Letter to the Editor Vox Sang lY92;63:2Y 1-292
A . G. Hadleya G . D . Poole' I. D . Fraser' a
International Blood Group Reference South Laboratory, Western andRegional Blood Transfusion Centre, Bristol, UK
Serological [l], quantitative [2] and monocyte- and lymphocytebased cellular assays [3,4] of alloantibodies in maternal serum have all been reported to predict the severity of haemolytic disease of the newborn (HDN) with varying degrees of accuracy. In a retrospective comparison of several assays [5], results from the chemiluminescence test (CLT), which measures the metabolic response of human monocytes to red cells sensitized with maternal serum [6], correlated with disease severity significantly better than AutoAnalyzer estimations of anti-D content. We have now confirmed this observation in an ongoing prospective study to evaluate the clinical usefulness of the CLT. HDN is rare at concentrations of anti-D in maternal serum of less than 5 IU/ml [2, 51. Over a 12-month period, therefore, serum samples containing over 5 IU/ml anti-D or IgG alloantibodies of other specificities with titres by indirect antiglobulin test of 64 or greater were collected by the S.W. Regional Blood Transfusion Centre, UK. Anti-D levels were measured by AutoAnalyzer within 3 days of sample collection. Using red cells heterozygous for each antibody specificity, sera were tested using the CLT
Predicting the Severity of Haemolytic Disease of the Newborn: Prospective Evaluation of the ChemiluminescenceTest as described previously [5], generally within 14 days of sample collection. In brief, red cells (5% v/v) were sensitized with an equal volume of serum, and then washed and incubated with mononuclear cells (isolated from blood pooled from 6 donors) and luminol. The CL response of monocytes was monitored for 60 min at 37°C using a luminometer (LKB Wallac). In each assay, CL responses were expressed as a percentage of a positive control (PPC) comprising Rlr cells sensitized with 10,000 IgGl monoclonal anti-D molecules/cell [ 5 ] . Sera were collected from 45 women. Clinical data (transfusion requirements and cord H b levels) were obtained on 29 babies; 5 were 'antigennegative', one died in utero from nonimmunological causes and one pregnancy resulted in twins. Disease severity in the remaining 22 was graded: 0 = unaffected (Hb > 14.0 g/dl); 1= top-up transfusion (Hb < 14.0 g/ dl); 2 = exchange transfusion (Hb < 14.0 g/dl); 3 = intrauterine transfusion (Hb < 12.5 g/dl). The interassay variations of two sera tested in 14 assays over a 12month period were 15.5 and11.5%, respectively. Figure 1 shows that when maternal serum samples were tested during the last 8 weeks of pregnancy,
results from the CLT predicted disease severity with significantly greater accuracy than the AutoAnalyzer. Consistent with retrospective studies [5], a PPC of greater than 20% discriminated all babies with moderate or severe H D N (grades 2 and 3, predictive value = loo%), while the usefulness of AutoAnalyzer estimations was again compromised by the wide range of results from mothers of unaffected babies (predictive value = 64%). H D N due to anti-K and antiFy" was also predicted using the CLT. Although results from the CLT correlated well with disease severity, two main obstacles t o the use of tests of maternal antibody to predict fetal outcome were again identified. High levels of functionally active antibodies were frequently detected in sera from mothers of 'antigen-negative' babies, and antibody levels may increase rapidly towards the end of pregnancy, leading t o severe disease, In summary, the CLT, which is less laborious to perform than monocytebased phagocytosis and cytotoxicity assays, appears to be a useful noninvasive test to complement other procedures for monitoring fetal progress such as amniotic fluid analysis, ultrasonography and fetal blood sampling [7].
Dr. Andrew G. Hadley lnternatioiial Blood Group Reference Laboratory South Western Regional Blood Transfusion Centre Southmead Rd. Bristol ( U K )
0 1992 S. Karger AG. Basel oO42-'xx)7/92/0634-02Y 1 $2.75/0
2oo
1
2oo
AutoAnalyzer
1
Chemiluminescence test
e
0
0
0
I
I
I
1
2
3
Disease severity
Fig. 1. Relationships between results of the AutoAnalyzer (t = 0.47, p < 0.01, Kendal's rank correlation) or CLT (t = 0.64, p
A . G. Hadleya G . D . Poole' I. D . Fraser' a
International Blood Group Reference South Laboratory, Western andRegional Blood Transfusion Centre, Bristol, UK
Serological [l], quantitative [2] and monocyte- and lymphocytebased cellular assays [3,4] of alloantibodies in maternal serum have all been reported to predict the severity of haemolytic disease of the newborn (HDN) with varying degrees of accuracy. In a retrospective comparison of several assays [5], results from the chemiluminescence test (CLT), which measures the metabolic response of human monocytes to red cells sensitized with maternal serum [6], correlated with disease severity significantly better than AutoAnalyzer estimations of anti-D content. We have now confirmed this observation in an ongoing prospective study to evaluate the clinical usefulness of the CLT. HDN is rare at concentrations of anti-D in maternal serum of less than 5 IU/ml [2, 51. Over a 12-month period, therefore, serum samples containing over 5 IU/ml anti-D or IgG alloantibodies of other specificities with titres by indirect antiglobulin test of 64 or greater were collected by the S.W. Regional Blood Transfusion Centre, UK. Anti-D levels were measured by AutoAnalyzer within 3 days of sample collection. Using red cells heterozygous for each antibody specificity, sera were tested using the CLT
Predicting the Severity of Haemolytic Disease of the Newborn: Prospective Evaluation of the ChemiluminescenceTest as described previously [5], generally within 14 days of sample collection. In brief, red cells (5% v/v) were sensitized with an equal volume of serum, and then washed and incubated with mononuclear cells (isolated from blood pooled from 6 donors) and luminol. The CL response of monocytes was monitored for 60 min at 37°C using a luminometer (LKB Wallac). In each assay, CL responses were expressed as a percentage of a positive control (PPC) comprising Rlr cells sensitized with 10,000 IgGl monoclonal anti-D molecules/cell [ 5 ] . Sera were collected from 45 women. Clinical data (transfusion requirements and cord H b levels) were obtained on 29 babies; 5 were 'antigennegative', one died in utero from nonimmunological causes and one pregnancy resulted in twins. Disease severity in the remaining 22 was graded: 0 = unaffected (Hb > 14.0 g/dl); 1= top-up transfusion (Hb < 14.0 g/ dl); 2 = exchange transfusion (Hb < 14.0 g/dl); 3 = intrauterine transfusion (Hb < 12.5 g/dl). The interassay variations of two sera tested in 14 assays over a 12month period were 15.5 and11.5%, respectively. Figure 1 shows that when maternal serum samples were tested during the last 8 weeks of pregnancy,
results from the CLT predicted disease severity with significantly greater accuracy than the AutoAnalyzer. Consistent with retrospective studies [5], a PPC of greater than 20% discriminated all babies with moderate or severe H D N (grades 2 and 3, predictive value = loo%), while the usefulness of AutoAnalyzer estimations was again compromised by the wide range of results from mothers of unaffected babies (predictive value = 64%). H D N due to anti-K and antiFy" was also predicted using the CLT. Although results from the CLT correlated well with disease severity, two main obstacles t o the use of tests of maternal antibody to predict fetal outcome were again identified. High levels of functionally active antibodies were frequently detected in sera from mothers of 'antigen-negative' babies, and antibody levels may increase rapidly towards the end of pregnancy, leading t o severe disease, In summary, the CLT, which is less laborious to perform than monocytebased phagocytosis and cytotoxicity assays, appears to be a useful noninvasive test to complement other procedures for monitoring fetal progress such as amniotic fluid analysis, ultrasonography and fetal blood sampling [7].
Dr. Andrew G. Hadley lnternatioiial Blood Group Reference Laboratory South Western Regional Blood Transfusion Centre Southmead Rd. Bristol ( U K )
0 1992 S. Karger AG. Basel oO42-'xx)7/92/0634-02Y 1 $2.75/0
2oo
1
2oo
AutoAnalyzer
1
Chemiluminescence test
e
0
0
0
I
I
I
1
2
3
Disease severity
Fig. 1. Relationships between results of the AutoAnalyzer (t = 0.47, p < 0.01, Kendal's rank correlation) or CLT (t = 0.64, p
