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Granulocyte kinetic studies in the elucidation of clinical problems
74
T H E R O Y A L COLLEGE O F P A T H O L O G I S T S O F A U S T R A L I A
T h e difference in survival between the group with deep clefts alone and the group without nucleoli or deep clefts was not statistically significant. These two groups were combined for comparison with all cases showing nucleoli. A highly significant difference was shown: (Wilcoxon rank test 2 a -= 0.01). Patients with presenting H b I' 11.0 g./lOO ml., with ESR > 50 mmlhr.. or with platelets 100,000' survived less well than appropriate control groups (Wilcoxon rank tests 2 u : 0.01, 0.01 and 0.1 respectively). No relationship was seen between height of WBC count, presence of lymphadenopathy or splenomegaly and survival. Ten patients out of 47 with lymphosarcoma showed a lymphocytosis. Only one case showed nucleolated lymphocytes in the blood. T h e mean survival of these 10 patients was 35 mth. compared with 23 mth. for the group as a whole. T h e prognostic value of these findings was considered. HAEMOPOIETIC STEM CELL STIMULATION IN SHORT-TERM TISSUE CULTURE
RICKARD, K. A,, MORLEY,A., HOWARD, D., GARRITY, MARIANNE & STOHLMAN, F., JR. St. Elizabeth's Hospital, Tuft's University School of Medicine, Massachusetts, U.S.A. Murine serum with high levels of agar colony stimulating factor (CSF) was examined for its ability to maintain myeloid stem cell and pluripotential stem cell numbers in a short term tissue culture of normal murine bone marrow. T h e serum was obtained from whole body irradiated (WBI) mice and had been previously shown to have high levels of CSF. T h e serum was mixed with normal bone marrow suspensions, whilst mixtures of normal serum and normal bone marrow suspensions served as controls. T h e content of pluripotential and myeloid stem cell compartments in these short term tissue cultures were assayed at regular intervals by bone marrow transplantation or the agar colony system respectively. I n the presence of WBI serum myeloid stem cell numbers were maintained for periods up to 72 hr., whilst their numbers decreased rapidly in the presence of normal serum. Similarly, but to a lesser extent, pluripotential stem cell numbers were maintained in the presence of WBI serum. These data suggest that C S F appearing in vivo has a selective stimulatory effect on committed myeloid stem cell compartments in vitro. It is postulated that WBI serum may contain a single substance which influences both proliferation and differentiation of committed myeloid stem cells, and thus may be a specific regulator of granulopoiesis at this level. T h e slower decline of pluripotential stem cells in the presence of a maintained myeloid stem cell compartment may be due to a lower demand for pluripotential stem cells rather than direct stimulation at this level. Such findings are in accord with the notion that C S F may be involved in the humoral regulation of granulopoiesis. GRANULOCYTE KINETIC STUDIES IN THE ELUCIDATION OF CLINICAL PROBLEMS
VINCENT, P. C., LEVI,J. A,, MCQUEEN, ANN& GUNZ,F. W. Sydney, New South Wales
Sydney Hospital,
Study of neutrophil granulocyte kinetics following the reinfusion of autologous DFP"'-labelled granulocytes in man allows one to interpret the dynamic abnormalities of granulopoiesis in a wide variety of clinical disorders. T h e size of the total blood granulocyte pool (TBGP) can be calculated from the number of labelled granulocytes infused and the number found initially in the circulation, using isotope dilution principles. T h e T B G P is always greater than the circulating granulocyte pool (CGP), due to the presence of granulocytes marginated on the walls of vessels (the margined granulocyte pool, MGP). Granulocytes disappear from the blood in s random fashion, which can be characterized by the half disappearance time ( T $ ) of labelled granulocytes in the blood. T h e granulocyte turnover rate (GTR) can be derived from the TBGP and the T i . In a steady-state situation the G T R is equal to the rate at which granulocytes are produced by the marrow. T h e authors used these techniques to study two groups of patients: (a) those with lymphoma, and (b) those with neutropenia due to non-malignant disease. Six of the 8 patients with lymphoma had splenamegaly at the time of study, and 4 of thesr were neutropenic. Five of the 7 other patients with neutropenia also had splenomegaly (three Felty's syndrome,
ABSTRACTS OF ANNUAL SCIENTIFIC MEETING
1971
75
one DLE, one portal hypertension). T h e majority of patients with splenomegaly had an increased M G P : C G P ratio, but none showed a significant shortening of granulocyte survival. In most cases, granulocytes were being produced at a normal or greater than normal rate. Two patients with splenomegaly (one with lymphoma, one with Felty's syndrome) underwent splenectomy, with an increase in circulating granulocytes in each case. Results in 2 patients with lymphoma without splenomegaly were essentially normal, while 2 patients with neutropenia without splenomegaly showed a normal granulocyte survival, a decrease in granulocyte production and an increase in the M G P CGP ratio. PROLONGATION OF QUICK'S ONE-STAGE PROTHROMBIN TIME BY A N INHIBITOR SPECIFIC FOR FACTOR V
HANDLEY, D. A . Australia
Iiistirure qf Medical mid Vererinarjl Science, Adelaide, Soutli
A 78-yr.-old man was proven by laparotomy to have a carcinoma of the prostate. Phenindione therapy was commenced post-operatively for a suspected pulmonary embolus, but was discontinued when the prothrombin activity was found to be less than 5",,. T h e prothrombin activity rose only slightly with vitamin K and has remained at about 15",, for over 12 mth. T h e addition of a 1 10 volume of normal plasma did not significantly affect the patient's prothrombin time and the incubation of the patient's serum with normal plasma led to marked prolongation, indicating the presence of an anti-coagulant. A clotting system based on the one-stage prothrombin time was prepared in which alumina-absorbed normal plasma was used as a source of factor V, and a concentrate of factors 11, VII, IX, and X (P.P.S.B., C.S.L., Melbourne) was used to supply the remaining clotting factors required. Pre-incubation of the patient's serum with the alumina plasma (factor V) led to marked prolongation of the clotting time, while pre-incubation with P.P.S.B. had no effect. This suggested thait the inhibitor was acting specifically on factor V. The inhibitory action was not immediate, but was maximal after about 5 min. incubation at 17 . On column chromatography using 'Sephadex' G 200 gel, the inhibitor appeared with the second protein peak, while traces of factors V I I and X were evident in the third peak. There are only 6 case reports of inhibitors specific for factor V in the literature, and all of these were transient, being present for days or weeks. T h e present case is unusual in that the inhibitor has remained unchanged for over a year. A surprising feature, also seen in the casc reported by Handley and Duncan is that, in spite of a gross abnormality on laboratory testing, the patient has not had excessive bleeding. CLINICAL A N D LABORATORY FINDINGS I N FACTOR XI DEFICIENCY
GRACE,C. S., KROKENBERG, H. & KICKARL), K. A . Svdnev, h'ew Sourli Wales
Royal P r i m e Alfred H o . p t d ,
T h e propositus was a 39-yr.-old woman who gave a history of excessive bleeding following surgery, but apart from frequent epistaxes as a child, no spontaneous haemorrhage. Her daughter, who also has a low level of Factor XI, had undergone an uneventful appendeci-omy and normal pregnancy. 1.aboratory studies revealed a normal bleeding time, a prolonged whole blood clotting time and a prolonged partial thromboplastin time with kaolin. T h e thromboplastin generation test was abnormal using the patient's serum or absorbed plasma while the patient's plasma corrected the defect in the plasma from a patient with Factor XI1 deficiency. T h e diagnosis of Factor XI deficiency was confirmed by the failure of her plasma to correct plasmas artificially depleted of Factor XI and from a patient with known Factor XI deficiency (Rosenthal's original patient). T h e therapeutic management of the patient undergoing hysterectomy was described. T h e postoperative course was punctuated by a severe hacmorrhage when the level of Factor XI dropped to below 20' ,. She obtained the calculated rise in Factor XI following transfusion with fresh frozen plasma and the half life of Factor XI was found to be about 40 hr.
T H E R O Y A L COLLEGE O F P A T H O L O G I S T S O F A U S T R A L I A
T h e difference in survival between the group with deep clefts alone and the group without nucleoli or deep clefts was not statistically significant. These two groups were combined for comparison with all cases showing nucleoli. A highly significant difference was shown: (Wilcoxon rank test 2 a -= 0.01). Patients with presenting H b I' 11.0 g./lOO ml., with ESR > 50 mmlhr.. or with platelets 100,000' survived less well than appropriate control groups (Wilcoxon rank tests 2 u : 0.01, 0.01 and 0.1 respectively). No relationship was seen between height of WBC count, presence of lymphadenopathy or splenomegaly and survival. Ten patients out of 47 with lymphosarcoma showed a lymphocytosis. Only one case showed nucleolated lymphocytes in the blood. T h e mean survival of these 10 patients was 35 mth. compared with 23 mth. for the group as a whole. T h e prognostic value of these findings was considered. HAEMOPOIETIC STEM CELL STIMULATION IN SHORT-TERM TISSUE CULTURE
RICKARD, K. A,, MORLEY,A., HOWARD, D., GARRITY, MARIANNE & STOHLMAN, F., JR. St. Elizabeth's Hospital, Tuft's University School of Medicine, Massachusetts, U.S.A. Murine serum with high levels of agar colony stimulating factor (CSF) was examined for its ability to maintain myeloid stem cell and pluripotential stem cell numbers in a short term tissue culture of normal murine bone marrow. T h e serum was obtained from whole body irradiated (WBI) mice and had been previously shown to have high levels of CSF. T h e serum was mixed with normal bone marrow suspensions, whilst mixtures of normal serum and normal bone marrow suspensions served as controls. T h e content of pluripotential and myeloid stem cell compartments in these short term tissue cultures were assayed at regular intervals by bone marrow transplantation or the agar colony system respectively. I n the presence of WBI serum myeloid stem cell numbers were maintained for periods up to 72 hr., whilst their numbers decreased rapidly in the presence of normal serum. Similarly, but to a lesser extent, pluripotential stem cell numbers were maintained in the presence of WBI serum. These data suggest that C S F appearing in vivo has a selective stimulatory effect on committed myeloid stem cell compartments in vitro. It is postulated that WBI serum may contain a single substance which influences both proliferation and differentiation of committed myeloid stem cells, and thus may be a specific regulator of granulopoiesis at this level. T h e slower decline of pluripotential stem cells in the presence of a maintained myeloid stem cell compartment may be due to a lower demand for pluripotential stem cells rather than direct stimulation at this level. Such findings are in accord with the notion that C S F may be involved in the humoral regulation of granulopoiesis. GRANULOCYTE KINETIC STUDIES IN THE ELUCIDATION OF CLINICAL PROBLEMS
VINCENT, P. C., LEVI,J. A,, MCQUEEN, ANN& GUNZ,F. W. Sydney, New South Wales
Sydney Hospital,
Study of neutrophil granulocyte kinetics following the reinfusion of autologous DFP"'-labelled granulocytes in man allows one to interpret the dynamic abnormalities of granulopoiesis in a wide variety of clinical disorders. T h e size of the total blood granulocyte pool (TBGP) can be calculated from the number of labelled granulocytes infused and the number found initially in the circulation, using isotope dilution principles. T h e T B G P is always greater than the circulating granulocyte pool (CGP), due to the presence of granulocytes marginated on the walls of vessels (the margined granulocyte pool, MGP). Granulocytes disappear from the blood in s random fashion, which can be characterized by the half disappearance time ( T $ ) of labelled granulocytes in the blood. T h e granulocyte turnover rate (GTR) can be derived from the TBGP and the T i . In a steady-state situation the G T R is equal to the rate at which granulocytes are produced by the marrow. T h e authors used these techniques to study two groups of patients: (a) those with lymphoma, and (b) those with neutropenia due to non-malignant disease. Six of the 8 patients with lymphoma had splenamegaly at the time of study, and 4 of thesr were neutropenic. Five of the 7 other patients with neutropenia also had splenomegaly (three Felty's syndrome,
ABSTRACTS OF ANNUAL SCIENTIFIC MEETING
1971
75
one DLE, one portal hypertension). T h e majority of patients with splenomegaly had an increased M G P : C G P ratio, but none showed a significant shortening of granulocyte survival. In most cases, granulocytes were being produced at a normal or greater than normal rate. Two patients with splenomegaly (one with lymphoma, one with Felty's syndrome) underwent splenectomy, with an increase in circulating granulocytes in each case. Results in 2 patients with lymphoma without splenomegaly were essentially normal, while 2 patients with neutropenia without splenomegaly showed a normal granulocyte survival, a decrease in granulocyte production and an increase in the M G P CGP ratio. PROLONGATION OF QUICK'S ONE-STAGE PROTHROMBIN TIME BY A N INHIBITOR SPECIFIC FOR FACTOR V
HANDLEY, D. A . Australia
Iiistirure qf Medical mid Vererinarjl Science, Adelaide, Soutli
A 78-yr.-old man was proven by laparotomy to have a carcinoma of the prostate. Phenindione therapy was commenced post-operatively for a suspected pulmonary embolus, but was discontinued when the prothrombin activity was found to be less than 5",,. T h e prothrombin activity rose only slightly with vitamin K and has remained at about 15",, for over 12 mth. T h e addition of a 1 10 volume of normal plasma did not significantly affect the patient's prothrombin time and the incubation of the patient's serum with normal plasma led to marked prolongation, indicating the presence of an anti-coagulant. A clotting system based on the one-stage prothrombin time was prepared in which alumina-absorbed normal plasma was used as a source of factor V, and a concentrate of factors 11, VII, IX, and X (P.P.S.B., C.S.L., Melbourne) was used to supply the remaining clotting factors required. Pre-incubation of the patient's serum with the alumina plasma (factor V) led to marked prolongation of the clotting time, while pre-incubation with P.P.S.B. had no effect. This suggested thait the inhibitor was acting specifically on factor V. The inhibitory action was not immediate, but was maximal after about 5 min. incubation at 17 . On column chromatography using 'Sephadex' G 200 gel, the inhibitor appeared with the second protein peak, while traces of factors V I I and X were evident in the third peak. There are only 6 case reports of inhibitors specific for factor V in the literature, and all of these were transient, being present for days or weeks. T h e present case is unusual in that the inhibitor has remained unchanged for over a year. A surprising feature, also seen in the casc reported by Handley and Duncan is that, in spite of a gross abnormality on laboratory testing, the patient has not had excessive bleeding. CLINICAL A N D LABORATORY FINDINGS I N FACTOR XI DEFICIENCY
GRACE,C. S., KROKENBERG, H. & KICKARL), K. A . Svdnev, h'ew Sourli Wales
Royal P r i m e Alfred H o . p t d ,
T h e propositus was a 39-yr.-old woman who gave a history of excessive bleeding following surgery, but apart from frequent epistaxes as a child, no spontaneous haemorrhage. Her daughter, who also has a low level of Factor XI, had undergone an uneventful appendeci-omy and normal pregnancy. 1.aboratory studies revealed a normal bleeding time, a prolonged whole blood clotting time and a prolonged partial thromboplastin time with kaolin. T h e thromboplastin generation test was abnormal using the patient's serum or absorbed plasma while the patient's plasma corrected the defect in the plasma from a patient with Factor XI1 deficiency. T h e diagnosis of Factor XI deficiency was confirmed by the failure of her plasma to correct plasmas artificially depleted of Factor XI and from a patient with known Factor XI deficiency (Rosenthal's original patient). T h e therapeutic management of the patient undergoing hysterectomy was described. T h e postoperative course was punctuated by a severe hacmorrhage when the level of Factor XI dropped to below 20' ,. She obtained the calculated rise in Factor XI following transfusion with fresh frozen plasma and the half life of Factor XI was found to be about 40 hr.