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Surgery for angiodysplasia of the gut in von willebrand's disease using D.D.A.V.P. alone
347
ABSTRACTS OF COMBINED ANNUAL MEETING
gene and gives the haplotype aaa42/l. This complements the previously described aaa37/haplotypeand taken in conjunction with the - a3' and - a42/deletions in a thalassemia ( a thalassemia-2) provides further evidence that interchromosomal crossing-over is the most likely mechanism for deletions in the a+ thalassemias. The second rearrangement demonstrated a triplication of the 7-globin gene to give the haplotype GyGyAy/. Family studies in the latter rearrangement have shown that an additional y-globin gene does not significantly alter the level of H b F. This provides an interesting contrast to what is observed when extensive deletions with the p-globin cluster (for example in (Sg) O thalassemia and HPFH) are associated with elevated levels of Hb F. +
USE OF RESTRICTION FRAGMENT LENGTH POLYMORPHISMS FOR THE DIAGNOSIS OF THALASSEMIA
Z. RUDZKI,A. IASIELLO& R. J. KIMBER Division of Haematologj, Institute of Medical and Veterinary Science. Adelaide
Restriction fragment length polymorphisms (RFLPs) are caused by variations in DNA nucleotide sequences randomly distributed throughout the entire human genome. Thus, DNA from different people will give different length fragments when digested with restriction endonucleases. If RFLPs are closely linked to a genetic disorder, the pattern can be used to identify the chromosome carrying the genetic disorder. This approach for the antenatal diagnosis of [I thalassemia only requires amniocentesis and not fetoscopy to obtain the specimen but family studies are required to obtain the necessary data. There are 7 polymorphic restriction enzyme sites known to be tightly linked to the globin gene and these sites have been used for antenatal diagnosis of p thalassemia. To test the applicability of this procedure to our local population, D N A specimens have been obtained from 6 families with a homozygous p thalassemic child. The Hind 111 polymorphic sites in the G y and *y globin genes were investigated in a retrospective manner with the intention of using the technique for antenatal diagnosis in the future. In 4 families. complete diagnosis was possible using the Hind 111 sites alone. In one of the 2 remaining families, diagnosis by exclusion would be possible in 509, of cases while in the last family no diagnosis was possible. However. other polymorphic restriction sites may allow diagnosis to be made and this work is currently being undertaken. Specimens from a further 6 families have recently become available for study. Thus the preliminary results available to date confirm overseas experience that RFLPs can be used for the antenatal diagnosis of homozygous p thalassemia in about 75% of families at risk for this disorder. EFFICACY OF FILTER COLUMNS IN THE MANAGEMENT OF TRANSFUSION REACTIONS IN CHRONICALLY TRANSFUSED THALASSEMIC PATIENTS
G. J . BRYANT & A . T. LAMMI Haematology Department. Royal Alexandra Hospital for Children, Sydney
Filter columns (Imugard IG 500 Filter) containing cotton wool (Gossypium barbadense) are used to produce leukocyte poor blood for transfusion. In this study, 5 patients with thalassemia major who require regular transfusion, were transfused for 1 yr with filtered, packed red cells. Three patients (adult group) had been receiving triple washed packed cells for over 5 yr to prevent febrile reactions. The other 2 patients (younger group) had been having an increasing number of febrile reactions with transfusion. Transfusion episodes over the year prior to the use of filters were used as the control for each patient. One hundred and sixty-nine units of packed red cells were filtered: 96.77; of leukocytes were removed on filtration. In 12 units (779, the leukocyte count was greater than 0.5 x 10y/l,while in 6 of these 12 units (3.5% overall), the leukocyte count was greater than 1 x 10y/l. Four febrile reactions (temperature > 37.5 "C)were recorded during the year (incidence of less than 3%). I n the 3 adult patients, 117 units of
packed cells were transfused each year. There was no significant difference in the number of febrile reactions; two with filtered blood compared with none with triple washed blood. In the younger two patients, 52 units of blood were transfused each year. There were fewer febrile reactions with the filtered blood (2 reactionsj as compared with 16 reactions with the unfiltered blood. Pretransfusion hemoglobin levels were higher (P i0.01) in each ofthe 3 patients receiving filtered cells, as compared with hemoglobin levels when triple washed cells were transfused. In the younger group, there was no difference ( P>0.10) in pretransfusion hemoglobins. with filtered and unfiltered blood. Column filters were efficient in the prophylaxis against febrile reactions in sensitized patients. Filtration did not increase the requirements for donor blood. QUINIDINE-INDUCED THROMBOCYTOPENIA AND LEUKOPENIA. IN VlTRO STUDIES OF THE QUINIDINE-DEPENDENT PLATELET AND GRANULOCYTE ANTIBODIES
B. H. CHONG,M. C. BERNDT.J. KOUTTS,T. I . ROBERTSON & P. A. CASTALDI Deparimrni oj. Medicine, Universirj,of Sydney. Westmeutl Centre, Sydney Quinidine-induced thrombocytopenia and leukopenia occurring together is very rare and has not been well documented. A 49-yr-old woman developed severe pancytopenia (WBC 0.6 x 10ytl, platelets 6 x 109;l and H b 8.1 g/dl) while receiving quinidine. Quinidinedependent platelet and granulocyte antibodies were detected in her serum by the immunofluorescence technique. Direct and indirect (with quinidine) Coornbs tests were negative. Laboratory tests showed iron deficiency and no hemolytic anemia. To immuno-chemically characterize the antibodies. fluoresceintagged specific anti-immunoglobulin and anti-complement reagents were used and the following results were obtained: Anti-(IgG, IgM & IgA) Anti-lgG Anti-lgM Anti-C', Platelets Granulocytes
+ +
+ +
~
+ -
To determine the identity of the platelet antigen involved, platelets were surface-labelled with periodate and ['HI sodium borohydride, solubilized with Triton X-100, and then incubated with patient's serum or purified IgG in the absence and presence of quinidine. The antigenantibody complexes were adsorbed onto protein A-containing. fixed Staphylococcus aicreus cells, which were washed and then eluted with SDS. SDS-polyacrylamide gel electrophoresis and fluorography revealed a quinidine-dependent band corresponding to platelet membrane glycoprotein Ib (GPIb). GPIb is not present on granulocytes (confirmed by using a mouse monoclonal antibody) suggesting that the quinidine-dependent platelet and granulocyte antibodies were distinct. T o confirm the presence of 2 antibodies, the immunofluorescence test was repeated with platelets and granulocytes from a normal individual and a patient with Bernard-Soulier Syndrome (BSS). Normal and BSS granulocytes and normal platelets gave positive reactions but BSS platelets (which lack GPIb) were negative. The combined results are consistent with an antibody which reacted with a quinidinc-GPIb complex on normal but nur on BSS platelets and a separate antibody which reacted with a complex formed between quinidine and a yet undefined membrane component on normal and BSS granulocytes. SURGERY FOR ANGIODYSPLASIA OF THE GUT IN VON WILLEBRAND'S DISEASE USING D.D.A.V.P. ALONE
K. R. ROMERIL, C. M. HICKTON & M. E . J . BEARD Department of Haemaiology. Chrisrchurch Hospital, Chrisrchurch A 69-yr-old woman with moderately severe V o n Willebrand's Disease (bleeding time > 25 min) presented with recurrent colonic bleeding. The bleeding site was identified by colonoscopy and she had a total colectomy performed under the sole cover of D.D.A.V.P. (Desmopressin) pre- and post-operatively to raise factor VIIl coagulant activity and ristocetin co-factor. D.D.A.V.P. had been shown pre-
348
Pathology (1983). 15, July
HSA & NZSH & ASBT
operatively to reduce the bleeding time from > 2 5 min to 18 min. The surgical procedure was uneventful and no abnormal bleeding ensued. The only complication of D.D.A.V.P. was slight fluid retention postoperatively. Histology confirmed the presence of angiodysplasia which has previously been described only once in patients with congenital Von Willebrand’s Disease. The significance of this finding will be discussed in relationship to the disorder of vascular endothelium in Von Willebrand’s Disease. AN ABNORMAL PLATELET a-GRANULE GLYCOPROTEIN IN A PATIENT WITH ESSENTIAL THROMBOCYTHEMIA
WILLIAM Boom, M. C. BERNDT& P. A. CASTALDIDepartment o j ’ Medicine, Universitj of Sydney, Westmead Centre, Sydney Essential thrombocythemia is one of the myeloproliferative syndromes characterized primarily by excessive production of megakaryocyteplatelet elements. Disturbed platelet function is believed to be responsible to some degree for the bleeding and thrombotic tendency commonly associated with this disorder. As part of a continuing investigation into the molecular basis of the defect. the platelet protein profile was analysed using SDS-polyacrylamide gel electrophoresis. In one patient with essential thrombocythemia, electrophoretic analysis revealed an additional protein band in the reduced M, = 170000 region. This protein, clearly identifiable in the patient’s unstimulated platelets, was actively secreted by a-thrombin along with the other r-granule protein constituents. One explanation for the occurrence of this band is that it represents cr-granule storage of a new protein. A more likely possibility is that the protein represents a modified form of a pre-existing a-granule constituent. Three lines of evidence are consistent with the abnormal protein being derived from the a-granule protein. glycoprotein G (GPG): (i) GPC (reduced M, = 185 000) in its native form is a disulfide-linked trimer. SDS-gel electrophoresis of the patient’s cr-granule proteins under non-reducing conditions indicated that the abnormal protein was also a disulfide-linked trimer. (ii) In the presence of the chelating agent, EDTA, all the a-granule proteins are secreted into the supernatant. However, in the presence of exogenous calcium, the released G P G and fibrinogen are partially bound and become major constituents of the activated platelet membrane surface. Control and patient platelets were aggregated in the presence of calcium using cc-thrombin and pulse-radioiodinatedwith lactoperoxidase. After separation of the platelets from supernatant by sucrose gradient centrifugation, SDS-gel electrophoresis and autoradiography confirmed that like G P G and fibrin, the abnormal protein shared this functional characteristic. (iii) The a-granule proteins of control and patient platelets were labelled using lactoperoxidase-catalyzed iodination. In the patient sample, affinity purified rabbit anti-GPG antibody immune precipitated both G P G and the abnormal protein. This constitutes the first secure example of an abnormality in an agranule constituent. Preliminary screening indicates that the abnormal protein is present in variable amounts in patients with essential thrombocythemia and polycythemia rubra Vera but not in patients with chronic granulocytic leukemia. Current evidence suggests that the platelet aggregation complex consists of membrane glycoproteins IIb and 111, calcium. fibrinogen and GPG. An abnormal form of this glycoprotein may therefore partially explain the aggregation defect seen in the myeloproliferative syndromes. RESULTS OF HEPARIN TREATMENT FOR VENOUS THROMBOEMBOLISM (VTE)
A. S. GALLUS. K . T. GOODALL. J. TILLETT&J . JACKAMAN Department of Huematologj, Flinders Medical Centre, Adelaide One hundred and nine patients treated for VTE were surveyed for recurrence or bleeding during admission to provide baseline information for clinical trials designed to evaluate new therapy.
Patients were studied if suspected venous thrombosis (VT) was confirmed by venography, or pulmonary embolism (PE) by ‘highprobability’ perfusionlventilation lung scanning. Surveillance was by repeated interview, IZsI-fibrinogen leg-scan. plethysmography and lung scan. Suspected recurrence was called definite (recurrent chest pain with new perfusion defect(s). or extension beyond the initiallydefined limits ofVT shown by leg-scan and’orrepeat venography), possible (return of pain and swelling within the initially defined limits of VT) or negative (chest pain without new perfusion defect). Bleeding was major (transfusion and/or interrupted treatment) or minor. Treatment guidelines included heparin by continuous intravenous infusion for 7 d (aiming for activated partial thromboplastin time (APTT) = 50-80 sec: General Diagnostics automated reagent). then warfarin (aiming for P T ratio = 2.0-3.5; Australian Reference Thromboplastin) with a 2-3 d overlap. Heparin was given for a mean of 9 d (range = 2-23 d) at an average dose of 1100 units’h. The 11 clinically suspected recurrences were found to be 3 definite (2.8”/,), 3 possible (2.8%) and 5 negative (4.6%). Most occurred during warfarin treatment after heparin was stopped (see table). Three patients (2.8%) had major and 7 (6.4%) minor bleeding during heparin therapy. Complications during heparin treatment occurred with the APTT within its therapeutic range. Three of 4 recurrences after heparin occurred with a sub-therapeutic PT ratio. Treatment
Heparin f warfarin warfarin alone
Recurrences
I
I 2
I
, 1 2
I
3 2
PLASMA LACTOFERRIN AS A MARKER OF NEUTROPHIL KINETICS
Ross D. BROWN,K. A. RICKARD& H. KRONENBERG Hoematolog!. Department, Royal Prince Alfred Hospital, S j d n e j Lactoferrin (LF), a non-heme iron binding protein present in many biological fluids and tissues, has a bacteriostatic function which has not yet been clearly defined. In hemopoietic cells, LF is found in the secondary granules of mature neutrophils. Thus L F is also a specific marker of neutrophilic differentiation. A 2-site solid phase immunoradiometric (IRMA) assay for plasma L F was developed and the L F concentration in over 1500 plasma samples was determined as a means of understanding the importance of L F and studying possible applications for this assay. The results indicate that EDTA is the anticoagulant of choice as both serum and heparinized samples had falsely high levels of LF which increased significantly in samples which were not separated immediately after collection. The correlation between plasma lactoferrin and absolute neutrophil count (r = 0.90) was studied in a large group of patients and the results suggested that plasma L F gives an indirect measure of the total blood granulocyte pool and granulocyte turnover rate in most patients. Regeneration of the myeloid series in patients recovering from aplasia was detected by plasma L F IRMA several days prior to any significant rise in the absolute neutrophil count. Thus plasma lactoferrin levels can indicate the size ofthe bone marrow reserve and play an important role in monitoring patients after chemotherapy or transplantation. The ratio between plasma L F and the absolute neutrophil count was increased in patients with gross splenomegaly and decreased in patients after splenectomy. These results probably indicate
ABSTRACTS OF COMBINED ANNUAL MEETING
gene and gives the haplotype aaa42/l. This complements the previously described aaa37/haplotypeand taken in conjunction with the - a3' and - a42/deletions in a thalassemia ( a thalassemia-2) provides further evidence that interchromosomal crossing-over is the most likely mechanism for deletions in the a+ thalassemias. The second rearrangement demonstrated a triplication of the 7-globin gene to give the haplotype GyGyAy/. Family studies in the latter rearrangement have shown that an additional y-globin gene does not significantly alter the level of H b F. This provides an interesting contrast to what is observed when extensive deletions with the p-globin cluster (for example in (Sg) O thalassemia and HPFH) are associated with elevated levels of Hb F. +
USE OF RESTRICTION FRAGMENT LENGTH POLYMORPHISMS FOR THE DIAGNOSIS OF THALASSEMIA
Z. RUDZKI,A. IASIELLO& R. J. KIMBER Division of Haematologj, Institute of Medical and Veterinary Science. Adelaide
Restriction fragment length polymorphisms (RFLPs) are caused by variations in DNA nucleotide sequences randomly distributed throughout the entire human genome. Thus, DNA from different people will give different length fragments when digested with restriction endonucleases. If RFLPs are closely linked to a genetic disorder, the pattern can be used to identify the chromosome carrying the genetic disorder. This approach for the antenatal diagnosis of [I thalassemia only requires amniocentesis and not fetoscopy to obtain the specimen but family studies are required to obtain the necessary data. There are 7 polymorphic restriction enzyme sites known to be tightly linked to the globin gene and these sites have been used for antenatal diagnosis of p thalassemia. To test the applicability of this procedure to our local population, D N A specimens have been obtained from 6 families with a homozygous p thalassemic child. The Hind 111 polymorphic sites in the G y and *y globin genes were investigated in a retrospective manner with the intention of using the technique for antenatal diagnosis in the future. In 4 families. complete diagnosis was possible using the Hind 111 sites alone. In one of the 2 remaining families, diagnosis by exclusion would be possible in 509, of cases while in the last family no diagnosis was possible. However. other polymorphic restriction sites may allow diagnosis to be made and this work is currently being undertaken. Specimens from a further 6 families have recently become available for study. Thus the preliminary results available to date confirm overseas experience that RFLPs can be used for the antenatal diagnosis of homozygous p thalassemia in about 75% of families at risk for this disorder. EFFICACY OF FILTER COLUMNS IN THE MANAGEMENT OF TRANSFUSION REACTIONS IN CHRONICALLY TRANSFUSED THALASSEMIC PATIENTS
G. J . BRYANT & A . T. LAMMI Haematology Department. Royal Alexandra Hospital for Children, Sydney
Filter columns (Imugard IG 500 Filter) containing cotton wool (Gossypium barbadense) are used to produce leukocyte poor blood for transfusion. In this study, 5 patients with thalassemia major who require regular transfusion, were transfused for 1 yr with filtered, packed red cells. Three patients (adult group) had been receiving triple washed packed cells for over 5 yr to prevent febrile reactions. The other 2 patients (younger group) had been having an increasing number of febrile reactions with transfusion. Transfusion episodes over the year prior to the use of filters were used as the control for each patient. One hundred and sixty-nine units of packed red cells were filtered: 96.77; of leukocytes were removed on filtration. In 12 units (779, the leukocyte count was greater than 0.5 x 10y/l,while in 6 of these 12 units (3.5% overall), the leukocyte count was greater than 1 x 10y/l. Four febrile reactions (temperature > 37.5 "C)were recorded during the year (incidence of less than 3%). I n the 3 adult patients, 117 units of
packed cells were transfused each year. There was no significant difference in the number of febrile reactions; two with filtered blood compared with none with triple washed blood. In the younger two patients, 52 units of blood were transfused each year. There were fewer febrile reactions with the filtered blood (2 reactionsj as compared with 16 reactions with the unfiltered blood. Pretransfusion hemoglobin levels were higher (P i0.01) in each ofthe 3 patients receiving filtered cells, as compared with hemoglobin levels when triple washed cells were transfused. In the younger group, there was no difference ( P>0.10) in pretransfusion hemoglobins. with filtered and unfiltered blood. Column filters were efficient in the prophylaxis against febrile reactions in sensitized patients. Filtration did not increase the requirements for donor blood. QUINIDINE-INDUCED THROMBOCYTOPENIA AND LEUKOPENIA. IN VlTRO STUDIES OF THE QUINIDINE-DEPENDENT PLATELET AND GRANULOCYTE ANTIBODIES
B. H. CHONG,M. C. BERNDT.J. KOUTTS,T. I . ROBERTSON & P. A. CASTALDI Deparimrni oj. Medicine, Universirj,of Sydney. Westmeutl Centre, Sydney Quinidine-induced thrombocytopenia and leukopenia occurring together is very rare and has not been well documented. A 49-yr-old woman developed severe pancytopenia (WBC 0.6 x 10ytl, platelets 6 x 109;l and H b 8.1 g/dl) while receiving quinidine. Quinidinedependent platelet and granulocyte antibodies were detected in her serum by the immunofluorescence technique. Direct and indirect (with quinidine) Coornbs tests were negative. Laboratory tests showed iron deficiency and no hemolytic anemia. To immuno-chemically characterize the antibodies. fluoresceintagged specific anti-immunoglobulin and anti-complement reagents were used and the following results were obtained: Anti-(IgG, IgM & IgA) Anti-lgG Anti-lgM Anti-C', Platelets Granulocytes
+ +
+ +
~
+ -
To determine the identity of the platelet antigen involved, platelets were surface-labelled with periodate and ['HI sodium borohydride, solubilized with Triton X-100, and then incubated with patient's serum or purified IgG in the absence and presence of quinidine. The antigenantibody complexes were adsorbed onto protein A-containing. fixed Staphylococcus aicreus cells, which were washed and then eluted with SDS. SDS-polyacrylamide gel electrophoresis and fluorography revealed a quinidine-dependent band corresponding to platelet membrane glycoprotein Ib (GPIb). GPIb is not present on granulocytes (confirmed by using a mouse monoclonal antibody) suggesting that the quinidine-dependent platelet and granulocyte antibodies were distinct. T o confirm the presence of 2 antibodies, the immunofluorescence test was repeated with platelets and granulocytes from a normal individual and a patient with Bernard-Soulier Syndrome (BSS). Normal and BSS granulocytes and normal platelets gave positive reactions but BSS platelets (which lack GPIb) were negative. The combined results are consistent with an antibody which reacted with a quinidinc-GPIb complex on normal but nur on BSS platelets and a separate antibody which reacted with a complex formed between quinidine and a yet undefined membrane component on normal and BSS granulocytes. SURGERY FOR ANGIODYSPLASIA OF THE GUT IN VON WILLEBRAND'S DISEASE USING D.D.A.V.P. ALONE
K. R. ROMERIL, C. M. HICKTON & M. E . J . BEARD Department of Haemaiology. Chrisrchurch Hospital, Chrisrchurch A 69-yr-old woman with moderately severe V o n Willebrand's Disease (bleeding time > 25 min) presented with recurrent colonic bleeding. The bleeding site was identified by colonoscopy and she had a total colectomy performed under the sole cover of D.D.A.V.P. (Desmopressin) pre- and post-operatively to raise factor VIIl coagulant activity and ristocetin co-factor. D.D.A.V.P. had been shown pre-
348
Pathology (1983). 15, July
HSA & NZSH & ASBT
operatively to reduce the bleeding time from > 2 5 min to 18 min. The surgical procedure was uneventful and no abnormal bleeding ensued. The only complication of D.D.A.V.P. was slight fluid retention postoperatively. Histology confirmed the presence of angiodysplasia which has previously been described only once in patients with congenital Von Willebrand’s Disease. The significance of this finding will be discussed in relationship to the disorder of vascular endothelium in Von Willebrand’s Disease. AN ABNORMAL PLATELET a-GRANULE GLYCOPROTEIN IN A PATIENT WITH ESSENTIAL THROMBOCYTHEMIA
WILLIAM Boom, M. C. BERNDT& P. A. CASTALDIDepartment o j ’ Medicine, Universitj of Sydney, Westmead Centre, Sydney Essential thrombocythemia is one of the myeloproliferative syndromes characterized primarily by excessive production of megakaryocyteplatelet elements. Disturbed platelet function is believed to be responsible to some degree for the bleeding and thrombotic tendency commonly associated with this disorder. As part of a continuing investigation into the molecular basis of the defect. the platelet protein profile was analysed using SDS-polyacrylamide gel electrophoresis. In one patient with essential thrombocythemia, electrophoretic analysis revealed an additional protein band in the reduced M, = 170000 region. This protein, clearly identifiable in the patient’s unstimulated platelets, was actively secreted by a-thrombin along with the other r-granule protein constituents. One explanation for the occurrence of this band is that it represents cr-granule storage of a new protein. A more likely possibility is that the protein represents a modified form of a pre-existing a-granule constituent. Three lines of evidence are consistent with the abnormal protein being derived from the a-granule protein. glycoprotein G (GPG): (i) GPC (reduced M, = 185 000) in its native form is a disulfide-linked trimer. SDS-gel electrophoresis of the patient’s cr-granule proteins under non-reducing conditions indicated that the abnormal protein was also a disulfide-linked trimer. (ii) In the presence of the chelating agent, EDTA, all the a-granule proteins are secreted into the supernatant. However, in the presence of exogenous calcium, the released G P G and fibrinogen are partially bound and become major constituents of the activated platelet membrane surface. Control and patient platelets were aggregated in the presence of calcium using cc-thrombin and pulse-radioiodinatedwith lactoperoxidase. After separation of the platelets from supernatant by sucrose gradient centrifugation, SDS-gel electrophoresis and autoradiography confirmed that like G P G and fibrin, the abnormal protein shared this functional characteristic. (iii) The a-granule proteins of control and patient platelets were labelled using lactoperoxidase-catalyzed iodination. In the patient sample, affinity purified rabbit anti-GPG antibody immune precipitated both G P G and the abnormal protein. This constitutes the first secure example of an abnormality in an agranule constituent. Preliminary screening indicates that the abnormal protein is present in variable amounts in patients with essential thrombocythemia and polycythemia rubra Vera but not in patients with chronic granulocytic leukemia. Current evidence suggests that the platelet aggregation complex consists of membrane glycoproteins IIb and 111, calcium. fibrinogen and GPG. An abnormal form of this glycoprotein may therefore partially explain the aggregation defect seen in the myeloproliferative syndromes. RESULTS OF HEPARIN TREATMENT FOR VENOUS THROMBOEMBOLISM (VTE)
A. S. GALLUS. K . T. GOODALL. J. TILLETT&J . JACKAMAN Department of Huematologj, Flinders Medical Centre, Adelaide One hundred and nine patients treated for VTE were surveyed for recurrence or bleeding during admission to provide baseline information for clinical trials designed to evaluate new therapy.
Patients were studied if suspected venous thrombosis (VT) was confirmed by venography, or pulmonary embolism (PE) by ‘highprobability’ perfusionlventilation lung scanning. Surveillance was by repeated interview, IZsI-fibrinogen leg-scan. plethysmography and lung scan. Suspected recurrence was called definite (recurrent chest pain with new perfusion defect(s). or extension beyond the initiallydefined limits ofVT shown by leg-scan and’orrepeat venography), possible (return of pain and swelling within the initially defined limits of VT) or negative (chest pain without new perfusion defect). Bleeding was major (transfusion and/or interrupted treatment) or minor. Treatment guidelines included heparin by continuous intravenous infusion for 7 d (aiming for activated partial thromboplastin time (APTT) = 50-80 sec: General Diagnostics automated reagent). then warfarin (aiming for P T ratio = 2.0-3.5; Australian Reference Thromboplastin) with a 2-3 d overlap. Heparin was given for a mean of 9 d (range = 2-23 d) at an average dose of 1100 units’h. The 11 clinically suspected recurrences were found to be 3 definite (2.8”/,), 3 possible (2.8%) and 5 negative (4.6%). Most occurred during warfarin treatment after heparin was stopped (see table). Three patients (2.8%) had major and 7 (6.4%) minor bleeding during heparin therapy. Complications during heparin treatment occurred with the APTT within its therapeutic range. Three of 4 recurrences after heparin occurred with a sub-therapeutic PT ratio. Treatment
Heparin f warfarin warfarin alone
Recurrences
I
I 2
I
, 1 2
I
3 2
PLASMA LACTOFERRIN AS A MARKER OF NEUTROPHIL KINETICS
Ross D. BROWN,K. A. RICKARD& H. KRONENBERG Hoematolog!. Department, Royal Prince Alfred Hospital, S j d n e j Lactoferrin (LF), a non-heme iron binding protein present in many biological fluids and tissues, has a bacteriostatic function which has not yet been clearly defined. In hemopoietic cells, LF is found in the secondary granules of mature neutrophils. Thus L F is also a specific marker of neutrophilic differentiation. A 2-site solid phase immunoradiometric (IRMA) assay for plasma L F was developed and the L F concentration in over 1500 plasma samples was determined as a means of understanding the importance of L F and studying possible applications for this assay. The results indicate that EDTA is the anticoagulant of choice as both serum and heparinized samples had falsely high levels of LF which increased significantly in samples which were not separated immediately after collection. The correlation between plasma lactoferrin and absolute neutrophil count (r = 0.90) was studied in a large group of patients and the results suggested that plasma L F gives an indirect measure of the total blood granulocyte pool and granulocyte turnover rate in most patients. Regeneration of the myeloid series in patients recovering from aplasia was detected by plasma L F IRMA several days prior to any significant rise in the absolute neutrophil count. Thus plasma lactoferrin levels can indicate the size ofthe bone marrow reserve and play an important role in monitoring patients after chemotherapy or transplantation. The ratio between plasma L F and the absolute neutrophil count was increased in patients with gross splenomegaly and decreased in patients after splenectomy. These results probably indicate