A comprehensive laboratory-based program for classification of variants of uncertain significance in hereditary cancer genes.

Genetic testing has the potential to guide the prevention and treatment of disease in a variety of settings, and recent technical advances have greatly increased our ability to acquire large amounts of genetic data. The interpretation of this data remains challenging, as the clinical significance of genetic variation detected in the laboratory is not always clear. Although regulatory agencies and professional societies provide some guidance regarding the classification, reporting, and long-term follow-up of variants, few protocols for the implementation of these guidelines have been described. Because the primary aim of clinical testing is to provide results to inform medical management, a variant classification program that offers timely, accurate, confident and cost-effective interpretation of variants should be an integral component of the laboratory process. Here we describe the components of our laboratory's current variant classification program (VCP), based on 20 years of experience and over one million samples tested, using the BRCA1/2 genes as a model. Our VCP has lowered the percentage of tests in which one or more BRCA1/2 variants of uncertain significance (VUSs) are detected to 2.1% in the absence of a pathogenic mutation, demonstrating how the coordinated application of resources toward classification and reclassification significantly impacts the clinical utility of testing.

Consequences for fetus and neonate of maternal red cell allo-immunisation.

AIMS: To study the distribution of clinically important red cell antibodies in pregnancy, and the associated fetal and neonatal morbidity and mortality. METHODS: The case notes of women with clinically important red cell antibodies identified in their serum during pregnancy were reviewed. RESULTS: During a 12 month period 22,264 women were referred for antenatal screening. Clinically important red cell antibodies were detected in 244 (1%). Of these, 100 were anti-D and 144 were non-RhD antibodies. There were three intrauterine deaths, three fetuses required intrauterine transfusion, 10 neonates were transfused, 27 others had phototherapy, and 27 with a positive direct antiglobulin test received no treatment. Early fetal losses occurred in the presence of both high and low levels of anti-D. CONCLUSIONS: Anti-D remains the most common clinically important antibody in pregnancy, and accounts for the greatest fetal and neonatal morbidity and mortality. Of the other antibodies detected, anti-c was associated with most neonatal morbidity. The production of many of the non-D antibodies detected could be avoided by the use of selected red cells when transfusing pre-menopausal women.

The use of pooled red cells and column techniques for routine red cell antibody detection.

The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.

Kleihauer Testing and Flow Cytometry. A Comparative Study for Assessment of Feto-Maternal Haemorrhage.

The Kleihauer test has been used world-wide for many years to quantify feto-maternal haemorhage, and to ensure that an appropriate dose of anti-D Immunoglobulin (anti-D Ig) is administered both ante-natally and postnatally to RhD negative women to prevent Rh alloimmunisation. Although apparently a simple test to perform, recent reports have suggested that unless meticulous attention is paid to both technique and interpretation, the accuracy of the test cannot be guaranteed. It is suggested that it should be replaced with a flow cytometric test for the presence of fetal RhD positive cells which would give more relevant and accurate results. Flow cytometers are not, however, available to all laboratories performing estimations of feto-matemal haemorrhage (FMH). This study was undertaken to assess the comparability of results obtained using a standardised Kleihauer technique with results obtained using a variety of techniques within hospital laboratories and with flow cytometry. A total of 957 samples were analysed, referring hospitals initially performing a routine Kleihauer test and then forwarding the same sample to the Mersey Transfusion Centre where a standardised Kleihauer test and flow cytometric analysis of FMH were performed. Our results showed that there is variation in Kleihauer results, even when the same sample is used, particularly in quantifying an FMH for which additional anti-D Ig may be required. The tendency however, appears to be to over-estimate the size of FMH and administer unnecessary anti-D Ig. Our results suggest that if careful attention is paid to performing a standardised Kleihauer test, then it is of value in estimating the size of FMH, and that flow cytometry may be of additional value for cases in which the Kleihauer result is equivocal or indicates that a large FMH has occurred which requires the administration of additional anti-D Ig.

Red cell antibody screening and identification: a comparison of two column technology methods.

Two commercial column techniques for use in antibody screening and identification procedures were tested in parallel with 1000 random samples sent for ante-natal serological investigation. The DiaMed ID microtyping system uses a sephadex gel contained in microtubes, either neutral or impregnated with anti-human globulin (AHG), for use in two-stage enzyme methods and LISS indirect antiglobulin testing (IAT) respectively. The Ortho Biovue technique consists of a slurry of micro glass spheres which act as the filter to retain haemagglutination reactions within the matrix. Columns containing AHG also possess a macromolecular density barrier to prevent test serum from passing into the column and neutralising the AHG. Both systems offer the advantage of 'no-wash' IAT, which minimises the potential for problems and errors associated with conventional spin-tube techniques. In this comparison of the two column methods, antibody detection rates were found to be similar and the sensitivity of both methods was comparable, although the Biovue technique was prone to exhibit equivocal results, particularly in the IAT.

Evaluation of the ID-gel test for antibody screening and identification.

A gel technique for the detection of red blood cell (rbc) antigen-antibody reactions was evaluated for use in antenatal antibody screening and identification procedures. The evaluation was undertaken on 3,900 random antenatal samples. Results obtained in the gel test system were compared with those obtained from parallel testing using conventional serological methods. The ID gel system detected 148 (3.7%) red cell antibodies, compared with 95 (2.4%) using traditional techniques. The number of non-specific antibodies and false-positive screens were reduced using the gel test system. Antibody titres performed using the gel system were more sensitive than with our tube IAT method. The gel system was easy to use and gave reliable, reproducible results. Antibody detection rates were enhanced compared with our existing routine techniques.