Acta haemat. 59: 73-79 (1978)
‘False Positive’ Acidified Serum Test in a Preleukemic Dyserythropoiesis G. H auptmann, D. Sondag , J. M. L ang and F. O berling ' Institut d’Hématologie, CHU Strasbourg; Centre deTransfusion Sanguine de Strasbourg (Director; Prof. S. M ayer), and Clinique des Maladies du Sang, CHU Strasbourg (Director: Prof.G. M ayer), Strasbourg
Key Words. Acidified serum test • Preleukemia • Dyserythropoiesis Abstract. In a case of preleukemic dyserythropoiesis, in vitro red cell lysis tests showed a positive acidified serum test whose characteristics are described. The posi tive acidified serum test occurred in 10 normal sera, in 1 serum with complete defi ciency of the fourth component of complement and in 1 serum with complete defi ciency of the second component of complement. The test was found negative with 2 hyperlipemic sera. The other in vitro red-cell lysis tests were negative. The results of the in vitro lysis tests were different from the results obtained in paroxysmal nocturnal hemoglobinuria and congenital dyserythropoietic anemias.
Introduction In paroxysmal nocturnal hemoglobinuria (PNH) the red cells are unu sually susceptible to lysis by complement. Lysis of PNH red cells may be obtained in vitro in fresh serum where the complement system is activat ed either through the classical pathway (cold antibody lysis test) or the al ternate pathway (acidified scrum test sensitized with magnesium, inulin test, cobra venom test) or both pathways (acidified serum test sensitized with thrombin, sucrose lysis test) [4,11,12]. The acidified serum test (Ham’s test) is widely used to detect the PNH abnormality and generally considered to be specific for this red cell defect
Received: May 31, 1977; accepted: July 1, 1977.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
1 The technical assistance of Mrs. Y. Blanzat and Mrs. P. C uny is gratefully acknowledged.
74
H auptmann/S ondag/L ang/O berling
[4]. However, this test is also positive in congenital dyserythropoietic ane mia type II or HEMPAS [3] but, in contrast to PNH, HEMPAS red cells undergo lysis in only a portion of normal sera and they do not undergo lysis in the patient’s own acidified serum and the sucrose lysis test re mains negative. A positive acidified serum test has also been observed in 2 cases of congenital dyserythropoietic anemia type I [1, 14] but in 1 case the positivity was obtained in heated sera and due to spherocytosis [1]. A positive acidified serum test and a positive sucrose lysis test occurred also in 2 cases of lymphoproliferative disease [7, 17]. The present report describes the characteristics of a positive acidified serum test in a case of preleukemic dyserythropoiesis.
A 27-year-old man without noticeable antecedents was admitted to the Hemato logical Clinic in January 1975 for an anemia and neutropenia discovered in Septem ber 1974. Physical examination revealed a paleness and no other abnormalities. Laboratory findings showed: hemoglobin llg/lOOml, RBC 3.32 million/mm3, WBC 3,600/mm3; leukocyte differential count: 19% neutrophils, 1% basophils, 6% mono cytes, 73% lymphocytes and 1% neutrophilic metamyelocytes; reticulocytes 33°/00. The peripheral blood showed anisocytosis, anisochromia, Howell-Jolly bodies, Ca bot rings and basophil stippling. Platelet count 210,000/mm3. The bone marrow was markedly hypercellular with 70% erythroblasts and numerous erythroblastic islands. On light microscopy a small population of polychromatic and orthochromatic eryth roblasts showed abnormalities usually observed in congenital dyserythropoietic anemias: bi- or multinuclearity, incomplete cell divisions. Electron microscopy con firmed the light-microscopic findings: there were erythroblasts with two nuclei or bilobulated nuclei, protrusion of nuclear material into the cytoplasm. Some eryth roblasts showed a marked irregularity and ‘spongy’ appearance of the nucleus with wide nuclear pores and ‘invasion’ of the cytoplasm into the nucleus. The char acteristic morphological feature of the cytoplasm was the presence of abnormal cisternae delimitated by a membrane located near the cell surface. Deposits of iron were often found in the enlarged nuclear pores, in the mitochondria and in the zone of adherence of two adjacent erythroblasts [15]. Hemoglobin study revealed an increased level of HbF (6%). Serum iron was 110,Mg/100 ml; total bilirubin 0.7mg/100ml. Iron-kinetic study indicated an hyper active erythropoiesis. The curve of cell incorporation of labelled iron showed a fall at the 4th day indicating an early destruction of some newly formed red cells. How ever, clinical or biological signs of intravascular hemolysis could not be detected. The patient’s group was 0 rhesus CcDee, Kell - with no irregular antibodies. Serum proteins showed a normal electrophoretic pattern. The acidified serum test was positive with the patient’s serum and the serum of several different normal do nors. The characteristics of this positive acidified serum test will be given thereafter.
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Patient Summary
False Positive Acidified Serum Test
75
There was increased i and I antigen on the patient’s red cells (agglutination titers with known anti-i serum: patient 1/16, adult control 1/2, cord control 1/256; with an anti-I serum: patient 1/2,058, adult control 1/128, cord control 1/32). A study of the red cell enzymes showed an increased level of hexokinase, glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione peroxidase, while the level of phosphohexose isomerase was reduced at 50°/o of the normal level (Prof. P. Boivin ). This defect was not observed in the other family members. The karyotype analysis showed no abnormality. The blood picture remained stable without specific treatment until March 1976. At that time Hb was 10 g/100 ml, WBC 5,000 with 16% neutrophils, 31% mono cytes, 42% lymphocytes, 4% neutrophilic myelocytes and 7% myeloblasts. Platelet count was 101,000/mm3. The anemia, thrombocytopenia and neutropenia persisted and worsened and the myeloblast percentage increased to a level of 53 in the periph eral blood and 28 in the bone marrow in November 1976 where the patient died, 24 months after the discovery of the anemia and 8 months after myeloblasts were first noted in the blood.
Materials and Methods Veronal-buffered saline (VBS) containing 0.00015 m calcium and 0.0005 m mag nesium ions, VBS containing 0.01 m ethylenediaminetetraacetic acid (EDTA), diso dium salt (EDTA-VBS) and Alsevcrs’s solution were prepared according to M ayer [13]. VBS-sucrose buffer (60% isotonic sucrose-40% VBS) was prepared as outlined by L ogue et at. [11]. Blood from the patient and from normal donors was collected in an equal vol ume of Alsever’s solution and stored at 4 °C until used. Normal compatible serum was obtained from donors and stored at -30 °C for a short period of time. Serum from a patient with total genetic deficiency of the fourth component of complement and from a patient with total genetic deficiency of the second component of com plement were used [5, 6]. Anti-I antibody was obtained from a patient with chronic cold agglutinin syndrome. Inulin was purchased from Prolabo (Paris), cobra venom factor was purchased from Cordis Lab. (Miami, Fla.). The acidified serum test sensitized by the addition of magnesium (0.005 m ) was performed according to May et al. [12], The cold antibody test, sucrose test, inulin test and cobra venom test were performed according to L ogue et al. [11]. Optimal concentration of inulin, cobra venom factor and cold antibody were defined in pre liminary tests using AET-treated red cells [16]. Normal cells were not lysed in any of these procedures at the concentrations of reactants used.
The results of the in vitro red cell lysis are given in table I. The acidi fied serum test was markedly positive with the patient’s own serum, with
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
Results and Discussion
76
H auptmann/S ondag/L ang/O berling
Table /. Results of in vitro red cell lysis tests in the patient Test
Lysis, %
Acidified serum Patient’s own serum Normal donor serum 1 2 3 4 5 6 7 8 9 10 11 (hyperlipemic) 12 (hyperlipemic)
47
C4-deficient serum C2-deficient serum
45 43 negative with all sera negative with all sera negative with all sera negative with all sera
the serum of 10 normal donors, with the C4-deficient serum and with the C2-deficient serum. The test was negative with the serum of 2 normal do nors and it was observed that these sera were turbid, i.e. hyperlipemic. The test was negative with all sera heated at 56 °C for 20 min. The other in vitro red cell lysis tests remained negative with all sera. The tests were performed on three occasions (March 1975, April 1975 and March 1976) and gave similar results. The positivity of the acidified serum test with the patient’s own serum and the negativity of the cold antibody test excluded a congenital dyseryth ropoietic anemia type II (HEMPAS). The negativity of the insulin test, sucrose test and cobra venom test excluded an unusual susceptibility of the patient’s red cells to lysis by complement as observed in PNH. De spite the fact that the acidified serum test was found negative after heat ing the sera at 56 °C for 20 min, there is little reason to believe that the complement system was involved in the lysis of the patient’s red cells be
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
Sucrose lysis Cold antibody lysis Inulin Cobra venom
51 55 44 60 52 70 45 45 20 51 0 0
77
cause the lysis was obtained with complement-deficient sera and because all the tests except the acidified serum test based on the activation of the complement system through the classical and/or alternate pathway re mained negative. Therefore, we considered the positive acidified serum test as ‘false positive’. The negativity of the acidified serum test with two hyperlipemic sera suggests that an excess of lipids or lipoproteins protect ed the patient’s red cells against lysis and that the abnormal susceptibility to lysis in the acidified serum was due to an abnormal lipid composition or to an abnormal lipid metabolism in the patient’s red cells. This hypo thesis could not be verified. In congenital dyserythropoietic anemia type I, a positive acidified se rum test was observed in 2 cases [1, 14], In 1 case [1] the lysis was ob tained in 9 of the 10 normal acidified sera and with the patient’s own aci dified serum. However, hemolysis was marked only with 1 of the tested sera and lysis did not seem to depend on the presence of complement since sera heated at 56 °C for 30 min produced the same degree of lysis as unheated fresh serum. Therefore, the positive acidified serum test was also considered to be ‘false positive’. In PNH the evolution toward an acute myeloblastic leukemia is rare. Three observations have been published in 1969 [8-10]. The acute mye loblastic leukemia occurred 3, 7 and 2 years, respectively, after the dis covery of the clinical and biological signs of PNH. The diagnosis of PNH was based on the recognition of episodes of hemoglobinuria, hemosiderinuria, on the positivity of the acidified serum test in the 3 cases and on the positivity of the sucrose test in 2 cases [9, 10], In our observation we have not ascertained clinical or biological signs of PNH and the charac teristics of the positive acidified serum test allowed us to exclude the diag nosis of PNH. C atovsky et al. [2] have performed in vitro red cell lysis tests in sev eral cases of acute myeloblastic leukemia and have found frequently a po sitivity of the cold antibody lysis test (58%) and less frequently a positivi ty of the sucrose lysis test, whereas the acidified serum test was never found positive. In the two cases of lymphoproliferative disease with a ‘PNH-like’ red cell abnormality reported [7, 17], the acidified serum test was positive in one case togehter with a positive sucrose lysis test [17] and positive in the other case together with a positive cold antibody lysis test and with a positive sugar lysis test [7]. Our observation of a preleukemic dyserythropoiesis confirms that a positive acidified serum lysis test is not sufficient to state a ‘PNH defect’
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
False Positive Acidified Serum Test
78
H auptmann/S ondag/L ang/O berling
in the red cells and that several tests are necessary to explore and charac terize the in vitro susceptibility of red cells to lysis in the presence of nor mal sera, complement-deficient sera and also sera with abnormal lipid or lipoprotein composition.
1 C astro, O.; N ash, I., and F inch Stuart, C.: Congenital dyserythropoietic ane mia type I. Report of a case with increased erythrocyte agglutinability by anti-i serum. Archs intern. Med. 134: 346-351 (1974). 2 C atovsky, D.; L ew is , S. M., and Sherman, D.: Erythrocyte sensitivity to in vi tro lysis in leukemia. Br. J. Haemat. 21: 541-550 (1971). 3 Crookston , J. H.; Crookston , M. C.; Burnie , K. L.; F rancome, W. H.; D acie, J. V.; D avis, A., and L ew is , S. M.: Hereditary erythroblastic multinuclearity as sociated with a positive acidified serum test: a type of congenital dyserythro poietic anemia. Br. J. Haemat. 17: 11-26 (1969). 4 D acie , J. V. and L ew is , S. M.: Laboratory methods used in investigation of the haemolytic anaemias. V. Paroxysmal nocturnal haemoglobinuria (PNH); in Practical haematology; 5th ed., pp. 305-313 (Churchill/Livingstone, London/ Edinburgh 1975). 5 H auptmann, G.; G rosshans, E. et H eid , E.: Lupus érythémateux aigus et défi cits héréditaires en complément. A propos d’un cas par déficit complet en C4. Annls Derm. Syph. 101: 479-496 (1974). 6 H auptmann, G.; T ongio , M. M.; G rosse-W ilde , H., and M ayer, S.: Linkage be tween C2 deficiency and the HLA-A10, B18, Dw2/Bfs haplotype in a french family. Immunogenetics 4: 557-565 (1977). 7 H ill , R. S.; C atovsky, D., and L ew is , S. M.: PNH-like red cell abnormality in a patient with lymphoproliférative disease. Br. med. J. 4: 649 (1972). 8 H olden , D . and L ichtman , H.: Paroxysmal nocturnal hemoglobinuria with acute leukemia. Blood 33: 283-286 (1969). 9 J enkins , D. E. and H artmann, R. C.: Paroxysmal nocturnal hemoglobinuria ter minating in acute myeloblastic leukemia. Blood 33: 274-282 (1969). 10 K aufmann, R. W.; Schechter , G. P., and M c F arland, W.: Paroxysmal noctur nal hemoglobinuria terminating in acute granulocytic leukemia. Blood 33: 287-291 (1969). 11 L ogue , G . L.; R osse, W. F., and A dams, J. P.: Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells. J. clin. Invest. 52: 1129-1137 (1973). 12 May, J. E.; R osse, W. F., and F rank, M. M.: Paroxysmal nocturnal hemoglobi nuria. Alternate complement pathway mediated lysis induced by magnesium. New Engl. J. Med. 289: 705-709 (1973). 13 M ayer, M. M.: Complement and complement fixation; in Experimental immunochemistry; 2nd ed., pp. 133-240 (Thomas, Springfield 1961). 14 M euret , G.; Boll , L; K eyserling , D. G ra f und H eissmeyer, H.: M orpholo-
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References
False Positive Acidified Serum Test
79
gische und kinetische Befunde bei einer kongenitalen dyserythropoetischen Anämie. Blut 21: 341-356 (1970). 15 O berung , F.: Preleukemic dyserythropoiesis with deficiency of phosphohexose isomerase and false positive acidified serum test (in preparation). 16 SiRCHiA, G.; F errone, S., and M ercuriali, F.: The action of two sulfhydryl compounds on normal human red cells. Relationship to red cells of paroxysmal nocturnal hemoglobinuria. Blood 25: 502-510 (1965). 17 W atson, D. K.; Barlow , A. M., and H ewer , E. M.: Paroxysmal nocturnal haemoglobinuria-like defect in red cells in chronic lymphatic leukemia. Br. med. J. 2: 1484 (1976).
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Dr. G. H auptmann, Centre de Transfusion Sanguine, 10, rue Spielmann, F-67085 Strasbourg Cédex (France)
‘False Positive’ Acidified Serum Test in a Preleukemic Dyserythropoiesis G. H auptmann, D. Sondag , J. M. L ang and F. O berling ' Institut d’Hématologie, CHU Strasbourg; Centre deTransfusion Sanguine de Strasbourg (Director; Prof. S. M ayer), and Clinique des Maladies du Sang, CHU Strasbourg (Director: Prof.G. M ayer), Strasbourg
Key Words. Acidified serum test • Preleukemia • Dyserythropoiesis Abstract. In a case of preleukemic dyserythropoiesis, in vitro red cell lysis tests showed a positive acidified serum test whose characteristics are described. The posi tive acidified serum test occurred in 10 normal sera, in 1 serum with complete defi ciency of the fourth component of complement and in 1 serum with complete defi ciency of the second component of complement. The test was found negative with 2 hyperlipemic sera. The other in vitro red-cell lysis tests were negative. The results of the in vitro lysis tests were different from the results obtained in paroxysmal nocturnal hemoglobinuria and congenital dyserythropoietic anemias.
Introduction In paroxysmal nocturnal hemoglobinuria (PNH) the red cells are unu sually susceptible to lysis by complement. Lysis of PNH red cells may be obtained in vitro in fresh serum where the complement system is activat ed either through the classical pathway (cold antibody lysis test) or the al ternate pathway (acidified scrum test sensitized with magnesium, inulin test, cobra venom test) or both pathways (acidified serum test sensitized with thrombin, sucrose lysis test) [4,11,12]. The acidified serum test (Ham’s test) is widely used to detect the PNH abnormality and generally considered to be specific for this red cell defect
Received: May 31, 1977; accepted: July 1, 1977.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
1 The technical assistance of Mrs. Y. Blanzat and Mrs. P. C uny is gratefully acknowledged.
74
H auptmann/S ondag/L ang/O berling
[4]. However, this test is also positive in congenital dyserythropoietic ane mia type II or HEMPAS [3] but, in contrast to PNH, HEMPAS red cells undergo lysis in only a portion of normal sera and they do not undergo lysis in the patient’s own acidified serum and the sucrose lysis test re mains negative. A positive acidified serum test has also been observed in 2 cases of congenital dyserythropoietic anemia type I [1, 14] but in 1 case the positivity was obtained in heated sera and due to spherocytosis [1]. A positive acidified serum test and a positive sucrose lysis test occurred also in 2 cases of lymphoproliferative disease [7, 17]. The present report describes the characteristics of a positive acidified serum test in a case of preleukemic dyserythropoiesis.
A 27-year-old man without noticeable antecedents was admitted to the Hemato logical Clinic in January 1975 for an anemia and neutropenia discovered in Septem ber 1974. Physical examination revealed a paleness and no other abnormalities. Laboratory findings showed: hemoglobin llg/lOOml, RBC 3.32 million/mm3, WBC 3,600/mm3; leukocyte differential count: 19% neutrophils, 1% basophils, 6% mono cytes, 73% lymphocytes and 1% neutrophilic metamyelocytes; reticulocytes 33°/00. The peripheral blood showed anisocytosis, anisochromia, Howell-Jolly bodies, Ca bot rings and basophil stippling. Platelet count 210,000/mm3. The bone marrow was markedly hypercellular with 70% erythroblasts and numerous erythroblastic islands. On light microscopy a small population of polychromatic and orthochromatic eryth roblasts showed abnormalities usually observed in congenital dyserythropoietic anemias: bi- or multinuclearity, incomplete cell divisions. Electron microscopy con firmed the light-microscopic findings: there were erythroblasts with two nuclei or bilobulated nuclei, protrusion of nuclear material into the cytoplasm. Some eryth roblasts showed a marked irregularity and ‘spongy’ appearance of the nucleus with wide nuclear pores and ‘invasion’ of the cytoplasm into the nucleus. The char acteristic morphological feature of the cytoplasm was the presence of abnormal cisternae delimitated by a membrane located near the cell surface. Deposits of iron were often found in the enlarged nuclear pores, in the mitochondria and in the zone of adherence of two adjacent erythroblasts [15]. Hemoglobin study revealed an increased level of HbF (6%). Serum iron was 110,Mg/100 ml; total bilirubin 0.7mg/100ml. Iron-kinetic study indicated an hyper active erythropoiesis. The curve of cell incorporation of labelled iron showed a fall at the 4th day indicating an early destruction of some newly formed red cells. How ever, clinical or biological signs of intravascular hemolysis could not be detected. The patient’s group was 0 rhesus CcDee, Kell - with no irregular antibodies. Serum proteins showed a normal electrophoretic pattern. The acidified serum test was positive with the patient’s serum and the serum of several different normal do nors. The characteristics of this positive acidified serum test will be given thereafter.
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
Patient Summary
False Positive Acidified Serum Test
75
There was increased i and I antigen on the patient’s red cells (agglutination titers with known anti-i serum: patient 1/16, adult control 1/2, cord control 1/256; with an anti-I serum: patient 1/2,058, adult control 1/128, cord control 1/32). A study of the red cell enzymes showed an increased level of hexokinase, glyceraldehyde 3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione peroxidase, while the level of phosphohexose isomerase was reduced at 50°/o of the normal level (Prof. P. Boivin ). This defect was not observed in the other family members. The karyotype analysis showed no abnormality. The blood picture remained stable without specific treatment until March 1976. At that time Hb was 10 g/100 ml, WBC 5,000 with 16% neutrophils, 31% mono cytes, 42% lymphocytes, 4% neutrophilic myelocytes and 7% myeloblasts. Platelet count was 101,000/mm3. The anemia, thrombocytopenia and neutropenia persisted and worsened and the myeloblast percentage increased to a level of 53 in the periph eral blood and 28 in the bone marrow in November 1976 where the patient died, 24 months after the discovery of the anemia and 8 months after myeloblasts were first noted in the blood.
Materials and Methods Veronal-buffered saline (VBS) containing 0.00015 m calcium and 0.0005 m mag nesium ions, VBS containing 0.01 m ethylenediaminetetraacetic acid (EDTA), diso dium salt (EDTA-VBS) and Alsevcrs’s solution were prepared according to M ayer [13]. VBS-sucrose buffer (60% isotonic sucrose-40% VBS) was prepared as outlined by L ogue et at. [11]. Blood from the patient and from normal donors was collected in an equal vol ume of Alsever’s solution and stored at 4 °C until used. Normal compatible serum was obtained from donors and stored at -30 °C for a short period of time. Serum from a patient with total genetic deficiency of the fourth component of complement and from a patient with total genetic deficiency of the second component of com plement were used [5, 6]. Anti-I antibody was obtained from a patient with chronic cold agglutinin syndrome. Inulin was purchased from Prolabo (Paris), cobra venom factor was purchased from Cordis Lab. (Miami, Fla.). The acidified serum test sensitized by the addition of magnesium (0.005 m ) was performed according to May et al. [12], The cold antibody test, sucrose test, inulin test and cobra venom test were performed according to L ogue et al. [11]. Optimal concentration of inulin, cobra venom factor and cold antibody were defined in pre liminary tests using AET-treated red cells [16]. Normal cells were not lysed in any of these procedures at the concentrations of reactants used.
The results of the in vitro red cell lysis are given in table I. The acidi fied serum test was markedly positive with the patient’s own serum, with
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
Results and Discussion
76
H auptmann/S ondag/L ang/O berling
Table /. Results of in vitro red cell lysis tests in the patient Test
Lysis, %
Acidified serum Patient’s own serum Normal donor serum 1 2 3 4 5 6 7 8 9 10 11 (hyperlipemic) 12 (hyperlipemic)
47
C4-deficient serum C2-deficient serum
45 43 negative with all sera negative with all sera negative with all sera negative with all sera
the serum of 10 normal donors, with the C4-deficient serum and with the C2-deficient serum. The test was negative with the serum of 2 normal do nors and it was observed that these sera were turbid, i.e. hyperlipemic. The test was negative with all sera heated at 56 °C for 20 min. The other in vitro red cell lysis tests remained negative with all sera. The tests were performed on three occasions (March 1975, April 1975 and March 1976) and gave similar results. The positivity of the acidified serum test with the patient’s own serum and the negativity of the cold antibody test excluded a congenital dyseryth ropoietic anemia type II (HEMPAS). The negativity of the insulin test, sucrose test and cobra venom test excluded an unusual susceptibility of the patient’s red cells to lysis by complement as observed in PNH. De spite the fact that the acidified serum test was found negative after heat ing the sera at 56 °C for 20 min, there is little reason to believe that the complement system was involved in the lysis of the patient’s red cells be
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
Sucrose lysis Cold antibody lysis Inulin Cobra venom
51 55 44 60 52 70 45 45 20 51 0 0
77
cause the lysis was obtained with complement-deficient sera and because all the tests except the acidified serum test based on the activation of the complement system through the classical and/or alternate pathway re mained negative. Therefore, we considered the positive acidified serum test as ‘false positive’. The negativity of the acidified serum test with two hyperlipemic sera suggests that an excess of lipids or lipoproteins protect ed the patient’s red cells against lysis and that the abnormal susceptibility to lysis in the acidified serum was due to an abnormal lipid composition or to an abnormal lipid metabolism in the patient’s red cells. This hypo thesis could not be verified. In congenital dyserythropoietic anemia type I, a positive acidified se rum test was observed in 2 cases [1, 14], In 1 case [1] the lysis was ob tained in 9 of the 10 normal acidified sera and with the patient’s own aci dified serum. However, hemolysis was marked only with 1 of the tested sera and lysis did not seem to depend on the presence of complement since sera heated at 56 °C for 30 min produced the same degree of lysis as unheated fresh serum. Therefore, the positive acidified serum test was also considered to be ‘false positive’. In PNH the evolution toward an acute myeloblastic leukemia is rare. Three observations have been published in 1969 [8-10]. The acute mye loblastic leukemia occurred 3, 7 and 2 years, respectively, after the dis covery of the clinical and biological signs of PNH. The diagnosis of PNH was based on the recognition of episodes of hemoglobinuria, hemosiderinuria, on the positivity of the acidified serum test in the 3 cases and on the positivity of the sucrose test in 2 cases [9, 10], In our observation we have not ascertained clinical or biological signs of PNH and the charac teristics of the positive acidified serum test allowed us to exclude the diag nosis of PNH. C atovsky et al. [2] have performed in vitro red cell lysis tests in sev eral cases of acute myeloblastic leukemia and have found frequently a po sitivity of the cold antibody lysis test (58%) and less frequently a positivi ty of the sucrose lysis test, whereas the acidified serum test was never found positive. In the two cases of lymphoproliferative disease with a ‘PNH-like’ red cell abnormality reported [7, 17], the acidified serum test was positive in one case togehter with a positive sucrose lysis test [17] and positive in the other case together with a positive cold antibody lysis test and with a positive sugar lysis test [7]. Our observation of a preleukemic dyserythropoiesis confirms that a positive acidified serum lysis test is not sufficient to state a ‘PNH defect’
Downloaded by: East Carolina University - Laupus Library 150.216.68.200 - 1/1/2019 6:28:31 PM
False Positive Acidified Serum Test
78
H auptmann/S ondag/L ang/O berling
in the red cells and that several tests are necessary to explore and charac terize the in vitro susceptibility of red cells to lysis in the presence of nor mal sera, complement-deficient sera and also sera with abnormal lipid or lipoprotein composition.
1 C astro, O.; N ash, I., and F inch Stuart, C.: Congenital dyserythropoietic ane mia type I. Report of a case with increased erythrocyte agglutinability by anti-i serum. Archs intern. Med. 134: 346-351 (1974). 2 C atovsky, D.; L ew is , S. M., and Sherman, D.: Erythrocyte sensitivity to in vi tro lysis in leukemia. Br. J. Haemat. 21: 541-550 (1971). 3 Crookston , J. H.; Crookston , M. C.; Burnie , K. L.; F rancome, W. H.; D acie, J. V.; D avis, A., and L ew is , S. M.: Hereditary erythroblastic multinuclearity as sociated with a positive acidified serum test: a type of congenital dyserythro poietic anemia. Br. J. Haemat. 17: 11-26 (1969). 4 D acie , J. V. and L ew is , S. M.: Laboratory methods used in investigation of the haemolytic anaemias. V. Paroxysmal nocturnal haemoglobinuria (PNH); in Practical haematology; 5th ed., pp. 305-313 (Churchill/Livingstone, London/ Edinburgh 1975). 5 H auptmann, G.; G rosshans, E. et H eid , E.: Lupus érythémateux aigus et défi cits héréditaires en complément. A propos d’un cas par déficit complet en C4. Annls Derm. Syph. 101: 479-496 (1974). 6 H auptmann, G.; T ongio , M. M.; G rosse-W ilde , H., and M ayer, S.: Linkage be tween C2 deficiency and the HLA-A10, B18, Dw2/Bfs haplotype in a french family. Immunogenetics 4: 557-565 (1977). 7 H ill , R. S.; C atovsky, D., and L ew is , S. M.: PNH-like red cell abnormality in a patient with lymphoproliférative disease. Br. med. J. 4: 649 (1972). 8 H olden , D . and L ichtman , H.: Paroxysmal nocturnal hemoglobinuria with acute leukemia. Blood 33: 283-286 (1969). 9 J enkins , D. E. and H artmann, R. C.: Paroxysmal nocturnal hemoglobinuria ter minating in acute myeloblastic leukemia. Blood 33: 274-282 (1969). 10 K aufmann, R. W.; Schechter , G. P., and M c F arland, W.: Paroxysmal noctur nal hemoglobinuria terminating in acute granulocytic leukemia. Blood 33: 287-291 (1969). 11 L ogue , G . L.; R osse, W. F., and A dams, J. P.: Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells. J. clin. Invest. 52: 1129-1137 (1973). 12 May, J. E.; R osse, W. F., and F rank, M. M.: Paroxysmal nocturnal hemoglobi nuria. Alternate complement pathway mediated lysis induced by magnesium. New Engl. J. Med. 289: 705-709 (1973). 13 M ayer, M. M.: Complement and complement fixation; in Experimental immunochemistry; 2nd ed., pp. 133-240 (Thomas, Springfield 1961). 14 M euret , G.; Boll , L; K eyserling , D. G ra f und H eissmeyer, H.: M orpholo-
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References
False Positive Acidified Serum Test
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gische und kinetische Befunde bei einer kongenitalen dyserythropoetischen Anämie. Blut 21: 341-356 (1970). 15 O berung , F.: Preleukemic dyserythropoiesis with deficiency of phosphohexose isomerase and false positive acidified serum test (in preparation). 16 SiRCHiA, G.; F errone, S., and M ercuriali, F.: The action of two sulfhydryl compounds on normal human red cells. Relationship to red cells of paroxysmal nocturnal hemoglobinuria. Blood 25: 502-510 (1965). 17 W atson, D. K.; Barlow , A. M., and H ewer , E. M.: Paroxysmal nocturnal haemoglobinuria-like defect in red cells in chronic lymphatic leukemia. Br. med. J. 2: 1484 (1976).
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Dr. G. H auptmann, Centre de Transfusion Sanguine, 10, rue Spielmann, F-67085 Strasbourg Cédex (France)
