Sullivan Nicolaides Pathology

Obstetrics and gynaecology

Clinical Articles

Fetal Fraction – The Essential Factor in Non-Invasive Prenatal Testing

Non-Invasive Prenatal Testing (NIPT), the cell-free DNA-based blood test that screens for fetal chromosomal abnormalities, is fast becoming a routine part of obstetric care. NIPT at a glance During pregnancy, maternal plasma contains fragments of DNA from the mother and from the placenta (fetal DNA). The proportion of DNA fragments from particular chromosomes is usually very stable throughout pregnancy. If there is an excess of fetal fragments from one chromosome, the proportion of fragments from that chromosome will be changed. Inconclusive tests A key reason that NIPTs should precisely measures the amount of fetal DNA in the sample – the fetal fraction – is if there is insufficient fetal DNA, the result may merely reflect the genetic status of the mother. NIPT assays should report a result only if there is sufficient fetal DNA to be confident of accuracy. Rarely, a test for trisomy 21,18 and 13 cannot be reported. This occurs in 3% of women tested by Sonic Genetics and is usually because there is insufficient fetal DNA compared with maternal DNA in the mother’s plasma. This low fetal fraction can be due to a relative excess of maternal DNA and this can vary over time. It is more common in women with increased body weight, and more likely in the presence of infection and inflammation, or after exercise. It also occurs if the mother or fetus has some subtle benign variations in chromosome structure (copy number variants) that make estimating the proportion of fragments from a chromosome unreliable. In some instances, the DNA in the sample has degraded during collection and shipping to the laboratory, and the quality is insufficient for a reliable result. These factors interfere with quality control of the test. Two thirds of women will get a result on re-testing. However, if the second test is inconclusive, it should not be repeated. This occurs in 1% of pregnant women screened. It is also not worth using another form of non-invasive prenatal test. Other tests do not estimate the fetal fraction accurately and may provide false reassurance. A decision about other test modalities (combined first trimester screen, second trimester serum screen, detailed ultrasonography or invasive genetic testing such as CVS/amniocentesis) should be based on assessment of all identified risk factors and may require specialist consultation. More rarely (in 0.5 –1% of women) the test reports a result for trisomy 21, 18 and 13 but not for fetal gender and sex chromosome abnormalities. It is unlikely that a repeat test will provide a result. A decision about using fetal ultrasound or invasive genetic testing to document fetal gender should be based on assessment of need and any identified risk factors. General Practice Pathology is a new regular column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial, free of charge as part of an educational initiative developed and coordinated by Sonic Pathology.

Genetics

Clinical Articles

Prenatal Screening for Chromosome Disorders

Prenatal screening for chromosome disorders by maternal serum screening, ultrasound and non-invasive prenatal tests, such as Harmony®, is an established part of reproductive care in Australia. The overall risk of chromosome disorders rises markedly with maternal age, as shown in Figure 1. (There are two exceptions: Monosomy X, also known as Turner syndrome, and microdeletions, such as 22q11.2, occur independently of maternal age). This does not mean that chromosome screening should be restricted to older mothers. Younger mothers have more babies than older mothers, and the overall outcome is that the majority of pregnancies with a serious chromosome disorder occur in mothers under 35 years of age. For this reason, screening for chromosome disorders in pregnancy should be offered to mothers of all ages. The great majority of these chromosome disorders are new abnormalities that have happened for the first time in this pregnancy. They are not inherited disorders, and genetic testing of the parents provides no information about the risk of such an abnormality. This provides another reason for offering screening for chromosome disorders to all mothers, irrespective of family history.

The frequency of single-gene disorders at birth

Chromosome disorders are not the only type of genetic condition which can affect the developing foetus. Many serious childhood disorders are due to recessive mutations that have been inherited from parents, with the parents being unaffected by these mutations. A parent who is a carrier of a recessive mutation, that is, having one normal and one abnormal copy of a gene, will not be affected by the abnormal gene. Everyone is a carrier for one or more disorders; this is of no immediate consequence and there usually is no family history of the disorder. The situation changes if both parents are carriers of mutations in the same gene located on one of the autosomes (chromosomes 1-22). The chance of their child inheriting the abnormal gene from each parent, and so developing an autosomal recessive disorder, is 25%. The situation is a little different for a woman with a recessive mutation on an X-chromosome: each of her sons is at 50% risk of inheriting the abnormal gene and being affected, and half of her daughters will be carriers. Overall, the risk of a woman who is an X-linked carrier having an affected child is approximately 25%. There are hundreds of inherited autosomal and X-linked recessive disorders that present in infancy and early childhood. These disorders are individually rare but, together, they are more common than the chromosome disorders for which prenatal screening is widely available and accepted. Further, the risk of these recessive disorders does not vary with maternal age (Figure 1). For mothers under 35 years of age, the risk of having a child with a serious childhood-onset recessive disorder is greater than the risk of having a child with a chromosome disorder.

Screening potential parents for recessive disorders

These disorders are inherited but there is usually no family history to provide a clue. Until recently, the only way of identifying a carrier of a rare recessive disorder was to diagnose the disorder in their affected child. This has now changed. It is possible to screen a couple for mutations in autosomal genes, and a woman for mutations in X-linked genes, to determine whether they are at 25% risk of having an affected child. This screening test is called ’reproductive carrier screening’. From both a technical and clinical perspective, the challenge lies in choosing which genes to analyse. A number of providers, including Sonic Genetics, offer reproductive carrier screening for mutations responsible for three common disorders: cystic fibrosis and spinal muscular atrophy (both autosomal recessive) and Fragile X syndrome (X-linked recessive). Approximately 6% of people are carriers of one or more of these conditions, and 0.6% (one in 160) couples are at 25% risk of having an affected child. Those couples who are identified as carriers can consider a variety of options, including IVF with a donor gamete, pre-implantation genetic diagnosis, prenatal diagnosis by CVS, or they may make an informed decision to accept the risk. RANZCOG recommends that couples be offered such screening. The cost of this three-gene panel is approximately $400* per person. There is no Medicare rebate for carrier screening; there are exceptions (and restrictions) for people with a documented family history of cystic fibrosis or Fragile X syndrome.

Expanded reproductive screening

If we were to screen more genes, we would identify more carriers. Sonic Genetics offers a screen of over 300 genes (autosomal and X-linked) which cause serious recessive childhood disorders. We estimate that approximately 70% of Australians are carriers for one or more conditions included in this screen and 3% (one in 30) couples are at 25% risk of having an affected child. This amounts to five times more information than is provided by the three-gene panel. This screen, the Beacon Expanded Carrier Screen, currently costs $995* per person or $1,750* for couples tested together. It is tempting to think that ‘more genes tested = more information for a couple’. This is not the case because the information provided by a carrier screen is also determined by the carrier frequency, mode of inheritance and detection rate of the assay for each gene. Some currently available screens of more than 100 genes provide less information than the three-gene screen described earlier.

Implementing reproductive screening

Before offering reproductive carrier screening to your patients, it is important to consider some of the nuances, particularly in relation to the Fragile X syndrome (some carriers will develop premature ovarian failure or a tremor/ataxia syndrome in later life) and when there is a family history of a recessive disorder (seek expert advice; do not rely on screening). It is also important to recognise that some couples will not want this carrier information – and others will demand it. Each person needs to be free to make their own decision about what information they wish to have. We provide information about the three-gene and Beacon screens for both requestors and patients on our website. Sonic Genetics also offers genetic counselling free-of-charge for couples who are identified by either of these reproductive carrier screens as being at high risk of having an affected child (see www.sonicgenetics.com.au/rcs/gc).

Conclusion

It is accepted practice that every woman is offered screening for chromosome disorders in pregnancy, irrespective of age and family history. In a similar vein, every couple should be offered reproductive carrier screening for recessive disorders, irrespective of age and family history. For women under 35 years, the risk of their child having a recessive disorder is greater than the risk of a chromosome disorder. Offering reproductive carrier screening simply represents good medical practice.

References

RANZCOG. Prenatal screening and diagnostic testing for fetal chromosomal and genetic conditions. 2018 Aug. 35 p. Available from: https://www.ranzcog.edu.au/RANZCOG_SITE/media/RANZCOG-MEDIA/Women%27s%20Health/Statement%20and%20guidelines/Clinical-Obstetrics/Prenatal-screening.pdf?ext=.pdf Archibald AD, Smith MJ, Burgess T, Scarff KL, Elliott J, Hunt CE, et al. Reproductive genetic carrier screening for cystic fibrosis, fragile X syndrome, and spinal muscular atrophy in Australia: outcomes of 12,000 tests. Genet Med. 2018; 20(5): 513-523 Available from https://www.ncbi.nlm.nih.gov/pubmed/29261177 doi:10.1038/gim.2017.134. Sonic Genetics [Internet]. c2015. Reproductive Carrier Screening; 2018. Available from: www.sonicgenetics.com.au/rcs General Practice Pathology is a new regular column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial, free of charge as part of an educational initiative developed and coordinated by Sonic Pathology.

Pathology

Clinical Articles

Infection Risk in Asplenia and Hyposplenism

It is estimated that up to 25,000 Australians are affected by asplenia or hyposplenism.¹ Many are unaware of the fact, and its potential consequences. The spleen plays an important role in immune function, in particular the prevention of infection due to some specific organisms (Table 1).

Infection Risk

Infection is a relatively common occurrence in those without a functioning spleen. Overwhelming post-splenectomy infection (OPSI), occurs in up to 5% of asplenic patients and has a mortality rate of over 50%. The risk is particularly high in children aged under five, and in the first three years post-splenectomy. However, the risk is lifelong.¹

Organisms of Concern

Table 1: Organisms of Concern

Agent	Comment
Streptococcus pneumoniae	Accounts for >50% of severe infections. Vaccine available and recommended
Neiserria meningitidis	Vaccine available and recommended.
Haemophilus influenza type B	Vaccine available and recommended.
Capnocytophagia species	Oral flora in animals. Risk of acquisition after animal bites.
Bordetella holmesii	Newly recognised pathogen.
Plasmodia species (Malaria) Babesia, Ehrlichia	Potential risk for travellers. Seek pre-travel advice.

Causes of Asplenia and Hyposplenism

Asplenia maybe congenital but is more often acquired as a result of trauma or the surgical removal of the spleen due to haematological conditions, or after incidental splenic damage incurred during intra-abdominal surgery. Functional hyposplenism also confers an increased risk of infection and may occur as a result of a number of medical conditions (Table 2). Table 2: Medical conditions associated with hyposplenism

Coeliac disease

SLE

Sickle Cell disease

Rheumatoid arthritis

Malignant infiltration e.g. lymphoma

Splenic infarction or radiation

Graft versus host disease

Detection of Asplenia and Hyposplenism

The presence of Howell-Jolly bodies in a blood film may be a clue to the presence of unrecognised asplenia or hyposplenism. Other investigations that may be of assistance in suspected cases are imaging studies such as ultrasound or CT.

Prevention of Infection

Evidence suggests it is possible to significantly decrease the incidence of infection in asplenic and or hyposplenic patients. Spleen Australia has recently demonstrated a 69% reduction in serious infections in patients on their registry.² The key strategies utilised by Spleen Australia include; 1. Education

Informing patients and their families of the risk of infection, signs and symptoms of sepsis and the need to develop a management plan should these occur:
- Importance of seeking urgent medical attention if symptomatic.
- Maintaining a standby supply of antibiotics for emergency use.
Provision of advice regarding travel and other potential exposure risks e.g. animal contact.
Encouraging the wearing of a Medical Alert bracelet.

2. Provision of advice regarding appropriate antibiotic therapy (as per Therapeutic Guidelines)

Consider antibiotic prophylaxis, particular in first three years post-splenectomy (With either penicillin or roxithromycin).
Maintain a standby emergency supply of antibiotics in case of sepsis (usually Amoxil 3g).

3. Provision of current, detailed, practical guidelines for vaccination (As per Immunisation Handbook)

Pneumococcal, Meningococcal, Haemophilus influenza type B vaccines - initial course and ongoing boosters as required.
Annual influenza vaccine - to minimise the chance of post-influenza bacterial infection.

Currently, persons resident in Victoria, Queensland and Tasmania are able to register with Spleen Australia and will then receive regular newsletters and reminders when vaccines are due. It is hoped that this service will be extended to other states. See www.spleen.org.au for details.

Key Messages

Infection is a significant, life-long risk in asplenic and hyposplenic patients.
The risks can be mitigated by:
- The early recognition of the underlying condition,
- Comprehensive patient education,
- Appropriate use of use of prophylactic and empirical antibiotics, and
- Ensuring that patients receive recommended initial and ongoing vaccinations.

Spleen Australia provides an excellent range of resources and is happy to assist in the management of these patients if required.

References

Spleen Australia. Welcome to Spleen Australia: a clinical service and registry for people with a non-functioning spleen. Melbourne VIC: Diabetes Australia. Available from: www.spleen.org.au
Arnott A, Jones P, Franklin LJ, Spelman D, Leder K, Cheng AC. A Registry for Patients With Asplenia/Hyposplenism Reduces the Risk of Infections With Encapsulated Organisms. Clin Infect Dis. 2018 Aug 1; 67(4): 557-61. Available from: https://doi.org/10.1093/cid/ciy141

General Practice Pathology is a new regular column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial, free of charge as part of an educational initiative developed and coordinated by Sonic Pathology.

Immunology and allergy

Clinical Articles

Guide to Allergy Testing

Allergic disorders result from an inappropriate, usually IgE-mediated, immune response upon exposure to either environmental or food allergens. Common manifestations of allergy include rhinoconjunctivitis, asthma, eczema, acute urticaria and anaphylaxis. Disorders, such as chronic urticaria, hereditary angioedema and T-cell contact dermatitis (metal allergy), while clinically similar in some ways, are not IgE-mediated. Allergic disease manifests in different ways through life and the likely causative agents can also change with age (see Table 1).

Tests used in the diagnosis of IgE-mediated allergy

Total IgE Higher levels of total IgE are often found in patients with allergic conditions. However, normal total IgE does not exclude allergy. Total IgE is also elevated in other conditions including parasitic infections and allergic bronchopulmonary aspergillosis. It is used increasingly in determining anti-IgE therapy in moderate to severe asthmatics. Allergen-specific IgE Allergen-specific IgE can be detected for a large variety of allergens. The presence of a specific IgE to allergen can suggest allergic disease and is detected via a blood test (RAST or radioallergosorbent test) or skin prick test. RAST tests detect many of the different proteins within an individual allergen. Recombinant allergen testing Of the many proteins within a substance, only a few may cause allergic symptoms. Recombinant allergen testing looks for specific characterised protein within an allergen. Interpretation of RAST tests The presence of detectable specific IgE to an allergen does not confirm the patient is allergic to that substance. All results must be interpreted in conjunction with the clinical history of the patient. Low levels of detectable specific IgE can confirm the presence of allergy in the right clinical context. RAST testing aids in the assessment of, and identification of allergic sensitisation, but is not to be used alone as the deciding factor for inclusion or exclusion of allergy. As the level of specific IgE increases, the likelihood of clinical relevance also increases. As shown in Table 2, different allergens have different specific IgE level cutoffs at which serious allergy is >95% likely (positive predictive value or PPV). The range of values is vastly different between allergens and is affected by age and also by geographic region. Table 2 defines levels at which exposure, or a challenge, would be highly hazardous for a patient. Importantly, many patients could have serious reactions at much lower levels. [table id=1 /] [table id=2 /]

RAST tests

RAST tests are available for a range of allergens, however Medicare criteria limits rebates based on the number, type and frequency of tests. Medicare Australia limits rebates for RAST tests to a maximum of four specific allergens and/or mixes per pathology request and a maximum of four RAST test episodes per year. When ordering RAST tests, it is advisable to include allergens the patient feels are relevant and those likely for the clinical scenario. For common clinical scenarios we recommend the following: Childhood eczema Age <2 years: Milk, Egg, Wheat, Peanut Age >2 years: Milk, Egg, Peanut, Dust mite Additional allergens or an extended RAST combined allergy panel may be ordered. Asthma and allergic rhinoconjunctivitis Dust mite, Grass mix, Animal dander Additional allergens may be ordered or substituted if relevant (e.g. cat dander instead of animal dander). An extended RAST inhalant panel is also available. Default panel if no allergens are specified and no clinical notes are provided Age <5 years: Dust mite, Grass mix, Food Mix Age >5 years: Dust mite, Grass mix, Animal Mix Anaphylaxis Anaphylaxis is a severe life-threatening allergic reaction. It is recommended these patients require specialist assessment by a clinical immunologist or allergist. Initial testing should look for the causative allergen if possible. It is important to note that a negative RAST test does not exclude the allergen tested. RAST testing recommendations

Test individual likely causative allergen i.e. food, stinging insect.
Tryptase, if done within 2-6 hours of reaction, can support the occurrence of an allergic reaction.
Useful as an assessment of mastocytosis (condition with increased numbers of mast cells)

Extended RAST panels

Extended RAST panels have been developed to represent the common allergens encountered clinically in practice. They are particularly relevant in our geographic region and replace the skin prick test panel which is no longer available. Additional allergens may also be requested. All results must be interpreted in conjunction with the patient’s clinical history. Extended RAST Food Panel

Covers common food-related allergens
Almond; Hazelnut; Sesame seed; Banana; Mango; Shrimp (prawn); Cashew; Milk (cow); Soybean; Codfish; Peanut; Walnut; Egg white; Rice; Wheat

Extended RAST Nut Allergy Panel

Broad collection of commonly consumed nuts, including peanuts
Individual nut testing with appropriate clinical history is preferred
Recommend to discuss results with a clinical immunologist or allergist
Almond; Macadamia; Pine nut; Brazil; Peanut; Sesame seed; Cashew; Peanut (Ara-h2); Walnut; Hazelnut; Pecan

Extended RAST Combined Allergy Panel

Combination of common food and environmental allergens
Replaces the skin prick test panel (no longer available)
Almond; Dust mite; Mould mix; Cashew nut; Egg white; Peanut; Cat dander; Grass mix; Shrimp (prawn); Codfish; Hazelnut; Soy; Dog dander; Milk (cow); Wheat
* Preferable for children (<12 years) due to low serum volume

Extended RAST Inhalant Panel

Covers common environmental allergens
Useful for asthma and allergic rhinitis
Acacia (wattle); Blomia tropicalis; Dust mite; Alternaria alternate; Cat dander; Eucalyptus; Aspergillus fumigatus; Cladosporium; Horse dander; Bahia grass; Common ragweed; Johnson grass; Bermuda grass; Dog dander; Perennial rye grass

Recombinant allergens

Omega-5 gliadin

A component of wheat
Associated with anaphylaxis
Often in the context of eating wheat and physical activity within 1-2 hours

Alpha-gal (mammalian meat allergy) 

Associated with anaphylaxis; often delayed following consumption of meat (beef, lamb, pork)
Related to tick bites

Peanut Allergy Risk Assessment

Peanuts like all food is made up of many different proteins
Ara-h2 is associated with anaphylaxis to peanut
Can assist with risk assessment and should be done in conjunction with a clinical immunologist or allergist
A negative Ara-h2 in peanut positive patient does not imply there is no risk to anaphylaxis
Results of RAST tests can also be of use in monitoring ongoing allergy in patients in conjunction with their treating clinician

How to order allergy tests

RAST tests - standard panels Medicare Australia limits rebates for RAST tests to a maximum of four specific allergens and/or mixes per pathology request and a maximum of four RAST test episodes per year. Extended RAST tests (Medicare rebate + $120* per panel)

Extended RAST Food
Extended RAST Nut
Extended RAST Combined
Extended RAST Inhalant

Please note, extended RAST panels are not bulk billed. Recombinant allergen tests (Medicare rebate + $60* each)

Alpha-gal
Omega-5 gliadin
Peanut (Ara-h2)
Peanut Allergy Risk Assessment

General Practice Pathology is a regular column each authored by an Australian expert pathologist on a topic of particular relevance and interest to practising GPs. The authors provide this editorial free of charge as part of an educational initiative developed and coordinated by Sonic Pathology.

Sullivan Nicolaides Pathology