7

Techniques for Hematological Disorders

Hematologic tests help diagnose diseases of the blood and bone marrow cells, such as anemia, infection, hemophilia, blood-clotting disorders, leukemia, lymphoma, and myeloma. Common hematology tests include the complete blood count (including red blood cells, white blood cells, platelet count, hemoglobin, hematocrit, red blood cell volume, differential white blood count, and other red blood cell indices), prothrombin time, partial thromboplastin time, and others. Bone marrow biopsies and aspirates are more specialized tests for diagnosing hematologic diseases. Blood and bone marrow diseases and treatments have serious health consequences and adverse effects on patient function and quality of life (NASEM, 2022).

Rapidly emerging scientific advances in molecular biology have enabled the development of newer diagnostic techniques that focus on individualized molecular diagnoses and targeted therapeutics, on flow cytometry to determine whether leukemia cells express the target protein for a particular targeted therapy, and on genetic testing for a particular gene mutation which can be targeted by a specific medication. This chapter provides information about select diagnostic techniques that have come into practice since 1990, focusing on those that show improvement over previous techniques or that are the first of their type such as, for example, genetic sequencing tests.

OVERVIEW OF SELECTED TECHNIQUES

Using the selection criteria discussed in Chapter 1, the committee focused its review on the hematologic techniques shown in Box 7-1. These

are techniques that assess molecular and physiologic functions. The committee notes that none of the hematological tests discussed in the chapter measure deficits in functioning; as diagnostic tests, they may identify disease or disorder that is likely to result in a physical impairment or loss of function, but the level of severity would have to be measured by other tests or metrics. The chapter discusses the evidence and information about the selected techniques and responds to the requested items (a)–(j) of the Statement of Task for each technique. Following those discussions, at the end of the chapter the committee highlights hematological tests that may become generally available in the next 5–10 years.

SELECTED DIAGNOSTIC TECHNIQUES FOR HEMATOLOGICAL DISORDERS

The advances in diagnostic techniques for assessing hematologic disorders shown in Box 7-1 are described below. The most significant leap has been in the area of genetic testing, which was in its infancy in 1990. In addition, advances in the use of antibodies for laboratory testing have also improved, which has affected a number of different tests. As noted below

under standard requirements for performing the selected tests, laboratories are approved under the Clinical Laboratory Improvement Amendments (CLIA). CLIA is a federal program to regulate laboratory testing, and it requires clinical laboratories to be certified by the Centers for Medicare & Medicaid Services before they can accept human samples for diagnostic testing (FDA, 2021).

Genetic Testing

As described in Chapter 3, genetic testing has become widely available for clinical diagnostic purposes, aiding clinicians in genetic counselling for families, monitoring affected patients, and determining treatment options. Over the past 30 years the genetic variants associated with many hematologic diseases have been identified. The committee discusses several of these genetic variants below.

The responses to the items in the statement of task are as follows:

1. Genetic testing is used to detect inherited variants in a patient’s DNA to help diagnose an inherited genetic disorder (such as hemoglobinopathy, Diamond-Blackfan anemia, Fanconi anemia, and many other inherited hematologic disorders). Genetic testing is also used to detect somatic (acquired) genetic variants in cancer, which helps to establish the diagnosis, determine the appropriate therapy to target those genetic changes, and monitor the efficacy of therapy by detecting minimal residual disease.
2. As discussed in Chapter 3, advances in molecular and genetic diagnostics have improved the detection of inherited and somatic diseases, such as the examples of hematological diseases identified above.
3. There are numerous examples of hematologic diseases for which genetic testing is used as part of the standard of care, including a few representative examples listed here. Genetic variants in hemoglobin genes are one example of germline genetic variants commonly detected in patients. Hemoglobinopathies are the most common single-gene disorders worldwide (occurring in over 330,000 births annually). Mutations in either alpha globin or beta globin genes can cause these diseases (Traeger-Synodinos and Harteveld, 2014). DNA sequencing analysis of the alpha or beta globin genes may be done in order to confirm or clarify the exact genetic variant and make a definitive diagnosis.

   Gene panel tests for hematological disorders include those that that search for inherited causes of neutropenia (low neutrophil count), thrombocytopenia (low platelet count), and hemolytic

anemia (low red blood cells). Children with possible Diamond-Blackfan anemia merit genetic testing via the sequencing of a panel of ribosomal genes that are known to cause this disorder. For patients presenting with pancytopenia (low white blood cell count, hemoglobin, and platelets), genetic testing for a panel of inherited bone marrow failure syndromes is commonly done in order to distinguish idiopathic aplastic anemia from inherited syndromes, since their treatment options may differ.

Finally, genetic testing for somatic (acquired) genetic variants is a standard part of the workup of many malignancies to help diagnose the type of cancer, provide guidance for prognosis, and inform treatment decisions. For example, identifying FMS‐like tyrosine kinase 3 mutations in acute myelogenous leukemia is generally part of a larger cancer-risk genetic panel.

4. Genetic testing became generally available in clinical practice around 2014 (Ee et al., 2015).
5. The committee is not aware of any disparities for these techniques aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. The new methods of gene sequencing are faster and in some cases able to detect genetic changes that were not possible to detect with the prior methods.
7. None of these methods assess deficits in functioning. They are useful only for diagnosis and disease monitoring.
8. Only CLIA-approved labs can perform these tests. A trained clinician should interpret the test, such as the patient’s clinical provider, genetic counselor, or a laboratory medicine provider who signs the test report.
9. There are minimal impediments to the widespread use of genetic testing techniques, as long as tissue or DNA samples can be shipped to specialized labs.
10. Assuming an optimal tissue sample, DNA sequencing tests done in CLIA-approved labs are highly accurate in identifying sequence variants. The accuracy, generally greater than 99 percent (Yin et al., 2021), is dependent on the tissue source, any possible DNA contamination from other cell types, and the coverage depth of sequencing for the specific test.

Phenotype Characterization

Immunophenotyping

Immunophenotyping is a widely used testing method for cell classification and diagnosis which uses antibodies targeted against certain antigens in specific tissues and cells to determine normal and malignant cell type and organ of origin (Magaki et al., 2019). By exploiting the specific binding of an antibody to its target antigen, laboratories can identify particular proteins expressed on the surface or inside cells from a patient blood or tissue sample. Immunohistochemistry is used to examine cells in tissue sections on microscope slides, and allows cells to be examined in the context of surrounding histologic tissues. Flow cytometry examines antibodies bound to cells in a liquid suspension, allowing the analysis of multiple markers simultaneously on each individual cell.

Immunohistochemistry

Immunohistochemistry (IHC) is a powerful technique that examines the binding between an antibody and antigen to detect and localize specific antigens in cells found in tissue sections on microscope slides. Unlike flow cytometry, IHC allows the cells of interest to be visualized in the context of the tissue as a whole, which provides useful diagnostic information. The particular immuno-phenotype as discovered by IHC is critical for making a diagnosis of lymphoma and other types of cancer, and it increasingly provides predictive and prognostic information. In addition, the expression of certain antigens determines whether a patient is eligible for immunotherapy treatments. For example, antibodies against programmed death-ligand 1 (PD-L1), CD19, and CD30 are being used as immunotherapy for lymphoma and other types of cancer (Cho, 2022). Expression of these proteins by the cancer must be verified before offering an immunotherapy treatment, as cancers that do not express the target antigen would not respond.

The responses to the items in the statement of task are as follows:

1. IHC is a diagnostic tool that can be used for a wide variety of lymphomas and other solid tumors and can be used to further define hematologic cancer cells (Herold and Mitra, 2022). It can also help support clinical diagnosis.
2. IHC has been expanded to assess predictive and prognostic biomarkers in many malignancies. This technique continues to provide advances every year as new biomarkers are identified and new antibodies are developed to identify them in tissues (Magaki et al., 2019).

3. The technique is used to assess predictive and prognostic biomarkers in many malignancies, including those related to the breast, gastrointestinal tract, lung, hematolymphoid, and central nervous system.
4. Clinical use of IHC has been around since the early 2000s.
5. The committee is not aware of any disparities for this techniques aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. Prior methods used cell and tissue staining along with light microscopy to examine cellular morphology in order to make a diagnosis. IHC expands on that method to examine the expression of specific proteins, thereby more accurately determining cell phenotypes, some of which were not possible to detect prior to the advent of this method.
7. These methods do not assess deficits in functioning. They are used for diagnosis and disease monitoring only.
8. Only CLIA-approved labs can perform these tests. Guidelines for the standardization and analytic validation of immunohistochemical tests have been established by the College of American Pathologists (Magaki, et al., 2019). These tests require the use of appropriate controls so that the test can be interpreted accurately by a trained pathologist (Magaki et al., 2020).
9. There are minimal impediments to widespread use of this technique, as long as tissue samples can be shipped to specialized labs.
10. IHC tests are extremely reliable when performed in a CLIA-approved laboratory with the appropriate control samples run simultaneously.

Flow Cytometry

Flow cytometry also takes advantage of the ability of an antibody to bind specifically to its target antigen. In this case, cocktails of antibodies, each labeled with a different fluorescent compound, are mixed with patient cells. This single cell suspension is then assessed by a flow cytometer to detect which cells emit the different fluorescent wavelengths, and the intensity of the fluorescence is correlated with the level of expression of the particular protein. The patterns of protein expression are used to identify and categorize the tagged cells. For example, this method is used to measure CD4+ T cell counts and other lymphocyte subsets when studying specific immunodeficiency disorders and immune-related diseases. Likewise flow cytometry is used to diagnose specific sub-types of leukemia (Weir and Borowitz, 2001).

The responses to the items in the statement of task are as follows:

1. Flow cytometry is a diagnostic tool that can be used for a wide variety of leukemias and immunodeficiency disorders and can be used to further define hematologic cancer cells (Herold and Mitra, 2022). It can also be used to monitor minimal residual disease in the bone marrow of leukemia patients.
2. Flow cytometry continues to expand the different predictive and prognostic biomarkers used for different malignancies. This technique continues to provide advances every year as new biomarkers are identified and new antibodies are developed to identify them in tissues (Magaki et al., 2019).
3. This method does not assess deficits in functioning and is only used for diagnostic purposes.
4. Clinical use of flow cytometry has been around since the 1980s (Picot et al., 2012).
5. The committee is not aware of any disparities for this technique aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. Prior methods used cell and tissue staining along with light microscopy to examine cellular morphology in order to make a diagnosis. Flow cytometry expands on that method to examine the expression of specific proteins, thereby more accurately determining cell phenotypes, some of which were not possible to detect prior to the advent of this method.
7. These methods do not assess deficits in functioning. They are used for diagnosis and disease monitoring only.
8. Only CLIA-approved labs can perform these tests. Guidelines for the standardization and analytic validation of flow cytometry tests have been established by the College of American Pathologists (Magaki, et al., 2019). These tests require the use of appropriate controls so that the test can be interpreted accurately by a trained pathologist (Magaki et al., 2020).
9. There are minimal impediments to the widespread use of this technique, as long as tissue samples can be shipped to specialized labs.
10. Flow cytometry tests are extremely reliable when performed in a CLIA-approved laboratory with the appropriate control samples run simultaneously.

Other Flow Cytometry Tests

Eosin-5-Maleimide Test for Hereditary Spherocytosis

Hereditary spherocytosis (HS) is clinically heterogeneous and characterized by mild to moderate hemolysis resulting from red cell membrane protein defects. Characteristic symptoms of HS are the destruction of red blood cells in the spleen and their removal from the blood stream (hemolytic anemia), a yellow tone to the skin (jaundice), and an enlarged spleen (splenomegaly). Symptoms can develop in infancy, but some people with HS have no symptoms or have minor symptoms and are diagnosed later in life. Diagnosis is confirmed based on blood tests. Surgical removal of the spleen (splenectomy) is recommended in the case of HS with severe anemia, as this effectively cures the patient of hemolytic anemia. Other treatments include folate supplementation and blood transfusions for patients with severe hemolysis.

People with HS may also have hemolytic, aplastic, and megaloblastic crises. Hemolytic crises are often triggered by a viral illness that causes an increased destruction of red blood cells. Blood transfusions may be needed, but hemolytic crises are typically mild. Aplastic crises are less common and more severe than hemolytic crises but are also triggered by viral illness (particularly parovirus B19).

The responses to the items in the statement of task are as follows:

1. The eosin-5-maleimide (EMA) test is used in the diagnosis of HS.
2. Previously, before the advent of the EMA test, diagnosis of HS was made using osmotic fragility testing. The osmotic fragility test assesses the resistance of red blood cells to hemolysis by subjecting cells to osmotic stress using various concentrations of sodium chloride solution. The test has good accuracy; however, the method is laborious and depends on those who administer it having well trained technical skills. The test is sensitive in detecting abnormalities only when at least 2 percent of the red blood cells are spherocytes. Thus, for milder or atypical forms of HS, the test may fail to show an abnormality (Chari and Prasad, 2018). There is a high degree of concurrence between osmotic fragility testing and the EMA binding test, but the latter demonstrated a higher sensitivity and specificity in screening for HS (Chari and Prasad, 2018). The EMA test takes advantage of flow cytometry technology to examine red blood cell membrane proteins, mainly band 3 protein. In patients with HS, the amount of the band 3 protein is markedly decreased in the red blood cells, and this is detected with great sensitivity using the EMA test.

3. The impairments that are more accurately assessed with this test are described above.
4. The test became generally available around 2015 (Christensen et al., 2015).
5. The committee is not aware of any disparities for this technique aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to this method. A flow cytometer may not be available in underresourced facilities (Chari and Prasad, 2018).
6. Compared to the previously used osmotic fragility tests, the EMA test is simple, cost effective, highly reproducible, and less labor intensive in the diagnosis of HS (Chari and Prasad, 2018).
7. This method does not assess deficits in functioning. It is used for diagnosis and disease-monitoring purposes only.
8. As for requirements, the EMA test must be performed by a CLIA-approved laboratory.
9. There are minimal impediments to the widespread use of this technique, as long as blood samples can be shipped to specialized labs.
10. A study comparing the sensitivity and specificity of HS screening tests reported that the EMA-binding test is the best screening test, but false negatives may occur. Careful clinical correlation is advised (Mothi et al., 2021).

GPI-Anchored Proteins in Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH), a rare condition, is an acquired hematopoietic stem cell disorder in which the affected stem cell clones are deficient in glycosylphosphatidyl-inositol (GPI)–anchored surface proteins. PNH is characterized clinically by bone marrow failure, thrombosis, and chronic hemolytic anemia.

The wide spectrum of clinical presentation and variable disease course provides challenges in establishing a diagnosis and managing patients. However, in the last 15 years advances have been made in understanding the molecular and cellular biology of PNH and in defining the molecular lesion responsible for the PNH abnormality (Richards et al., 2000).

Molecular biology techniques have uncovered the genotypic lesion in PNH, while the use of monoclonal antibodies and flow cytometry has made significant contributions in defining phenotypic abnormalities in PNH. Since 1985 flow cytometry has become established as a reliable diagnostic procedure for PNH and for measuring the extent of the PNH clone within various hematopoietic cell lineages.

As noted, there is a great degree of heterogeneity in the patterns and levels of expression of the GPI-linked proteins in the various cell types as well as possible heterogeneity in lineage. The different patterns of expression of GPI-linked proteins should be considered when using flow cytometry to diagnose PNH. Interpreting results in PNH is dependent on having a detailed knowledge of the cellular distribution of GPI-linked antigens and their expression at the different stages of hematopoietic cell differentiation (Richards et al., 2000).

The responses to the items in the statement of task are as follows:

1. Typically, analysis of GPI-linked proteins is used in the diagnosis of PNH.
2. Analysis of GPI-linked proteins on red cells and granulocytes by flow cytometry provides a specific and sensitive screening technique for diagnosing PNH and monitoring PNH clones. Prior to this test, the diagnosis was made based on the sucrose lysis test or the acidified serum test (Ham test), both of which are less accurate and more time consuming to perform.
3. As noted above, flow cytometry can more accurately diagnose PNH and measure the extent of the PNH clone within various hematopoietic cell lineages.
4. It has been possible to analyze GPI-linked proteins since 1985.
5. The committee is not aware of any disparities for this technique aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. Previously PNH was diagnosed using the sucrose lysis test and acidified serum lysis (i.e., the Ham test), both of which are now obsolete, having been replaced approximately 20 years ago by flow cytometry to test for GPI-linked proteins (CD55 and CD59) on the surface of blood cells.
7. These methods are used for diagnosis and disease-monitoring purposes only, and do not provide information about the severity of deficits in functioning.
8. For the diagnosis and monitoring of PNH, flow cytometry is the preferred method, and it must be performed by a CLIA-approved laboratory.
9. There are minimal impediments to widespread use of this technique, as long as blood samples can be shipped to specialized labs.
10. Concerning the known limitations of analyzing GPI-linked proteins, recent transfusions can decrease the sensitivity of this test and interfere with its accuracy (Mayo Clinic, 2023).

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) is a cytogenetic technique developed in the early 1980s. FISH uses fluorescent DNA probes to target specific chromosomal locations within the nucleus and produces colored signals that can be detected using a fluorescent microscope. FISH has taken on an increasingly important role in detecting specific biomarkers and genetic translocations in solid and hematologic neoplasms. It combines standard microscopic cytogenetic analysis with molecular methods and has been an important part of the developing field of personalized medicine. After its development in the 1980s, the applications of FISH have broadened to include more genetic diseases, hematologic malignancies, and solid tumors (Hu et al., 2014). Recent advances in FISH applications include both de novo discovery and the routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies (Hu et al., 2014).

The responses to the items in the statement of task are as follows:

1. FISH is used for both de novo discovery and routine detection of chromosomal rearrangements, amplifications, and deletions that are associated with the pathogenesis of various hematopoietic and non-hematopoietic malignancies. For example, the BCR–ABL fusion protein is known to drive the pathogenesis of chronic myelogenous leukemia and results from a chromosomal translocation t(9:22) that is readily detectable by FISH. The use of FISH to detect this translocation is critical for monitoring disease status, as this is more sensitive than standard chromosomal karyotyping.
2. FISH does not require cell culturing and can directly use fresh or paraffin-embedded interphase nuclei for a rapid evaluation (Hu et al., 2014).
3. FISH is used in the detection of multiple myeloma, myelodysplastic syndrome, and hematopoietic and non-hematopoietic malignancies (e.g., lung cancer, prostate cancer, breast cancer, melanoma) (Hu et al., 2014).
4. FISH was developed in the early 1980s and became widely available as a clinical test in the late 1980s (Hu et al., 2014).
5. The committee is not aware of any disparities for these techniques aside from generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. FISH analysis can be performed on more cells than standard cytogenetics and is thus able to detect small populations of neoplastic

cells (Sabath, 2004). FISH is considered the gold standard cytogenetic method for the detection of diseased or malignant cells containing chromosomal rearrangements or gene aberrations (Huber et al., 2018).

7. These methods do not assess deficits in functioning. They are used for diagnosis and disease-monitoring purposes only.
8. FISH must be performed by a CLIA-approved laboratory.
9. There are minimal impediments to the widespread use of this technique as long as tissue samples can be shipped to specialized labs.
10. This test is extremely reliable when performed in a CLIA-approved laboratory with the appropriate control samples run simultaneously. However, the ability of a FISH test to detect a particular genetic abnormality depends on the design of the DNA probe for that test. Only known genetic aberrations are detected, as long as the specific probe is available, and other genetic abnormalities would likely not be detected. (Bishop, 2010).

Functional Assays

Viscoelastic Hemostatic Assays

Viscoelastic hemostatic assays (VHAs) such as thromboelastograpy (TEG) and rotational thromboelastometry (ROTEM) are tests that quantitatively measure the ability of whole blood to form and dissolve a clot. Major bleeding is a serious medical complication which may be caused by external trauma, surgery, an invasive procedure, or an underlying medical condition such as an aneurysm rupture or peptic ulcer. Several congenital disorders associated with a coagulation factory deficiency such as von Willebrand disease or hemophilia A or B may cause significant bleeding even with minor injuries. Furthermore, prescribed anticoagulants and anti-platelet agents may create a coagulopathic state that may lead to excessive bleeding associated with trauma or medical procedures. Additionally, major acute blood loss can lead to coagulopathy due to a loss of coagulation factors. Patients with ongoing or expected major bleeding would benefit from an accurate assessment of the functional state of the hemostatic system so that their providers can provide optimal care (Shaydakov et al., 2022). Venous thromboembolism is another condition associated with abnormal blood coagulation. Several commonly used blood tests assess blood coagulation, including prothrombin time, international normalized ration, activated partial thromboplastin time, platelet count, fibrinogen concentration, and D-dimer. Those tests are used for the clinical diagnosis of coagulopathy, to monitor anticoagulation therapy, and to assist in treating bleeding episodes (Shaydakov et al., 2022).

The responses to the items in the statement of task are as follows:

1. VHAs are useful in determining a diagnosis of coagulopathy or prothombotic state. TEG and ROTEM are used in cardiovascular surgery, liver transplantation, and trauma care and for patients on continuous dialysis or cardiac extracorporeal membrane oxygenation. The tests are also used in patients with post-partum hemorrhage, gastrointestinal bleeding, and severe epistaxis and also in selected post-operative patients (Morcom, 2018). ROTEM has been used as a point-of-care test to optimize hemostatic resuscitation in trauma patients. In trauma, ROTEM parameters are used for treatment decision. ROTEM provides information on platelet function and fibrinogen reserve (Veigas et al., 2016).¹
2. VHAs have been promoted as an improvement over traditional coagulation tests in the management of trauma patients due to the quick turnaround time in receiving results and the comprehensive evaluation of clot formation and thrombolysis.
3. Some examples of specific impairments that are accurately assessed through VHAs are coagulopathy and prothrombotic state; the test is also valuable in the prediction of massive transfusion and in transfusion guidance (Veigas et al., 2016).
4. TEG was developed in 1948. Since that time TEG has evolved into a more commonly used noninvasive test that quantitatively measures the ability of the whole blood to form and dissolve a clot. In the 1980s, TEG was found to be useful in liver transplant patients, and in the 1990s it was shown to be useful in cardiac surgery (Shaydakov et al., 2022).
5. The committee is not aware of any known disparities for these techniques aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. Previously existing methods are used in conjunction with viscoelastic assays such as TEG and ROTEM and have not been replaced by them. The results of viscoelastic assays are available more quickly than standard coagulopathy tests such as prothrombin time and partial prothrombin time, so TEG and ROTEM are most useful when lab results are needed quickly for making treatment decisions.
7. These methods do not assess deficits in functioning. They are used for diagnosis and disease-monitoring purposes only.

___________________

¹ The FDA-approved patient populations for TEG and for ROTEM are not the same, and FDA has not currently approved some of the populations listed.

8. These tests must be performed by a CLIA-approved laboratory.
9. These tests are performed on site and may not be available at all institutions, although both TEG and ROTEM have been widely adopted across clinical sites.
10. There are no known limitations as long as the test is performed by a CLIA-certified lab.

Platelet Function Testing

Platelet function analysis (PFA-100) uses a microprocessor-controlled instrument/cartridge system (PFA-100^TM) designed to assess primary, platelet-related hemostasis in routinely collected whole blood. Platelet dysfunction is a potential cause of bleeding diathesis, especially in critically ill patients who may develop life-threatening hemorrhages. Additionally, the test is used to identify acetylsalicyclic acid–induced platelet dysfunction (Mammen et al., 1998).

The responses to the items in the statement of task are as follows:

1. Platelet function analysis is used for patients with bleeding symptoms in order to aid in the diagnosis of platelet dysfunction, or pre-operatively to identify bleeding risk prior to surgery.
2. Some of the advances associated with PFA-100 are that it only requires small volumes of blood, it can be used by non-skilled personnel, and it is rapid and automated.
3. The PFA-100 is used as a screening test as the result can be obtained quickly. When the test is abnormal, formal platelet aggregation studies are required to be performed by a skilled laboratory medicine technician in order to confirm the diagnosis.
4. Since the early 1970s, attempts have been made to find a device that would globally measure platelet-related primary hemostasis.
5. The committee is not aware of any disparities for this technique aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. The PFA-100 test examines platelet aggregation in response to collagen and adenosine diphosphate (ADP) or collagen and epinephrine. In contrast, formal platelet aggregation studies examine these and several other substances that activate platelets and the response of platelets to different concentrations of those substances.
7. These methods do not assess deficits in functioning. They are used for diagnosis and disease-monitoring purposes only.
8. PFA-100 must be performed by a CLIA-approved laboratory.

9. There are minimal impediments to widespread use of this technique, as long as blood samples can be shipped to specialized labs.
10. The PFA-100 is limited in its testing of only certain platelet agonists (specifically epinephrine and ADP) and therefore is generally used as a screening test. Formal platelet aggregation studies using light transmission aggregometry are generally done to confirm a positive result with the PFA-100 if a congenital platelet disorder is suspected (e.g., Bernard Soulier syndrome or Glanzmann’s thrombasthenia). Light transmission aggregometry tests a full panel of platelet agonists, including ADP, arachidonic acid, collagen, epinephrine, and high-and low-dose ristocetin, among others.

Anti-Phospholipid Antibody Testing

Anti-phospholipid antibodies (APLAs), auto-antibodies that activate the formation of abnormal blood clots, are usually found in people with autoimmune diseases. Antiphospholipid antibodies are usually made in the process of a normal immune response, when the immune system generates an antibody against phospholipids found in the cell membrane of cells lining blood vessels (called endothelial cells). Normally clots form when these endothelial cells are damaged. However, when these antibodies bind to the phospholipids on endothelial cells, they can activate the formation of an abnormal clot in the absence of damage.

The diagnosis of antiphospholipid syndrome relies on the detection of circulating APLAs alongside other clinical manifestations. APLA testing is done when patients have developed blood clots or have an unexpectedly prolonged partial thromboplastin time. The testing is useful in determining the cause of recurrent miscarriages or helping diagnose or evaluate an autoimmune disorder. APLAs are also called lupus anticoagulants as they are commonly found in patients who have been diagnosed with systemic lupus erythematosus; this name, however, is a misnomer as APLA are also found in people who do not have lupus and are actually pro-thrombotic, not anticoagulants.

People with recurrent venous thromboembolism or other abnormal clotting, repeated miscarriages, or autoimmune diseases such as systemic lupus erythematosus and multiple sclerosis often have antiphospholipid antibodies. In some cases, cancer patients also have those antibodies, which fade away when the cancer is treated. Antiphospholipid antibodies are associated with an increased risk of clotting and with the risk of recurrent miscarriages, premature labor, and pre-eclampsia. One or more antiphospholipid antibodies have been identified in people with autoimmune diseases (e.g., lupus, rheumatoid arthritis, systemic sclerosis), infections (e.g., HIV, mononucleosis, rubella), and cancers (e.g., solid tumors, leukemias,

lymphomas) as well as in individuals who have used certain drugs (e.g., procainamide, phenothiazines, oral contraceptives) (Labcorp, 2022).

The responses to the items in the statement of task are as follows:

1. The tests are used for people who have abnormal blood clots, repeated miscarriages, or autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, or systemic sclerosis.
2. Progress has been made on the standardization of tests and on guidelines for lupus anticoagulant testing and immunological assays for anticardiolipin and antibeta2-glycoprotein1 antibodies (Devreese et al., 2020).
3. This test does not evaluate for deficits in functioning, but rather aids in the diagnosis of antiphospholipid syndrome in patients with abnormal blood clots, repeated miscarriages, or autoimmune diseases.
4. The anticardiolipin antibody test, which is one type of antiphospholipid antibody test, has been widely used since the mid-1980s for diagnosing patients with antiphospholipid syndrome (Pierangeli, 2006).
5. The committee is not aware of any disparities for these techniques aside from the generally known disparities in health care. Ethnic minorities and people of low socioeconomic status who have impaired access to health care in general would hence also have less access to these methods.
6. Consensus guidelines and proposals for antiphospholipid antibodies testing have been published over the last 27 years or so, which has led to substantial improvement in the standardization of testing, which in turn has had a positive effect on test accuracy. (Devreese et al., 2020).
7. These methods do not assess deficits in functioning. They are used for diagnosis and disease-monitoring purposes only.
8. Lupus anticoagulants are identified using plasma-based clotting assays, and solid-phase antibodies are identified using enzyme immunoassays and include anticardiolipin antibodies, antibodies to β2-glycoprotein 1 (β2GP1), and other antibodies. In order to determine the presence of antiphospholipid syndrome, both lupus anticoagulants and immunoassays for anticardiolipin and β2GP1 antibodies should be performed (Labcorp, 2022). These tests must be performed by a CLIA-approved laboratory.
9. There are minimal impediments to widespread use of this technique, as long as blood samples can be shipped to specialized labs.
10. Anticoagulant therapies may produce false-positive lupus anticoagulants results (Labcorp, 2022).

EMERGING DIAGNOSTIC TECHNIQUES

In the upcoming years breakthroughs in genetic sequencing tests are expected to continue to improve the diagnosis and assessment of hematological disorders. As more genetic syndromes are identified, these tests will continue to become more accurate in their diagnoses. In addition, the technology to perform sequencing tests more quickly and with smaller samples will continue to improve. Finally, tests are currently being developed to sequence cell-free DNA in the blood as a mechanism to monitor for genetic mutations found in cancer cells throughout the body. Currently these cell-free DNA tests are being used in the research setting but are expected to affect diagnosis and disease monitoring in such a way as to enhance the clinical care of patients in the future.

REFERENCES