- Email: [email protected]
FALSE-POSITIVE RESULTS IN ANTENATAL DIAGNOSIS OF NEURAL-TUBE DISORDERS
345
advantages of most commercially available R.P.H.A. tests. However, a major problem in all R.P.H.A. tests, as emphasised in yourleader,18is the detection of false-positive reactions. False positivity is usually lower than 1%,16, 19, 20 but figures as high as 18% have also been reported." We have evaluated an R.P.H.A. test for HBAg using sheep erythrocytes coated with sheep HBAb (’ Hepanosticon ’, Organon), performing it in parallel with counterelectrophoresis (C.E.P.). All sera found positive by c.E.P. were strongly positive by R.P.H.A., in titres usually higher than 1 in 64. False-positive reactions rarely had a titre higher than 1 in 8, and they could be easily neutralised by a hightitre human HBAb or absorbed by sheep erythrocytes coated with normal sheep y-globulin. The frequency of false-positive reactions was 0.6% among 7295 blood-donors and other healthy controls and 27% among 2641 patients with miscellaneous hepatic and other diseases.19 Although these frequencies may seem rather high, a much higher frequency of true HBAg-positive reactions not detectable by c.E.P. was discovered by R.P.H.A.
ing procedures have had any influence on the incidence of post-transfusion hepatitis. 24-26 Since only relatively few hepatitis infections can be related to transfusion blood, epidemiological efforts to reduce the incidence of viral hepatitis should be concentrated on other sources of infection. This is especially the case in the hospital environment, where possibilities of infection
are
many among transfused
the
at
same
time that,
at
least with the reagents used in
this study,
false-positive reactions are not very common and can be clearly recognised by relatively simple specificity controls. Second Department of Medicine,
Hippokration Hospital, Athens 610, Greece.
S.
J.
as
non-
population. A. ARNDT-HANSER , R. ANANTHAKRISHNAN Blood-transfusion Centre and
Department of Internal Medicine, University Clinic, Mainz, Germany,
Landesuntersuchungsamt, Münster, Germany. Department of Internal Medicine, St. Elisabeth County Hospital and Blood-bank, Bispebjerg Hospital,
respectively). These findings confirm the value of HBAg screening by reverse hsmagglutination in several situations, indicating
well
Compulsory screening of donors should not be introduced unless there is strong evidence that the number of units of infectious blood can be reduced.23 Even the best donorscreening procedure could not be expected to reduce significantly the incidence of viral hepatitis in the general-
Red Cross Blood-transfusion Centre and Department of Virology,
Thus, in our series the ratio of true/false-positive reactions was 11/1, whereas in other countries, with a low overall prevalence of HBAg, this ratio has been found as low as 1/18.15, 20 We have found screening by R.P.H.A. very valuable in liver patients, especially those with low serum-HBAg titres. For example, in acute type-B hepatitis in the recovery phase, HBAg could be still clearly detected by R.P.H.A. in many sera (21/39 or 53%) found already negative by C.E.P. Similarly, the frequency of HBAg discovered by R.P.H.A. in cirrhosis (111/256 or 43.3%) and liver-cell carcinoma (51/86 or 593%) was much higher than that detected by C.E.P. (90/256 or 35-2% and 39/86 or 453%,
as
transfused persons.
Blood-transfusion Centre,
Vicenza, Italy. Red Cross Blood-transfusion Centre,
Leuven, Belgium.
K. EWE K.-H. HOLTERMÜLLER A. KÖHLER.
H. FIEDLER G. MAASS.
V. REINICKE E. DYBKJÆR. G. ROSSI. C. VERMYLEN.
FALSE-POSITIVE RESULTS IN ANTENATAL DIAGNOSIS OF NEURAL-TUBE DISORDERS
SIR,-Recent communications 27 have demonstrated the value of amniotic-fluid &agr;1-fetoprotein (A.F.P.) levels in the early antenatal diagnosis of open neural-tube anomalies. In interpreting the results obtained from such an investigation, it must be remembered that, whilst the A.F.P. level in the amniotic fluid is in the µg per ml. range, that in fetal blood is in the mg. per ml. range, and that in the maternal blood in the ng. per ml. range. These widely divergent levels in three body-fluids in such close physical proximity may, on occasions, lead to significant contamination and false assay results as seen in two recent cases. The two cases, shown as open triangles in fig. 1, gave
HADZIYANNIS. 24. 25.
PREVENTION OF POST-TRANSFUSION HEPATITIS
Arndt-Hanser, A., Pyka, R. Vox sang. 1973, suppl. 24, 72. Reinicke, V., Dybkjær, E., Poulsen, H., Banke, O., Lylloff, K., Nordenfeldt, E. New Engl. J. Med. 1972, 286, 867. 26. Reinicke, V., Banke, O., Dybkjær, E. Vox sang. 1973, suppl. 24, 65. 27. Lancet, 1974, i, 907.
SIR There is no conclusive evidence to support the assumption that transfusion blood plays an important part in the general spreading of viral hepatitis.21-23 Consequently, the entire range of screening procedures to prevent post-transfusion hepatitis (exclusion of potential donors with a previous history of hepatitis, liver-function tests, Australia-antigen determinations with different techniques, and so on) have not led to a clear-cut reduction in the general spread of viral hepatitis. It is even disputed whether screenLancet, 1974, i, 1202. 19. Hadziyannis, S., Karamanos, B. Iatriki, 1973, 24, 482. 20. Schuurs, A., Kacaki, J. 9th int. Congr. trop. Med. Malaria, Athens, 1973. 21. Fiedler, H. Münch. med. Wschr. 1972, 114, 549. 22. Reinicke, V. Scand. J. infect. Dis. (in the press). 23. Arndt-Hanser, A., Fiedler, H. Biotest Mitt. 1973, 32, 15, 110. 18.
Fig. 1—Amniotic-fluid A.F.P. levels in
haemorrhage.
two cases
(•)
of fetal
346 A.F.P.
levels of 45 and 41 µg. per ml.
at
15 and 16 weeks’
gestation respectively. The solid line represents the upper normal limit (mean+2 log S.D.) for A.F.P. in amniotic fluid for the relevant period of gestation. The second patient underwent a repeat amniocentesis at 18 weeks’ gestation, when the A.F.P. level was shown to be 15 µg. per ml. The initial amniotic-fluid sample in both cases was bloodstained ; examination of the amniotic fluid and maternal blood for fetal haemoglobin was positive in both cases, indicating fetal haemorrhage into the amniotic fluid and a significant fetomaternal transfusion. Using the tables for amniotic fluid volume 28 together with the mean levels of A.F.P. in amniotic fluid and fetal blood, it is possible to calculate the volume of fetal blood necessary to produce any given degree of elevation of the amniotic-fluid A.F.P. Fig. 2 shows that a fetal bleed of only 1 ml. will increase the A.F.P. above the norm at 14 weeks’ gestation, whilst fetal bleeds of 2-5 ml. and 5 ml. are required at 15--and 17 weeks respectively to give similar elevations. These figures do assume, however, total mixing of fetal blood in amniotic fluid-a situation which is unlikely to occur in practice. With incomplete mixing the volumes of fetal bleed would be considerably smaller.
RAPID DIAGNOSIS OF DOWN’S SYNDROME
SIR,-We read with interest the letter by Dr Smithies and Dr Valman (May 25, p. 1056) in which they report a technique for rapid chromosome analysis by short-term culture of bone-marrow cells in one newborn baby with Down’s syndrome. We have been utilising a similar for technique, especially quick determination of genetic sex in newborn babies with ambiguous external genitalia and for rapid confirmation or denial of the clinical diagnosis of 13 or 18 trisomy syndromes. Chromosome results have been available within two to five hours and the findings have assisted in an early decision as to sex of rearing in the former cases and have allowed for early counselling toward a lack of medical intervention in the latter disorders. With our technique bone-marrow is aspirated into a heparinised syringe from the medial aspect of the proximal tibia. The aspirate is placed immediately into culturemedium and rushed to the laboratory. The cells are then treated with prewarmed (37 °C) 0075M hypotonic potassium chloride for 20 to 25 minutes. Thereafter the cells are fixed and processed for chromosomal studies in the same manner as with a standard cultured peripheral-blood-
leucocyte preparation. Peripheral-blood-leucocyte cultures are set up concurrently in order to ensure a complete chromosome study, lest the marrow sample is not adequate. Rapid chromosome results were obtained in seven of nine cases studied, the two failures being due to an inadequate sample of marrow cells. We completely agree with Dr Smithies on the value of this simple technique for rapid chromosome diagnosis in the newborn baby. Department of Pediatrics, Dysmorphology Unit, School of Medicine, University of Washington Seattle, Washington 98195, U.S.A.
BRADLEY T. GONG KENNETH L. JONES DAVID W. SMITH.
TRANSCERVICAL FETOSCOPY
SIR,-The experience of Dr Lawrence and his colleagues (June 1, p. 1120) using transabdominal fetoscopy to
Fig. 2-Volumes (in ml.) of fetal bleed mean
amniotic-fluid A.F.P. level
necessary to increase the the normal range
(•) above
(mean + 2 log S.D.).
From these two cases, and the theoretical evidence, we suggest that amniotic-fluid samples should be screened for the presence of fetal erythrocytes by the Kleihauer technique. In this way one may become aware of possible contamination of the sample, and one cause of false-positive results in this very useful investigation can be eliminated. Protein Reference Unit, Department of Immunology, Hallamshire Hospital,
Sheffield.
A. MILFORD WARD.
University Department of Obstetrics and Gynæcology,
Jessop’s Hospital, Sheffield. 28.
C. R. STEWART.
Emery, A. E. H. Modem Trends in Human Genetics, vol. I, p. 267. London, 1970.
demonstrate fetal abnormality during the second trimester of pregnancy is of considerable interest and illustrates some of the problems involved in this diagnostic procedure. My personal experience of fetoscopy using a 5 mm. flexible-tip bronchofiberscope (Olympus BF type 5B) and a cold light source (Olympus CLE-3) has been obtained, passing the endoscope transcervically into the extraamniotic space, in 28 patients admitted for termination of pregnancy between 8 and 20 weeks’ gestation. In each instance using aseptic precautions, the endoscope was passed without anaathesia or premedication and with minimal discomfort to the patients; no cervical dilatation was necessary. Intermittent injections of small volumes of physiological saline solution through the operating limb of the bronchoscope were necessary to keep the distal lens clear. The view of the fetus obtained through the intact fetal membranes varied. Though definition was usually good (20 cases) difficulty was encountered in being sure which fetal parts were seen, because of marked fetal movement; on no occasion were the facial features seen with certainty. Such activity was not commented on by Dr Lawrence, perhaps because the fetus was sedated by maternal medication in his cases. In the present series, traumatic rupture of the membranes did not occur but in 2 cases bleeding through the cervix was observed after the instrument was withdrawn. The transcervical extra-amniotic technique has the advantage of obviating the need for a general anæsthetic while it also avoids penetration of the fetal membranes;
advantages of most commercially available R.P.H.A. tests. However, a major problem in all R.P.H.A. tests, as emphasised in yourleader,18is the detection of false-positive reactions. False positivity is usually lower than 1%,16, 19, 20 but figures as high as 18% have also been reported." We have evaluated an R.P.H.A. test for HBAg using sheep erythrocytes coated with sheep HBAb (’ Hepanosticon ’, Organon), performing it in parallel with counterelectrophoresis (C.E.P.). All sera found positive by c.E.P. were strongly positive by R.P.H.A., in titres usually higher than 1 in 64. False-positive reactions rarely had a titre higher than 1 in 8, and they could be easily neutralised by a hightitre human HBAb or absorbed by sheep erythrocytes coated with normal sheep y-globulin. The frequency of false-positive reactions was 0.6% among 7295 blood-donors and other healthy controls and 27% among 2641 patients with miscellaneous hepatic and other diseases.19 Although these frequencies may seem rather high, a much higher frequency of true HBAg-positive reactions not detectable by c.E.P. was discovered by R.P.H.A.
ing procedures have had any influence on the incidence of post-transfusion hepatitis. 24-26 Since only relatively few hepatitis infections can be related to transfusion blood, epidemiological efforts to reduce the incidence of viral hepatitis should be concentrated on other sources of infection. This is especially the case in the hospital environment, where possibilities of infection
are
many among transfused
the
at
same
time that,
at
least with the reagents used in
this study,
false-positive reactions are not very common and can be clearly recognised by relatively simple specificity controls. Second Department of Medicine,
Hippokration Hospital, Athens 610, Greece.
S.
J.
as
non-
population. A. ARNDT-HANSER , R. ANANTHAKRISHNAN Blood-transfusion Centre and
Department of Internal Medicine, University Clinic, Mainz, Germany,
Landesuntersuchungsamt, Münster, Germany. Department of Internal Medicine, St. Elisabeth County Hospital and Blood-bank, Bispebjerg Hospital,
respectively). These findings confirm the value of HBAg screening by reverse hsmagglutination in several situations, indicating
well
Compulsory screening of donors should not be introduced unless there is strong evidence that the number of units of infectious blood can be reduced.23 Even the best donorscreening procedure could not be expected to reduce significantly the incidence of viral hepatitis in the general-
Red Cross Blood-transfusion Centre and Department of Virology,
Thus, in our series the ratio of true/false-positive reactions was 11/1, whereas in other countries, with a low overall prevalence of HBAg, this ratio has been found as low as 1/18.15, 20 We have found screening by R.P.H.A. very valuable in liver patients, especially those with low serum-HBAg titres. For example, in acute type-B hepatitis in the recovery phase, HBAg could be still clearly detected by R.P.H.A. in many sera (21/39 or 53%) found already negative by C.E.P. Similarly, the frequency of HBAg discovered by R.P.H.A. in cirrhosis (111/256 or 43.3%) and liver-cell carcinoma (51/86 or 593%) was much higher than that detected by C.E.P. (90/256 or 35-2% and 39/86 or 453%,
as
transfused persons.
Blood-transfusion Centre,
Vicenza, Italy. Red Cross Blood-transfusion Centre,
Leuven, Belgium.
K. EWE K.-H. HOLTERMÜLLER A. KÖHLER.
H. FIEDLER G. MAASS.
V. REINICKE E. DYBKJÆR. G. ROSSI. C. VERMYLEN.
FALSE-POSITIVE RESULTS IN ANTENATAL DIAGNOSIS OF NEURAL-TUBE DISORDERS
SIR,-Recent communications 27 have demonstrated the value of amniotic-fluid &agr;1-fetoprotein (A.F.P.) levels in the early antenatal diagnosis of open neural-tube anomalies. In interpreting the results obtained from such an investigation, it must be remembered that, whilst the A.F.P. level in the amniotic fluid is in the µg per ml. range, that in fetal blood is in the mg. per ml. range, and that in the maternal blood in the ng. per ml. range. These widely divergent levels in three body-fluids in such close physical proximity may, on occasions, lead to significant contamination and false assay results as seen in two recent cases. The two cases, shown as open triangles in fig. 1, gave
HADZIYANNIS. 24. 25.
PREVENTION OF POST-TRANSFUSION HEPATITIS
Arndt-Hanser, A., Pyka, R. Vox sang. 1973, suppl. 24, 72. Reinicke, V., Dybkjær, E., Poulsen, H., Banke, O., Lylloff, K., Nordenfeldt, E. New Engl. J. Med. 1972, 286, 867. 26. Reinicke, V., Banke, O., Dybkjær, E. Vox sang. 1973, suppl. 24, 65. 27. Lancet, 1974, i, 907.
SIR There is no conclusive evidence to support the assumption that transfusion blood plays an important part in the general spreading of viral hepatitis.21-23 Consequently, the entire range of screening procedures to prevent post-transfusion hepatitis (exclusion of potential donors with a previous history of hepatitis, liver-function tests, Australia-antigen determinations with different techniques, and so on) have not led to a clear-cut reduction in the general spread of viral hepatitis. It is even disputed whether screenLancet, 1974, i, 1202. 19. Hadziyannis, S., Karamanos, B. Iatriki, 1973, 24, 482. 20. Schuurs, A., Kacaki, J. 9th int. Congr. trop. Med. Malaria, Athens, 1973. 21. Fiedler, H. Münch. med. Wschr. 1972, 114, 549. 22. Reinicke, V. Scand. J. infect. Dis. (in the press). 23. Arndt-Hanser, A., Fiedler, H. Biotest Mitt. 1973, 32, 15, 110. 18.
Fig. 1—Amniotic-fluid A.F.P. levels in
haemorrhage.
two cases
(•)
of fetal
346 A.F.P.
levels of 45 and 41 µg. per ml.
at
15 and 16 weeks’
gestation respectively. The solid line represents the upper normal limit (mean+2 log S.D.) for A.F.P. in amniotic fluid for the relevant period of gestation. The second patient underwent a repeat amniocentesis at 18 weeks’ gestation, when the A.F.P. level was shown to be 15 µg. per ml. The initial amniotic-fluid sample in both cases was bloodstained ; examination of the amniotic fluid and maternal blood for fetal haemoglobin was positive in both cases, indicating fetal haemorrhage into the amniotic fluid and a significant fetomaternal transfusion. Using the tables for amniotic fluid volume 28 together with the mean levels of A.F.P. in amniotic fluid and fetal blood, it is possible to calculate the volume of fetal blood necessary to produce any given degree of elevation of the amniotic-fluid A.F.P. Fig. 2 shows that a fetal bleed of only 1 ml. will increase the A.F.P. above the norm at 14 weeks’ gestation, whilst fetal bleeds of 2-5 ml. and 5 ml. are required at 15--and 17 weeks respectively to give similar elevations. These figures do assume, however, total mixing of fetal blood in amniotic fluid-a situation which is unlikely to occur in practice. With incomplete mixing the volumes of fetal bleed would be considerably smaller.
RAPID DIAGNOSIS OF DOWN’S SYNDROME
SIR,-We read with interest the letter by Dr Smithies and Dr Valman (May 25, p. 1056) in which they report a technique for rapid chromosome analysis by short-term culture of bone-marrow cells in one newborn baby with Down’s syndrome. We have been utilising a similar for technique, especially quick determination of genetic sex in newborn babies with ambiguous external genitalia and for rapid confirmation or denial of the clinical diagnosis of 13 or 18 trisomy syndromes. Chromosome results have been available within two to five hours and the findings have assisted in an early decision as to sex of rearing in the former cases and have allowed for early counselling toward a lack of medical intervention in the latter disorders. With our technique bone-marrow is aspirated into a heparinised syringe from the medial aspect of the proximal tibia. The aspirate is placed immediately into culturemedium and rushed to the laboratory. The cells are then treated with prewarmed (37 °C) 0075M hypotonic potassium chloride for 20 to 25 minutes. Thereafter the cells are fixed and processed for chromosomal studies in the same manner as with a standard cultured peripheral-blood-
leucocyte preparation. Peripheral-blood-leucocyte cultures are set up concurrently in order to ensure a complete chromosome study, lest the marrow sample is not adequate. Rapid chromosome results were obtained in seven of nine cases studied, the two failures being due to an inadequate sample of marrow cells. We completely agree with Dr Smithies on the value of this simple technique for rapid chromosome diagnosis in the newborn baby. Department of Pediatrics, Dysmorphology Unit, School of Medicine, University of Washington Seattle, Washington 98195, U.S.A.
BRADLEY T. GONG KENNETH L. JONES DAVID W. SMITH.
TRANSCERVICAL FETOSCOPY
SIR,-The experience of Dr Lawrence and his colleagues (June 1, p. 1120) using transabdominal fetoscopy to
Fig. 2-Volumes (in ml.) of fetal bleed mean
amniotic-fluid A.F.P. level
necessary to increase the the normal range
(•) above
(mean + 2 log S.D.).
From these two cases, and the theoretical evidence, we suggest that amniotic-fluid samples should be screened for the presence of fetal erythrocytes by the Kleihauer technique. In this way one may become aware of possible contamination of the sample, and one cause of false-positive results in this very useful investigation can be eliminated. Protein Reference Unit, Department of Immunology, Hallamshire Hospital,
Sheffield.
A. MILFORD WARD.
University Department of Obstetrics and Gynæcology,
Jessop’s Hospital, Sheffield. 28.
C. R. STEWART.
Emery, A. E. H. Modem Trends in Human Genetics, vol. I, p. 267. London, 1970.
demonstrate fetal abnormality during the second trimester of pregnancy is of considerable interest and illustrates some of the problems involved in this diagnostic procedure. My personal experience of fetoscopy using a 5 mm. flexible-tip bronchofiberscope (Olympus BF type 5B) and a cold light source (Olympus CLE-3) has been obtained, passing the endoscope transcervically into the extraamniotic space, in 28 patients admitted for termination of pregnancy between 8 and 20 weeks’ gestation. In each instance using aseptic precautions, the endoscope was passed without anaathesia or premedication and with minimal discomfort to the patients; no cervical dilatation was necessary. Intermittent injections of small volumes of physiological saline solution through the operating limb of the bronchoscope were necessary to keep the distal lens clear. The view of the fetus obtained through the intact fetal membranes varied. Though definition was usually good (20 cases) difficulty was encountered in being sure which fetal parts were seen, because of marked fetal movement; on no occasion were the facial features seen with certainty. Such activity was not commented on by Dr Lawrence, perhaps because the fetus was sedated by maternal medication in his cases. In the present series, traumatic rupture of the membranes did not occur but in 2 cases bleeding through the cervix was observed after the instrument was withdrawn. The transcervical extra-amniotic technique has the advantage of obviating the need for a general anæsthetic while it also avoids penetration of the fetal membranes;