Table 1.

Standard work-up in the evaluation of a patient with suspected AA being considered for transplantation

Test	At diagnosis	Purpose	Notes	References15.19
Medical and family history assessment61,64,68,81,*
Heme Long-standing cytopenia(s) or macrocytosis?   Unexplained cytopenia(s) or macrocytosis, AA, MDS, or AML in 1 or more close relative(s)?	X	Could suggest IBMFD if chronic cytopenias at early age are found in the patient or cytopenias or blood cancers cluster in the family		61,68,80
Developmental Short stature, physical anomalies (especially thumb/radial ray, cardiac, or renal)?	X	FA, STS, DBA, or thrombocytopenia-absent radius
Immunologic/infectious disease Severe, recurrent, or atypical infections (eg, mycobacterial, viral, fungal)?	X	Could suggest GATA2 or other primary immunodeficiency	If present, consider referral to immunology and immunocompromised infectious disease specialists
Dermatologic Gray hair prior to 25? Leukoplakia or nail dysplasia? Reticulated skin pigmentation, café au lait macules?	X	Could suggest STS; Cafe au lait macules could suggest FA
Pulmonary Pulmonary fibrosis and/or early-onset emphysema, pulmonary alveolar proteinosis, fungal or mycobacterial infection?	X	Could suggest STS or GATA2
Abdominal Pancreatic insufficiency, liver fibrosis, renal anomaly or malplacement?	X	Could suggest SDS, STS, FA, respectively
Neurologic Ataxia, nystagmus? Cognitive dysfunction?	X	Could suggest SAMD9L, multiple IBMFD, respectively
Cardiac/lymphatic Cardiac anomaly, lymphedema?	X	Could suggest multiple IBMFD, GATA2, respectively
Oncologic H&N or anogenital SCC, early-onset GI cancers or multiple cancers in patient or close relatives?	X	Could suggest STS, Li Fraumeni, other hereditary cancer syndromes
Laboratory studies on peripheral blood
Complete blood count with differential and blood smear review	X	Assess severity of cytopenias and for alternative etiologies	Monocytopenia should prompt consideration of GATA2-deficiency syndrome. However, many severely neutropenic SAA and VSAA patients are also monocytopenic, so it is not specific to GATA2 deficiency.	81
Reticulocyte count	X	Assess marrow response to anemia and use in AA severity assessment		44
Percentage of hemoglobin F	X	Elevated levels can indicate that an IBMFD may be present.		82
Vitamin B12, folate, copper, zinc, ferritin	X	Rule out vitamin or mineral deficiencies as cause or contributor to cytopenias
Hepatitis A/B/C, HIV, EBV, parvovirus, and CMV serologies	X	Rule out infectious disease contributors to cytopenias and identify comanagement needs during treatment and transplant
LDH, haptoglobin	X	Rule out a hemolysis component to anemia
PNH clone	X	Assess presence or absence of GPI-anchored protein expression	Clone sizes vary (in monocytes and granulocytes) in acquired SAA, but larger clone sizes (>10%) are often associated with response to IST.	26,29,83
ANA with reflex to anti–double-stranded DNA if positive	X	Assess for systemic lupus erythematosus
Immunoglobulins A, G, and M quantification	X	Assess for additional immune deficits	Severe deficits and/or combination with B-, T-, or NK cell deficits may warrant work-up for GATA2-deficiency syndrome and/or autoimmunity/primary immunodeficiency disorders. Consider referral to immunologist.	81,84,85
Flow cytometry to assess B, T, and NK cell numbers	X	Assess for additional immune deficits	Severe or combination B-, T-, and NK cell deficits may warrant work-up for GATA2 deficiency syndrome and/or autoimmunity/primary immunodeficiency disorders. Consider referral to immunologist.	81,84,85
Telomere length measurement of peripheral blood lymphocytes by Flow-FISH	X (age ≤40 y or those proceeding to BMT)	Determine lymphocyte telomere length by Flow-FISH; if less than first percentile, patient may have an STS and be at risk for increased transplant-related toxicity with standard preparative regimens, and an STS-specific regimen should be considered.	Lymphocyte telomere lengths less than first percentile are highly sensitive and specific for an STS diagnosis in young patients with AA. Caveats: Other IBMFDs can have lengths less than or equal to the first percentile; individuals with pathogenic telomere gene mutations can have lengths in the normal range, usually between the first and tenth percentiles; and short telomeres can be seen in acquired AA with reduced stem cell reserve.	66-68,86
Chromosome breakage analysis on peripheral blood	X (age ≤40 y or those proceeding to BMT)	Evaluate for FA; if test is positive and consistent with a diagnosis of FA, patient is at increased risk for transplant-related toxicity, and an FA-specific regimen should be used.	If results are normal but clinical suspicion remains high, this test can be performed on cultured skin fibroblasts to rule out a false negative in the peripheral blood.	87
Bone marrow aspirate and biopsy	X	Assess cellularity, iron stores, and reticulin fibrosis and rule out other marrow pathologies.
Conventional karyotyping	X		Most often AA patients have normal cytogenetics but there are some cytogenetic abnormalities seen in AA that are not considered adverse or indicative of MDS (in the absence of dysplasia). These include del13q, trisomy 8, loss of heterozygosity of short arm of chromosome 6, among others. If <20 metaphases are obtained, perform an MDS FISH panel; a microarray can be considered as an alternative in these cases. Monosomy 7, especially in young patients, increases suspicion for an IBMFD.	40,88
HLA typing	X	Determine HLA profile for stem cell donor search.
Myeloid malignancy gene sequencing from peripheral blood or bone marrow	Strongly consider if any concern for possible hypoplastic MDS	Evaluate for mutations in genes recurrently mutated in AA (eg, PIGA, BCOR, BCORL1) and/or MDS (eg, epigenetic mutations, TP53).	Identification of mutations should not necessarily be used as a discriminating tool between AA and hypoplastic MDS, because most MDS-associated mutations (including BCOR, BCORL1, and epigenetic mutations, such as DNMT3A, TET2, ASXL1, and others) are seen in AA, MDS, and aging-related clonal hematopoiesis and have poor discriminating power for AA or MDS in this context. Acquired mutation panel may identify a subset of AA likely to progress to MDS/AML. A portion of the genes on these acquired panels overlap with inherited marrow failure gene panels but they should not be considered adequate testing for IBMFD as a stand-alone test.	52,56,89
Inherited BMF gene panel sequencing	Patients aged ≤40 y or if clinical picture or screening tests warrant	Evaluate for multiple IBMFDs at once. Overlapping phenotypes and lack of physical features and family history in a substantial subset of those with IBMFD makes universal testing of young patients warranted. Tissue source for this testing should ideally be cultured skin fibroblasts (see text for discussion).	Yield in adult patients with AA aged 18-40 y is 5-15%. Yield increases with the presence of phenotypic features or family history or in cases where hypoplastic MDS is a consideration (eg, monosomy 7).	13,14
Erythrocyte adenosine deaminase	If clinical picture warrants	Screen for Diamond-Blackfan anemia		90
Serum pancreatic isoamylase (age > 3 y)	If clinical picture warrants	Screen for pancreatic insufficiency suggestive of Shwachman-Diamond syndrome		91
Fecal elastase	If clinical picture warrants	Screen for pancreatic insufficiency suggestive of Shwachman-Diamond syndrome		91

AML, acute myeloid leukemia; ANA, anti-nuclear antibodies; CMV, cytomegalovirus; EBV, Epstein-Barr virus; GI, gastrointestinal; GPI, glycosylphosphatidylinositol; H&N, head and neck; LDH, lactate dehydrogenase; MDS, myelodysplastic syndrome; NK, natural killer; SCC, squamous cell carcinoma; VSAA, very severe AA.

^*These questions are intended to evaluate for signs and symptoms of the known IBMFD. This is not an all-encompassing list, but it does include the more common features that should prompt a more detailed evaluation for an IBMFD, additional specialized screening tests if indicated, and strong consideration of germline genetic testing via a panel approach per below.