleeding after tonsillectomy, and adenoidectomy; was stopped after tamponade. At age 12 years, platelets were 69,300-82,390 per c.mm. Splenectomy was considered, but was refused by the parents. Since then there has been no serious hsemorrhagic
episodes. Case3 The 22-year-old paternal uncle of case 1. He has always bruised easily, and during childhood had frequent epistaxes often lasting several days. At age 13 years he had been admitted for epistaxis, which ceased after tamponade and transfusion of 1 litre of blood. Laboratory findings were: Hb, 5-5 g. per 100 ml.; platelets, 22,950 per c.mm.; Duke bleeding-time more than 10 minutes. Recalcification-time and capillary fragility were normal. During the next 8 months, platelet-counts varied between 73,240 and 238,510 with a mean of 135,740 per c.mm. The bleeding-time was always prolonged and unrelated to platelet-count. Splenectomy was considered, but instead cortisone therapy was tried during a few weeks without affecting the thrombocytopenia or the bleeding-time. At age 20 he was readmitted, for extraction of wisdom teeth. Before and after the extractions plasma infusions were given and no serious bleeding occurred. Laboratory findings were: platelet-count, 262,000, 97,000, 252,000, and 223,000 per c.mm.; Duke bleeding-time more than 30 minutes; A.H.G. concentration, 12, 24, 15, and 20%. Case 4 55-year-old paternal grandmother of case 1. She has bruised easily since childhood and had prolonged bleeding after small skin lesions and extraction of teeth. The menstruations were prolonged and profuse. She had four children. The first delivery was accompanied by excessive bleeding, the others were uncomplicated. At age 37, carcinoma of the neck of the uterus was diagnosed, and radiation therapy was instituted; the
platelet-count
was
74,400-53,110 per
c.mm.
Aged 40, profuse
hasmaturia necessitated transfusion of 2 litres of blood. At age 53, uneventful tooth-extractions were carried out under cover of plasma infusions. Laboratory findings were: Hb, 11-0 g. per 100 ml.; leucocytes, 2000-5200 per c.mm. with a normal differential count. Erythrocyte-sedimentation rate, 90-110 mm. in lst hour (Westergren); Duke bleeding-time 18 minutes; platelet-counts, 47,000, 61,000, 66,000, 140,000, 36,000, and 75,000 per c.mm.; recalcification and prothrombinproconvertin times were normal; plasma-fibrinogen, 0-23 g. per 100 ml.; A.H.G. 38, 50, 50, 56%. Serum-proteins were 8-23 g. per 100 ml. with slight hypergammaglobulinxmia; flocculation test for syphilis was positive, but Wassermann’s reaction was negative.
Methods The Duke bleeding-time was used. Platelet-counts were performed on platelet-rich plasma from sequestrene/heparinblood (1 drop of 15% sequestrene, 1 drop of heparin /500 units
mi./and 2 ml. of blood) using a Thoma counting chamber. Capillary fragility was estimated by the tourniquet test described by Biggs and Macfarlane (1962). The partial thromboplastin (kaolin) time and the assay for A.H.G. concentration have previously been described (Laursen et al. 1967). The methods for determining clot retraction, recalcificationtime, thromboplastin generation, Quick’s time, plasmathrombin time, fibrinogen concentration, and euglobulin fibrinolysis time were those described by Dybkjaer et al. (1964). In addition, the thromboplastin-generation test of Biggs and Macfarlane (1962) and the adenosine-diphosphate platelet aggregation test described by Hutton (1966) were used. Fibrinolytic activity was also estimated with the fibrin-plate methods described by Astrup and Mullertz (1952) and Lassen (1952). Plasminogen was measured by the method of Biggs and Macfarlane (1962). per
Results
The family members with a history of bleeding tendency investigated in greater detail in March, 1967 (table i). The abnormalities observed were’ a prolonged bleedingwere
time, prolonged partial-thromboplastin time, delayed generation of thromboplastin, and decreased A.H.G. levels. Cases 1 and 4 also had low platelet-counts, which easily explains the decreased prothrombin consumption, diminished clot retraction, and decreased response to A.D.P. of the platelet-rich plasma. Consistent with the low A.H.G. levels, thromboplastin generation was delayed when the aluminium-hydroxide treated plasma came from the patients. In contrast, the patients’ sera responded normally in this test, as did the platelets from case 4 (after concentration of the platelet-rich plasma to normal platelet-counts) and from case 3. There was not enough platelet-rich plasma from the other two patients for this test. No morphological platelet abnormalities could be observed in peripheral blood-smears, and the leucocytes contained no May-Hegglin inclusions. Fibrinolytic activity and plasminogen activity were normal. The coincidence of prolonged bleeding-time and low A.H.G. levels suggested von Willebrand’s disease, and so we tested the effect of fraction i-o on the bleeding-time of case 2. Before the infusion, bleeding had not ceased within 13 minutes; infusion of 300 ml. fraction 1-0 (obtained from the Danish State Serum Institute) with an A.H.G. concentration of 345% was started, and 7 minutes TABLE II-CASE
2:
EFFECT OF FRACTION 1-0 INFUSION
968 later the bleeding stopped. After the infusion the bleedingtime was 4 minutes. The results are shown in table n. Unfortunately, it was not possible to follow the A.H.G. values over a longer period. The serum of case 4 contained the irregular red-cell antibody, anti-S, and leucoagglutinins, lymphocytotoxic antibodies, and platelet-agglutinins. The direct Coombs’ test on her red cells was weakly positive. These tests were all negative with blood from the other patients. The Rose-Waaler test and Hyland’s rheumatoid arthritis and lupus erythamatosus latex tests were negative throughout. Discussion
According Nilsson and Blomback (1963) a diagnosis of von Willebrand’s disease requires demonstration of A.H.G. deficiency, prolonged bleeding-time, and an autosomal dominant hereditary pattern. Our patients fulfil these criteria, except that the pedigree does not (yet) exclude the possibility of an X-linked dominant inheritance. Moreover, the correcting effect of fraction 1-0 on the bleeding-time seems definitely to confirm the diagnosis. We could exclude thrombocytopenia as the cause of the prolonged bleeding-time, at least in cases 2 and 3. The possibility of a thrombasthenia or a thrombopathy seems to be ruled out by the observation that the platelets had normal morphology, exhibited normal clot retraction, responded adequately to A.D.P., and showed normal to
thromboplastin generation. We think it reasonable to classify the hxmorrhagic disorder in this family as von Willebrand’s disease accompanied by intermittent thrombocytopenia. The thrombo-
cytopenia may be independently inherited, inherited in close linkage with von Willebrand’s disease, or be part of the von Willebrand’s disease. The first possibility cannot be completely excluded, but seems rather unlikely since the two disorders coincided in all four patients. The second proposal cannot be discounted since the defects might have arisen by a structural, gametic mutation affecting closely linked genes in one of the grandmother’s chromosomes. The third possibility, that the thrombocytopenia is a result of the von Willebrand disorder, is challenged by numerous published case-reports with normal platelet-counts. Furthermore, Nilsson (1967) has found normal platelet-counts in about two hundred Swedish patients with this disease. At least two cases of transient thrombocytopenia in patients most probably having von Willebrand’s disease have been published (Sharp and Ellis 1960, Karaca and Stefanini 1963), and rather low platelet-counts have been reported (Raccuglia and Neel 1960, Cornu et al. 1963). We do not think it impossible that the thrombocytopenia observed intermittently in our patients is a consequence of the von Willebrand disorder. If so, a tentative explanation could be that, owing to decreased platelet adhesiveness resulting from lack of a plasma factor (Meyer et al. 1967), every bleeding episode tends to cause an unusual loss of platelets. During prolonged haemorrhage this loss might exceed the capacity of the bone-marrow to produce new platelets, and thrombocytopenia would result. Accordingly, one should expect the periods with low platelet-counts to be preceded by bleeding episodes, and this was usually so in our patients and in the patient described by Sharp and Ellis (1960). Our case 4 presented signs of " autoimmune " disease, and it may be of interest that Karaca and Stefanini (1963) suggested that the thrombocytopenia in their patient was due
to
"
autoimmune " mechanisms. However,
none
of
patients in our family presented evidence of autoimmunity ". Whatever the explanation of the intermittent thrombocytopenia in this family, its mere presence will clearly tend to aggravate the bleeding tendency caused by the von Willebrand disorder. Furthermore, the case-reports stress the importance of performing detailed coagulation studies whenever hsemorrhagic disorders are encountered. These patients were initially assumed to be instances of thrombocytopenia alone. However, treatment with platelet concentrates will not correct the abnormality in von Willebrand’s disease, which should be treated with A.H.G. preparations (Nilsson and Blomback 1963).
the other "
We thank Miss Grethe Krogh for skilled technical assistance. Requests for reprints should be addressed to A. S. REFERENCES
Astrup, T., Mullertz, S. (1952) Archs Biochem. 40, 346. Biggs, R., Macfarlane, R. G. (1962) Human Blood Coagulation and its Disorders. Oxford. Borchgrevink, C. F. (1961) Acta med. scand. 170, 231. Cornu, P., Larrieu, M. J., Caen, J., Bernard, J. (1963) Br. J. Hœmat. 9, 189. Dybkjær, E., Berg, E., Kissmeyer-Nielsen, F. (1964) Acta chir. scand. 128, 350. Hutton, R. A. (1966) J. med. Lab. Technol. 23, 161. Karaca, M., Stefanini, M. (1963) Acta hœmat. 29, 102. Lassen, M. (1952) Acta physiol. scand. 27, 37. Laursen, N. P. R., Glavind-Kristensen, S., Kissmeyer-Nielsen, F. (1967) Dan. med. Bull. 14, 49. Macfarlane, R. G. (1941) Q. Jl Med. 1941, 10, 1. Meyer, D., Larrieu, M. J., Maroteaux, P., Caen, J. P. (1967) J. clin. Path. 20, 190. Nilsson, I. M. (1967) Personal communication. Blombäck, M. (1963) in Heparin and Thromboplastin (edited by E. Koller); p. 103. Stuttgart. Blombäck, B. (1959) Acta med. scand. 164, 263. O’Brien, J. R., Heywood, J. B. (1967) J. clin. Path. 20, 56. Raccuglia, G., Neel, J. V. (1960) Blood, 15, 807. Sharp, A. A., Ellis, H. (1960) Br. med. J. ii, 356. Willebrand, E. A. von, Jurgens, R. (1933) Dt. Arch. klin. Med. 175, 453. Zucker, M. B. (1963) Nature, Lond. 197, 601. —
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A TEST FOR THE BACTERICIDAL ACTIVITY OF EYE-DROPS M.D. SENIOR
KEVIN ANDERSON Lond., M.R.A.C.P., M.C.P.A., M.C.Path.
MEDICAL
BACTERIOLOGIST, INSTITUTE OF MEDICAL SCIENCE, ADELAIDE, SOUTH AUSTRALIA
AND
VETERINARY
M.B.
DAVID CROMPTON Adelaide, D.O. Melb., F.R.A.C.S.
HONORARY EYE
SURGEON, ROYAL ADELAIDE HOSPITAL, ADELAIDE, SOUTH AUSTRALIA
The bactericidal efficacy of eye-drops in which sterility has been preserved by adding chlorbutol, benzalkonium chloride, or mercurial or combined preservatives or drops with no stated preservative can be checked by a simple test in which viable cells of Pseudomonas aeruginosa are added. After filtration, the membrane filters are transferred on to neutralising medium and incubated for ten days. Growth on the membrane is recorded: a figure of not less than 99.99% destruction is accepted as evidence of bactericidal activity. The ability to destroy an experimental inoculum of Ps. aeruginosa varied with the active ingredient, even when identical preservatives had been incorporated. Summary
Introduction
THE preservation of ophthalmic solutions after terminal sterilisation is an accepted principle, and many agents are used for this purpose. Ideally, the chosen substance