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RAPIDLY ADHERING AMNIOTIC-FLUID CELLS AND PRENATAL DIAGNOSIS OF NEURAL-TUBE DEFECTS
99
RAPIDLY ADHERING AMNIOTIC-FLUID CELLS AND PRENATAL DIAGNOSIS OF NEURAL-TUBE DEFECTS our first observations of rapidly cells in cultures of amniotic fluid from two pregnancies at risk for neural-tube defects (NTD). Both samples contained normal levels of a-fetoprotein (AFP). One fetus (case 1) was morphologically normal; the other fetus (case 2) had a large skin-covered occipital encephalocele. Case 1.-A 30-year-old woman was seen for prenatal diagnosis in her third pregnancy. She had had one baby with a large, open meningomyelocele and a neurologically normal girl with a unilateral cleft lip and palate. Transabdominal amniocentesis with placental localisation by sonography was unsuccessul at 15 weeks’ gestation. At 16 weeks a second attempt produced grossly bloody amniotic fluid; the blood was 98% fetal in origin. A sample of amniotic fluid was sent to Dr Aubrey Milunsky at the E. K. Shriver Center for AFP assay. The remainder of the sample was placed in culture. 10 days after the flasks were set up, cells of types that had not been observed in more than 300 previous amniotic-fluid cultures was noted. These included long bipolar cells, cells with vacuolated cytoplasm, multinucleated cells, and cells with multiple cytoplasmic processes, all of which resembled the rapidly adhering cells described by Gosden and Brock1.2 in the amniotic fluid of fetuses with open NTD. Uncertainty over the origin of the cells and their significance was heightened when the AFP level was reported to be 1.62 mg/dl, a normal value for gestational age. The patient and her husband were advised of this finding and the possible significance of such cells, and a decision was made to repeat the amniocentesis. 20 ml of darkly stained amniotic fluid was obtained without difficulty at 18 weeks’ gestation; no fresh blood was noted in the sample. A second culture was established and examined at 20 h. Large numbers of adherent cells were observed with morphological characteristics similar to those in the first culture. The AFP assay was again normal for gestational age (0.66 mg/dl). The parents elected to terminate the pregnancy at 20 weeks’ gestation. The 590 g fetus had no internal or external abnormalities, and no puncture marks, ecchymoses, or hxmatomas on skin or placenta were noted. Case 2.-A 27-year-old woman was seen for prenatal diagnosis in her second pregnancy. Her first pregnancy had resulted in the birth of an anencephalic male infant. Sonography was done at 15 weeks’ gestation with good visualisation of the fetal head. After a second normal sonographic examination at 16 weeks, the parents decided not to proceed with amniocentesis, but agreed to continued monitoring by sonography. At 19 weeks, sonography revealed lack of fetal head growth and a cystic structure in the lower occipital area. The parents agreed to further diagnostic studies. Amniocentesis was done and diatrizoate sodium was injected into the amniotic cavity. A large occipital mass was visualised radiographically. The parents elected to terminate the pregnancy at 19-Lweeks. The 320 g female fetus had a congested, soft occipital encephalocele 3.5cm in diameter, completely covered by intact skin. The left occipital pole and what seemed to be cerebellar tissue, were contained within the encephalocele. Amniotic fluid removed during amniography was placed in culture and examined at 20 h. Many adherent cells similar in morphology to those seen in case 1 were noted. AFP was normal for gestational age (0-66
SIR,-We record here
adhering
mg/dl). The observation
by Sutherland
et
al. 3.4 of large numbers of
DJH, Gosden C. Early antenatal diagnosis of small open spina bifida lesions. Br Med J 1977; ii: 934. 2. Gosden C, Brock DJH. Morphology of rapidly adhering amniotic-fluid cells as an aid to the diagnosis of neural-tube defects. Lancet 1977; i: 919-22. 3. Sutherland GR, Brock DJH, Scrimgeour JB. Amniotic fluid macrophages and anencephaly. Lancet 1973; ii: 1098. 4. Sutherland GR, Brock DJH, Scrimgeour JB. Amniotic fluid macrophages and the antenatal diagnosis of anencephaly and spina bifida. J Med Genet 1. Brock
1975; 12: 135-7.
macrophage-like cells in amniotic-fluid cultures from anencephalic fetuses has led to efforts to quantitate and characterise such cells as an adjunct to sonography and AFP assay in the prenatal diagnosis of open NTD. The large number of unusual cells in the original culture of amniotic fluid in case 1 was unprecedented in our experience. Although not quantitated, the large numbers of similar cells observed at 20 h of culture in the second sample of fluid indicated that these were indeed "rapidly adhering cells".’ Contamination of the fluid by fetal blood suggested a possible source of the cells, but their morphology was unlike that of placental cells2.5.6 and strongly resembled that of cells from fetuses with open NTD. 5-7 The observation of morphologically identical cells at 20 h of culture in case 2 in which an occipital encephalocele was demonstrated suggested that the cells were neural in origin in both cultures. The termination of pregnancy in case 1 was based on parental anxiety because we were unable to rule out an NTD. An additional concern was the possibility that the source of both the fetal blood and the rapidly adhering cells might have been trauma to the fetal brain or spinal cord during amniocentesis. No evidence was found at necropsy to support this explanation. The pregnancy in case 2 was terminated before the normal AFP was known. The intact skin covering the encephalocele explains the normal AFP in the amniotic fluid but obscures the origin of the rapidly adhering cells, which to our knowledge have not previously been reported with closed lesions. Our experience both confirms the value of rapidly adhering cells as an adjunct to other techniques for diagnosis of NTD and indicates that the presence of such cells must be interpreted with caution. Cecil H. and Ida Green Center for Reproductive Biology Sciences and Department of Obstetrics and Gynecology, University of Texas Health Science Center, Dallas, Texas 75235, U.S.A.
Department of Pathology, University of Texas Health Science Center
M. J. E. HARROD J. M. FRIEDMAN
J. JIMENEZ R. SANTOS-RAMOS J. B. BYRNE V.G. DEV A. G. WEINBERG
RUBELLA ANTIBODY SCREENING
SIR,-In the face of growing demands for rubella antibody I have tested a commercial kit (’Rubascreen’; Northumbria Biologicals Ltd) which utilises the established technique of single radial haemolysis.
screening,
The principle is simple. Red blood-cells are coated with rubella-specific antigen and suspended in a mixture of agarose and complement which is poured into a petri dish. The kit provides two plates for each set of sera-a test plate containing antigen and a control plate without antigen. Both plates are ready punched with wells, allowing 25 sera to be tested simultaneously. 5 pi of serum (inactivated at 56OC) is placed in a well of the test and control plates, which are then incubated overnight in a wet chamber at 37°C. Antibody diffuses out into the medium and reacts with the antigen, and a clear zone of haemolysis forms around the well. The control well should show little or no
haemolysis. 389 sera were tested in parallel by single radial haemolysis and hsemaggtutination inhibition (HAI)8with day-old chick cells and serum absorption by 25% kaolin. Rubella HA antigen and commercial controls were supplied by Wellcome Reagents Ltd. With dilutions of both a commercial control and a laboratory pooled 5. Gosden C, Brock DJM. Combined use of alpha fetoprotein and amniotic fluid cell morphology in early prenatal diagnosis of fetal abnormalities. J Med Genet 1978; 15: 262-70. 6. Gosden C, Brock DJH, Eason P. The origin of the rapidly adhering cells found in the amniotic fluids from foetuses with neural tube defects. Clin
Genet 1977; 12: 193-201.
Voigtlander T, Muller U, Schroeder TM. Rapidly adhering cells in two cases of anencephaly. Hum Genet 1978; 45: 107-10. 8. Stewart GL, Parkman PD, Hopps HE, Douglas RD, Hamilton JP, Meyer HM Jr. Rubella-virus hæmagglutination-inhibition test. N Engl J Med
7.
1967; 276: 554-7.
RAPIDLY ADHERING AMNIOTIC-FLUID CELLS AND PRENATAL DIAGNOSIS OF NEURAL-TUBE DEFECTS our first observations of rapidly cells in cultures of amniotic fluid from two pregnancies at risk for neural-tube defects (NTD). Both samples contained normal levels of a-fetoprotein (AFP). One fetus (case 1) was morphologically normal; the other fetus (case 2) had a large skin-covered occipital encephalocele. Case 1.-A 30-year-old woman was seen for prenatal diagnosis in her third pregnancy. She had had one baby with a large, open meningomyelocele and a neurologically normal girl with a unilateral cleft lip and palate. Transabdominal amniocentesis with placental localisation by sonography was unsuccessul at 15 weeks’ gestation. At 16 weeks a second attempt produced grossly bloody amniotic fluid; the blood was 98% fetal in origin. A sample of amniotic fluid was sent to Dr Aubrey Milunsky at the E. K. Shriver Center for AFP assay. The remainder of the sample was placed in culture. 10 days after the flasks were set up, cells of types that had not been observed in more than 300 previous amniotic-fluid cultures was noted. These included long bipolar cells, cells with vacuolated cytoplasm, multinucleated cells, and cells with multiple cytoplasmic processes, all of which resembled the rapidly adhering cells described by Gosden and Brock1.2 in the amniotic fluid of fetuses with open NTD. Uncertainty over the origin of the cells and their significance was heightened when the AFP level was reported to be 1.62 mg/dl, a normal value for gestational age. The patient and her husband were advised of this finding and the possible significance of such cells, and a decision was made to repeat the amniocentesis. 20 ml of darkly stained amniotic fluid was obtained without difficulty at 18 weeks’ gestation; no fresh blood was noted in the sample. A second culture was established and examined at 20 h. Large numbers of adherent cells were observed with morphological characteristics similar to those in the first culture. The AFP assay was again normal for gestational age (0.66 mg/dl). The parents elected to terminate the pregnancy at 20 weeks’ gestation. The 590 g fetus had no internal or external abnormalities, and no puncture marks, ecchymoses, or hxmatomas on skin or placenta were noted. Case 2.-A 27-year-old woman was seen for prenatal diagnosis in her second pregnancy. Her first pregnancy had resulted in the birth of an anencephalic male infant. Sonography was done at 15 weeks’ gestation with good visualisation of the fetal head. After a second normal sonographic examination at 16 weeks, the parents decided not to proceed with amniocentesis, but agreed to continued monitoring by sonography. At 19 weeks, sonography revealed lack of fetal head growth and a cystic structure in the lower occipital area. The parents agreed to further diagnostic studies. Amniocentesis was done and diatrizoate sodium was injected into the amniotic cavity. A large occipital mass was visualised radiographically. The parents elected to terminate the pregnancy at 19-Lweeks. The 320 g female fetus had a congested, soft occipital encephalocele 3.5cm in diameter, completely covered by intact skin. The left occipital pole and what seemed to be cerebellar tissue, were contained within the encephalocele. Amniotic fluid removed during amniography was placed in culture and examined at 20 h. Many adherent cells similar in morphology to those seen in case 1 were noted. AFP was normal for gestational age (0-66
SIR,-We record here
adhering
mg/dl). The observation
by Sutherland
et
al. 3.4 of large numbers of
DJH, Gosden C. Early antenatal diagnosis of small open spina bifida lesions. Br Med J 1977; ii: 934. 2. Gosden C, Brock DJH. Morphology of rapidly adhering amniotic-fluid cells as an aid to the diagnosis of neural-tube defects. Lancet 1977; i: 919-22. 3. Sutherland GR, Brock DJH, Scrimgeour JB. Amniotic fluid macrophages and anencephaly. Lancet 1973; ii: 1098. 4. Sutherland GR, Brock DJH, Scrimgeour JB. Amniotic fluid macrophages and the antenatal diagnosis of anencephaly and spina bifida. J Med Genet 1. Brock
1975; 12: 135-7.
macrophage-like cells in amniotic-fluid cultures from anencephalic fetuses has led to efforts to quantitate and characterise such cells as an adjunct to sonography and AFP assay in the prenatal diagnosis of open NTD. The large number of unusual cells in the original culture of amniotic fluid in case 1 was unprecedented in our experience. Although not quantitated, the large numbers of similar cells observed at 20 h of culture in the second sample of fluid indicated that these were indeed "rapidly adhering cells".’ Contamination of the fluid by fetal blood suggested a possible source of the cells, but their morphology was unlike that of placental cells2.5.6 and strongly resembled that of cells from fetuses with open NTD. 5-7 The observation of morphologically identical cells at 20 h of culture in case 2 in which an occipital encephalocele was demonstrated suggested that the cells were neural in origin in both cultures. The termination of pregnancy in case 1 was based on parental anxiety because we were unable to rule out an NTD. An additional concern was the possibility that the source of both the fetal blood and the rapidly adhering cells might have been trauma to the fetal brain or spinal cord during amniocentesis. No evidence was found at necropsy to support this explanation. The pregnancy in case 2 was terminated before the normal AFP was known. The intact skin covering the encephalocele explains the normal AFP in the amniotic fluid but obscures the origin of the rapidly adhering cells, which to our knowledge have not previously been reported with closed lesions. Our experience both confirms the value of rapidly adhering cells as an adjunct to other techniques for diagnosis of NTD and indicates that the presence of such cells must be interpreted with caution. Cecil H. and Ida Green Center for Reproductive Biology Sciences and Department of Obstetrics and Gynecology, University of Texas Health Science Center, Dallas, Texas 75235, U.S.A.
Department of Pathology, University of Texas Health Science Center
M. J. E. HARROD J. M. FRIEDMAN
J. JIMENEZ R. SANTOS-RAMOS J. B. BYRNE V.G. DEV A. G. WEINBERG
RUBELLA ANTIBODY SCREENING
SIR,-In the face of growing demands for rubella antibody I have tested a commercial kit (’Rubascreen’; Northumbria Biologicals Ltd) which utilises the established technique of single radial haemolysis.
screening,
The principle is simple. Red blood-cells are coated with rubella-specific antigen and suspended in a mixture of agarose and complement which is poured into a petri dish. The kit provides two plates for each set of sera-a test plate containing antigen and a control plate without antigen. Both plates are ready punched with wells, allowing 25 sera to be tested simultaneously. 5 pi of serum (inactivated at 56OC) is placed in a well of the test and control plates, which are then incubated overnight in a wet chamber at 37°C. Antibody diffuses out into the medium and reacts with the antigen, and a clear zone of haemolysis forms around the well. The control well should show little or no
haemolysis. 389 sera were tested in parallel by single radial haemolysis and hsemaggtutination inhibition (HAI)8with day-old chick cells and serum absorption by 25% kaolin. Rubella HA antigen and commercial controls were supplied by Wellcome Reagents Ltd. With dilutions of both a commercial control and a laboratory pooled 5. Gosden C, Brock DJM. Combined use of alpha fetoprotein and amniotic fluid cell morphology in early prenatal diagnosis of fetal abnormalities. J Med Genet 1978; 15: 262-70. 6. Gosden C, Brock DJH, Eason P. The origin of the rapidly adhering cells found in the amniotic fluids from foetuses with neural tube defects. Clin
Genet 1977; 12: 193-201.
Voigtlander T, Muller U, Schroeder TM. Rapidly adhering cells in two cases of anencephaly. Hum Genet 1978; 45: 107-10. 8. Stewart GL, Parkman PD, Hopps HE, Douglas RD, Hamilton JP, Meyer HM Jr. Rubella-virus hæmagglutination-inhibition test. N Engl J Med
7.
1967; 276: 554-7.